WO2025049305A1

WO2025049305A1 - Viral strain serology assays

Info

Publication number: WO2025049305A1
Application number: PCT/US2024/043635
Authority: WO
Inventors: Jacob N. Wohlstadter; George Sigal; Jeffery Debad; Hans Biebuyck; Priscilla KRAI; Alan Kishbaugh; Leonid DZANTIEV; Christopher SHELBURNE; Seth B. Harkins; John Fulkerson; Jocelyn Jean JAKUBIK; Daniel Douek; Sandeep NARPALA; Ananda CHOWDHURY; Alicen SPAULDING; Leonid SEREBRYANNYY; Adrian Mcdermott; Sarah O'connell
Original assignee: Meso Scale Technologies, Llc.; The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
Priority date: 2023-08-25
Filing date: 2024-08-23
Publication date: 2025-03-06

Abstract

The invention relates to methods and kits for determining a SARS-CoV-2 strain in a sample. The invention also provides methods and kits for detecting a single nucleotide polymorphism (SNP) in a target nucleic acid, wherein the target nucleic acid is a SARS-CoV-2 nucleic acid. The invention further provides methods and kits for detecting one or more antibody biomarkers in a sample.

Description

VIRAL STRAIN SEROLOGY ASSAYS

INCORPORATION BY REFERENCE

[0001] Reference is made to U.S. Publication No. 2022/0003766; U.S. Publication No. 2021/0349104; PCT Publication No. WO 2021/222827; PCT Publication No. WO 2021/222830; PCT Publication No. WO 2021/222832; U.S. Publication No. 2022/0404360; U.S. Publication No. 2022/0381780; PCT Publication No. WO 2022/246213; PCT Publication No. WO 2022/246215; and International Application No. PCT/US2023/068614, the contents of each of which is incorporated by reference in its entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said Sequence Listing XML, created on August 5, 2022. is named 0076-0031PR33. xml and is 975,567 bytes in size.

FIELD OF THE INVENTION

[0003] The invention relates to methods and kits for determining a SARS-CoV-2 strain in a sample. The invention also provides methods and kits for detecting a single nucleotide polymorphism (SNP) in a target nucleic acid, wherein the target nucleic acid is a SARS-CoV-2 nucleic acid. The invention further provides methods and kits for detecting one or more antibody biomarkers in a sample.

BACKGROUND

[0004] Respiratory viruses, including coronaviruses, can cause outbreaks of severe respiratory illnesses that place great burden on communities and healthcare systems. During an outbreak, large-scale tests arc needed to identify infected but asy mptomatic or mildly ill individuals, which can mitigate widespread disease transmission.

[0005] The COVID-19 pandemic created an urgent need for assays for multiple reasons, for example; to detect infection, to determine the stage of infection, e.g., viral load, to determine transmissibility of the virus, to determine presence or absence of virus, e.g., on surfaces, to aid in the development of vaccines, for epidemiological studies, to follow the immune status and past viral exposure of individuals, for research into factors contributing to morbidity and mortality of viral infection. Although some assays were developed early in the pandemic, they were slow or low throughput, lacked sensitivity, were inaccurate, were expensive, or otherwise inadequate. For example, current PCR-based tests, e.g., for SARS-CoV-2, are analytically sensitive but require a lengthy, complex, and expensive sample processing procedure, and may be difficult to run at the scale needed to screen large populations. Moreover, accurate and sensitive serologytests can be useful for epidemiological studies and to identify individuals who are immune or at low risk of infection. Thus, high-quality assays are desperately needed to address the pandemic.

SUMMARY OF THE INVENTION

[0006] In embodiments, the invention provides a method for determining a SARS-CoV-2 strain in a sample, comprising: (a) detecting at least a first antibody biomarker in the sample that binds to an antigen, e.g., an S protein, N protein, and/or S-RBD, from a first SARS-CoV-2 strain and at least a second antibody biomarker in tire sample that binds to an antigen, e.g.. an S protein, N protein, and/or S-RBD, from a second SARS-CoV-2 strain, wherein the detecting comprises contacting the sample with a surface comprising one or more binding domains, wherein the S pro tern from the first SARS-CoV-2 strain is immobilized on a first binding domain, and the S protein from the second SARS-CoV-2 strain is immobilized on a second binding domain; and (b) determining a ratio of the first antibody biomarker to the second antibody biomarker, thereby determining the SARS-CoV-2 strain. In embodiments, the detecting comprises forming a binding complex in each binding domain that comprises an antibody biomarker and the antigen, e.g., the S protein, N protein, or S-RBD; contacting the binding complex in each binding domain with a detection reagent; and measuring concentration of the antibody biomarker in each binding complex.

[0007] In embodiments, the invention provides method for detecting a single nucleotide polymorphism (SNP) in a target nucleic acid, wherein tire target nucleic acid is a SARS-CoV-2 nucleic acid, comprising: (a) contacting a sample comprising the target nucleic acid with (i) a targeting probe, wherein tire targeting probe comprises a first region complementary to a polymorphic site of the target nucleic acid that comprises die SNP, and wherein the targeting probe comprises an oligonucleotide tag; and (ii) a detection probe, wherein the detection probe comprises a second region complementary' to an adjacent region of the target nucleic acid comprising die polymorphic site, and wherein the detection probe comprises a detectable label; (b) hybridizing the targeting and detection probes to the target nucleic acid; (c) ligating the targeting and detection probes that hybridize w ith perfect complementarity at the polymorphic site to form a ligated target complement comprising die oligonucleotide tag and the detectable label; (d) contacting die product of (c) with a surface comprising an immobilized binding reagent, wherein the binding reagent comprises an oligonucleotide complementary to the oligonucleotide tag; (e) forming a binding complex on die surface, wherein the binding complex comprises the binding reagent and the ligated target complement; and (I) detecting the binding complex, thereby detecting the SNP at the polymorphic site.

10008| In embodiments, the invention provides a kit for detecting one or more antibody biomarkers of interest in a sample, the kit comprising, in one or more vials, containers, or compartments: (a) a surface comprising one or more binding domains, wherein each binding domain comprises an antigen immobilized thereon; and (b) one or more detection reagents, wherein each detection reagent comprises a detection antibody, a detection antigen, or an ACE detection reagent.

[0009] In embodiments, the invention provides a method of detecting one or more antibody biomarkers of interest in a sample, comprising: (a) contacting the sample with a surface comprising one or more binding domains, wherein each binding domain comprises an antigen immobilized thereon; (b) forming a binding complex in each binding domain, wherein the binding complex comprises the antigen and an antibody biomarker that binds to the antigen; (c) contacting the binding complex in each binding domain with a detection reagent; and (d) detecting the binding complexes on the surface, thereby detecting the one or more antibody biomarkers in the sample.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The following drawings form part of the present specification and are included to further demonstrate exemplary' embodiments of certain aspects of the present invention. [0011] FIGS. 1A-1C show SARS-CoV-2 strains and associated single nucleotide polymorphisms (SNPs) as described in embodiments herein. FIG. 1 A shows SARS-CoV-2 SNPs. the corresponding amino acid change, and strains associated with the SNPs. FIG. IB shows mutations of SARS-CoV-2 Spike (S) and nucleocapsid (N) proteins and strains associated with the mutations. FIG. 1C shows additional SARS-CoV- 2 SNPs and the corresponding amino acid changes.

[0012] FIG. 2 relates to Example 1 . FIG. 2 shows the results of an embodiment of a bridging serology assay described herein. SARS-CoV-2 S-RBD was immobilized as binding reagent, and labeled S-RBD was used as detection reagent. The bridging serology assay was tested on serum samples from COVID-19 positive (red circles) and normal (non-COVID-19) (blue circles) patients, diluted 10-fold or 100-fold. Higher signal indicates increased number of antibodies bound to the immobilized antigen.

[0013] FIG. 3 relates to Example 2. FIG. 3 shows the results of an embodiment of a neutralization serology assay described herein. SARS-CoV-2 S protein was immobilized as binding reagent, and labeled ACE2 was added as a competitor to SARS-CoV-2 antibodies that may be present. The neutralization serology assay was tested on scrum samples from COVID-19 positive (red circles) and normal (non- COVID-19) (blue circles) patients, diluted 10-fold or 100-fold. Lower signal (generated by competitor) indicates increased number of antibodies bound to the immobilized antigen.

[0014] FIGS. 4A-4D illustrate an embodiment of the methods described herein for detecting a single nucleotide polymorphism (SNP) in a viral nucleic acid. In FIGS. 4A-4C, a target nucleic acid (1) that comprises an SNP (2) is contacted with: a targeting probe (3) that comprises an oligonucleotide tag (4) and a sequence that is complementary to the SNP, and a detection probe (5) that comprises detectable label (6). The targeting and detection probes (3, 5) hybridize to the target nucleic acid, and the targeting and detection probes that hybridize with perfect complementarity at the SNP are ligated to form a ligated target complement (11) comprising the oligonucleotide tag and detectable label. The reaction mixture containing the ligated target complement is contacted with a surface comprising one or more binding reagents (7) immobilized in one or more binding domains (9). A signal (10) is detected if the ligated target complement is immobilized on the surface via hybridization of the complementary oligonucleotides in the oligonucleotide tag and the binding reagent. In FIG. 4D, the targeting probe has a mismatch with the SNP in the target nucleic acid, and thus, hybridization and ligation do not occur.

[0015] FIG. 5 illustrates an embodiment of the methods described herein for detecting a viral nucleic acid. RNA is extracted from a sample containing an RNA virus (e.g.. SARS-CoV-2), and the extracted RNA is converted to cDNA. A "Master Mix" is prepared by combining a forward primer comprising a 5' binding reagent complement sequence and a cDNA complement sequence, a reverse primer comprising a cDNA reverse complement sequence and a 3' binding partner of a detectable label, and other PCR components such as dNTPs and DNA polymerase. The cDNA and Master Mix are combined, and PCR is performed for 30 to 40 cycles to form a plurality of PCR products, each PCR product comprising tire 5' binding reagent complement sequence and 3' binding partner of a detectable label. Each PCR product hybridizes to a binding reagent on a surface. The surface is then contacted with a detectable label, which binds to the PCR product. The PCR product bound to the detectable label is then subjected to detection as described herein.

[0016] FIG. 6 relates to Example 3 and shows the correlation between embodiments of serology assays described herein.

[0017] FIG. 7 relates to Example 4. FIG. 7 shows the assay performance (sensitivity at early and late infections and specificity) of IgG indirect serology assay and IgM indirect serology assay and ACE2 competitor assay.

[0018] FIGS. 8-10 relate to Example 5. FIG. 8 shows the results from an exemplary oligonucleotide ligation assay (OLA) for detection of SARS-CoV-2 single nucleotide polymorphisms (SNPs) at genome locations 8782, 11083, 23403. and 28144, with a synthetic template oligonucleotide.

[0019] FIG. 9 shows the results of an exemplary singleplex OLA assay for detecting SARS-CoV-2 SNPs at genome locations 8782, 11083, 23403. and 28144, with samples obtained from SARS-CoV-2 positive patients.

[0020] FIG. 10 shows the results of an exemplary multiplex OLA assay for detecting SARS-CoV-2 SNPs at genome locations 8782, 11083, 23403, and 28144, with samples obtained from SARS-CoV-2 positive patients.

[0021] FIGS. 11-13 relate to Example 6. FIG. 11 shows the results of an exemplary' assay for measuring the concentration (fg/mL) of SARS-CoV-2 nucleocapsid (N) protein from the following samples: nasopharyngeal swabs from 12 patients who tested positive for CO VID-19, nasopharyngeal swabs from 6 patients who tested negative for CO VID-19, and normal (CO VID-19 negative) human saliva, serum, and EDTA plasma.

[0022] FIG. 12 shows the percent recovery results of an exemplary test to assess dilution linearity of the SARS-CoV-2 N protein detection assay. The nonnal human serum, EDTA plasma, saliva, and COVID-19 negative human nasopharyngeal swab samples were spiked with calibrator and tested at different dilutions. [0023] FIG. 13 shows the percent recovery results of an exemplary test to assess spike recovery of the SARS-CoV-2 N protein detection assay. The normal human serum, EDTA plasma, saliva, and COVID-19 negative human nasopharyngeal swab samples were spiked with calibrator at three levels.

[0024] FIG. 14 relates to Example 7 and shows the results of an exemplary serology assay performed on samples obtained from SARS-CoV-2-infected individuals in the United States during early 2020 (known to be infected with wild-type SARS-CoV-2 ("Wuhan")); SARS-CoV-2 -infected individuals in the United Kingdom (dominating strain: SARS-CoV-2 strain B.1.1.7); or SARS-CoV-2 -infected individuals in South Africa (dominating strain: SARS-CoV-2 strain 501Y.V2, also known as B.1.351). The measured ratios of antibodies against wild-type SARS-CoV-2 versus SARS-CoV-2 strain B.1.1.7 were plotted on the x-axis, and the measured ratios of antibodies against wild-type SARS-CoV-2 versus SARS-CoV-2 strain 501Y.V2 were plotted on the y-axis.

[0025] FIGS. 15 and 16 relate to Example 8. FIG. 15 shows the results of an exemplary serology' assay to determine correlation in reactivity to SARS-CoV-2 antigens measured in self-collected saliva versus fingcr-stick blood from subjects described in die Example. [0026] FIG. 16 shows the relative reactivity to the SARS-CoV-2 nucleocapsid (N) and spike (S) antigens measured in finger-stick blood and saliva from subjects whose levels of anti-spike IgG exceeded the 98^th percentile threshold for presumed naive (PN) donors. Spearman coefficient = 0.95, p = 0.001. The dashed line has slope of 1 and represents the expected correlation.

[0027] FIGS. 17 and 18 relate to Example 9. FIG. 17 shows the results of exemplary indirect IgG serology and ACE2 competition assays using a 10-spot SARS-CoV-2 S-RBD antigen panel. The graph shows tire signals for each of the antigens in the S-RBD antigen panel (identified in the inset table) after normalization to the signal from the wild-type SARS-CoV-2 S-RBD antigen spot.

[0028] FIG. 18 shows a subset of the data in FIG. 17, with signals from two spots in the 10-spot S-RBD antigen panel. Each dot represents one individual.

[0029] FIG. 19 relates to Example 10 and show the results of an exemplary biomarker assay to assess levels of IL-6, IL-10, IL-12p70, IL-4, TNF-a, IL-2, IL- I [L IFN-y, and IL-17A, performed on cerebrospinal fluid (CSF) and serum samples from acute COVID-19 patients and non-CO VID-19 control subjects.

[0030] FIGS. 20A and 20B illustrate exemplary assay surfaces described in embodiments herein. FIG. 20 A shows a well of an exemplar}' 384-well assay plate, comprising four distinct binding domains ("spots"). FIG. 20B shows a well of an exemplary' 96-well assay plate, comprising ten distinct binding domains ("spots").

DETAILED DESCRIPTION OF THE INVENTION

[0031] Certain inventions disclosed herein were made jointly under Research Collaboration Agreement 2020-0351 between the National Institute of Allergy' and Infectious Diseases (NIAID), which is a component of the National Institutes of Health (NIH). which is an agency of the U.S. Department of Health and Human Services, and Meso Scale Diagnostics, LLC., which is an affiliate of Meso Scale Technologies, LLC.

[0032] The disclosed embodiments fulfill the urgent need for high-quality viral assays and methods useful for the CO VID-19 pandemic. Disclosed embodiments have been widely adopted for COVID-19 research, epidemiology, and vaccine development and have had a significant impact on tire CO VID-19 public health response. For example, serology embodiments are widely used (e.g., Johnson M et al. J Clin Virol 2020; 130: 104572; Corbett KS et al. N Engl J Med 2020;383:1544-55; Folegatti PM et al. The Lancet 2020;396:467-78; Ramasamy MN et al. The Lancet 2020;396:1979-93; Goldblatt D et al. J Hosp Infect 2021;110:60-6; Majdoubi A et al. JCI Insight 2021, doi.org/10.1172/jci.insight.146316: Amjadi MF et al. MedRxiv 2021:2021.01.05.21249240. doi.org/10.1101/2021.01.05.21249240; Grandjean L et al. MedRxiv 2020:2020.07.16.20155663, doi.org/10.1101/2020.07.16.20155663; Majdoubi A et al. MedRxiv 2020:2020.10.05.20206664, doi.org/10.1101/2020.10.05.20206664). Certain embodiments disclosed herein were chosen by the United States government initiative. Operation Warp Speed, as the basis of its standard binding assay for immunogenicity assessments in all funded Phase III clinical trials of vaccines. Serology assay embodiments (e.g., assays to detect immunoglobulin(s) conducted on non-bodily samples or bodily samples (e.g., serum, plasma, saliva)) disclosed herein aid in assessing human immune responses to COVID-19 infection and vaccination and in understanding the interplay between CO VID-19 and immunity to other coronaviruses and respiratory pathogens. The disclosed nucleic acid detection embodiments have advantages over PCR methods, e.g.. in their speed, simplicity, cost, and high throughput. The disclosed intact virus detection embodiments provide improved accuracy and specificity of an active infection diagnosis as compared to detection of an individual viral component. Serology assays, nucleic acid detection assays, and other embodiments related to mutations and variants of SARS-CoV-2 are proving important as new mutations and variants arise. Other biomarker detection embodiments disclosed herein, e.g.. detection of inflammatory and/or tissue damage response biomarkers and/or extracellular vesicles, e.g.. from virus-infected cells, have wide applicability, regardless of viral mutation status, to studies on morbidity and mortality to understand factors underlying severe illness, death, and persistent symptoms following acute infection and may lead to better interventions. Data showing the high-quality nature of the disclosed embodiments are described in the Examples and elsewhere herein.

[0033] Immunoassays described herein for the detection of respiratory viruses, including coronaviruses, provide numerous advantages compared with nucleic acid amplification (e.g.. PCR) based detection methods. For example, immunoassays arc conducted in a simple and streamlined format with improved sensitivity . Improved sensitivity with immunoassays occurs because these assays not only detect viral particles, but also individual viral proteins in damaged tissue being cleared by the body at the site of infections. Moreover, immunoassays for biomarkers produced by tire body in response to infection (e.g., antibodies against tire virus or inflammatory factors associated with tire host response to infection) take advantage of die natural amplification associated with the immune response.

[0034] Unless otherwise defined herein, scientific and technical terms used in the present disclosure shall have the meanings that are commonly understood by one of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more dran one element.

[0035] The use of the term "or" in the claims is used to mean "and/or." unless explicitly indicated to refer only to alternatives or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or."

[0036] As used herein, the terms "comprising" (and any variant or form of comprising, such as "comprise" and "comprises"), "having" (and any variant or form of having, such as "have" and "has"), "including" (and any variant or form of including, such as "includes" and "include") or "containing" (and any variant or form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited, elements or method steps.

[0037] The use of the term "for example" and its corresponding abbreviation "e.g." (whether italicized or not) means that the specific terms recited are representative examples and embodiments of the invention drat are not intended to be limited to the specific examples referenced or cited unless explicitly stated otherwise. [0038] As used herein, "between" is a range inclusive of the ends of the range. For example, a number between x andy explicitly includes the numbers x and y, and any numbers that fall within x and y.

Respiratory Virus Detection

[0039] In embodiments, the invention provides an immunoassay method for detecting at least one respiratory virus, including a coronavirus, in a biological sample. As used herein, a "respiratory virus" refers to a virus that can cause a respirator,' tract infection, e.g., in a human. Exemplary respiratory viruses include, but are not limited to, coronavirus, influenza virus, respiratory syncytial virus (RSV), paramyxovirus, adenovirus, parainfluenza virus (PIV), bocavirus, metapneumovirus (MPV). orthopneumovirus. enterovirus, rhinovirus (RV). parechovirus (PeV), and the like. Respiratory virus infections can be difficult to diagnose because different viruses can often cause similar symptoms in a patient. For example, coughing and low-grade fever are typical symptoms of early disease progression or mild cases of a coronavirus infection (e.g., COVID-19), as well as influenza or a respiratory syncytial virus (RSV) infection. An assay that can simultaneously test for several potential causes of infection would advantageously allow a respiratory virus infection to be correctly and efficiently diagnosed in a single assay run and utilizing a single patient sample. In embodiments, the methods herein distinguish between and among different types of a given virus (e.g., distinguishing PIV-1, PIV-2. PIV-3, and PIV-4 from each other or influenza A from influenza B from each other), as well as between and among different subtypes or strains (e.g., distinguishing influenza A (H1N1) from influenza A (H3N2)).

[0040] In embodiments, the invention provides an immunoassay method for detecting at least one respiratory' virus in a biological sample, comprising: (a) contacting the biological sample with a binding reagent that specifically binds a component of at least one respiratory' virus in the biological sample; (b) forming a binding complex comprising the binding reagent and the respiratory' virus component; and (c) detecting the binding complex, thereby detecting die at least one respiratory' virus in die biological sample. [0041] In embodiments, the at least one respiratory' virus comprises a coronavirus, an influenza virus, a paramyxovirus, an adenovirus, a bocavirus, a pneumovirus, an enterovirus, a rhinovirus, or a combination thereof. Exemplary coronaviruses and metiiods for their detection are described herein and include, but are not limited to, SARS-CoV (also known as SARS-CoV-1), MERS-CoV, SARS-CoV-2, HCoV-OC43, HCoV-229E, HCoV-NL63. and HCoV-HKUl. In embodiments, the method detects a coronavirus by detecting a coronavirus nonstructural protein, e.g., nspl, nsp2. nsp3, nsp4, nsp5, nsp6. nsp7, nsp8, nsp9, nsplO, nspl l, nsp!2. nsp!3, nsp!4, nsp!5. or nsp!6. In embodiments, the method detects a coronavirus by detecting a coronavirus structural protein, e.g., the E, S (including SI, S2, S-NTD, S-ECD, and S-RBD), M, HE. or N proteins. Coronaviruses and their proteins are further described herein.

[0042] Exemplary influenza viruses include, but are not limited to, influenza A (Flu A), influenza B (Flu B), and influenza C (Flu C). Typically, the seasonal flu is caused by Flu A and/or Flu B. Flu A viruses can be further characterized into various subtypes based on the hemagglutinin (HA) and neuraminidase (N) proteins present on the surface of the viral particle, e.g., H1N1, H1N2, H2N2. H3N2, H5N1, H7N2. H7N3, H7N7. H9N2, and H10N7. Flu A strains include, e.g.. Hl strains (such as Hl/Michigan strain, Hl/Wisconsin strain (also referred to as Hl/Wisconsin 2019. Hl/Wisconsin/588/2019 or H1N1)), H3 strains (such as H3/Hong Kong strain. H3/Darwin strain (also referred to as H3/Darwin. H3/Darwin/9/2021 or H3N2)), H7 strains (such as H7/Shanghai strain), and the like. Flu B viruses can be further characterized into genetic lineages, e.g.. the Flu B/Victoria lineage (including, e.g., the strain B/Austria/1359417/2021) or Flu B/Yamagata lineage (including, e.g., the strain B/Phuket/3073/2013). In embodiments, the immunoassay detects an influenza virus component, e g., an influenza virus-specific protein. In embodiments, the immunoassay detects an influenza structural protein. In embodiments, the immunoassay detects an influenza nonstructural protein. In embodiments, the immunoassay detects an influenza virus by detecting the influenza HA protein. In embodiments, the immunoassay detects an influenza virus by detecting the influenza N protein. In embodiments, the immunoassay detects an influenza virus by detecting an influenza nucleoprotein (NP). In embodiments, the immunoassay detects a Flu A virus and is further capable of determining the subtype of the FluA virus. In embodiments, tire immunoassay detects a FluB virus and is further capable of determining the lineage of the FluB virus.

[0043] In embodiments, the method detects SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKUl, influenza A, influenza B, RSV, or a combination thereof. In embodiments, the method is a multiplexed method capable of simultaneously detecting one or more of SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKUl, influenza A, influenza B, and RSV. In embodiments, the method further comprises repeating one or more of the method steps described herein to detect one or more respiratory viruses in the sample. In embodiments, the method further comprises repeating steps (a)-(c) of the method described herein, wherein each detected respiratory virus comprises a component that binds to a different binding reagent, thereby detecting the at least one respiratory virus. In embodiments, each of steps (a)-(c) is performed for each respiratory' virus in parallel.

[0044] As used herein, the term "simultaneous" in reference to one or more events (e.g., detection of one or more viruses, viral components, or biomarkers as described herein) means that the events occur at exactly the same time or at substantially the same time. e.g.. simultaneous events described herein can occur less than or about 30 minutes apart, less than or about 20 minutes apart, less than or about 15 minutes apart, less than or about 10 minutes apart, less than or about 5 minutes apart, less than or about 2 minutes apart, less than or about 1 minute apart, or less than or about 30 seconds apart. In the context of embodiments of multiplexed immunoassays provided herein, "simultaneous" refers to detecting a on single surface (e.g.. a particle, an assay plate, an assay cartridge, or a well of a multi-well assay plate) the presence of one or more viruses, viral components or biomarkers described herein. In embodiments, a multiplexed assay is performed on a single assay plate. In embodiments, a multiplexed assay is performed in a single well of an assay plate. In embodiments, a multiplexed assay is performed in a single assay cartridge. In embodiments, a multiplexed immunoassay is performed on more than one assay plates. In embodiments, more than one multiplexed immunoassay (e.g., wherein each multiplexed immunoassay detects a combination of biomarkers and/or viral components as described herein) is performed on a single surface, e.g., a single well of an assay plate or a single assay cartridge. The number of assay wells and/or assay plates that may be required to perform a multiplexed assay can be determined, e.g., based on the number of substances of interest to be detected in one or more samples (e.g.. a multiplex of about 2 to about 100. or about 2 to about 90, or about 2 to about 80, or about 2 to about 70, or about 2 to about 60. or about 2 to about 50, or about 2 to about 40, or about 2 to about 35, about 2 to about 30, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19. 20, 21, 22, 23. 24, 25, 26. 27. 28, 29, 30. or more viruses, viral components, and/or biomarkers described herein); the number of samples being assayed (e g., from one or more subjects); the number of calibration reagents being measured to generate a calibration curve (e g., 3, 4, 5. 6. 7, 8, 9, 10, 11. 12, or more); the number of control reagents being measured (e.g., 0, 1. 2, 3, or more); the number of replicates for each sample, calibration reagent, and/or control reagent being measured (e.g., singlicate, duplicate, triplicate, or more); and the number of wells per assay plate (e.g., 6, 12. 48, 96, 384, or 1536 wells per assay plate). When multiplexed immunoassay is conducted on multiple assay plates, the assay plates can be read simultaneously or at different tunes. The timing of reading the assay plates can be determined, e.g., based on the capacity of the assay reader instrument (e.g., capable of reading 1, 2, 3, 4, or more plates at once); the read-time of the assay reader instrument (e.g., about 1 s to about 600 s, about 10 s to about 500 s, about 20 s to about 300 s, about 30 s to about 180 s, about 60 s to about 120 s, about 70 s, or about 90 s per assay plate); the time required to prepare the assay components (e.g., about 10 s, 20 s, 30 s, 1 min, 2 min, 5 min, 10 min, 15 min, 30 min, 1 hr. or more per plate); and the equipment for performing die assay (e.g., a singlechannel pipettor may require a longer time for pipetting the assay components as compared to a multichannel pipettor; handling liquids from different containers, e.g., tubes, vials, or plates, may require different lengths of time). In embodiments, "simultaneous" refers to events occurring with respect to a single sample (e.g., a biological sample in a single vial or container from a single subject) or replicates or dilutions of a single sample. Factors affecting die timing of simultaneous events include the following: the number of multiplexed assays being performed at the same time on a single sample (e.g., a multiplex of or about 2 to about 100. or about 2 to about 90, or about 2 to about 80, or about 2 to about 70, or about 2 to about 60, or about 2 to about 50, or about 2 to about 40. or about 2 to about 35, about 2 to about 30, or 2, 3.

4. 5, 6, 7, 8, 9. 10, 11, 12. 13. 14, 15, 16. 17, 18, 19, 20. 21, 22, 23. 24. 25, 26, 27. 28, 29, 30, or more assays in a single well or cartridge); the number of assay modules in a panel (e.g.. 1, 2, 3, or more plates or cartridges in a panel); the number of samples being assayed at the same time (e.g.. a number of samples capable of being assayed in one kit or more than one kit); the number of points on a calibration curve (e.g.,

5, 6. 7. 8, 9, 10. 12, or more); the presence and number of controls (e.g.. 0, 1, 2, 3, or more controls); the read-time of the instrument (e.g., about 1 s to about 600 s, about 10 s to about 500 s, about 20 s to about 300 s, about 30 s to about 180 s, about 60 s to about 120 s. about 70 s, or about 90 s); the number of replicates of each calibrator, control, or sample (e.g., singlicate, duplicate, triplicate, or more); the number of wells per plate (e.g., 6. 12, 48, 96, 384, or 1536 wells per plate); and/or the type of equipment for performing tire assay (e.g., a single channel or a multi channel pipettor, tubes or plates for dilution).

[0045] In embodiments, the binding reagent that specifically binds to the respiratory virus component described herein is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer. In embodiments, the binding reagent is an antibody or a variant thereof, including an antigcn/cpitopc-binding portion thereof, an antibody fragment or derivative, an antibody analogue, an engineered antibody, or a substance that binds to antigens in a similar manner to antibodies. In embodiments, the binding reagent comprises at least one heavy or light chain complementarity determining region (CDR) of an antibody. In embodiments, the binding reagent comprises at least two CDRs from one or more antibodies. In embodiments, the binding reagent is an antibody or antigen-binding fragment thereof. In embodiments, the binding reagent is a receptor for the respiratory virus component. In embodiments, the binding reagent is a binding partner of the respiratory virus component. In embodiments, the binding reagent is angiotensin-converting enzyme 2 (ACE2). In embodiments, the binding reagent is a neuropilin (NRP) receptor. In embodiments, the binding reagent is NRP1. In embodiments, the binding reagent is NRP2.

Coronavirus Detection

[0046] Coronaviruses, which belong to the Coronaviridae family of viruses, are enveloped viruses with a positive-sense single-stranded RNA genome and a nucleocapsid of helical geometry. A characteristic feature of coronaviruses is the club-shaped spikes that project from the virus surface. In general, a coronavirus particle is assembled from its structural proteins, including an envelope (E), a spike glycoprotein (S), which includes SI and S2 subunits that form the ectodomain (S-ECD), a viral membrane protein (M), a hemagglutinin-esterase dimer (HE), nucleocapsid (N), and RNA. The S protein comprises a N-tenninal domain (N-Tenn or NTD). The SI subunit comprises a receptor binding domain (S-RBD), which binds a host receptor (e.g., ACE2) during infection. The SI subunit can also bind to the cell surface neuropilin-1 (NRP1) receptor. See, e.g., Daly et al., bioRxiv 2020.06.05. 134114 (2020) doi: 10.1101/2020.06.05.134114. In embodiments, coronavirus S proteins, including recombinantly expressed S proteins and variants thereof, are further described, e.g., in WO 2018/081318. For example, tw o variants of SARS-CoV-2 each has a single polynucleotide morphism (SNP) at genome location 23403, which is in the gene encoding the S protein, resulting in a different amino acid at position 614 of the S protein: D614 and G614 (denoted as S: 23403 A>G, D614G; see, e.g., Korber et al., bioRxiv 2020.04.29. 069054 (2020) doi: 10.1101/2020.04.29.069054; also published as Korber et al.. Cell 182(4):P812-827 (2020)), referred to herein respectively as S-D614 and S-D614G. Further mutations of the SARS-CoV-2 S protein are described in FIGS. 1A and IB. Sequence alignments between die genetic material of various coronavirus species have also revealed additional conserved open reading frames for Coronaviruses also encode a number of nonstructural proteins (NSPs), which are expressed in infected cells but are generally not incorporated into the viral particle itself. Exemplary coronavirus NSPs include, but are not limited to, nspl, nsp2, nsp3, nsp4. nsp5, nsp6, nsp7, nsp8. nsp9 (replicase). nsplO, nspl l, nspl2 (multi-domain RNA polymerase), nspl3 (helicase. RNA 5' triphosphatase), nspl4 (N7-methyl transferase, exonuclease), nspl5 (endoribonuclease), nsp!6 (2'-O-methyl transferase), and the like. See. e.g.. Snijder et al., Adv Virus Res 96:59-126 (2016); Fehr et al.. Coronaviruses 1281 :1-23 (2015). Sequence alignments between the genetic material of various coronavirus species have revealed conserved open reading frames for several structural and nonstructural proteins, e.g., N, M, S, nspl, nsp3, nsp6, nsp7, and nsp8. See, e.g.. Grifoni et al., bioRxiv 2020.02.12.946087 (2020) doi: 10.1101/2020.02.12. [0047] While assays for a specific coronavirus species can identify infection by that particular coronavirus, such assays may have limited usefulness when new strains of infectious coronaviruses emerge. In embodiments, the invention provides a method for detecting a coronavirus in a sample by detecting a conserved coronavirus component, e.g., a protein that is generally conserved across all coronavirus species. Such a method would enable detection of novel coronaviruses of interest.

[0048] In embodiments, the invention provides an immunoassay method for detecting a coronavirus in a biological sample, comprising: a) contacting the biological sample with a binding reagent that specifically binds a component of the coronavirus; b) forming a binding complex comprising the binding reagent and the coronavirus component; and c) detecting the binding complex, thereby detecting tire coronavirus in the biological sample. In embodiments, the method detects SARS-CoV, MERS-CoV, SARS-CoV-2. HcoV- OC43, HcoV-229E, HcoV-NL63, HcoV-HKUl, or a combination thereof. In embodiments, the biological sample is saliva.

[0049] In embodiments, the coronavirus component is on the outer surface of the viral particle. In embodiments, the coronavirus component is integrated in the membrane of the viral particle. In embodiments, the coronavirus component is a protein. In embodiments, the coronavirus component comprises a sugar, e.g., a glycoprotein. In embodiments, the coronavirus component is a structural protein. In embodiments, the coronavirus component is an envelope (E) protein, hr embodiments, tire coronavirus component is a spike glycoprotein (S) or a variant or subunit thereof, e g., S-D614, S-D614G, or any of the S protein variants in FIGS. 1A and IB, subunit 1 (SI), subunit 2 (S2), ectodomain (S-ECD), N-tenninal domain (S-NTD or S-N-Term), or receptor binding domain (S-RBD). In embodiments, the S protein subunit (e g., SI. S2, S-ECD, S-NTD, or S-RBD) comprises a mutation as described in FIGS. 1A and IB. In embodiments, the coronavirus component is a viral membrane (M) protein, hr embodiments, the coronavirus component is a hemagglutinin-esterase dimer (HE). In embodiments, the coronavirus component is a nucleocapsid (N) protein. In embodiments, the coronavirus component comprises a mutation as described in FIG. 1A.

[0050] In embodiments, the coronavirus component is a non-structural protein. In embodiments, the coronavirus component is nspl, nsp2, nsp3, nsp4, nsp5, nsp6, nsp7, nsp8, nsp9, nsplO. nspl 1, nspl2, nsp!3. nsp!4, nspl5, or nsp!6. In embodiments, the coronavirus component is a protein substantially conserved across coronaviruses. It will be understood by one of ordinary skill in the art that a protein that is "substantially conserved" across a viral family, e.g.. the coronavirus family, means that at least 50%, at least 60%, at least 70%, at least 80%, at least 90%. or at least 95% of species in the viral family contains a protein with at least 50%, at least 60%. at least 70%, at least 80%, at least 90%, or at least 95% sequence similarity, structural similarity, or both. Methods and tools for determining sequence and/or structural similarity are known in the field and include, e.g.. algorithms such as Align, BLAST, and CLUSTAL for sequence similarity, and TM-align, DALI, STRUCTAL, and MINRMS.

[0051] In embodiments, the immunoassay method detects a coronavirus by detecting the coronavirus E protein. In embodiments, the immunoassay method detects a coronavirus by detecting the coronavirus S protein, hr embodiments, die immunoassay method detects a coronavirus by detecting the coronavirus SI protein subunit. In embodiments, the immunoassay method detects a coronavirus by detecting the coronavirus S2 protein subunit. In embodiments, the immunoassay method detects a coronavirus by detecting the coronavirus S-ECD. In embodiments, the immunoassay method detects a coronavirus by detecting the coronavirus S-RBD. In embodiments, the immunoassay method detects a coronavirus by detecting the coronavirus S-NTD. In embodiments, the immunoassay method detects a coronavirus by detecting the coronavirus M protein. In embodiments, the immunoassay method detects a coronavirus by detecting the coronavirus HE protein. In embodiments, the immunoassay method detects a coronavirus by detecting the coronavirus N protein. In embodiments, the immunoassay method detects a coronavirus by detecting one or more of the coronavirus nspl, nsp2, nsp3, nsp4, nsp5, nsp6, nsp7, nsp8, nsp9, nsplO. nspl 1. nsp!2, nspl3, nsp!4, nsp!5, or nsp!6. In embodiments, the immunoassay detects a coronavirus by detecting a combination of the coronavirus proteins described herein. In embodiments, the coronavirus is SARS-CoV-2. In embodiments, the immunoassay method detects SARS-CoV-2 by detecting SARS-CoV-2 N protein. In embodiments, the immunoassay method detects SARS-CoV-2 by detecting SARS-CoV-2 S protein and/or S-RBD. In embodiments, tire immunoassay method detects SARS-CoV-2 by detecting any of the SARS-CoV-2 S protein and/or S-RBD variants in FIGS. 1A and IB. In embodiments, the immunoassay method detects SARS-CoV-2 by detecting SARS-CoV-2 E protein. In embodiments, the immunoassay method detects SARS-CoV-2 by detecting SARS-CoV-2 M protein, hr embodiments, tire immunoassay detects SARS-CoV-2 by detecting SARS-CoV-2 N protein and S protein. In embodiments, the immunoassay method detects SARS-CoV-2 by detecting SARS-CoV-2 S protein, N protein, E protein, and M protein. SARS-CoV-2 nonstructural proteins include the Orfla and Orflab replicase/transcriptase proteins; the Orf3a protein; the Orf6a protein; the Orf7a and Orf7b accessory proteins; tire Orf8 protein monomer, which is known to form oligomers; and the OrflO protein. SARS-CoV-2 nonstructural proteins are further described in, e.g., Khailany et al., Gene Rep 19:100682 (2020); and Flower et al.. Proc Nat Acad Sci 118(2): e2021785118 (2021). In embodiments, the immunoassay detects SARS-CoV-2 by detecting any of SARS-CoV-2 Orfla, Orflab. Orf3a, Orf6a, Orf7a. Orf7b, Orf8 monomer, Orf8 oligomer. OrflO, RNA- dependent RNA polymerase (RdRp), or a combination thereof. In embodiments, the immunoassay method detects SARS-CoV-2 by detecting any of the SARS-CoV-2 protein variants in FIG. 1A.

[0052] In embodiments, the immunoassay method for detecting SARS-CoV-2 comprises: a) contacting the biological sample with a binding reagent that specifically binds a SARS-CoV-2 S, N. E, or M protein; b) forming a binding complex comprising the binding reagent and the SARS-CoV-2 S, N, E. or M protein; and c) detecting the binding complex, thereby detecting SARS-CoV-2 in the biological sample. In embodiments, the SARS-CoV-2 S protein is SARS-CoV-2 S-D614. In embodiments, the SARS-CoV-2 S protein is SARS-CoV-2 S-D614G. In embodiments, the SARS-CoV-2 S protein comprises any of the mutations shown in FIGS. 1A and IB. In embodiments, the SARS-CoV-2 N protein comprises any of the mutations shown in FIG. 1A. In embodiments, the SARS-CoV-2 E protein comprises any of the mutations shown in FIG. 1 A. In embodiments, tire binding complex further comprises a detection reagent that specifically binds to the SARS-CoV-2 S, N, E, or M protein. In embodiments, the detection reagent comprises a detectable label. In embodiments, the detection reagent comprises a nucleic acid probe. Detection reagents are further described herein. In embodiments, the biological sample is saliva. [0053] In humans, coronaviruses can cause respiratory tract infections ranging from mild to lethal. Infection by the coronaviruses SARS-CoV. MERS-CoV, and SARS-CoV-2 can cause severe respiratory illness symptoms, i.e., severe acute respiratory syndrome (SARS), Middle East respiratory' syndrome (MERS), or coronavirus disease 2019 (COVID-19), respectively. Infection by the coronaviruses HcoV- OC43. HcoV-229E, HcoV-NL63, or HcoV-HKUl can lead to mild respiratory illness symptoms, e.g., the common cold. Coronaviruses can also cause disease in animals such as cats, birds, chickens, cows, and pigs. As used herein, "respiratory tract infection" or "respiratory' infection" can refer to an upper respiratory' tract infection (URI or URTI) or a lower respiratory' tract infection (LRI or LRTI). URTIs include infection of the nose, sinuses, pharynx, and lary nx, e.g., tonsillitis, pharyngitis, laryngitis, sinusitis, otitis media, and tire common cold. LRTIs include infection of the trachea, bronchial tubes, bronchioles, and the lungs, e.g., bronchitis and pneumonia. Symptoms of illnesses caused by coronaviruses include, e.g., fever, cough, shortness of breath, fatigue, congestion, chills, muscle pain, headache, sore throat, loss of taste or smell, diarrhea, etc.

[0054] In embodiments, the coronavirus component is a fragment of any of the proteins described herein, e.g., a structural or non-structural coronavirus protein. In embodiments, the fragment comprises a domain of the full length protein. For example, the S protein includes an N-terminal domain (S-NTD) and an ectodomain (S-ECD), which includes the spike SI and S2 subunits. The SI subunit also includes a receptor binding domain (S-RBD), which is responsible for binding the host receptor (e.g., ACE2 and/or NRP1). In some embodiments, the immunoassay detects a coronavirus by detecting the coronavirus SI subunit. In some embodiments, the immunoassay detects a coronavirus by detecting the coronavirus S2 subunit. In some embodiments, the immunoassay method detects a coronavirus by detecting the coronavirus S-NTD. In some embodiments, the immunoassay method detects a coronavirus by detecting the coronavirus S-ECD. In some embodiments, the immunoassay method detects a coronavirus by detecting the coronavirus S-RBD. In embodiments, the S protein subunit (e.g.. SI. S2, S-ECD, S-NTD, or S-RBD) comprises a mutation as described in FIGS. 1A and IB. In embodiments, the immunoassay detects a coronavirus by detecting a combination of the coronavirus proteins described herein. In embodiments, the coronavirus is SARS-CoV-2. In embodiments, the immunoassay method detects SARS-CoV-2 by detecting SARS-CoV-2 N protein. In embodiments, the immunoassay method detects SARS-CoV-2 by detecting SARS-CoV-2 S protein and/or S-RBD. In embodiments, the immunoassay method detects SARS-CoV-2 by detecting any' of tire SARS-CoV-2 S protein and/or S-RBD variants in FIGS. 1A and IB. In embodiments, the immunoassay detects SARS-CoV-2 by' detecting SARS-CoV-2 N protein and S protein. [0055] In embodiments, the coronavirus component is a nucleic acid. As used herein in tire context of viral components, a viral nucleic acid refers to a viral genome or portion thereof. The viral nucleic acid can encode a viral protein, or the viral nucleic acid can be a non-coding sequence. In embodiments, detection of a viral nucleic acid comprises detecting a sequence that is present in the viral genome, but not in the host genome. In embodiments, the coronavirus component is DNA or RNA. In embodiments, the coronavirus component comprises a nucleic acid secondary structure, e.g.. an RNA loop. In embodiments, the coronavirus component is a lipid, e.g.. that forms part of the viral envelope.

[0056] In embodiments, the invention provides methods for distinguishing between strains of a coronavirus. In embodiments, the coronavirus is SARS-CoV-2. In embodiments, the invention provides methods for assessing the transmissibility of a CO VID- 19 infection outbreak by determining the SARS- CoV-2 strain. In embodiments, the invention provides methods for assessing the virulence of a SARS-CoV- 2 strain by determining the SNPs in the strain. In embodiments, the invention provides methods for assessing effectiveness of a vaccine against a particular strain of SARS-CoV-2. The term "strain" is used interchangeably herein with "variant," "lineage." and "type." In embodiments, a mutant strain or variant of a virus described herein, e.g., SARS-CoV-2, comprises one or more mutations relative to a reference or parent or wild-type strain of the virus. As referred to throughout this application, the SARS-CoV-2 NC 045512 strain is the "reference" or "wild-ty pe" strain, and all SNPs described herein are attributed to one or more "mutant" strains or "variants." In embodiments, tire invention provides methods to trace the lineage of a coronavirus in a population. For example, two strains of SARS-CoV-2 have been identified, referred to as the "L" strain (also known as "lineage B") and "S" strain (also known as "lineage A"). The L strain can be differentiated from the more ancestral S strain based on two different SNPs that show nearly complete linkage: one at location 8782 (orflab T8517C, synonymous) and one at location 28144 (ORF8: C25 IT, S84L). See, e.g., Tang et al., Natl Sci Rev, nwaa036; doi: 10.1093/nsr/nwaa036 (3 Mar 2020).

Moreover, as discussed herein, two SARS-CoV-2 strains have been identified to contain an SNP at genome location 23403, which encodes the S protein, and are referred to herein as die "S-D614" and "S-D614G" strains. A further SARS-CoV-2 SNP of interest is at location 11083, where the 11083G to T mutation (denoted as "11083G>T") is associated with asymptomatic presentation. In embodiments, the SARS-CoV-2 reference strain comprises the "L strain" SNP at genome locations 8782 and 28144, the "S-D614" SNP at genome location 23403, and a G nucleotide at genome location 11083.

[0057] Mutations in the SARS-CoV-2 S protein can affect, e.g., binding to the ACE2 receptor, overall structure and antibody recognition, and/or protein conformation. Critical residues in the SARS-CoV-2 S- RBD for binding to the ACE2 receptor include, e.g.. K417. N439, Y453, L452, S477, T478, E484, Q493, and N501. See. e.g.. Lan et al.. Nature 581:215-220 (2020). In embodiments, mutations in the SARS-CoV-2 S protein alter binding of the S protein to its host binding partner, e.g., ACE2. In embodiments, mutations in the SARS-CoV-2 S protein affect transmissibility' of the virus. In embodiments, mutations in the SARS- CoV-2 S protein affect vaccine effectiveness against the virus. In embodiments, SARS-CoV-2 strains are characterized by SNPs in the coding sequence of the S protein. Such SARS-CoV-2 strains include, e.g..

A.23.1 (also referred to as the "Uganda strain"); A.VOI.V2 (also referred to as the "Tanzania strain"); B. l;

B.1.1.519 (also referred to as the "Mexico/Texas BV-2 strain"); B.l.1.529 (also referred to as the "Omicron variant" or "BA.l," which comprises sub-lineages BA.2 and BA.3); B.l.1.7 (also referred to as the "UK strain" or "Alpha variant"); B.l.351 or 501Y.V2 (referred to as the "South Africa strain" or "Beta variant"); B.l.429 or Cal.20C (referred to as the "California strain" or "Epsilon variant"); B.l.525 (also referred to as die "Nigeria strain" or "Eta variant"); B.1.526 (also referred to as the "New York strain" or "Iota variant"); B.1.617 (also referred to as the "India strain"); the B.1.617.1 strain (also referred to as the "Kappa strain"); B.l.617.2 (also referred to as the "Delta variant"), which has been further reclassified into sub-lineages designated as "AY"; B.l.617.3; Texas BV-1; B.l.621 (also referred to as the "Mu variant"); C.37 (also referred to as the "Chile/Peru strain" or "Lambda variant"); P.l (also referred to as the "Brazil strain" or "Gamma variant"); P.2 (also referred to as the "Zeta variant"); P.3 (also referred to as the "Philippines strain"); and R.1 (also referred to as the Kentucky strain). The B.1 .1 .529 strain comprises the following mutations in the S protein: A67V, A69-70, T95I, G142D/A143-145, A211/L212I, ins214EPE, G339D, S371L, S373P. S375F, K417N, N440K, G446S, S477N, T478K. E484A. Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F. of which G339D, S371L, S373P, S375F. K417N. N440K. G446S, S477N, T478K, E484A, Q493R, G496S. Q498R, N501Y, and Y505H are in the S-RBD. The B. l.1.7 strain is characterized by the following mutations in the S protein: a deletion of amino acid residues 69-70, E484K. N501Y. D614G. and P681H. The 501Y.V2 strain is characterized by the following mutations in the S protein: D215G, K417N, E484K, N501Y, and D614G. The P.1 strain is characterized by the following mutations in the S protein: K417T, E484K, N501Y, and D614G. The Cal.20C strain is characterized by a L452R mutation in the S protein. The B.l.526 strain comprises the following mutations in the S protein: L5F, T95I, D253G, D614G, A701V, and either E484K or S477N. The B.l.526 strain comprising E484K is referred to herein as "B.l.526" or "B.1.526/E484K" and tire B.1.526 strain comprising S477N is referred to herein as "B.1.526.2" "B.1.526/S477N."

[0058] In embodiments, mutations in SARS-CoV-2 proteins, e.g., S protein, result from genetic recombination between two or more SARS-CoV-2 variants. For example, a host subject may be simultaneously infected by two variants, e.g., die B.l.617.2/ AY.4 ("Delta") and B.1.1.529ZBA.1 ("Omicron") variants, which may recombine when replicating in the host to produce a recombinant variant. The recombinant variant may be designated as the cross between its parent variants. For example, the recombinant variant resulting from Delta (AY.4) and Omicron (BA.l) variants is designated as the BA.l x AY.4 recombinant.

[0059] As used herein, all strain designations include all of its sub-strains. For example, the B.l.526 strain includes the B.1.526, B. l.526.1, and the B.1.526.2 strains, and the B.1.617 strain includes the B.1.617. B.1.617.1. B.1.617.2. and B.1.617.3 strains. The B.1.617.2 strain ("Delta variant") includes all "AY" sub-lineage designations, including AY.l, AY.2, AY.3, AY.4, AYA, AY.6. AY.7. AY.8, AY.9, AY.10, AY.l l. AY.12, AY.13, AY.14, AY.15. AY.16, AY.17, AY.18. AY.19, AY.20, AY.21. AY.22, AY.23, AY.24, AY.25, and all sub-lineages thereof (e.g., AY.4.2). As used herein, strains "characterized" by particular mutations include at least those particular mutations and may include additional mutations. These strains and associated mutations are summarized in FIG. 1 A. Additional variants of SARS-CoV-2 comprise mutations in the S protein as shown in FIG. IB and Table 1A and are further described, e.g., in Faria et al., “Genomic characterisation of an emergent SARS-CoV-2 lineage in Manaus: preliminary’ findings" (2020). Accessed at virological.org/t/586; Wu et al., bioRxiv doi:10.1101/2021.01.25.427948 (2021); Guruprasad, Proteins 2021: 1-8 (2021); Zhou et al., bioRxiv doi: 10.1101/2021.03.24.436620 (2021). Further strains and mutations of SARS-CoV-2 are provided in the PANGO lineages database (cov- lineages.org); the Nextstrain database (nextstrain.org); the Global Evaluation of SARS-CoV-2/hCoV-19 Sequences (GESS) database provided by Fang et al.. Nucleic Acid Res 49(Dl):D706-D714 (2021) (wan- bioinfo.shinyapps.io/GESS); and the SARS-CoV-2 Mutation Browser provided by Rakha et al., bioRxiv doi: 10.1101/2020.06.10.145292 (2020) (covid-19.dnageography.com). The mutations denoted as "del" or "A" indicate a deletion of the indicated amino acid residues present in the reference sequence. For example, a variant S protein comprising a "A69-70" mutation means that amino acid residues at positions 69 and 70 of the wild-type S protein are deleted. The mutations denoted as "ins" indicates an insertion of one or more amino acid residues at the indicated amino acid position. For example, a variant S protein comprising an "insl46N" mutation means the variant S protein comprises an asparagine residue at amino acid position 146 of the variant S protein. The mutations denoted as (X1-X2)->Y denotes that the amino acid residues X1-X2 indicated in the parentheses are mutated to a single amino acid Y. For example, a variant S protein comprising a "(L24-A27)->S" mutation means die variant S protein comprises a replacement of the amino acid residues at positions 24 to 27 with a serine residue.

[0060] Throughout this application, when referring to an S protein comprising a specific mutation, the mutation is relative to the SARS-CoV-2 reference strain NC 045512. The S protein from the SARS-CoV-2 reference strain is also known as the "wild-type" S protein. For example, the S-D614G protein from SARS- CoV-2 comprises D to G substitution at amino acid residue 614 relative to the wild-type S protein from SARS-CoV-2.

[0061] Further SARS-CoV-2 SNPs have been identified, for example, at the genome locations listed in FIG. 1C, e.g., locations 3036, 8782 18060, 11083, 1397, 2891, 14408. 17746, 17857, 23403, 26143, 28144, and 28881. See. e.g., Pachetti et al., J TranslMed 18: 179 (2020); Banerjee et al.. bioRxiv, doi.org/10.1101/2020.04.06.027854 (9 Apr 2020); Alouane et al., bioRxiv doi.org/10.1101/2020.06.20.163188 (21 Jun 2020); Brufsky, J Med Virol 2020:1-5 (2020); and Mishra et al., bioRxiv doi.org/10.1101/2020.05.07.082768 (12 May 2020). The ability to determine viral strain and/or trace viral lineage in a population provides valuable epidemiological insight into the spread and evolution of the virus. Determining the particular viral strain that has infected a patient also allows more comprehensive treatment. For example, the patient can be treated with a strain-specific drug. If a particular strain is more transmissible and/or more likely to cause severe illness, early interventions can be provided to the patient.

Table 1A. Selected SARS-CoV-2 Strains and S Protein Mutations

Alternative mutations for the S protein of SARS-CoV-2 strains AY.l, AY.2, and B.1.617.2 are listed as "Alt Seq #."

Table IB. Selected SARS-CoV-2 Strains and S-RBD Mutations

[0062] In embodiments, the invention provides a method for detecting a coronavirus in a biological sample, comprising: a) contacting the biological sample with a binding reagent that specifically binds a nucleic acid of the coronavirus; b) forming a binding complex comprising the binding reagent and the coronavirus nucleic acid; and c) detecting the binding complex, thereby detecting the coronavirus in the biological sample. In embodiments, the coronavirus nucleic acid is RNA. In embodiments, the coronavirus is SARS-CoV-2. In embodiments, the binding reagent comprises an oligonucleotide comprising a sequence complementary to the coronavirus nucleic acid sequence. In embodiments, the binding reagent binds to a nucleic acid from a specific strain of the coronavirus, e.g., a SARS-CoV-2 strain as described herein. In embodiments, the binding reagent binds to a SARS-CoV-2 nucleic acid encoding the N protein (i.e. , the N gene). The SARS-CoV-2 N gene can be detected at three different regions: N 1 , N2, and N3. The N 1 and N2 regions are specific to SARS-CoV-2, and the N3 region is universal to the coronaviruses in the same clade as SARS-CoV-2 (e.g., clade 2 and 3 viruses within the subgenus Sarbecovirus, including SARS-CoV-2, SARS-CoV, and bat- and civet-SARS-like CoVs. See, e.g., Lu et al.. Emerg Infect Dis 26(8): 1654-1665 (2020)). hr embodiments, tire binding reagent binds to SARS-CoV-2 N1 region. N2 region, N3 region, or a combination thereof. In embodiments, the biological sample is saliva, the coronavirus is SARS-CoV-2 and die nucleic acid is RNA.

[0063] In embodiments, the coronavirus is capable of infecting a human. In embodiments, the coronavirus causes a respiratory tract infection in a human. In embodiments, the coronavirus is SARS-CoV, MERS-CoV, SARS-CoV-2, HcoV-OC43, HcoV-229E, HcoV-NL63, HcoV-HKUl, or a combmation thereof, hi embodiments, die mediod detects a coronavirus component that is substantially conserved in SARS-CoV, MERS-CoV, SARS-CoV-2, HcoV-OC43, HcoV-229E. HcoV-NL63, and HcoV-HKUl. In embodiments, the mediod detects a protein or peptide fragment that is substantially conserved in SARS- CoV, MERS-CoV. SARS-CoV-2. HcoV-OC43, HcoV-229E, HcoV-NL63, and HcoV-HKUl.

[0064] In embodiments, the immunoassay described herein is a multiplexed immunoassay method. A multiplexed immunoassay can simultaneously detect multiple substances of interest, e.g., coronavirus components, in a sample. A multiplexed immunoassay can also use multiple binding reagents that specifically bind a substance of interest, e.g.. a coronavirus component, in a sample. Multiplexed immunoassays can provide reliable results while reducing processing time and cost. In embodiments, a multiplexed immunoassay for detecting a coronavirus comprises multiple binding reagents, each of which binds to a different coronavirus component, e.g., a conserved coronavirus protein. In embodiments, a multiplexed immunoassay comprising binding reagents that each specifically binds a different coronavirus component provides improved detection accuracy, e.g., over a singleplex method utilizing a single binding reagent. In embodiments, the immunoassay method detects a coronavirus by detecting one or more of the coronavirus E protein, S protein, including S 1 and S2 subunits. S-NTD. S-ECD. and S-RBD. M protein, HE protein, N protein, nspl, nsp2. nsp3, nsp4, nsp5, nsp6. nsp7, nsp8, nsp9, nsplO, nspl l. nspl2, nspl3, nspl4. nspl5, and nspl6. In embodiments, the coronavirus is SARS-CoV-2. In embodiments, the coronavirus is SARS-CoV-2. In embodiments, the immunoassay method detects SARS-CoV-2 by detecting SARS-CoV-2 N protein. In embodiments, the immunoassay method detects SARS-CoV-2 by detecting SARS-CoV-2 S protein and/or S-RBD. In embodiments, the immunoassay method detects SARS-CoV-2 by detecting any of tire SARS-CoV-2 S protein and/or S-RBD variants in FIGS. 1A and IB. In embodiments, the immunoassay detects SARS-CoV-2 by detecting SARS-CoV-2 N protein and S protein. In embodiments, the immunoassay detects SARS-CoV-2 by detecting any combmation of the SARS-CoV- 2 N protein, S protein, E protein, and M protein. In embodiments, the immunoassay detects SARS-CoV-2 by detecting SARS-CoV-2 N pro tern, S protein, E protein, and M protein. In embodiments, the immunoassay detects SARS-CoV-2 by detecting any of the SARS-CoV-2 protein variants in FIG. 1A. [0065] In embodiments, the immunoassay method is a multiplexed method comprising: contacting tire biological sample with a surface comprising a binding reagent in each binding domain on the surface, wherein the binding reagent in each binding domain independently binds to a viral protein selected from SARS-CoV-2 N protein, SARS-CoV-2 S protein, SARS-CoV-2 S-RBD, SARS-CoV-2 E protein, SARS- CoV-2 M protein, or a combmation thereof; forming a binding complex in each binding domain comprising the viral protein and the binding reagent that binds to the viral protein; and measuring the concentration of the viral protein in each binding complex. In embodiments, the SARS-CoV-2 S protein comprises any of the mutations shown in FIGS. 1A and IB. In embodiments, each binding complex further comprises a detection reagent that specifically binds to the viral protein of the binding complex. Detection reagents are further described herein.

[0066] In embodiments, the immunoassay method is a multiplexed method capable of simultaneously detecting multiple coronaviruses in a biological sample. In embodiments, the multiplexed method is capable of simultaneously detecting one or more of SARS-CoV. MERS-CoV, SARS-CoV-2, HcoV-OC43, HcoV- 229E, HcoV-NL63, and HcoV-HKUl.

[0067] In embodiments, the binding reagent and/or the detection reagent that specifically binds to the coronavirus component described herein is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope. or aptamer. In embodiments, the binding reagent and/or the detection reagent is an antibody or a variant thereof, including an antigen/epitope-binding portion thereof, an antibody fragment or derivative, an antibody analogue, an engineered antibody, or a substance that binds to antigens in a similar maimer to antibodies. In embodiments, the binding reagent and/or the detection reagent comprises at least one heavy or light chain complementarity determining region (CDR) of an antibody. In embodiments, the binding reagent and/or the detection reagent comprises at least two CDRs from one or more antibodies. In embodiments, the binding reagent and/or the detection reagent is an antibody or antigen-binding fragment thereof. In embodiments, the binding reagent and/or the detection reagent is a receptor for the coronavirus component. In embodiments, the binding reagent and/or the detection reagent is a receptor for the coronavirus S protein. In embodiments, the binding reagent and/or the detection reagent is angiotensinconverting enzyme 2 (ACE2). In embodiments, the binding reagent and/or the detection reagent is neuropilin-1 (NRP1). In embodiments, tire binding reagent and/or the detection reagent is CD147.

[0068] In embodiments where the method comprises detecting one or more variants of an SARS-CoV-2 protein (e.g., an S protein comprising a mutation shown in FIGS. 1A and IB or an Orflab, E, Orf8, or N protein comprising a mutation shown in FIG. 1 A), the binding reagent comprises an antibody or antigenbinding fragment thereof that is capable of specifically binding the wild-type, protein variant(s), or both the protein variant and the wild-type, and the detection reagent comprises an antibody or antigen-binding fragment thereof that is capable of binding die wild-type, protein variants), or both the wild-type and variant forms of the protein. In embodiments, the SARS-CoV-2 protein is an S protein, an N protein, an E protein, an Orflab protein, an Orf8 protein, or a combination thereof. In embodiments, the SARS-CoV-2 protein is an S protein.

[0069] In embodiments, the method is capable of detecting about 1 fg/mL to about 1 ng/mL, about 1 fg/mL to about 0.8 ng/mL, about 1 fg/mL to about 0.5 ng/mL, about 1 fg/mL to about 0.1 ng/mL, about 1 fg/mL to about 50 pg/mL, about 1 fg/mL to about 20 pg/mL, about 1 fg/mL to about 10 pg/mL, about 1 fg/mL to about 5 pg/mL, about 1 fg/mL to about 2 pg/mL, about 1 fg/mL to about 1 pg/mL, about 5 fg/mL to about 100 fg/mL, about 7 fg/mL to about 75 fg/mL, or about 10 fg/mL to about 50 fg/mL of a virus (e.g.. a coronavirus such as SARS-CoV-2). In embodiments, the method is capable of detecting less than or about 5 pg/mL, less than or about 2 pg/mL, less than or about 1 pg/mL, less than or about 500 fg/mL, less than or about 100 fg/mL, less than or about 75 fg/mL, less than or about 50 fg/mL. or less tiian or about 10 fg/mL of a virus (e.g., a coronavirus such as SARS-CoV-2). In embodiments, the method is capable of detecting less than or about 10⁹ viral particles per mL. less than or about 10⁸ viral particles per mL, less than or about 10⁷ viral particles per mL, less than or about 10⁶ viral particles per mL, less than or about 100000 viral particles per mL, less than or about 10000 viral particles per mL. less than or about 1000 viral particles per mL, or less than or about 100 viral particles per mL. In embodiments where the method detects a viral nucleic acid, one viral particle is one viral genome equivalent. In embodiments, the method is capable of detecting less than or about 10⁹ viral genome equivalents per mL, less than or about 10⁸ viral genome equivalents per mL, less than or about 10⁷ viral genome equivalents per mL, less than or about 10⁶ viral genome equivalents per mL, less than or about 100000 viral genome equivalents per mL, less than or about 10000 viral genome equivalents per mL, less than or about 1000 viral genome equivalents per mL, or less than or about 100 viral genome equivalents per mL.

Biomarkers

[0070] In embodiments, the invention provides a method for detecting a biomarker that is produced by a host (e.g., a human subject) in response to a viral infection, e.g., by a respiratory virus, including coronaviruses such as SARS-CoV-2. As used herein, "host" refers to a subject who has been infected with or suspected of being infected with a virus described herein, e.g., a coronavirus such as SARS-CoV-2. Unless otherwise specified, the biomarkers described herein are produced by a host, e.g.. a human subject, in response to viral exposure and/or infection as described herein. In embodiments, the biomarker is an immune response biomarker. In embodiments, the biomarker is an antibody. The terms "antibody biomarker" and "antibody" are used interchangeably throughout the present disclosure. In embodiments, the biomarker is an inflammation response biomarker. In embodiments, the biomarker is a damage response biomarker. In embodiments, the method is used to assess the severity and/or prognosis of a viral infection in a subject. In embodiments, the method is used to determine whether a subject has been previously exposed to a virus. In embodiments, the method is used to estimate the time of virus exposure and/or infection. In embodiments, the method is used to determine whether a subject has immunity to a virus. In embodiments, tire virus is a coronavirus. In embodiments, the virus is SARS-CoV-2.

[0071] As used herein, the term "biomarker" refers to a biological substance that is indicative of a normal or abnormal process, e.g., disease, infection, or environmental exposure. Biomarkers can be small molecules such as ligands, signaling molecules, or peptides, or macromolecules such as antibodies, receptors, or proteins and protein complexes. A change in the levels of a biomarker can correlate with the risk or progression of a disease or abnormality or with tire susceptibility or responsiveness of tire disease or abnormality to a given treatment. A biomarker can be useful in the diagnosis of disease risk or the presence of disease in an individual, or to tailor treatments for the disease in an individual (e.g., choices of drug treatment or administration regimes). In evaluating potential drug therapies, a biomarker can be used as a surrogate for a natural endpoint such as survival or irreversible morbidity. If a treatment alters a biomarker that has a direct connection to improved health, die biomarker serves as a "surrogate endpoint" for evaluating clinical benefit. Biomarkers are further described in, e.g., Mayeux, NeuroRx 1(2): 182-188 (2004); Strimbu et al., Curr Opin HIV AIDS 5(6): 463-466 (2010): and Bansal et al., Statist Med 32: 1877- 1892 (2013). The term "biomarker," when used in the context of a specific organism (e.g.. human, nonhuman primate or another animal), refers to die biomarker native to diat specific organism. Unless specified otherwise, the biomarkers referred to herein encompass human biomarkers.

[0072] As used herein, the term "level" in the context of a biomarker refers to the amount, concentration, or activity of a biomarker. The term "level" can also refer to the rate of change of the amount, concentration, or activity of a biomarker. A level can be represented, for example, by the amount or synthesis rate of messenger RNA (mRNA) encoded by a gene, the amount or synthesis rate of polypeptide corresponding to a given amino acid sequence encoded by a gene, or the amount or synthesis rate of a biochemical form of a biomarker accumulated in a cell, including, for example, the amount of particular post-synthetic modifications of a biomarker such as a polypeptide (e.g.. an antibody), nucleic acid, or small molecule. "Level" can also refer to an absolute amount of a biomarker in a sample or to a relative amount of tire biomarker, including amount or concentration determined under steady -state or non-steady -state conditions. "Level" can further refer to an assay signal that correlates with the amount, concentration, activity or rate of change of a biomarker. The level of a biomarker can be determined relative to a control marker in a sample.

[0073] Measurement of biomarker values and levels before and after a particular event e.g.. cellular or environmental event, may be used to gain information regarding an individual's response to the event. For example, samples or model organisms can be subjected to stress- or disease-inducing conditions, or a treatment or prevention regimen, and a particular biomarker can then be detected and quantitated in order to determine its changes in response to the condition or regimen. However, the opposite, i.e., measuring biomarker values and levels to determine whether an organism has been subjected to stress- or disease- inducing condition, tends to be much more complicated, as changes in the levels of a single biomarker are sometimes not definitively associated with a particular condition.

[0074] In embodiments, the measured levels of the one or more biomarkers described herein provides information regarding infection and immune response to infection, e.g., the course or maturity of infection, tire etiology of severe illness, and the potential severity’ of illness. In embodiments, the measured levels of die one or more biomarkers described herein provides information regarding a subject's antibody response, cytokine response, neutrophil, macrophage, and/or monocyte production, complement activation, B cell and/or T cell activation, or a combination thereof.

[0075] In embodiments, detection and/or measurement of a single biomarker is sufficient to provide a prediction and/or diagnosis of a disease or condition. In embodiments, combinations of biomarkers are used to provide a strong prediction and/or diagnosis. Although a linear combination of biomarkers (i.e., the combination comprises biomarkers that individually provide a relatively strong correlation) can be utilized, linear combinations may not be available in many situations, for example, when there are not enough biomarkers available and/or with strong correlation. In alternative approaches, a biomarker combination is selected such that the combination is capable of achieving improved performance (i.e., prediction or diagnosis) compared with any of the individual biomarkers, each of which may not be a strong correlator on its own. Biomarkers for inclusion in a biomarker combination can be selected for based on their performance in different individuals, e.g., patients, wherein the same biomarker may not have the same performance in different individuals, but when combined with the remaining biomarkers, provide an unexpectedly strong correlation for prediction or diagnosis in a population. For example. Bansal et al.. Statist Med 32: 1877-1892 (2013) describe methods of determining biomarkers to include in such a combination, noting in particular that optimal combinations may not be obvious to one of skill in the art, especially when subgroups are present or when individual biomarker correlations are different between cases and controls. Thus, selecting a combination of biomarkers for providing a consistent and accurate prediction and/or diagnosis can be particularly challenging and unpredictable.

[0076] Even when a suitable combination of biomarkers is determined, utilizing the combination of biomarkers in an assay poses its own set of difficulties. For example, detecting and/or quantitating each biomarker in the combination in its own separate assay may not be feasible with small samples, and using a separate assay to measure each biomarker in a sample may not provide consistent and comparable results. Furthermore, running an individual assay for each biomarker in a combination can be a cumbersome and complex process that can be inefficient and costly.

[0077] A multiplexed assay that can simultaneously measure the concentrations of multiple biomarkers can provide reliable results while reducing processing time and cost. Challenges of developing a multibiomarker assay (such as, e.g.. a multiplexed assay described in embodiments herein) include, for example, determining compatible reagents for all of the biomarkers (e.g., capture and detection reagents described herein should be highly specific and not be cross-reactive; all assays should perform well in the same diluents); determining concentration ranges of the reagents for consistent assay (e.g., comparable capture and detection efficiency for the assays described herein); having similar levels in the condition and sample type of choice such that the levels of all of the biomarkers fall within the dynamic range of the assays at the same dilution; minimizing non-specific binding between the biomarkers and binding reagents thereof or other interferents; and accurately and precisely detecting a multiplexed output measurement.

[0078] In embodiments, the invention provides methods of assessing an individual's immune response to a viral infection, hr embodiments, the invention provides methods of assessing a group of individuals immune response to a viral infection. In embodiments, assessing an immune response comprises determining die type and/or strength of the immune response, e.g., detecting the molecular components produced in response to a viral infection (e.g., acute phase reactants, antibodies, cytokines, etc.) and measuring the amounts of each component produced. In embodiments, the invention provides methods of assessing die differences in immune responses by age, race, ethnicity, socioeconomic backgrounds, and/or underlying conditions, e.g., lung disease, diabetes, cancer, etc., which may be associated with poor clinical outcomes. In embodiments, the invention provides methods of determining the epidemiology of diseases caused by the viruses described herein, e.g., CO VID-19. In embodiments, the virus is a coronavirus. In embodiments, the virus is SARS-CoV-2.

|0079| In embodiments, the invention provides methods of assessing cross-reactivity of an individual's immune response between different coronaviruses (e.g.. SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV- OC43, HCoV-229E. HCoV-NL63, and HCoV-HKU 1). In embodiments, the invention provides methods of mapping the epitopes recognized by an individual's immune response, e.g., epitopes on a coronavirus S protein. In embodiments, the invention provides methods of assessing the individual's clinical outcome based on the mapped epitopes of immune responses. In embodiments, the invention provides methods of assessing an individual's immune response by detecting different IgG classes and/or subclasses. In embodiments, the invention provides methods of assessing the individual's clinical outcome based on the IgG classes and/or subclasses. In embodiments, the invention provides methods of assessing the affinity and/or avidity of an individual's immune response to different viral antigens. In embodiments, the invention provides methods of assessing the strength of an immune response, e.g., measuring the total antibody concentration or the concentration of different classes or subclasses of antibodies in an individual. In embodiments, the invention provides methods of determining the natural interacting partner(s) of tire virus, e.g.. a coronavirus such as SARS-CoV-2. As used in the context of viral infections, a "natural interacting partner" refers to a substance in die host cell (e.g., proteins or carbohydrate moictics on a host cell surface) that interacts with a viral component described herein. Natural interacting partners of viruses are further described in. e.g., Brito et al.. Front Microbiol 8: 1557 (2017). Natural interacting partners of SARS-CoV-2 include, e.g., ACE2, NRP1, and CD147, and are further described in Gordon et al.. bioRxiv 2020.03.22.002386vl (2020) doi: 10.1 l()l/2020.03.22.002386vl. Daly et al., bioRxiv 2020.06.05. 134114 (2020) doi: 10.1101/2020.06.05.134114, and Bojkova et al., Nature Research (Pre-Print 11 Mar 2020) doi:10.21203/rs.3.rs-17218/vl . In embodiments, the invention provides a competitive assay for SARS- CoV-2 utilizes ACE2, NRP1, CD147, or different sialic acid-containing substances to determine the interacting partner(s) of the SARS-CoV-2 S protein.

[0080] In embodiments, the invention provides methods of assessing changes in the immune response over time. In embodiments, the invention provides methods of assessing an individual's immune response at different time points after infection and/or after tire first onset of a symptom. In embodiments, the invention provides methods of assessing the cytokines present in an individual at different time points after infection and/or after the first onset of a symptom. Symptoms of viral infections are described herein. In embodiments, the invention provides methods of assessing the long-term effects of an infection on an individual. For example, the coronavirus SARS-CoV-2 can cause post-acute CO VID-19 syndrome (also known as post-CO VID syndrome or "long COVID"), in which symptoms of the infection, including fatigue, headaches, shortness of breath, anosmia, muscle weakness, low fever, and cognitive dysfunction, persist for weeks or months after the typical convalescence period of CO VID-19. In embodiments, the invention provides methods of assessing an individual's immune response at different time points after vaccination. In embodiments, the invention provides methods of determining the immune response components that provide immunity to a viral infection. In embodiments, the invention provides methods of assessing an individual's immune response at different time points after receiving a treatment for the viral infection. In embodiments, the invention provides methods of assessing the effect of convalescent serum treatment in an individual, e.g., comprising measuring the individual's immune response after administration of the convalescent serum. In embodiments, the invention provides methods of assessing the immune response components (e.g.. antibodies) present in a convalescent serum sample, e.g., comprising determining its elfectiveness, half life, and/or functional window of treatment in an individual. In embodiments, the invention provides methods of assessing the effectiveness, half life, and/or functional window of protection of a therapeutic antibody treatment. In embodiments, the virus is a coronavirus. In embodiments, the virus is SARS-CoV-2.

[0081] In embodiments, the invention provides methods of assessing an individual's immune response, e.g.. an antibody, to a coronavirus (e.g., an endemic coronavirus such as HCoV-OC43. HCoV-229E, HCoV-NL63, and HCoV-HKU) to determine a clinical outcome of infection by a different coronavirus, e.g.. SARS-CoV-2. In embodiments, the invention provides methods of assessing an individual's immune response, e.g., an antibody, to a respiratory virus (e.g., influenza or RSV) to determine a clinical outcome of infection by a different respiratory virus, e.g., SARS-CoV-2. In embodiments, the invention provides methods of assessing an individual's immune response and/or clinical outcome in a SARS-CoV-2 infection by determining a ratio of the individual's antibody level against the SARS-CoV-2 N protein to the individual's antibody level against the SARS-CoV-2 S protein. In embodiments, the antibody levels are measured in a blood sample. In embodiments, the antibody levels are measured in a saliva sample.

[0082] In embodiments, the invention provides a serology assay for determining the SARS-CoV-2 strain that has infected an individual. Currently, the only available methods for determining SARS-CoV-2 strain are nucleic acid-based methods such as PCR or sequencing, which typically require a nasopharyngeal or oropharyngeal sample from a subject. Assessment of the subject's antibody or immune response, as described herein, would require a further serology sample. Thus, a serology assay that determines SARS- CoV-2 strain reduces the amount and type of sample required from the subject, thereby reducing sample collection and processing time, and stress on the subject. It was surprisingly discovered that antibody biomarkers from an individual infected with a particular SARS-CoV-2 strain had highly specific activity against the S protein and/or S-RBD of that particular strain as compared to other strains. This unexpected result demonstrates a population-wide immune response (e.g., antibody response) bias for strain-specific epitopes on the SARS-CoV-2 S protein and/or S-RBD, which was not observed with other infectious diseases, e.g., other viruses, which ty pically have highly variable immune responses (e.g., antibody responses) between infected individuals that do not correlate with the viral strain, hr embodiments, tire invention provides methods of assessing an individual's immune response to different strains or variants of a coronavirus, e.g., SARS-CoV-2. In embodiments, the invention provides methods of mapping SARS- CoV-2 strain-specific epitopes on the SARS-CoV-2 S protein and/or S-RBD. Such methods are also useful for epidemiological studies to determine circulating variants in a population or geographical region.

[0083] In embodiments, the invention provides a method of determining the SARS-CoV-2 strain that has infected one or more individuals, comprising: performing a multiplexed serology assay on a sample obtained from the one or more individuals to detect one or more antibody biomarkers against S proteins and/or S-RBD from multiple SARS-CoV-2 strains; and dilferentiating the detected antibody biomarker(s) based on binding of the antibody biomarker(s) to the S protein and/or S-RBD from each SARS-CoV-2 strain. In embodiments, the differentiating comprises determining a ratio of: a first antibody biomarker that binds an S protein and/or S-RBD from a first SARS-CoV-2 strain (e.g., wild-type SARS-CoV-2), to a second antibody biomarker that binds an S protein and/or S-RBD from a second SARS-CoV-2 strain (e.g.. SARS-CoV-2 strain B.1.1.7). SARS-CoV-2 strains are further described herein, e.g.. in FIG. 1A. Multiplexed serology assays are further described herein. In embodiments, the multiplexed serology assay detects one or more antibody biomarkers that binds to one or more of: an S protein from wild-type SARS- CoV-2, an S protein from SARS-CoV-2 strain P.l, an S protein from SARS-CoV-2 strain B. l.1.7, and an S protein from SARS-CoV-2 strain 501Y.V2. In embodiments, the multiplexed serology assay detects one or more antibody’ biomarkers that binds to one or more of: an S protein from wild-ty pe SARS-CoV-2, an S- D614G from SARS-CoV-2. an S protein from SARS-CoV-2 strain P.l, an S protein from SARS-CoV-2 strain B.l.1.7, an S protein from SARS-CoV-2 strain 501Y.V2, and an S-RBD from wild-type SARS-CoV- 2. In embodiments, the multiplexed serology assay detects one or more antibody biomarkers that binds to one or more of: an S protein from wild-ty pe SARS-CoV-2, an S-RBD from wild-type SARS-CoV-2, an S protein from SARS-CoV-2 strain B.l.1.7, an S-RBD from SARS-CoV-2 strain B.l.1.7, an S protein from SARS-CoV-2 strain 501Y.V2, an S-RBD from SARS-CoV-2 strain 501Y.V2, an S protein from SARS- CoV-2 strain P. l, and an S-RBD from SARS-CoV-2 strain P. l. In embodiments, the multiplexed serology assay detects one or more antibody biomarkers that binds to one or more of: an S protein from wild-type SARS-CoV-2, an S-RBD from wild-type SARS-CoV-2. an S protein from SARS-CoV-2 strain B.1.429, an S-RBD from SARS-CoV-2 strain B.1.429, an S protein from SARS-CoV-2 strain B.1.526/E484K, an S- RBD from SARS-CoV-2 strain B.1 .526/E484K, an S protein from SARS-CoV-2 strain B.1.526/S477N, and an S-RBD from SARS-CoV-2 strain B.1.526/S477N. In embodiments, the multiplexed serology assay detects one or more antibody biomarkers drat binds to one or more S proteins or subunit or fragment thereof that comprises any of the mutations shown in FIGS. 1A and IB. In embodiments, the multiplexed serology assay is a classical serology assay, bridging serology assay, or competitive serology assay as described herein.

[0084] In embodiments, the invention provides a method of determining one or more SARS-CoV-2 strains in a sample. The method described herein is useful for tracking spread of one or more SARS-CoV-2 strains. The method provided herein is further useful for tracking the spread of one or more SARS-CoV-2 strains in one or more geographical regions and/or for tracking the spread of one or more SARS-CoV-2 strains over tune. In embodiments, the invention provides a method for determining a SARS-CoV-2 strain in a sample, comprising: detecting at least a first antibody biomarker in the sample that binds to an antigen from a first SARS-CoV-2 strain and at least a second antibody biomarker in tire sample that binds to an antigen from a second SARS-CoV-2 strain, wherein tire detecting comprises contacting the sample with a surface comprising at least two binding domains, wherein the antigen from the first SARS-CoV-2 strain is immobilized on a first binding domain, and the antigen from the second SARS-CoV-2 strain is immobilized on a second binding domain; and determining a ratio of the first antibody biomarker to the second antibody biomarker, thereby determining the SARS-CoV-2 strain.

|0085| In some embodiments, the sample is from one or more individuals, wherein the one or more individuals are currently infected with SARS-CoV-2. In some embodiments, the sample is from one or more individuals, wherein the one or more individuals were previously infected with SARS-CoV-2. In some embodiments, the sample is from at least two individuals, wherein at least one individual is currently infected with SARS-CoV-2 and at least one individual was previously infected with SARS-CoV-2. In some embodiments, the sample is from at least one individual, wherein the individual is currently infected and was previously infected with SARS-CoV-2. In embodiments, the sample is from one or more individuals, wherein the one or more individuals are located in one or more geographical regions. In embodiments, the sample is from one or more individuals obtained at different time points. In embodiments, the sample comprises a pooled sample from at least two individuals. Pooled samples are further described herein.

[0086] In embodiments, the method further comprises detemrining the SARS-CoV-2 from one or more samples. In embodiments, the one or more samples are from one or more individuals as described herein. In embodiments, the method further comprises comparing the SARS-CoV-2 in one or more samples from one or more individuals located in one or more geographical regions, thereby tracking spread of the SARS- CoV-2 strain in the one or more geographical regions. [0087] In embodiments, the SARS-CoV-2 strain is determined by inputting the ratio of the first antibody biomarker to die second antibody biomarker into a classification algorithm. Classification algorithms are further described herein. In embodiments, the method further comprises training a classification algorithm. In embodiments, the training comprises: measuring the amount of antibody biomarkers in a sample from a subject infected with a known SARS-CoV-2 strain that bind to an antigen from one or more SARS-CoV-2 strains, wherein the one or more SARS-CoV-2 strains comprise the known SARS-CoV-2 strain; normalizing the amount of measured antibody biomarker that bind to an antigen from the known SARS- CoV-2 strain against the amount of measured antibody biomarker that bind to an antigen from a further SARS-CoV-2 strain; and providing the normalized antibody biomarker amount to the classification algorithm.

[0088] In embodiments, the invention provides a method for differentiating infection associated with different SARS-CoV-2 strains. In embodiments, the method comprises training a classification algorithm. In embodiments, die method comprises obtaining a sample from a subject infected with a known SARS- CoV-2 strain; and measuring the amount of antibody biomarkers in the sample that bind to the S protein and/or S-RBD from multiple SARS-CoV-2 strains. In embodiments, die measuring comprises performing a multiplexed serology assay, e.g., a classical, bridging, or competitive multiplexed serology assay as described herein. In embodiments, the measured antibody biomarker amount for a particular strain is normalized against the measured antibody biomarker amount for a different strain. In embodiments, the normalized antibody biomarker amount is used to train the classification algoridun. In embodiments, normalized antibody biomarker amounts from multiple subjects, each infected with a known SARS-CoV-2 strain, are used to train the classification algorithm. Classification algorithms are known in the field and include but are not limited to, e.g.. linear regression, logistic regression, random forest, support vector machine, and neural network. In embodiments, the invention provides a method of determining the SARS- CoV-2 strain that has infected one or more individuals, comprising: performing a multiplexed serology assay on a sample obtained from the one or more individuals to detect one or more antibody biomarkers against S proteins and/or S-RBD from multiple SARS-CoV-2 strains as described herein; and applying the classification algorithm described herein to determine the SARS-CoV-2 strain.

[0089] Serology tests that assess the presence of an antibody biomarker against a SARS-CoV-2 antigen have received U.S. FDA Emergency Use Authorization (EUA) with specificity of 95%. In embodiments, the invention provides improved sensitivity and/or specificity in determining whether a subject is currently infected or has previously been infected with a virus, e.g., a coronavirus such as SARS-CoV-2. In embodiments, the invention provides improved sensitivity and/or specificity in determining whether a subject has immunity to a virus, e.g.. a coronavirus such as SARS-CoV-2. In embodiments, the methods herein have a sensitivity of greater than 90%. greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, greater than 99.5%, or greater than 99.9%. In embodiments, the methods herein have a specificity⁷ of greater than 90%. greater than 95%, greater than 96%. greater than 97%, greater than 98%, greater than 99%, greater than 99.5%, or greater than 99.9%. Assays with high sensitivity' and specificity arc important to correctly diagnose active infections and to correctly determine whether an individual has been previously exposed and/or immune to a virus, e.g.. a coronavirus such as SARS-CoV-2. In particular, assays with high specificity are useful for conducting epidemiological studies in populations with low disease prevalence. Moreover, assays with high specificity are important for individual assessment due to the high risk of a false positive to the individual and the individual's community; individuals who received a false positive serology test result for SARS-CoV-2 may believe themselves to be immune and therefore erroneously engage in activity that can increase the likelihood of infection and spread of the virus.

Antibody Biomarkers

[0090] In embodiments, the invention provides a method for detecting a respiratory virus, e.g., a coronavirus such as SARS-CoV-2, in a biological sample, by detecting a biomarker produced in response to an infection by the virus. In embodiments, the biomarker produced in response to a viral infection is an antibody.

[0091] In embodiments, the invention provides a method for detecting a biomarker that is capable of binding to a viral antigen in a biological sample. As used herein, a virus or viral antigen is any component or secretion of a virus that prompts an immune response in a host (e.g., a human). In embodiments, the viral antigen is a viral protein or fragment thereof. In embodiments, the viral antigen is a virus structural protein. In embodiments, the viral antigen is a virus nonstructural protein. Structural and nonstructural proteins of viruses, e.g., respiratory viruses such as coronaviruses, are described herein. In embodiments, the method is capable of determining whether a subject has been exposed to a particular virus, e.g., a coronavirus such as SARS-CoV-2. In embodiments, the method is capable of determining whether a subject is at risk of being infected by a particular virus, e.g., a coronavirus such as SARS-CoV-2. In embodiments, the method is capable of determining whether a subject has immunity' to a particular virus, e.g., a coronavirus such as SARS-CoV-2.

[0092] In embodiments, die invention provides an immunoassay⁷ metiiod comprising: quantify ing the amounts of one or more biomarkers capable of binding to a respiratory virus antigen in a biological sample, wherein die respiratory virus is a coronavirus, an influenza virus, a paramyxovirus, an adenovirus, a bocavirus, a pneumovirus, an enterovirus, a rhinovirus, a parainfluenza virus (PIV), a metapneumovirus (MPV), a parechovirus (PeV), RSV, or a combination diereof, wherein the quantifying comprises measuring the concentrations of each of the one or more biomarkers in an immunoassay. In embodiments, the immunoassay comprises detecting one or more biomarkers diat bind to a panel of respiratory virus antigens in a biological sample, wherein the respiratory viruses comprise a coronavirus, an influenza virus, and RSV. In embodiments, the immunoassay comprises detecting one or more biomarkers that bind to a panel of respiratory virus antigens in a biological sample, wherein the respiratory viruses comprise an enterovirus, MPV, RSV, an influenza virus, a rhinovirus, a coronavirus, a PIV, and a parechovirus. In embodiments, the immunoassay comprises detecting one or more biomarkers that bind to a panel of respiratory' virus antigens in a biological sample, wherein the respiratory viruses comprise an influenza virus and a PIV.

[0093] In embodiments, the coronavirus antigen comprises a Spike (S) protein or fragment thereof, e.g., S-RBD, or an N protein. In embodiments, the RSV antigen comprises a pre-fusion F protein. In embodiments, the MPV antigen comprises an F protein (e.g., a pre-fusion F protein). In embodiments, the PIV antigen comprises an F protein (e.g., a pre-fusion F protein). In embodiments, the influenza virus antigen comprises a hemagglutinin (HA) protein. In embodiments, the enterovirus antigen comprises a virus-like particle (VLP). In embodiments, the VLP comprises one or more enterovirus proteins (e.g.. a capsid protein, e.g.. VP1, VP2, VP3. VP4, or combination thereof). In embodiments, the rhinovirus antigen comprises a capsid protein, e.g., capsid protein VPO. In embodiments, the parechovirus antigen comprises a VPO protein.

[0094] In embodiments, the immunoassay method comprises: contacting the biological sample with a surface comprising a viral antigen in a binding domain on the surface; forming a binding complex in the binding domain comprising the viral antigen and a biomarker that binds to the viral antigen; and measuring the concentration of the biomarker in the binding complex. In embodiments, the biomarker is IgG, IgA, IgM, or combination thereof. In embodiments, the concentration of the biomarker is measured by contacting the binding complex with a detection reagent that specifically binds IgG, IgA, or IgM. In embodiments, the biomarkcr is a human biomarkcr, a mouse biomarkcr, a rat biomarkcr, a ferret biomarkcr, a minx biomarker, a bat biomarker, or a combination thereof. In embodiments, the biomarker is human IgG, IgA, or IgM. In embodiments, the biomarker is mouse IgG, IgA, or IgM. In embodiments, the biomarker is rat IgG, IgA, or IgM. In embodiments, the biomarker is ferret IgG, IgA, or IgM. In embodiments, the biomarker is minx IgG. IgA, or IgM. In embodiments, the biomarker is bat IgG, IgA, or IgM. Detection reagents are further described herein.

[0095] In embodiments, a binding domain comprises a mixture of antigens from more than one strain or type of virus, e.g., more than one strain of influenza, PIV, or SARS-CoV-2. hr embodiments, the method comprises forming one or more binding complexes in the binding domain, wherein the binding complex comprises one of the antigens and a biomarker, wherein the biomarker binds specifically to the one antigen, or wherein die biomarker is capable of binding to more than one of the antigens. In embodiments, the binding domain comprises HA proteins from multiple influenza strains, e.g., at least one HA from an influenza A strain and at least one HA from an influenza B strain. In embodiments, the binding domain comprises F proteins from multiple parainfluenza virus (PIV) strains, e.g.. at least one F protein from each of PIV1. PIV2. PIV3. and PIV4. In embodiments, the binding domain comprises S proteins and/or S-RBDs from multiple SARS-CoV-2 strains, e.g.. as described in FIGS. 1A-1C and Table 1A.

[0096] In embodiments, the immunoassay method detects a biomarker that binds to an N protein from SARS-CoV-2. In embodiments, the immunoassay method detects a biomarker that binds to a S protein from SARS-CoV-2. In embodiments, the immunoassay method detects a biomarker that binds to SI, S2, S- ECD, S-NTD, or S-RBD from SARS-CoV-2. In embodiments, the SARS-CoV-2 S protein or subunit or fragment thereof comprises a mutation as shown in FIGS. 1A and IB. In embodiments, the SARS-CoV-2 N protein comprises a mutation as shown in FIG. 1 A. In embodiments, the immunoassay method comprises: contacting the biological sample with a surface comprising a viral antigen in a binding domain on the surface; forming a binding complex in the binding domain comprising the viral antigen and a biomarkcr that binds to the viral antigen; and measuring the concentration of the biomarkcr in the binding complex. In embodiments, the biomarker is IgG, IgA, IgM, or combination thereof. In embodiments, the biomarker is an IgG. IgA, and/or IgM from a human, mouse, rat, ferret, minx, bat, or combination thereof. In embodiments, the concentration of the biomarker is measured by contacting the binding complex with a detection reagent that specifically binds IgG, IgA. or IgM as described herein. In embodiments, the immunoassay method is a classical serology assay. In embodiments, the immunoassay method is a bridging serology assay. In embodiments, the immunoassay method is a competitive serology assay. In embodiments, the detection reagent comprises a labeled competitor of the biomarker. In embodiments, the competitor is ACE2. Classical, bridging, and competitive serology’ assays are described herein.

[0097] In embodiments, the method is a multiplexed method capable of simultaneously detecting and/or quantifying the amounts of the one or more biomarkers that bind to a respiratory virus antigen. As discussed herein, a method that is capable of simultaneously testing for several potential causes of infection (e.g., multiple different viruses) can advantageously allow a respiratory’ virus infection to be correctly and efficiently diagnosed in a single assay run and utilizing a single patient sample. Such a method can also be useful for assessing a patient's immune response to different respiratory’ virus infections.

[0098] In embodiments, die multiplexed method simultaneously detects and/or quantifies one or more biomarkers that binds to S proteins from different strains SARS-CoV-2. In embodiments, the multiplexed method is capable of determining the SARS-CoV-2 strain that has infected an individual and/or the SARS- CoV-2 strain that is circulating in a population or geographical region, as described herein. As discussed herein, when referring to an S protein comprising a specific mutation, die mutation is relative to die SARS- CoV-2 reference strain NC 045512. and the S protein from the SARS-CoV-2 reference strain is also known as the "wild-type" S protein. Moreover, an S protein (or subunit thereof) referred to herein as being from a specific SARS-CoV-2 strain includes all of the S protein mutations of that strain as described herein. [0099] In embodiments, the invention provides a method for determining a SARS-CoV-2 strain in a sample, comprising detecting at least a first antibody biomarker in the sample that binds to an antigen, e.g., an S protein. N protein, and/or S-RBD, from a first SARS-CoV-2 strain and at least a second antibody biomarker in die sample that binds to an antigen, e.g.. an S protein, N protein, and/or S-RBD, from a second SARS-CoV-2 strain, wherein the detecting comprises contacting the sample with a surface comprising one or more binding domains, wherein the antigen, e.g., the S protein, N protein, or S-RBD from the first SARS-CoV-2 strain is immobilized on a first binding domain, and the antigen, e.g., the S protein. N protein, or S-RBD from the second SARS-CoV-2 strain is immobilized on a second binding domain; and determining a ratio of the first antibody biomarker to the second antibody biomarker, thereby determining the SARS-CoV-2 strain. In embodiments, the detecting comprises performing a multiplexed method described herein. In embodiments, the multiplexed method simultaneously detects and/or quantifies one or more biomarkers that bind to an antigen, e.g.. an S-protein, N protein, and/or an S-RBD from two or more SARS-CoV-2 strains as shown in FIG. 1A, Table 1A, and/or Table IB. In embodiments, the sample is a biological sample. In embodiments, the sample is from one or more individuals as described herein. In embodiments, the sample is a saliva sample. [00100] In embodiments, each antigen is immobilized on a distinct binding domain on the surface, wherein the antigens comprise an S protein, an N protein, and/or an S-RBD from a SARS-CoV-2 strain described herein. In embodiments, the antigens comprise an S protein, an N protein, and/or an S-RBD from a SARS-CoV-2 strain selected from: an S protein, an S-RBD, and/or an N protein from a SARS-CoV-2 strain selected from: wild-type; P.l; P.2; P.3; B. l.1.519; B.l.1.529; B. l.1.529 (+R346K); B.l.1.529 (+L452R); B A.1 ; BA.1 .1 ; B A.2; BA.3 ; B.1 .1.7; B.1.1.7 (+E484K); B.1.258.17; B.1.351 ; B.1.3 1.1 ;

B.l.429; B.1.466.2; B.1.525; B.1.526/E484K; B.1.526/S477N; B.l.526.1; B.1.617; B.l.617.1; B.l.617.2;

B.l.617.2 (+AY144); B.l.617.2 (+E484K); B. l.617.2 (+E484K/N501Y); B. l.617.2 (+K417N/N439K/E484K/N501Y); B.l.617.2 (+K417N/E484K/N501Y); AY. l; AY.2; AY.3, AYA; AY.5, AY.6, AY.7. AY.4.2; AY.12; AY.14; B.l.617.3; B.1.618; B.1.620; B.1.621; B.l.640.2; BV-1; A.23.1; A.VOI.V2; C.37; and R.1; and/or an S protein and/or an S-RBD from SARS-CoV-2 comprising one or more mutations selected from: R346K, V367F; Q414K, K417N, K417T, N439K, N450K, L452R, L452Q, S477N, T478K, T478R, E484K, E484Q, F490S, Q493R, N501Y.

[00101] In embodiments, die multiplexed method simultaneously detects and/or quantifies one or more biomarkers that binds to a panel of antigens, wherein the panel of antigens comprises antigens from one or more respiratory viruses described herein. In embodiments, die multiplexed method simultaneously detects and/or quantifies one or more biomarkers that binds to a panel of SARS-CoV-2 antigens as shown in Tables 2A-2H. In embodiments, die S protein mutations from the SARS-CoV-2 strains of Tables 2A-2H are described in Table 1A. In embodiments, the S-RBD mutations from the SARS-CoV-2 strains of Tables 2A-2H are described in Table IB. In embodiments, the SARS-CoV-2 antigens are immobilized on a surface. In embodiments, the surface comprises a well of a multi-well assay plate as described herein, hi embodiments, the surface, e.g., well of a multi-well plate, comprises ten distinct binding domains ("Spots"), e.g., as shown in FIG. 20B. In embodiments, the SARS-CoV-2 antigens are immobilized on a surface comprising Spots 1-10 as shown in FIG. 20B, wherein the antigens are arranged as shown in Tables 2A- 2H. In embodiments, the one or more biomarkers is IgG, IgA. IgM, or combination thereof. In embodiments, the IgG. IgA, and/or IgM is from a human, mouse, rat, ferret, minx, bat, or combination thereof. The Spots indicated with "BSA" in Tables 2A-2H below indicate an immobilized bovine serum albumin.

[00102] In embodiments, the multiplexed method simultaneously detects and/or quantifies one or more biomarkers that binds to a panel comprising one or more SARS-CoV-2 antigens and one or more antigens from a different respiratory virus, e.g., a different coronavirus than SARS-CoV-2 (e.g.. SARS-CoV, MERS-CoV. HCoV-OC43, HCoV-229E, HCoV-NL63. or HCoV-HKUl), an influenza virus, an enterovirus, MPV, RSV, a rhinovirus, PIV. or a parechovirus.

[00103] In embodiments, the multiplexed method simultaneously detects and/or quantifies one or more biomarkers that binds to a panel of respiratory virus antigens as show n in Table 21. In embodiments, the S protein mutations from the SARS-CoV-2 strains of Table 21 are described in Table 1A. In embodiments, the respiratory virus antigens are immobilized on a surface. In embodiments, the surface comprises a well of a multi-well assay plate as described herein. In embodiments, tire surface, e.g., well of a multi-wcll plate, comprises ten distinct binding domains ("Spots"), e.g., as shown in FIG. 20B. In embodiments, the respiratory virus antigens are immobilized on a surface comprising Spots 1-10 as shown in FIG. 20B, wherein the antigens are arranged as shown in Table 21. In embodiments, the one or more biomarkers is IgG. IgA, IgM. or combination thereof. In embodiments, the IgG, IgA, and/or IgM is from a human, mouse, rat, ferret, minx, bat, or combination thereof. The Spots indicated with "BSA" in Table 21 below indicate an immobilized bovine serum albumin.

Table 2A. SARS-CoV-2 Antigen Panels

Table 2B. SARS-CoV-2 Antigen Panels

Table 2C. SARS-CoV-2 Antigen Panels

Table 2D. SARS-CoV-2 Antigen Panels

Table 2E. SARS-CoV-2 Antigen Panels

Table 2F. SARS-CoV-2 Antigen Panels

Table 2G. SARS-CoV-2 Antigen Panels

Table 2H. SARS-CoV-2 Antigen Panels

Table 21. Respiratory' Virus Antigen Panels

[00104] In embodiments, the multiplexed method comprises: contacting the biological sample with a surface as described herein that comprises an immobilized viral (e.g.. SARS-CoV-2) antigen in each binding domain on the surface; forming a binding complex in each binding domain comprising the viral antigen and a biomarker that binds to the viral antigen; and measuring the concentration of the biomarker in each binding complex. In embodiments, biomarker is IgG. IgA, IgM. or combination thereof. In embodiments, the IgG, IgA. and/or IgM is from a human, mouse, rat. ferret, minx. bat. or combmation thereof. In embodiments, the concentration of the biomarker is measured by contacting the binding complex with a detection reagent that specifically binds IgG. IgA, or IgM. Detection reagents are further described herein. In embodiments, the detection reagent is an antibody or antigen-binding fragment thereof. In embodiments, the detection reagent is a detectably labeled viral antigen. In embodiments, the immunoassay method is a classical serology assay. In embodiments, tire immunoassay method is a bridging serology assay. In embodiments, the immunoassay is a competitive serology assay. Classical, bridging, and competitive serology assays are provided herein. In embodiments, the competitor is ACE2. In embodiments, the competitor is NRP1. In embodiments, the surface comprises a single assay plate. In embodiments, the surface comprises a multi-well assay plate, wherein each well comprises four distinct binding domains. In embodiments, the surface comprises a single assay plate, hr embodiments, tire surface comprises a multi-well assay plate, wherein each well comprises ten distinct binding domains.

[00105] In embodiments, the immunoassay method comprises detecting one or more viral antigens that are specific to SARS-CoV-2. As discussed herein, SARS-CoV-2 causes the respiratory’ illness COVID-19, which can cause mild to severe symptoms in patients. Sensitive and specific detection of SARS-CoV-2 is important for providing an accurate diagnosis, identify ing asymptomatic infected individuals, and tracking spread of the disease. A method that detects biomarkers produced by an individual in response to a SARS- CoV-2 infection (e.g., antibodies) is also useful for identifying those who may be immune to the virus and therefore may be at lower risk when interacting with tire general public or infected patients, and also may be potential candidates for plasma transfusions. In embodiments, the one or more biomarkers is capable of binding to a SARS-CoV-2 S-D614 protein, S-D614G, SI subunit, S2 subunit, S-NTD, S-RBD, M protein, E protein, N protein, or a combination thereof. In embodiments, the SARS-CoV-2 S protein or subunit or fragment thereof comprises a mutation as shown in FIGS. 1 A and IB. In embodiments, the SARS-CoV-2 N protein comprises a mutation as shown in FIG. 1 A.

|00106] In embodiments, the multiplexed method is capable of simultaneously quantifying die one or more biomarkers that bind to a SARS-CoV-2 antigen. In embodiments, the immunoassay comprises: (a) contacting the biological sample with the viral antigen that specifically binds to a first biomarker of die one or more biomarkers; (b) forming a binding complex comprising the viral antigen and the first biomarker; and (c) measuring the concentration of the first biomarker in die binding complex. In embodiments, the method further comprises repeating one or more of the method steps described herein to quantify’ the amounts of one or more biomarkers in the sample. In embodiments, the method further comprises repeating steps (a)-(c), wherein each biomarker specifically binds to a different viral antigen, thereby quantifying one or more biomarkers. In embodiments, each of steps (a)-(c) is performed for each biomarker in parallel. [00107] In embodiments, the multiplexed method is capable of simultaneously quantifying at least two biomarkers in the biological sample, wherein each of the at least two biomarkers is independently capable of binding to a viral antigen, e.g., any of HA, F, S, SI, S2, S-NTD, S-ECD, S-RBD, M, E, or N as described herein. In embodiments, the multiplexed method is capable of simultaneously quantifying two, tirree, four, five, or more than five biomarkers in the biological sample, wherein each biomarker is independently capable ofbindmg to a viral antigen, e.g., any of HA, F, S, SI, S2, S-NTD, S-ECD, S-RBD, M, E, or N as described herein. In embodiments, die multiplexed method comprising quantifying a combination of the biomarkers provided herein has improved sensitivity and/or dynamic range, compared to a method in which only a single biomarker is quantified. For example, a multiplexed method can provide earlier and more sensitive detection compared to a method that detects a single biomarker, since responses to each viral antigen may vary between individuals. Moreover, the ability to simultaneously measure antibody responses against multiple similar viruses, e.g.. a newly-emerged coronavirus such as SARS-CoV-2 and similar coronaviruses viruses such as hCoV-OC43. hCoV-HKUl. and hCoV-NL63, which have been circulating in the general population, improves understanding of how an individual's prior exposure to similar circulating viruses affects the individual's response to the newly-emerged virus of interest.

[00108] In embodiments, the method is used to diagnose whether a subject is infected with a virus, e.g., SARS-CoV-2. In embodiments, the method is used to assess the severity⁷ and/or prognosis of a viral infection in a subject. In embodiments, the method is used to determine whether a subject has been previously exposed to a virus. In embodiments, the method is used to estimate the time of virus exposure and/or infection. In embodiments, tire method is used to determine whether a subject has immunity to a virus, hr embodiments, the virus is a coronavirus. In embodiments, the virus is SARS-CoV-2.

[00109] In embodiments, the method is used to identify individuals with previous virus exposure for epidemiological studies (e.g., to understand true disease prevalence and evaluate the efficacy of infection control measures). In embodiments, the method is used to identify individuals at lower risk of future infection. Moreover, the method can be an important tool in the research, development, and validation of a vaccine for the virus. In embodiments, the method is used to assess differences in immune responses (e.g., antibody response) between individuals whose immunity is achieved by natural infection or vaccination. For example, a multiplexed method differentiates an individual's response to vaccination with different constructs of a viral antigen (e.g., different fragments of the S protein), compared with tire individual's response to natural infection by the virus. Such a method can advantageously distinguish between individuals with biomarkers produced in response an active infection and are potentially contagious and individuals with biomarkers produced in response to the vaccine. In embodiments, the virus is a coronavirus. In embodiments, the virus is SARS-CoV-2.

[00110] In embodiments, the biomarker capable of binding to a viral antigen is an immune biomarker. In embodiments, the biomarker is an antibody or antigen-binding fragment thereof. In embodiments, the biomarker is an immunoglobulin A (IgA), immunoglobulin G (IgG; including IgG subclasses IgGl , IgG2. IgG3, and IgG4). immunoglobulin M (IgM), immunoglobulin E (IgE), or immunoglobulin D (IgD), or antigen-binding fragments thereof capable of binding to S. SI, S2, S-NTD, S-ECD, S-RBD, M, E, and/or N. In embodiments, the IgG, IgA, IgM, IgD, and/or IgE is from a human, mouse, rat, ferret, minx, bat, or combination thereof. In embodiments, the biomarker is an IgA or antigen-binding fragment thereof capable of binding to S, SI. S2, S-NTD, S-ECD, S-RBD, M, E, and/or N. In embodiments, the biomarker is an IgG or antigen-binding fragment thereof capable of binding to S, SI, S2, S-NTD, S-ECD, S-RBD, M, E, and/or N. In embodiments, the biomarker is an IgGl or antigen-binding fragment thereof capable of binding to S, SI, S2, S-NTD, S-ECD, S-RBD, M, E, and/or N. In embodiments, the biomarkcr is an IgG2 or antigen- binding fragment thereof capable of binding to S, SI. S2, S-NTD, S-ECD, S-RBD, M, E, and/or N. In embodiments, the biomarker is an IgG3 or antigen-binding fragment thereof capable of binding to S. SI, S2, S-NTD, S-ECD, S-RBD, M, E, and/or N. In embodiments, the biomarker is an IgG4 or antigen-binding fragment thereof capable of binding to S. SI, S2, S-NTD, S-ECD, S-RBD, M. E, and/or N. In embodiments, the biomarker is an IgM or antigen-binding fragment thereof capable of binding to S. SI. S2, S-NTD, S-ECD, S-RBD, M, E, and/or N. In embodiments, the biomarker is an IgE or anti gen -binding fragment thereof capable of binding to S. SI, S2, S-NTD, S-ECD, S-RBD, M. E, and/or N. In embodiments, the biomarker is an IgD or antigen-binding fragment thereof capable of binding to S. SI, S2, S-NTD, S-ECD, S-RBD, M, E, and/or N. In embodiments, the viral antigen is a coronavirus antigen. In embodiments, the coronavirus is SARS-CoV-2. In embodiments, the biomarker binds to SARS-CoV-2 S- D614. In embodiments, tire biomarker binds to SARS-CoV-2 S-D614G. In embodiments, the biomarker binds to a SARS-CoV-2 S protein or subunit or fragment thereof that comprises a mutation as shown in FIGS. 1 A and IB. hr embodiments, tire biomarker binds to a SARS-CoV-2 N protein drat comprises a mutation as shown in FIG. 1A.

[00111] In embodiments, the biomarker to be detected is an antibody biomarker, and the binding reagent is a viral antigen that is bound by the antibody biomarker. In embodiments, the binding reagent is a viral protein described herein, e.g., HA, F, S, SI, S2, S-NTD, S-ECD, S-RBD, M, E, N.

[00112] In embodiments, the binding reagent is a peptide antigen. Peptide antigens are short peptides of a native, full-length protein that include the antibody binding epitope. Peptide antigens can be easier to produce and provide greater flexibility in performing an immunoassay to detect an antibody biomarker. Peptide antigens can also have higher specificity to the antibody biomarker compared with a full-length viral protein or domain described herein. In embodiments, an immunoassay utilizing a peptide antigen as the binding reagent has reduced cross-reactivity with antibody biomarkers for a different virus that are present in the biological sample. For example, an immunoassay utilizing a SARS-CoV-2 peptide antigen can have reduced cross-reactivity for antibodies that may be present in a subject for a circulating coronavirus.

[00113] In embodiments, the peptide antigen is a fragment of a viral protein, e.g., a coronavirus protein. In embodiments, the peptide antigen comprises about 10 to about 100 amino acids. In embodiments, the peptide antigen comprises about 20 to about 80 amino acids. In embodiments, the peptide antigen comprises about 30 to about 60 amino acids. In embodiments, the peptide antigen comprises about 40 to about 50 amino acids. In embodiments, the peptide antigen is a fragment of S. SI, S2, S-NTD, S-ECD, S- RBD, M, E, or N. In embodiments, the peptide antigen comprises an immunodominant region (IDR) of a viral protein. In embodiments, the peptide antigen comprises amino acids 1-49 of the N protein IDR. In embodiments, the peptide antigen comprises amino acids 340-390 of tire N protein IDR. In embodiments, the peptide antigen comprises amino acids 192-220 of the of the N protein IDR. In embodiments, the peptide antigen comprises amino acids 182-216 of the M protein IDR.

[00114] IgA. IgG (and subclasses thereof), IgM, IgE, and IgD are different isotypes of antibodies that have different immunological properties and functional locations. For example, IgA is ty pically found in the mucosal areas, such as the respiratory and gastrointestinal tracts, saliva, and tears and can prevent colonization by pathogens. IgG. the most abundant antibody isotype, has four subclasses as described herein and is found in all bodily fluids and provides the majority of antibody-based immunity against pathogens. IgM is mainly found in the blood and lymph fluid and is typically the first antibody made by the body to fight a new infection. IgE is mainly associated with allergic reactions (e.g., as part of aberrant immune response) and is found in the lungs, skin, and mucous membranes. IgD mainly functions as an antigen receptor on B cells and may activate basophils and mast cells to produce antimicrobial factors. Based on the timing and/or ty pe of infection, different amounts of each isotype are produced.

[00115] In embodiments, the method is a multiplexed immunoassay method capable of quantifying the amount of each isotype of antibodies, e.g., IgG. IgA, IgE, and IgM, present in tire biological sample. In embodiments, the amounts of the different isotypes of antibodies measured in a biological sample, e.g., the amounts of each of IgG, IgA, IgE, and IgM, can be used to determine whether a subject has been previously exposed to a virus. In embodiments, the amounts of the different isotypes of antibodies measured in a biological sample, e.g., the amounts of each of IgG, IgA, IgE, and IgM, can be used to estimate the time of virus exposure and/or infection. In embodiments, the amounts of tire different isotypes of antibodies measured in a biological sample, e.g., the amounts of each of IgG, IgA, IgE, and IgM, can be used to determine whether a subject has immunity to a virus, e.g., a coronavirus such as SARS-CoV-2.

[00116] In embodiments, the method comprises: (a) contacting tire biological sample with: at least a first, second, third, and fourth viral antigens, wherein each viral antigen specifically binds to IgG, IgA, IgE, and IgM, respectively; (b) fanning at least a first, second, third, and fourth binding complex comprising the viral antigens and IgG, IgA, IgE, or IgM; and (c) measuring the concentration of IgG, IgA, IgE, or IgM in each of the binding complexes. In embodiments, each viral antigen is independently S, SI. S2. S-NTD. S- ECD, S-RBD, M, E, N, or a peptide antigen described herein. In embodiments, the IgG. IgA, IgE, and/or IgM is from a human, mouse, rat, ferret, minx, bat, or combination thereof.

[00117] IgG is further divided into four subclasses. IgGl. IgG2, IgG3, and IgG4, based on properties such as ability to activate complement, bind to macrophages, and/or pass through the placenta. Each subclass also has a distinct biological function. For example, the response to protein antigens is primarily mediated by IgGl and IgG3, while IgG2 primarily mediates the response to polysaccharide antigens. IgG4 plays a role in protection against certain hypersensitivity reactions and pathogenesis of some autoimmune diseases. IgG subclass screening is performed to monitor a subject's infection response and/or determine whether a subject has antibody deficiency, and/or assess a subject's risk of an adverse response to infection. In embodiments, the method comprises determining the amount of IgGl, IgG2, IgG3, and IgG4 in the biological sample. In embodiments, the IgG is from a human, mouse, rat, ferret, minx, bat, or combination thereof.

[00118] In embodiments, the method comprises: (a) contacting the biological sample with: at least a first, second, third, and fourth viral antigens, wherein each viral antigen specifically binds to IgGl, IgG2, IgG3, and IgG4 respectively; (b) forming at least a first, second, third, and fourth binding complex comprising the viral antigens and IgGl, IgG2, IgG3, or IgG4; and (c) measuring the concentration of IgGl, IgG2, IgG3, or IgG4 in each of the binding complexes. In embodiments, each viral antigen is independently S, SI. S2, S- NTD. S-ECD. S-RBD. M, E. N, or a peptide antigen described herein. In embodiments, the IgG is from a human, mouse, rat, ferret, minx, bat, or combination thereof.

[00119] In embodiments, the method comprises: (a) contacting the biological sample with: a plurality of viral antigens, wherein each viral antigen specifically binds to an immunoglobulin selected from IgGl, IgG2, IgG3, IgG4, IgA. IgE, and IgM; (b) forming a plurality of binding complexes comprising the viral antigens and immunoglobulins; and (c) measuring the concentration of the immunoglobulin in each of the binding complexes. In embodiments, each viral antigen is independently S, SI, S2, S-NTD, S-ECD, S- RBD, M, E, N. or a peptide antigen described herein. In embodiments, the IgG, IgA, IgE, and/or IgM is from a human, mouse, rat, ferret, minx, bat, or combination thereof.

Inflammatory/Tissue Damage Response Biomarkers

[00120] In embodiments, the invention provides a method for detecting a biomarker in a subject to detect a viral infection, e.g., by a respiratory virus, including coronaviruses such as SARS-CoV-2. In embodiments, the invention provides a method for detecting a biomarker in a subject to assess the severity and/or prognosis of a viral infection, e g., by a respiratory virus, including coronaviruses such as SARS- CoV-2. In embodiments, the biomarker is produced in response to the viral infection. In embodiments, the biomarker is a stress response protein. In embodiments, the biomarker is an inflammatory response biomarker. In embodiments, the biomarker is a tissue damage response biomarker. In embodiments, the biomarker is a T cell activation biomarker. In embodiments, the biomarker is an extracellular vesicle.

[00121] In embodiments, the binding reagent that specifically binds the biomarker described herein is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or aptamer. In embodiments, the binding reagent is an antibody or a variant thereof, including an antigen/epitope-binding portion thereof, an antibody fragment or derivative, an antibody analogue, an engineered antibody, or a substance that binds to antigens in a similar manner to antibodies. In embodiments, the binding reagent comprises at least one heavy or light chain complementarity determining region (CDR) of an antibody. In embodiments, the binding reagent comprises at least two CDRs from one or more antibodies, hi embodiments, the binding reagent is an antibody or antigen-binding fragment thereof.

Extracellular Vesicles

[00122] In embodiments, the biomarker is an extracellular vesicle. Extracellular vesicles, also known as EVs or exosomes, are small membrane vesicles released by most cell types. For example, virus-infected cells release EVs that can mediate further in vivo viral spread in a variety of ways and produce other pathogenic effects. For example, EVs have been shown to transfer membrane-associated viral proteins, viral cargo proteins or RNAs, indirectly assist pathogens in escaping die immune system, or inhibit an immune response. EVs can also transfer viral genes from SARS-CoV-2 infected to non-infected cells and can induce inflammation in the absence of direct viral infection.

[00123] In embodiments, detecting EVs from infected cells is used to identify reservoirs of infection. In embodiments, EV populations in a biological sample are analyzed to determine the mechanism of infection, disease prognosis, and adaptive immunity. In embodiments, an EV released from a particular cell, e.g., an immune cell, comprises one or more of the same surface marker as that cell. In embodiments, the biomarker is an EV comprising an inflammatory damage and/or a tissue damage protein as described herein, on the surface of the EV.

Viral Component and Biomarker Detection

[00124] In embodiments, the invention provides a method comprising simultaneously detecting a host biomarker (e.g., an antibody biomarker or inflammatory and/or tissue damage response biomarker) described herein and a viral component described herein. A method that simultaneously determines, from a single sample, whether a subject is infected by a virus (e.g., a coronavirus such as SARS-CoV-2) and assesses the subject's immune response is capable of determining the subject's disease prognosis, for example, determining whether the subject will likely have poor disease progression and increased likelihood of intensive care treatment. Thus, the method enables preparation of an early response to a potentially serious illness.

[00125] In embodiments, the method is a multiplexed immrmoassay method. In embodiments, the multiplexed immunoassay method detects a viral nucleic acid, a host antibody biomarker, a host inflammatory and/or tissue damage response biomarker, or a combination thereof.

[00126] In embodiments, a subject's infection status, disease progression, prognosis, or combination thereof is assessed by simultaneously detecting (1) a viral component, (2) a host antibody biomarker, and (3) a host inflammatory and/or tissue damage response biomarker as described herein. For example, Table 3 provides exemplary outcomes and assessments based on the combined detection for diagnosis and prognosis of CO VID-19, the disease caused by SARS-CoV-2 infection.

Table 3. Exemplary Scenarios and Predicted Outcomes

Samples and Assay Devices

[00127] In embodiments, the viruses, viral components, and/or biomarkers described herein are measured in a biological sample. In embodiments, the biological sample comprises a mammalian fluid, secretion, or excretion. In embodiments, the sample is a purified mammalian fluid, secretion, or excretion. In embodiments, the mammalian fluid, secretion, or excretion is whole blood, plasma, serum, sputum, lachrymal fluid, lymphatic fluid, synovial fluid, pleural effusion, urine, sweat, cerebrospinal fluid, ascites, milk, stool, a respiratory sample, bronchial/bronchoalveolar lavage, saliva, mucus, oropharyngeal swab, sputum, endotracheal aspirate, pharyngeal/nasal swab, throat swab, amniotic fluid, nasal secretions, nasopharyngeal wash or aspirate, nasal mid-turbinate swab, vaginal secretions, a surface biopsy, sperm, semen/seminal fluid, wound secretions and excretions, ear secretions or discharge, or an extraction, purification therefrom, or dilution thereof. In embodiments, the biological sample is diluted such that the assay signal is within tire upper and lower detection limits of the assay, hr embodiments, tire biological sample is diluted to achieve a desired assay sensitivity. Further exemplary biological samples include but are not limited to physiological samples, samples containing suspensions of cells such as mucosal swabs, tissue aspirates, endotracheal aspirates, tissue homogenates, cell cultures, and cell culture supernatants. In embodiments, the biological sample is a respiratory sample obtained from the respiratory' tract of a subject. Examples of respiratory' samples include, but arc not limited to, bronchial/bronchoalveolar lavage, saliva, mucus, endotracheal aspirate, sputum, nasophary ngcal/nasal swab, throat swab, orophary ngeal swab and die like. In embodiments, die biological sample is whole blood, serum, plasma, cerebrospinal fluid (CSF), urine, saliva, sputum, endotracheal aspirate, nasopharyngeal/nasal swab, bronchoalveolar lavage, or an extraction or purification therefrom, or dilution thereof. In embodiments, the biological sample is blood that has been dried and reconstituted. In embodiments, the biological sample is serum or plasma. In embodiments, the plasma is in EDTA, heparin, or citrate. In embodiments, the biological sample is saliva. In embodiments, the biological sample is endotracheal aspirate. In embodiments, the biological sample is a nasal swab. In embodiments, the virus, viral component, and/or biomarkers described herein have substantial levels in the saliva or endotracheal aspirate of a subject. In embodiments, the virus, viral components, and/or biomarkers described herein are present in higher amounts in certain bodily fluids (e.g., saliva) compared to others (e.g.. throat swab). In embodiments, certain antibody biomarker levels, e.g.. IgG (including subclasses thereof) and IgA, are substantially similar in blood and saliva of a subject. In embodiments, the ratio of antibody levels to different components from a virus (e.g.. SARS-CoV-2 S and N proteins) are highly correlated in blood and saliva of a subject. In embodiments, tire ratio of antibody levels to different components from a virus, e.g., the ratio of the antibody levels against the SARS-CoV-2 S protein and the SARS-CoV-2 N protein is used to assess the immune response and/or clinical outcome of a subject infected with SARS-CoV-2.

[00128] In embodiments, the biological sample is from an animal. In embodiments, the biological sample from an animal is useful for animal model studies, e.g., for vaccine and/or drug research and development, and/or to better understand disease progression and infection lethality. Exemplary animals that are usefill for animal model studies include, but are not limited to, mouse, rat, rabbit, pig, primate such as monkey , and the like.

[00129] In embodiments, the biological sample is from a human or an animal subject. In embodiments, die subject is susceptible or suspected to be susceptible to infection by the viruses described herein. In embodiments, the subject is known or suspected to transmit the viruses described herein. Virus transmission may occur among the same species (e.g., human-to-human) or inter-species (e.g.. bat-to-human). Nonlimiting examples of animal subjects include domestic animals, such as dog. cat. horse, goat, sheep, donkey, pig, cow. chicken, duck, rabbit, gerbil, hamster, guinea pig, and the like; non-human primates (NHP) such as macaque, baboon, marmoset, gorilla, orangutan, chimpanzee, monkey, and the like; big cats such as tiger, lion, puma, leopard, snow leopard, and the like; and other mammals such as bats and pangolins. In embodiments, the biological sample is from a human, a mouse, a rat, a ferret, a minx, or a bat. In embodiments, the subject is a host that has been exposed to and/or infected by a virus as described herein. In embodiments, the biological ample comprises a plasma (e.g.. in EDTA, heparin, or citrate) sample from a subject. In embodiments, the biological sample comprises a serum sample from a subject. In embodiments, the biological sample is from a healthy subject. In embodiments, the biological sample is from a subject known to never have been exposed to a virus described herein. In embodiments, the biological sample is from a subject known to be immune to a virus described herein. In embodiments, the biological sample is from a subject known to be infected with a virus described herein. In embodiments, the biological sample is from a subject suspected of having been exposed to a virus described herein. In embodiments, the biological sample is from a subject at risk of being exposed to a virus described herein. In embodiments, the virus is a coronavirus. In embodiments, the virus is SARS-CoV-2.

[00130] In embodiments, the sample is an environmental sample. In embodiments, tire environmental sample is aqueous, including but not limited to, fresh water, drinking water, marine water, reclaimed water, treated water, desalinated water, sewage, wastewater, surface water, ground water, runoff, aquifers, lakes, rivers, streams, oceans, and other natural or non-natural bodies of water. In embodiments, the aqueous sample contains bodily solids or fluids (e.g., feces or urine) from subjects who have been exposed to or infected with a virus herein (e.g., a coronavirus such as SARS-CoV-2). In embodiments, the environmental sample is from a air filtration device, e.g., air filters in a healthcare or long-term care facility or other communal places of gathering. Detection of a virus described herein (e.g., a coronavirus such as SARS- CoV-2) in an environmental sample can provide early identification and/or tracing of an outbreak or potential outbreak, thereby allowing a more prompt and robust response. Moreover, detection of a biomarker, e.g.. one or more antibody biomarkers that specifically binds a viral antigen (e.g., from a coronavirus such as SARS-CoV-2) in an environmental sample can provide an estimation of the percentage of a population with detectable antibodies against the virus (i.e., seroconversion), which is useful for epidemiology studies. In some embodiments, tire sample comprises wastewater. Detection of SARS-CoV-2 in wastewater is described, e.g., in U.S. Publication No. 2022/0003766 and U.S. Publication No. 2021/0349104.

[00131] Wastewater samples are also uselul for determining the viral strain, i.e.. the genotype, of SARS- CoV-2 in a population. SARS-CoV-2 strains are further described herein and include, e.g., the L strain and tire S strain, which differ at genome locations 8782 and 28144; and the S-D614 strain and the S-D614G strain, which differ by a single polynucleotide at genome location 23403, and the strains described in FIG. 1A, e.g., strains B.l.1.7, 501Y.V2, P.l, and Cal.20C. In embodiments, the invention provides a method for detecting SARS-CoV-2 nucleic acid in a wastewater sample, comprising: a) contacting the wastewater sample with a binding reagent that specifically binds a SARS-CoV-2 nucleic acid; b) forming a binding complex comprising the binding reagent and the SARS-CoV-2 nucleic acid; and c) detecting the binding complex, thereby detecting the SARS-CoV-2 nucleic acid in the wastewater sample. In embodiments, the SARS-CoV-2 nucleic acid comprises a SARS-CoV-2 single nucleotide polymorphism (SNPs) or mutation as described herein, e.g., in FIGS. 1 A and 1 C. In embodiments, the method is a multiplexed method that simultaneously detects one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more SARS-CoV-2 SNPs. Methods of detecting SNPs in viral nucleic acids, e.g., SARS-CoV-2 RNA, are provided herein. In embodiments, levels of IgA, IgG, and/or IgM in wastewater samples are used as controls for normalizing tire detected amount of viral protein and/or genetic material (e.g., RNA) in the waste water sample.

[00132] In embodiments where the sample comprises a liquid (e.g., endotracheal aspirate, saliva, blood, serum, plasma and the like), the sample is about 0.05 mL to about 50 mL, about 0.1 mL to about 10 ink, about 0.2 mL to about 5 mL, or about 0.3 mL to about 3 mL. In embodiments where the sample is solid or semi-solid (e.g., a swab such as a nasopharyngeal swab or oropharyngeal swab, mucus, sputum and the like), the sample is provided into a storage liquid of about 0.05 mL to about 50 mL, about 0.1 mL to about 10 mL, about 0.2 mL to about 5 mL, or about 0.3 mL to about 3 mL. In embodiments, the storage liquid is Viral Transport Medium (VTM), Amies transport medium, or sterile saline. In embodiments, the storage liquid comprises a substance for stabilizing nucleic acids, e.g.. EDTA. In embodiments, the storage liquid comprises a reagent for inactivating live virus as described herein.

[00133] In embodiments, the sample comprises saliva. In embodiments, the invention provides a method of identifying a saliva sample in which the viral component and/or biomarker of interest has degraded, i.e., a low qualify saliva sample. In embodiments, a low quality saliva sample is not suitable for the assays described herein. In embodiments, a low qualify saliva sample comprises low levels of total antibodies as compared to a freshly obtained sample and/or as compared to a threshold total antibody level. In embodiments, a low qualify saliva sample comprises low levels of IgA as compared to a freshly obtained sample and/or as compared to a threshold antibody level. In embodiments, the threshold antibody level is determined based on the average of an aggregate of samples. In embodiments, a low qualify saliva sample comprises low levels of antibodies against circulating coronaviruses (e.g.. hCoV-NL63, hCoV-HKUl, hCoV-229E, and/or hCoV-OC43) as compared to a freshly obtained sample and/or a threshold antibody level. In embodiments, identifying die low qualify saliva sample comprises determining the total antibody level in a sample and. if the sample has low antibody levels as compared to a freshly isolated control sample and/or as compared to a threshold total antibody level, identify ing the sample as a low qualify saliva sample. In embodiments, identifying the low qualify saliva sample comprises determining the IgA level in a sample and, if the sample has low IgA levels as compared to a freshly isolated control sample and/or as compared to a threshold antibody level, identify ing the sample as a low qualify saliva sample. In embodiments, identify ing the low qualify' saliva sample comprises determining the levels of antibodies against one or more circulating coronaviruses in a sample and, if the sample has low antibody levels against the one or more circulating coronaviruses as compared to a freshly isolated control sample and/or a threshold antibody level, identifying the sample as a low quality saliva sample.

[00134] In embodiments, the sample comprises an extracellular vesicle. As described herein, extracellular vesicles (also known as EVs or exosomes) are small membrane vesicles released by most cell types, including immune cells and infected cells (e.g., by a respiratory virus described herein such as SARS-CoV- 2). Detection and analysis of EVs are further described, e.g., in US 2022/0003766; US 2021/0349104; WO 2019/222708; and WO 2020/086751.

[00135] In embodiments, the sample is pretreated prior to being subjected to the methods provided herein. In embodiments, the sample is pretreated prior to being handled by, processed by, or in contact with laboratory' and/or clinical personnel. In embodiments, pretreating the sample comprises subjecting the sample to conditions sufficient to inactivate live virus in the sample. Inactivation of live virus that may be present in the sample reduces the risk of infection of the laboratory and/or clinical personnel handling and/or processing the sample, e.g., by performing the methods described herein on the sample. In embodiments, pretreating the sample comprises heating die sample to at least 55 °C, at least 56 °C, at least 57 °C, at least 58 °C, at least 59 °C, at least 60 °C, at least 65 °C, at least 70 °C, at least 75 °C, at least 80 °C, at least 85 °C, at least 90 °C, at least 95 °C, or at least 100 °C. In embodiments, the sample is heated for about 10 minutes to about 4 hours, about 20 minutes to about 2 hours, or about 30 minutes to about 1 hour. In embodiments, the sample is heated to about 65 °C for at least 10 minutes. In embodiments, the sample is heated to about 65 °C for at least 30 minutes. In embodiments, the sample is heated to about 58 °C for at least 1 horn.

[00136] In embodiments, pretreating the sample comprises contacting the sample with an inactivation reagent. In embodiments, the inactivation reagent comprises a detergent, a chaotropic agent, a fixative, or a combination thereof. Non-limiting examples of detergents include sodium dodecyl sulfate and TRITON™ X-100. Non-limiting examples of chaotropic agents include guanidium thiocyanate, guanidium isothiocyanate, and guanidium hydrochloride. Non-limiting examples of fixatives include formaldehyde, formalin, paraformaldehyde, and glutaraldehyde. In embodiments, pretreating the sample comprises subjecting the sample to UV or gamma irradiation. In embodiments, pretreating the sample comprises subj ectmg the sample to a highly alkaline (e.g., above pH 10. above pH 11, or above pH 12) condition. In embodiments, pretreating the sample comprises subjecting the sample to a highly acidic (e.g.. below pH 4. below pH 3, below pH 2) condition. Additional methods of pretreating samples, e.g., containing the viruses described herein, is further discussed in Bain et al., Curr Protoc Cy tometry 93:e77 (2020).

[00137] In embodiments, the sample comprises a viral nucleic acid. In embodiments, the sample comprising the viral nucleic acid is pretreated with a reagent that stabilizes and/or prevents degradation of the viral nucleic acid. In embodiments, the pretreating comprises removing and/or inhibiting activity of a nuclease, e.g., an RNase, in the sample. In embodiments, the viral nucleic acid is SARS-CoV-2 RNA. [00138] In embodiments, the sample comprises an RT-PCR product. In embodiments, the RT-PCR product comprises a cDNA that is generated from a viral RNA. In embodiments, the sample comprising the RT-PCR product is pretreated to remove tire viral RNA and/or a reagent used in the RT-PCR. In embodiments, the pretreating comprises contacting the sample with RNase. In embodiments, the pretreating comprises heating the sample, e.g., as described herein. In embodiments, the viral RNA is SARS-CoV-2 RNA.

[00139] In embodiments, the sample is pretreated immediately after being collected, e.g.. from a subject described herein. Sample collection methods are provided herein. In embodiments, the sample is pretreated while being transported to a facility, e.g.. a laboratory, for processing and analyzing the sample, e g. using the methods described herein. In embodiments, the sample is pretreated after arrival at a facility, e.g., a laboratory, for processing and analyzing the sample, e.g. using the methods described herein. In embodiments, the sample is pretreated prior to being stored. In embodiments, tire sample is stored prior to processing and analysis, e.g. using the methods described herein. In embodiments, the sample is stored at about -80 °C to about 30 °C, about -70 °C to about 25 °C, about -60 °C to about 20 °C, about -20 °C to about 15 °C, about 0 °C to about 10 °C, about 2 °C to about 8 °C, or about 4 °C to about 12 °C. Methods and conditions for storing the samples described herein are know n to one of ordinary’ skill in the art.

[00140] As used herein, the term "exposure," in the context of a subject being exposed to a virus, refers to die introduction of a virus into the subject's body. "Exposure" does not imply any particular am omit of virus; introduction of a single viral particle into the subject's body can be referred to herein as an "exposure" to the virus. As used herein, the term "infection," in the context of a subject being infected with a virus, means that die virus has penetrated a host cell and has begun to replicate, assemble, and release new viruses from the host cell. The term "infection" can also be used to refer to an illness or condition caused by a virus, e.g., respiratory tract infection as described herein.

[00141] In embodiments, the virus, viral component, and/or biomarker are detectable in a subject immediately (e.g., within seconds) after the subject is exposed to the virus and/or infected with the virus. In embodiments, the virus, viral component, and/or biomarker are detectable in a subject within about 5 minutes to about 1 year, about 1 hour to about 9 months, about 6 hours to about 6 months, about 12 horns to about 90 days, about 1 day to about 60 days, about 2 days to about 50 days, about 3 days to about 40 days, about 4 days to about 30 days, about 5 days to about 28 days, about 6 days to about 25 days, about 7 days to about 22 days, or about 8 days to about 20 days after the subject is exposed to the virus and/or infected with the virus. In embodiments, the virus, viral component, and/or biomarker are detectable in a subject within about 5 minutes, about 1 hour, about 3 hours, about 6 hours, about 12 hours, about 1 day. about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 14 days, about 21 days, about 1 month, about 2 months, about 3 months, about 6 months, about 1 year, or more than 1 year after the subject is exposed to the virus and/or infected with the virus. Different biomarkers, e.g., antibody biomarkers or inflammatory or tissue damage response biomarkers, in the same subject may have a varying magnitude of change in response to virus exposure and/or infection, for example, depending on whether the biomarker is an acute response biomarker or a biomarker related to a long-term effect. For some viral infections, the antibody biomarker IgG ty pically plateaus after 10 days of disease onset and persist (e.g., potentially signifying longer-term immunity ); the antibody biomarkers IgA and IgM are detectable within 6 days of disease onset, peak around 10 days, and diminish after approximately 14 days (e.g., as part of tire initial infection response). Different viruses can trigger biomarker responses at different times. For example, antibodies to SARS-CoV-2 may not be consistently detected in a subject until about three weeks after infection, which is longer than the typical timing for other types of viral infections. The timing of producing the same biomarker type, e.g., IgM or IgG antibody, can also vary widely among different subjects. Thus, in embodiments, the methods for multiplexed assays for a combination of biomarkers disclosed herein includes a determination or consideration of the response timing of each of the biomarkers. [00142] In embodiments, the biological sample is obtained from a subject who has not been exposed to the virus. In embodiments, the biological sample is obtained from a subject immediately (e.g., within seconds) after the subject is known or suspected to be exposed to the virus. In embodiments, the biological sample is obtained from a subject within about 5 minutes to about 1 year, about 1 hour to about 9 months, about 6 hours to about 6 months, about 12 horns to about 90 days, 1 day to about 60 days, about 2 days to about 50 days, about 3 days to about 40 days, about 4 days to about 30 days, about 5 days to about 28 days, about 6 days to about 25 days, about 7 days to about 22 days, or about 8 days to about 20 days after the subject is known or suspected to be exposed to the virus. In embodiments, the biological sample is obtained from a subject within about 5 minutes, about 1 hour, about 3 horns, about 6 horns, about 12 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 14 days, about 21 days, about 1 month, about 2 months, about 3 months, about 6 months, about 1 year, or more than 1 year after the subject is known or suspected to be exposed to the virus.

[00143] In embodiments, the biological sample is obtained from a subject prior to the subject showing any symptoms of a viral infection. In embodiments, the biological sample is obtained from a subject immediately (e.g., within seconds) after the subject begins to show symptoms of a viral infection, hr embodiments, the biological sample is obtained from a subject within about 5 minutes to about 1 year, about 1 hour to about 9 months, about 6 horns to about 6 months, about 12 horns to about 90 days, about 1 day to about 60 days, about 2 days to about 50 days, about 3 days to about 40 days, about 4 days to about 30 days, about 5 days to about 28 days, about 6 days to about 25 days, about 7 days to about 22 days, or about 8 days to about 20 days after the subject begins to show symptoms of a viral infection. In embodiments, the biological sample is obtained from a subject within about 5 minutes, about 1 hour, about 3 hours, about 6 horns, about 12 hours, about 1 day. about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 14 days, about 21 days, about 1 month, about 2 months, about 3 months, about 6 months, about 1 year, or more than 1 year after the subject begins to show symptoms of a viral infection. Symptoms of a viral infection are described herein and include, e.g., cough, shortness of breath, fever, and fatigue.

[00144] In embodiments, the biological sample is obtained from a subject after the subject is diagnosed with a viral infection. As described herein, the SARS-CoV-2 virus can cause post-acute COVID-19 syndrome, with certain symptoms persisting weeks or months after the initial illness period. In embodiments, the biological sample is obtained from a subject after about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 9 months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, or more than 10 years after the subject is diagnosed with the viral infection. [00145] In embodiments, the biological sample is obtained from a subject prior to the subject being administered with a vaccine or a treatment for the virus described herein. In embodiments, the biological sample is obtained from a subject immediately (e.g.. within seconds) after a vaccine or a treatment is administered to the subject. In embodiments, the biological sample is obtained from a subject within about 12 hours to about 90 days, about 1 day to about 60 days, about 2 days to about 50 days, about 3 days to about 40 days, about 4 days to about 30 days, about 5 days to about 28 days, about 6 days to about 25 days, about 7 days to about 22 days, or about 8 days to about 20 days after a vaccine or a treatment is administered to the subject. In embodiments, the biological sample is obtained from a subject within about 5 minutes, about 1 hour, about 3 horns, about 6 hours, about 12 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 14 days, about 21 days, about 1 month, about 2 months, about 3 months, about 6 months, about 1 year, or more than 1 year after a vaccine or a treatment is administered to the subject.

Sample Pooling

[00146] Samples may be obtained from a single source described herein, or may contain a mixture from two or more sources, e.g.. pooled from one or more individuals who may have been exposed to or infected by a particular virus in a similar manner. Sample pooling strategies are further described, e.g., in U.S. Publication No. 2022/0003766 and U.S. Publication No. 2021/0349104. For example, tire individuals may live or have lived in the same household, visited the same location(s), and/or associated with the same people. In embodiments, samples are pooled from two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, 50 or more, 100 or more, 150 or more, 200 or more, 300 or more, 400 or more, 500 or more, 1000 or more, 5000 or more, or 10000 or more individuals. For example, a "negative" result for an active viral infection from a pooled sample indicates that none of the individuals from die pooled sample have an active infection, which can significantly reduce the number of tests needed to test every individual in a population. In embodiments, die sample comprises a respiratory sample, e.g., bronchial/bronchoalveolar lavage, saliva, mucus, orophary ngeal swab, sputum, endotracheal aspirate, pharyngeal/nasal swab, throat swab, nasal secretion, or combmation thereof. In embodiments, the sample comprises saliva. In embodiments, die sample comprises blood. In embodiments, the sample comprises serum or plasma. In embodiments, die virus is a coronavirus. In embodiments, the virus is SARS-CoV-2. In embodiments, a "positive" result for an active viral infection in the pooled sample prompts or indicates a need for further testing using the methods and/or kits provided by the invention of individual samples comprised in the pool of samples.

[00147] In embodiments, the pooled sample is subjected to a single layer pooling strategy. A "single layer pooling strategy," as used herein, refers to testing a pooled sample, and if the result of the pooled sample is "positive" for an active viral infection, each individual sample comprised in the pooled sample is then individually tested, e.g.. using the methods and/or kits provided in the invention. In embodiments, the pooled sample is subjected to a multi-layer pooling strategy, e.g., a two-layer pooling strategy. In a "multilayer pooling strategy," a pooled sample containing n number of individual sample is tested in a first round, and if the result of the first round is "positive" for an active viral infection, then the pooled sample is divided into smaller pools, e.g., wherein each smaller pool comprises a number of individual samples equal to the square root of n, and re-tested in a second round. The smaller pool(s) with the "positive" results can be further divided into even smaller pools for one or more additional rounds of testing until the positive individual samples are identified. In an exemplary two-layer pooling strategy, a pooled sample containing 100 individual samples is tested in a first round, and if the pooled sample is tested to be "positive" for an active viral infection, then the pooled sample is divided into pools containing 10 individual samples. Each individual sample comprised in any 10-sample pools that tested "positive" are then tested.

[00148] In embodiments, the invention provides a method for determining the number of individual samples to be included in a pooled sample. In embodiments, the number of individual samples included in a pooled sample is based on disease prevalence in a population. For example, if disease prevalence is high, die likelihood of a pooled sample, containing a large number of individual samples, testing "positive" is also high, which reduces the benefits of testing pooled samples because additional tests are required to determine the positive individual samples.

[00149] In embodiments, each individual sample is about 0.1 mL to about 10 mL, about 0.2 mL to about 5 mL, or about 0.3 mL to about 3 mL. In embodiments, about 1%, about 2%, about 3%, about 4%, about 5%. about 10%, or about 20% of die total volume of each individual sample is added to the pooled sample. In embodiments, about 1 pL to about 100 pL, about 5 pL to about 50 pL. or about 10 pL to about 20 pL of each individual sample is added to die pooled sample. In embodiments, the amount of each individual sample not added to the pooled sample is sufficient for one or more additional roimds of testing (e.g.. in a multi-layered pooling strategy as described herein).

Collection and Assay Devices

[00150] In embodiments, die biological sample is a liquid sample. In embodiments, die biological sample is in contact with a sample collection device. In embodiments, the sample collection device is an applicator stick. In embodiments, the sample collection device comprises an elongated handle (e g., a rod or a rectangular prism) and a sample collection head configmed to collect sample from a biological tissue (e.g., from a subject's nasal or oral cavity) or a surface. In embodiments, the sample collection head comprises an absorbent material (e.g., cotton) or a scraping blade. In embodiments, the sample collection device is a swab. In embodiments, the sample collection device is a tissue scraper. In embodiments, the sample collection device is capable of collecting a sample described herein that may contain analytes at a concentration too low to support an accurate or reliable analysis result.

[00151] In embodiments, the sample collection device or the liquid sample is contacted with an assay cartridge. Assay cartridges are further described in. e.g.. U.S. Publication No. 2022/0003766 and U.S. Publication No. 2021/0349104. Assay cartridges may be used with assay cartridge readers known in the art. An exemplary assay cartridge reader is the MSD® Cartridge Reader instrument. Further exemplary' assay cartridges and assay cartridge readers are described, e.g., in US 9,921,166; US 10,184,884; US 9,731,297; US 8.343.526; US 10,281,678; US 10.272.436; US 2018/0074082; and US 2019/0391170.

[00152] In embodiments, the method is performed in an assay plate. Assay plates are known in the art and described, e.g.. in U.S. Publication No. 2022/0003766 and U.S. Publication No. 2021/0349104. Further exemplary assay plates are disclosed in, e g., US 7,842,246; US 8,790,578; and US 8,808,627. In embodiments, the assay plate result is read in a plate reader, e.g.. the MESO® QUICKPLEX® or MESO® SECTOR® instruments.

[00153] In embodiments, the method is performed on a particle. Particles known in the art. e.g., as described in U.S. Publication No. 2022/0003766 and U.S. Publication No. 2021/0349104, can be used in conjunction with the methods and kits described herein. In embodiments, tire particle comprises a microsphere.

[00154] Further exemplary devices for performing the methods herein include, but are not limited to, cassettes, measurement cells, dipsticks, reaction vessels, and assay modules described in, e.g., US 8,298,934 and US 9,878,323.

Assay Methods and Components

[00155] The viruses, viral components, and/or biomarkers described herein can be measured using a number of techniques available to a person of ordinary skill in the art, e.g., direct physical measurements (e.g., mass spectrometry) or binding assays (e.g., immunoassays, agglutination assays and immunochromatographic assays). Exemplary methods are described in, e.g., U.S. Publication No. 2022/0003766 and U.S. Publication No. 2021/0349104.

[00156] Exemplary binding assay methods include sandwich or competitive binding assays. Examples of sandwich immunoassays are described in US 4,168,146 and US 4,366,241. Examples of competitive immunoassays include those described in US 4,235,601; US 4,442,204; and US 5,208,535.

[00157] Multiple viruses, viral components, and/or biomarkers can be measured using a multiplexed assay format, e.g., as described in US 2022/0003766; US 2021/0349104; US 2003/0113713; US 2003/0207290; US 2004/0022677; US 2004/0189311; US 2005/0052646; US 2005/0142033; US 2006/0069872; US 5,807,522; US 6,110,426; US 6,977,722; US 7,842,246; US 10,189,023; and US 10,201,812.

[00158] The methods herein can be conducted in a single assay chamber, such as a single well of an assay plate. The methods herein can also be conducted in an assay chamber of an assay cartridge as described herein. The assay modules, e.g.. assay plates or assay cartridges, methods and apparatuses for conducting assay measurements suitable for die present invention, are described, e.g., in US 8,343,526; US 9,731,297; US 9.921.166; US 10,184,884; US 10.281.678; US 10,272,436; US 2004/0022677; US 2004/0189311; US 2005/0052646; US 2005/0142033; US 2018/0074082; and US 2019/0391170.

Binding

[00159] Binding reagents that specifically bind to viruses, viral components, and/or biomarkers are described herein and. e.g., in U.S. Publication No. 2022/0003766 and U.S. Publication No. 2021/0349104. In embodiments where the method comprises quantifying the amounts of one or more biomarkers capable of binding to a viral antigen (e.g., an antibody biomarker), the binding complex comprises the binding reagent and the antibody biomarker. In embodiments, the binding reagent is immobilized on a binding domain. In embodiments, the binding complex is formed on the binding domain.

[00160] In embodiments where the method is a multiplexed immunoassay method, more than one binding complex is formed, and each binding complex comprises a different binding reagent and its binding partner (e.g., a biomarker described herein). Multiplexed immunoassay methods are described herein and, e g., in U.S. Publication No. 2022/0003766 and U.S. Publication No. 2021/0349104. In embodiments, each of the binding reagents are immobilized on separate binding domains. In embodiments, each binding domain comprises a targeting agent capable of binding to a targeting agent complement, wherein the targeting agent complement is connected to a linking agent, and each binding reagent comprises a supplemental linking agent capable of binding to the linking agent.

[00161] In embodiments, an optional bridging agent, which is a binding partner of both the linking agent and die supplemental linking agent, bridges the linking agent and supplemental linking agent, such diat die binding reagents, each bound to its respective targeting agent complement, arc contacted with the binding domains and bind to their respective targeting agents via the bridging agent, the targeting agent complement on each of the binding reagents, and die targeting agent on each of the binding domains.

[00162] In embodiments, the targeting agent and targeting agent complement, and the linking agent and supplemental linking agent, are each two members of a binding partner pair selected from avidin-biotin, streptavidin-biotin. antibody -hapten, antibody -antigen, antibody -epitope tag, nucleic acid-complementary nucleic acid, aptamer-aptamer target, and receptor-ligand. In embodiments, the targeting agent and targeting agent complement are cross-reactive moieties, e.g., thiol and maleimide or iodoacetamide: aldehyde and hydrazide: or azide and alkyne or cycloalkyne. In embodiments, the targeting agent is biotin, and the targeting agent complement is avidin or streptavidin. In embodiments, the linking agent is avidin or streptavidin, and the supplemental linking agent is biotin. In embodiments, the targeting agent and targeting agent complement are complementary oligonucleotides. In embodiments, tire targeting agent complement is streptavidin, the targeting agent is biotin, and the linking agent and the supplemental linking agent are complementary oligonucleotides.

[00163] In embodiments, each binding domain is an element of an array of binding elements. In embodiments, the binding domains are on a surface. In embodiments, the surface is a plate. In embodiments, the surface is a well in a multi-well plate. In embodiments, the array of binding elements is located within a well of a multi-well plate. Non-limiting examples of plates include the MSD® SECTOR™ and MSD QUICKPLEX® assay plates, e.g., MSD® GOLD™ 96-well Small Spot Streptavidin plate. In embodiments, the surface is a particle. In embodiments, the particle comprises a microsphere. In embodiments, the particle comprises a paramagnetic bead. In embodiments, each binding domain is positioned on one or more particles. In embodiments, the particles are in a particle array. In embodiments, tire particles are coded to allow for identification of specific particles and distinguish between each binding domain. In embodiments, the surface is an assay cartridge surface. In embodiments, each binding domain is positioned in a distinct location on the assay cartridge surface. Detection

[00164] In embodiments, the method further comprises detecting the binding complex described herein. In embodiments, the binding complex comprising a binding reagent and its binding partner (e.g., a biomarker described herein) further comprises a detection reagent. In embodiments, the detection reagent specifically binds to the biomarker described herein. Detection methods are know n in the art and further described, e.g.. in U.S. Publication No. 2022/0003766 and U.S. Publication No. 2021/0349104.

[00165] In embodiments, the method comprises contacting the binding reagent with its binding partner and the detection reagent simultaneously or substantially simultaneously to form a binding complex. In embodiments, the method comprises contacting the binding reagent with its binding partner and the detection reagent sequentially to form a binding complex. In embodiments, the method comprises contacting the detection reagent with its binding partner and die binding reagent sequentially. In embodiments, the binding partner comprises a biomarker, e.g.. antibody biomarker described herein. [00166] In embodiments, the detection reagent is an antibody, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotopc, or aptamer. In embodiments, the detection reagent is an antibody or a variant diereof, including an antigen/epitope-binding portion thereof, an antibody fragment or derivative, an antibody analogue, an engineered antibody, or a substance that binds to antigens in a similar maimer to antibodies. In embodiments, detection reagent comprises at least one heavy or light chain complementarity determining region (CDR) of an antibody. In embodiments, the detection reagent comprises at least two CDRs from one or more antibodies. In embodiments, the detection reagent is an antibody or antigenbinding fragment thereof. In embodiments where the method comprises detecting and/or quantify ing the amounts of one or more biomarkers capable of binding to a viral antigen (e.g., an antibody biomarker), the detection reagent comprises an antigen (e.g., a viral protein described herein).

[00167] In embodiments, the detection reagent comprises a detectable label. In embodiments, measuring the concentration of the biomarkers in each of the binding complexes comprises measuring the presence and/or amount of the detectable label. In embodiments, the detectable label is measured by light scattering, optical absorbance, fluorescence, luminescence, chemiluminescence, electrochemiluminescence (ECL), bioluminescence, phosphorescence, radioactivity, magnetic field, or combination thereof. In embodiments, the detectable label comprises an electrochemiluminescence label. In embodiments, the detectable label comprises ruthenium. In embodiments, measuring the concentration of the biomarkers comprises measuring the presence and/or amount of the detectable label by electrochemiluminescence. In embodiments, the measuring of the detectable label comprises measuring an electrochemiluminescence signal.

[00168] In embodiments, detection reagent comprises a nucleic acid probe. In embodiments, the immunoassay further comprises binding the nucleic acid probe to a template oligonucleotide and extending the nucleic acid probe to form an extended sequence. In embodiments, the extended sequence binds to an anchoring reagent immobilized on the surface comprising the binding reagent. In embodiments, the virus, viral component, and/or biomarker is detected and/or quantified by detecting or quantify ing the amount of extended sequence bound to the surface. In embodiments, die surface is contacted w ith a labeled probe that binds to the extended sequence, wherein the labeled probe comprises a detectable label. [00169] In embodiments, the binding complex comprising the binding reagent and its binding partner (e.g., a biomarker described herein) further comprises a first detection reagent and a second detection reagent. In embodiments, the first detection reagent comprises a first nucleic acid probe, and the second detection reagent comprises a second nucleic acid probe. In embodiments, the immunoassay method further comprises binding the first and second nucleic acid probes to a template oligonucleotide and extending the second nucleic acid probe to form an extended sequence. In embodiments, the extended sequence binds to an anchoring reagent immobilized on the surface comprising the binding reagent. In embodiments, the biomarker is detected and/or quantified by detecting or quantifying the amount of extended sequence bound to tire surface. In embodiments, the surface is contacted with a labeled probe that binds to the extended sequence, wherein the labeled probe comprises a detectable label. Detection methods are further described, e.g.. in WO2014/165061; WO2014/160192; WO2015/175856; W02020/ 180645; US9618510;

US10908157; and US10114015.

[00170] In embodiments, the immunoassay is described in U.S. Publication No. 2022/0003766 and U.S. Publication No. 2021/0349104 and comprises:

(a) contacting a biotinylated binding reagent and a biotinylated anchoring reagent with a surface comprising streptavidin or avidin, e.g., for about 30 minutes to about 2 hours at room temperature, or about 6 hours to about 12 horns at 4°C; and optionally washing the surface to remove unbound binding reagent and/or anchoring reagent;

(b) contacting a sample comprising the analyte of interest (e.g., biomarker described herein) with the surface, e.g., for about 1 horn to about 2 hours at about 20°C to about 35°C (e g., about 27°C); and optionally washing the surface to remove rmbound analyte;

(c) contacting a detection reagent comprising a nucleic acid probe with the surface, e.g., for about 30 minutes to about 1 hour at about 20°C to about 35°C (e.g., about 27°C). thereby forming a binding complex comprising the binding reagent, die analyte, and the detection reagent; and optionally washing the surface to remove unbound detection reagent;

(d) contacting a template oligonucleotide with the surface and ligating the template oligonucleotide to form a circular template, e.g.. for about 10 minutes to about 30 minutes at about 20°C to about 35°C (e.g., about 27°C). thereby hybridizing the nucleic acid probe to the circular template; and optionally washing the surface to remove excess template oligonucleotide;

(e) incubating the surface under conditions sufficient to perform rolling circle amplification, e g., for about 5 minutes to about 30 minutes, thereby forming an extended sequence that binds to the anchoring reagent;

(f contacting a labeled probe comprising a detectable label with tire surface, e.g., for about 1 hour to about 2 horns at about 20°C to about 35°C (e.g., about 27°C), thereby binding the labeled probe to the extended sequence; and optionally washing the surface to remove excess labeled probe; and

(g) measuring the amount of extended sequence by quantifying the amount of detectable label, thereby detecting and/or measuring the amount of analyte (e.g., biomarker described herein) in tire sample. [00171] In embodiments, the surface comprising the binding domains described herein comprises an electrode. In embodiments, the electrode is a carbon ink electrode. In embodiments, the measuring of the detectable label comprises applying a potential to the electrode and measuring electrochemiluminescence. In embodiments, applying a potential to the electrode generates an electrochemiluminescence signal. In embodiments, the strength of the electrochemiluminescence signal is based on the amount of detected analyte, e.g., biomarker described herein, in the binding complex.

[00172] In embodiments, the immunoassay described herein further comprises measuring the concentration of one or more calibration reagents. In embodiments, a calibration reagent comprises a know n concentration of a biomarker described herein. In embodiments, the calibration reagent comprises a mixture of known concentrations of multiple biomarkers. Measurement of calibration reagents is known in the art and further described, e.g., in U.S. Publication No. 2022/0003766 and U.S. Publication No. 2021/0349104.

Competitive Assays

[00173] In embodiments, the methods provided herein are in a competitive assay format. In general terms, a competitive assay, e.g., a competitive immunoassay or a competitive inhibition assay, an analyte (e.g., a biomarker described herein) and a competitor compete for binding to a binding reagent (e.g., a viral antigen described herein). In such assays, the analyte is ty pically indirectly measured by’ directly measuring the competitor. As used herein, "competitor" refers to a compound capable of binding to tire same binding reagent as an analyte, such that the binding reagent can only bind either the analyte or the competitor, but not both. In embodiments, competitive assays are used to detect and measure analytes that are not capable of binding more than one binding reagents, e.g., small molecule analytes or analytes that do not have more than one distinct binding sites. Examples of competitive immunoassays include those described in US 4,235,601; US 4,442,204; and US 5,028,535.

Assays for Antibody Biomarkers

[00174] In embodiments where the biomarker detected is an antibody, e.g., an antibody capable of binding to a viral antigen such as S, SI, S2, S-NTD, S-ECD. S-RBD. M, E, or N, the binding reagent is an antigen that is bound by the antibody biomarker. In embodiments, antibody biomarkers are detected using a bridging serology assay. In a bridging serology assay, the binding complex further comprises a detection reagent described herein, and both the binding reagent and the detection reagent are an antigen that that is bound by the antibody biomarker. Since antibodies are typically bivalent, tire antibody biomarker can bind both the binding reagent antigen and tire detection reagent antigen.

[00175] In embodiments, antibody biomarkers are detected using a regular bridging serology assay. In a regular bridging serology assay, the antibody biomarker, binding reagent antigen, and detection reagent antigen are incubated together to form a complex where die antibody biomarker bivalently binds bodi the binding reagent antigen and the detection reagent antigen, e.g., a bridged complex. The incubation can be perfonned in any appropriate container, for example, in the well of a polypropylene plate, or in a chamber of an assay cartridge. In embodiments, the binding reagent antigen is conjugated to a biotin, and the bridged complex solution can be transferred to contact a surface comprising streptavidin, e.g., a streptavidin plate. In this embodiment, the biotin conjugated to the binding reagent antigen binds to the streptavidin plate, causing the entire bridged complex to be immobilized on the streptavidin plate.

[00176] In embodiments, antibody biomarkers are detected using a stepwise bridging serology assay. In a fest step of a stepwise bridging serology assay, the binding reagent antigen is first immobilized on a surface. In embodiments where the binding reagent antigen is conjugated to biotin, the binding reagent antigen can be immobilized on a streptavidin plate. In a second step, after the binding reagent antigen is immobilized on the surface, a solution containing the antibody biomarker is contacted with the surface, allowing the first bivalent position on the antibody biomarker to bind the binding reagent antibody. In a third step, the detection reagent antigen is then contacted with the surface, allowing the second bivalent position on the antibody to bind the detection reagent antibody. In this stepwise method, the bridging complex is formed stepwise on the surface, rather than forming the entire bridging complex before immobilization, as is done in the regular bridging assay described above. In the stepwise bridging assay, the surface may optionally be rinsed or washed betw een any of the steps.

[00177] In cither of tire regular bridging serology assay or stepwise bridging serology assay, a method may be used where tire detectable label is not directly conjugated to the detection reagent antigen but is instead attached to the detection antigen reagent using a binding complex such as streptavidin/biotin or other binding pair. The advantage of using this method is drat it is not necessary to prepare separately conjugated binding reagent antigen and detection reagent antigen. In a non -limiting example of this method, a biotin conjugated antigen is prepared. Some of this biotin conjugated antigen is then incubated with a detectable label conjugated with streptavidin. The binding of biotin to streptavidin causes the detectable label to become attached to the biotin conjugated antigen, creating a detection reagent antigen comprising a detectable label as follows:

Antigen - biotin - streptavidin - detectable label

In embodiments, additional free biotin is added to the antigen - detectable label reagent to fully occupy the streptavidin binding sites and prevent other biotin conjugates from binding to the antigen - detectable label reagent. An additional amount of the biotin conjugated antigen, which is not attached to a detectable label, is then used as the binding reagent antigen. Binding reagent antigen and detection reagent antigen prepared in this way may be used in any of the assay methods described herein.

[00178] In embodiments, the antibody biomarker is detected using a classical serology' assay. In embodiments of a classical serology' assay, the binding reagent is an antigen that is bound by the antibody biomarker. After the antibody’ biomarker is bound by the binding reagent antigen, the binding complex is detected using a detection reagent antibody that binds the antibody biomarker. In embodiments, the detection reagent antibody is an anti-human antibody' that binds human antibody biomarkers. In embodiments, the detection reagent antibody is an anti-human IgG, an anti-human IgM or an anti-human IgA isotype antibody. In embodiments, the detection reagent antibody is an anti-mouse antibody that binds mouse antibody biomarkers, or an anti-rat antibody that binds rat antibody biomarkers, or an anti-ferret antibody that binds ferret antibody biomarkers, or an anti-minx antibody that binds minx antibody biomarkers, or an anti-bat antibody that binds bat antibody biomarkers. In embodiments, the detection reagent antibody is an anti-mouse IgG, IgM. or IgA antibody, an anti-rat IgG, IgM, or IgA antibody, an anti-ferret IgG, IgM. or IgA antibody, an anti-minx IgG, IgM. or IgA antibody, or an anti-bat IgG, IgM, or IgA antibody.

[00179] In embodiments, the antibody biomarker is detected using a competitive serology assay (also termed a neutralization serology assay). Competitive immunoassays are described herein. In embodiments of a competitive serology⁷ assay, the binding reagent is an antigen that is bound by the antibody biomarker and by a competitor. In embodiments, the competitor is a substance that binds a specific region of the viral antigen. In embodiments, the competitor is a recombinant antibody or antigen-binding fragment thereof drat binds specifically to an epitope of the viral antigen, e.g., a neutralizing epitope. In embodiments, the competitor is a monoclonal antibody against an epitope of the viral antigen, e.g., a neutralizing epitope. In embodiments, the competitor comprises a detectable label described herein. For example, the biomarker can be an antibody that binds specifically to a coronavirus spike protein, and the competitor can be the ACE2 receptor, NRP1 receptor, or CD147, i.e., natural interaction partners of die spike protein. In embodiments, die competitor is the ACE2 receptor. In embodiments, the receptor is the NRP1 receptor. In embodiments, die competitor is CD 147. In embodiments, die competitor comprises a sialic acid. In embodiments, the binding reagent is a substance that binds a viral antigen (e.g., ACE2, NRP1, or CD147), and the competitor is the viral antigen (e.g., spike protein or a variant thereof described herein, such as, e.g., SI, S2, S-NTD, S- ECD, or S-RBD). hi embodiments, the coronavirus is SARS-CoV-2. In embodiments, a competitive serology assay as described herein is used to assess a potential protective serological response, e.g., the ability of the immune response to block binding of a viral antigen to its host cell receptor such as ACE2, NRP1, or CD147.

[00180] In embodiments, the antibody biomarker serology assay (either bridging, classical, or competitive) described herein comprises measuring the concentration of one or more calibration reagents. In embodiments, the calibration reagent is a positive control. In embodiments, the positive control comprises an antigen for which an antibody is known or expected to be present in the biological sample. In embodiments, the positive control comprises an antigen from a prevalent influenza strain, to which most subjects are expected to have antibodies. In embodiments, the positive control is an antigen from the Hl Michigan influenza virus. In embodiments, the positive control is immobilized in a binding domain of a surface that further comprises one or more viral antigens immobilized thereon in one or more additional binding domains, as described herein. In embodiments, antibody biomarker serology assay further comprises measuring the total levels of a particular antibody, e.g., total IgG. IgA, or IgM.

[00181] In embodiments, the calibration reagent is a negative control. In embodiments, the negative control comprises an antigen for which no antibodies are expected to be present in the biological sample. In embodiments, the negative control comprises a substance obtained from a non-human subject, and the biological sample is obtained from a human subject. In embodiments, the negative control comprises bovine serum albumin (BSA). In embodiments, the negative control, e.g., BSA, is immobilized in a binding domain of a surface that further comprises one or more viral antigens immobilized thereon in one or more additional binding domains, as described herein. [00182] In embodiments, the calibration reagent comprises a combination of biological samples from subjects known to be infected or exposed to a virus described herein. In embodiments, the calibration reagent comprises a pooled sample of serum and/or plasma from subjects known to be infected or exposed to a virus described herein. In embodiments, the calibration reagent is the same biological material as the sample to be assayed. For example, if the biological sample for the antibody biomarker serology assay is a serum sample, then the calibration reagent is a pooled serum sample. Similarly, if the biological sample for the antibody biomarker serology assay is a plasma sample, then the calibration reagent is a pooled plasma sample. In embodiments, the pooled sample comprises a known amount of IgG. IgA, and/or IgM that specifically bind to one or more viral antigens of interest. Methods of measuring IgG, IgA. and/or IgM concentration in a serum or plasma sample is known in the art. e.g., as described in Quataert et al., Clinical and Diagnostic Laboratory Immunology 2(5):590-597 (1995). In embodiments, the antibody biomarker serology assay comprises measuring the concentration of viral antigen-specific IgG, IgA, and/or IgM in multiple pooled samples to provide a calibration cur e. In embodiments, the antibody biomarker serology assay comprises measuring the concentration of viral antigen-specific IgG, IgA, and/or IgM in multiple pooled samples, wherein the multiple pooled samples correspond to high, medium, and low levels of viral antigen-specific IgG, IgA, and/or IgM (referred to herein as "high pooled sample," "medium pooled sample," and "low pooled sample," respectively). In embodiments, the pooled sample comprises serum and/or plasma from subjects known to never have been exposed to a virus described herein, i.e., a negative pooled sample. Pooled samples as calibration reagents allow baseline immune response thresholds to be defined and provides a better understanding of the levels of antibody response to a viral infection. In embodiments, the virus is a coronavirus. In embodiments, the virus is SARS-CoV-2.

[00183] In embodiments, the biological sample for the antibody biomarker serology assay is a saliva sample, and the calibration reagent comprises a calibration saliva sample. In embodiments, the calibration saliva sample contains a known amount of viral antigen-specific IgG, IgA, and/or IgM. In embodiments, the calibration saliva sample comprises serum from a subject known to be infected or exposed to a virus described herein. In embodiments, the calibration saliva sample comprises about 0.1% to about 1% of high pooled serum sample described herein. In embodiments, the calibration saliva sample comprises about 0.1%, about 0.2%, about 0.3%, about 0.4%. or about 0.5% of high pooled serum sample described herein. In embodiments, the calibration saliva sample comprises levels of viral antigen-specific IgG. IgA, and/or IgM equivalent to a 1 :500 dilution of the high pooled serum sample as described herein. In embodiments, the calibration saliva sample is obtained from a subject known to never have been exposed to a virus described herein, i.e., a negative saliva sample. In embodiments, the calibration saliva sample provides a consistent threshold for comparing viral antigen-specific IgG, IgA, and/or IgM levels in saliva samples. In embodiments, the virus is a coronavirus. In embodiments, the virus is SARS-CoV-2.

[00184] In embodiments, the calibration reagents, e.g., the pooled sample and/or the calibration saliva sample described herein, is subjected to an antibody biomarker serology assay, e.g., the classical, bridging, and/or competitive serology' assays described herein. In embodiments, the assay comprises measuring the total amount of IgG, IgA, and/or IgM in a dilution scries of the calibration reagent. In embodiments, the assay further comprises generating a standard curve based on the measured amounts of IgG. IgA, and/or IgM in the calibration reagent dilution series. In embodiments, the assay comprises determining the amount of IgG. IgA, and/or IgM in a biological sample based on the standard curve. In embodiments, the IgG, IgA, and/or IgM is from a human, a mouse, a rat, a ferret, a minx, a bat, or a combination thereof.

[00185] An exemplary multiplexed classical or bridging serology assay detecting human IgG and/or IgM against SARS-CoV-2 antigens, and/or an exemplary multiplexed competitive serology assay detecting human neutralizing antibodies (also known as blocking antibodies) against SARS-CoV-2 antigens, as described in embodiments herein, comprises:

[00186] 1. Preparation of assay plate. In embodiments, the assay plate is a 384-well assay plate. In embodiments, the assay plate is a 96-well assay plate. In embodiments, each well comprises four distinct binding domains, e.g., as shown in FIG. 20A. In embodiments, each well comprises ten distinct binding domains, e.g., as shown in FIG. 20B.

[00187] In embodiments, each well of the assay plate comprises ten distinct binding domains, wherein each binding domain comprises an immobilized viral (e.g., a respiratory’ virus such as SARS-CoV-2) antigen as described herein, hr embodiments, tire viral antigens, e.g., SARS-CoV-2 antigens, are immobilized on a surface comprising Spots 1-10 as shown in FIG. 20B, wherein the antigens are arranged as shown in Tables 2A-2I. In embodiments, the S protein mutations from the SARS-CoV-2 strains of Tables 2A-2I are described in Table 1A. hi embodiments, tire S-RBD mutations from the SARS-CoV-2 strains of Tables 2A-2I are described in Table IB.

[00188] In embodiments, about 1 LIL to about 200 pL, about 3 pL to about 150 pL, about 5 pL to about 100 pL, about 10 pL to about 90 pL, about 15 pL to about 80 pL, about 20 pL to about 70 pL, about 30 pL to about 60 pL, about 50 pL, or about 150 pL of a blocking solution to each well of the plate. In embodiments, the plate is sealed or covered, e.g.. with an adhesive seal or a plate cover. In embodiments, the plate is incubated at about 15 °C to about 30 °C, about 18 °C to about 28 °C, about 20 °C to about 26 °C, or about 22 °C to about 24 °C. In embodiments, the plate is incubated for about 10 minutes to about 6 hours, or about 30 minutes to about 4 hours, or about 45 minutes to about 2 hours, or about 1 hour. In embodiments, the plate is incubated at about room temperature (e.g., about 22 °C to about 28 °C) for at least 30 minutes. In embodiments, the plate is incubated at about room temperature (e.g.. about 22 °C to about 28 °C) for about 1 hour. In embodiments, the plate is incubated without shaking. In embodiments, the plate is incubated with shaking, e.g., at about 500 to 1000 rpm. In embodiments, the plate is incubated with shaking at about 700 rpm.

[00189] 2. Preparation of reagents. In embodiments, the assay comprises measuring the amount of one or more calibration reagents. In embodiments, the calibration reagent comprises a known quantity of IgG and/or IgM. In embodiments, the calibration reagent comprises a blank solution containing no IgG or IgM. In embodiments, the assay comprises measuring the amount of multiple calibration reagents, e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 calibration reagents. In embodiments, the assay comprises generating a standard curve from the multiple calibration reagents. In embodiments, the multiple calibration reagents comprise a range of concentrations of IgG and/or IgM. In embodiments, the assay comprises diluting a concentration reagent to provide multiple calibration reagents comprising a range of concentrations. In embodiments, the calibration reagent is diluted 1: 10, 1 :20, 1 :30. 1 :40. 1 :50. 1:60, 1:70, 1:80, 1:90, 1 :100, 1: 140, 1:160. 1 :200, 1:300, 1 :400, 1 :500. 1:600, 1:700, 1:800, 1 :900. 1:1000, 1 : 1500, 1:2000. 1:2500. 1:3000, 1:3500, 1 :4000, 1:4500. 1:5000. 1:5500, 1:6000, 1:6500, 1 :7000, 1:7500, 1:8000. 1:8500, 1:9000, 1 :9500, 1 :10000, 1:20000. 1:30000, 1 :40000. or 1:50000 to provide multiple concentrations of the calibration reagent. Calibration reagents are further described herein. [00190] In embodiments, the assay comprises measuring the amount of one or more control reagents. In embodiments, the control reagent comprises a known quantity of IgG and/or IgM against the specific viral antigens in the assay, e.g.. SARS-CoV-2 S. SARS-CoV-2 N, and/or SARS-CoV-2 S-RBD. In embodiments, the one or more control reagents comprises a first control reagent obtained from a subject known to never have been exposed to SARS-CoV-2, a second control reagent obtained from a subject during an early stage of infection by SARS-CoV-2, a third control reagent obtained from a subject during a late stage infection by SARS-CoV-2, a fourth control reagent obtained from a subject who has recovered from an infection by SARS-CoV-2, or a combination thereof. Control reagents arc further described herein. [00191] Examples of samples, e.g., biological samples, are provided herein. In embodiments, the sample is diluted about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 30-fold, about 40-fold, about 50- fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 250-fold, about 500- fold, about 750-fold, about 1000-fold, about 1500-fold, about 2000-fold, about 2500-fold, about 3000-fold, about 3500-fold, about 4000-fold, about 4500-fold, or about 5000-fold for use in the assay.

[00192] 3. Addition of samples and reagents. In embodiments, the assay plate is washed at least once, at least twice, at least three times, at least four times, or at least five times with a wash buffer after incubation with the blocking solution. In embodiments, the assay plate is washed with at least about 10 pL. at least about 20 pL, at least about 30 pL. at least about 40 pL, at least about 50 pL, at least about 60 pL. at least about 70 pL, at least about 80 pL. at least about 90 pL, at least about 100 pL. at least about 150 pL, or at least about 200 pL of wash buffer.

[00193] 3A. Classical or Bridging Serology Assay: In embodiments, after the washing, the sample, one or more calibration reagents, and one or more control reagents are added to their respectively designated wells of the plate. In embodiments, about 5 pL to about 50 pL. about 10 pL to about 40 pL. about 20 pL to about 30 pL, about 15 pL. about 25 pL, or about 50 pL of the sample, calibration reagent, or control reagent is added to each well.

[00194] 3B. Competitive Serology Assay: In embodiments, after the washing, the sample and one or more calibration reagents are added to their respectively designated wells of the plate. In embodiments, about 5 pL to about 50 pL, about 10 pL to about 40 pL, about 20 pL to about 30 pL, about 15 pL, about 25 pL, or about 50 pL of the sample or calibration reagent is added to each well.

[00195] In embodiments, the plate is sealed or covered, e.g., with an adhesive seal or a plate cover. In embodiments, the plate is incubated at about 15 °C to about 30 °C, about 18 °C to about 28 °C, about 20 °C to about 26 °C, or about 22 °C to about 24 °C. In embodiments, the plate is incubated while shaken at about 500 rpm to about 3000 rpm, about 800 rpm to about 2000 rpm, about 1000 rpm to about 1800 rpm, about 500 rpm to about 1000 rpm, or about 1200 rpm to about 1600 rpm. In embodiments, the plate is incubated for about 10 minutes to about 12 hours, or about 30 minutes to about 8 hours, or about 45 minutes to about 6 horns, or about 1 hour, or about 4 hours. In embodiments, the plate is incubated at about room temperature (e.g.. about 22 °C to about 28 °C) while shaken at about 1500 rpm for about 4 hours. In embodiments, the plate is incubated at about room temperature (e.g., about 22 °C to about 28 °C) while shaken at about 700 rpm for about 1 hour.

[00196] 4A. Classical or Bridging Serology Assay - Addition of detection reagent. In embodiments, the detection reagent is diluted from a stock solution of detection reagent to obtain a solution comprising a working concentration of detection reagent. Detection reagents are further described herein.

[00197] 4B. Competitive Serology Assay - Addition of ACE2 detection reagent. In embodiments, the ACE2 detection reagent is diluted from a stock solution of detection reagent to obtain a solution comprising a working concentration of ACE2 detection reagent. ACE2 is further described herein.

[00198] In embodiments, the assay plate is washed at least once, at least tw ice, at least three times, at least four times, or at least five times with a wash buffer after incubation with the sample, calibration reagent, or control reagent. In embodiments, the assay plate is washed with at least about 10 pL. at least about 20 pL, at least about 30 pL, at least about 40 pL, at least about 50 pL, at least about 60 pL, at least about 70 pL, at least about 80 pL, at least about 90 pL, at least about 100 pL, at least about 150 pL, or at least about 200 pL of wash buffer.

[00199] In embodiments, after the washing, the detection reagent solution for the classical or bridging serology assay, or the ACE2 detection reagent solution for the competitive serology assay, is added to each well of the plate. In embodiments, about 5 pL to about 50 pL, about 10 pL to about 40 pL. about 10 pL to about 20 pL, about 20 pL to about 30 pL, about 15 pL, about 25 pL. or about 50 pL of the detection reagent solution or the ACE2 detection reagent solution is added to each well.

100200] In embodiments, the plate is sealed or covered, e.g.. with an adhesive seal or a plate cover. In embodiments, the plate is incubated at about 15 °C to about 30 °C. about 18 °C to about 28 °C, about 20 °C to about 26 °C, or about 22 °C to about 24 °C. In embodiments, the plate is incubated while shaken at about 500 rpm to about 3000 rpm. about 800 rpm to about 2000 rpm, about 1000 rpm to about 1800 rpm, about 500 rpm to about 1000 rpm. or about 1200 rpm to about 1600 rpm. In embodiments, the plate is incubated for about 10 minutes to about 6 horns, or about 30 minutes to about 4 hours, or about 45 minutes to about 2 hours, or about 1 hour. In embodiments, the plate is incubated at about room temperature (e.g., about 22 °C to about 28 °C) while shaken at about 1500 rpm for about 1 hour. In embodiments, the plate is incubated at about room temperature (e.g., about 22 °C to about 28 °C) while shaken at about 700 rpm for about 1 hour. [00201] 5. Addition of read buffer. In embodiments, tire assay plate is washed at least once, at least twice, at least three times, at least four times, or at least five tunes with a wash buffer after incubation with the detection reagent. In embodiments, the assay plate is washed with at least about 10 pL, at least about 20 pL, at least about 30 pL, at least about 40 pL, at least about 50 pL, at least about 60 pL, at least about 70 pL, at least about 80 pL, at least about 90 pL, at least about 100 pL, at least about 150 pL, or at least about 200 pL of wash buffer. [00202] In embodiments, the read buffer is added to each well of die plate. Read buffers are further described herein.

[00203] 5A. Classical or Bridging Serology Assay: In embodiments, about 5 pL to about 200 pL. about 5 pL to about 150 pL. about 5 pL to about 100 pL, about 10 pL to about 80 pL, about 20 pL to about 60 pL, about 40 pL, about 50 pL. about 100 pL, or about 150 L of the read buffer is added to each well.

[00204] 5B. Competitive Serology Assay: In embodiments, the read buffer is added to each well of the plate. Read buffers are further described herein. In embodiments, about 5 pL to about 200 pL, about 5 pL to about 150 pL, about 5 pL to about 100 pL, about 10 pL to about 80 pL, about 20 pL to about 60 pL, or about 40 pL of tire read buffer is added to each well.

[00205] In embodiments, the assay comprises reading the plate, e.g., on a plate reader as described herein. In embodiments, the assay comprises reading the plate immediately following addition of the read buffer. [00206] A further exemplary serology’ assay for detecting an antibody biomarker that binds to a SARS- CoV-2 antigen comprises:

(a) mixing (i) a coating solution comprising a binding reagent bound to a linking agent and (ii) a detection reagent, wherein each of the binding reagent and the detection reagent comprises a SARS-CoV-2 antigen, and wherein the detection reagent comprises a detectable label;

(b) contacting a surface with: (i) a sample comprising the antibody biomarker, (ii) a calibration reagent, or (iii) a control reagent, wherein the surface comprising one or more binding domains, wherein each binding domain comprises a targeting agent;

(c) contacting the surface with the mixture of (a);

(d) measuring the amount of detectable label on die surface, thereby detecting and/or measuring the amount of the antibody biomarker. In embodiments, the SARS-CoV-2 antigen is SARS-CoV-2 N protein. In embodiments, the SARS-CoV-2 antigen is SARS-CoV-2 S protein. In embodiments, the SARS- CoV-2 antigen is SARS-CoV-2 S-RBD. In embodiments, the SARS-CoV-2 antigen comprises the SARS- CoV-2 N protein and SARS-CoV-2 S-RBD. and the assay is a multiplexed assay that detects antibody biomarkers that bind to the SARS-CoV-2 N protein and the SARS-CoV-2 S-RBD.

[00207] A further exemplary serology assay for detecting an antibody biomarker that binds to a SARS- CoV-2 antigen comprises:

(a) mixing (i) a biotinylated binding reagent and (ii) a detection reagent, wherein each of the binding reagent and the detection reagent comprises a SARS-CoV-2 antigen, and wherein the detection reagent comprises a detectable label;

(b) contacting a surface with: (i) a sample comprising the antibody⁷ biomarker, (ii) a calibration reagent, or (iii) a control reagent, wherein the surface comprising one or more binding domains, wherein each binding domain comprises avidin or streptavidin;

(c) contacting the surface with tire mixture of (a);

(d) measuring the amount of detectable label on tire surface, thereby detecting and/or measuring the amount of the antibody biomarker. In embodiments, the SARS-CoV-2 antigen is SARS-CoV-2 N protein, hi embodiments, the SARS-CoV-2 antigen is SARS-CoV-2 S protein. In embodiments, the SARS- CoV-2 antigen is SARS-CoV-2 S-RBD. In embodiments, the SARS-CoV-2 antigen comprises the SARS- CoV-2 N protein and SARS-CoV-2 S-RBD. and the assay is a multiplexed assay that detects antibody biomarkers that bind to the SARS-CoV-2 N protein and the SARS-CoV-2 S-RBD.

[00208] A further exemplary competitive serology assay for detecting an antibody biomarker that binds to a SARS-CoV-2 antigen comprises:

(a) contacting a coating solution comprising a binding reagent bound to a linking reagent with a surface comprising one or more binding domains, wherein each binding domain comprises a targeting agent, and wherein the binding reagent is a SARS-CoV-2 antigen;

(b) contacting each binding domain with: (i) a sample comprising the antibody biomarker, (ii) a calibration reagent, or (iii) a control reagent;

(c) contacting each binding domain with an ACE2 detection reagent comprising a detectable label;

(d) measuring the amount of detectable label on tire surface, thereby detecting and/or measuring die amount of the antibody biomarkcr. In embodiments, the SARS-CoV-2 antigen is SARS-CoV-2 N protein, hi embodiments, die SARS-CoV-2 antigen is SARS-CoV-2 S protein. In embodiments, the SARS- CoV-2 antigen is SARS-CoV-2 S-RBD. In embodiments, the SARS-CoV-2 antigen comprises die SARS- CoV-2 N protein and SARS-CoV-2 S-RBD, and the assay is a multiplexed assay that detects antibody biomarkers diat bind to the SARS-CoV-2 N protein and the SARS-CoV-2 S-RBD.

[00209] A further exemplary competitive serology assay for detecting an antibody biomarker that binds to a SARS-CoV-2 antigen comprises:

(a) contacting a biotinylated binding reagent with a surface comprising one or more binding domains, wherein each binding domain comprises avidin or streptavidin, and wherein the binding reagent is a SARS-CoV-2 antigen;

(d) measuring the amount of detectable label on the surface, thereby detecting and/or measuring the amount of the antibody biomarker. In embodiments, the SARS-CoV-2 antigen is SARS-CoV-2 N protein. In embodiments, the SARS-CoV-2 antigen is SARS-CoV-2 S protein. In embodiments, the SARS- CoV-2 antigen is SARS-CoV-2 S-RBD. In embodiments, the SARS-CoV-2 antigen comprises the SARS- CoV-2 N protein and SARS-CoV-2 S-RBD, and the assay is a multiplexed assay that detects antibody biomarkers that bind to the SARS-CoV-2 N protein and the SARS-CoV-2 S-RBD.

[00210] In embodiments, the surface is a multi-well plate. In embodiments, the assay further comprises a wash step prior to one or more of the assay steps. In embodiments, die wash step comprises washing the assay plate at least once, at least twice, at least three times, at least four times, or at least five times with a wash buffer. In embodiments, the assay plate is washed with at least about 10 pL, at least about 15 pL. at least about 20 pL, at least about 25 pL, at least about 30 pL, at least about 40 pL, at least about 50 pL, at least about 60 pL, at least about 70 L. at least about 80 pL. at least about 90 pL, at least about 100 pL. at least about 150 pL. or at least about 200 pL of wash buffer. In embodiments, the classical or bridging serology assay does not comprise a wash step prior to any of steps (a), (b). or (c). In embodiments, the competitive serology assay does not comprise a wash step prior to any of steps (a), (b). or (c).

[00211] In embodiments, prior to step (a), a blocking solution is added to the plate to reduce non-specific binding of the coating solution or the biotinylated binding reagent to the surface. In embodiments, about 50 pl. to about 250 pL, about 100 pL to about 200 pL, or about 150 pL of blocking solution is added per well of the plate. In embodiments, the plate is incubated at about 15 °C to about 30 °C, about 18 °C to about 28 °C, about 20 °C to about 26 °C, or about 22 °C to about 24 °C. In embodiments, the plate is incubated while shaken at about at about 500 rpm to about 2000 rpm, about 600 rpm to about 1500 rpm, or about 700 rpm to about 1000 rpm. In embodiments, the method comprises incubating the blocking solution on the plate for about 10 minutes to about 4 hours, about 20 minutes to about 3 horns, or about 30 minutes to about 2 minutes. In embodiments, the plate is incubated at about room temperature (e.g., about 22 °C to about 28 °C) while shaken at about 700 rpm for about 30 minutes to about 2 hours.

[00212] In embodiments comprising a coating solution, die assay further comprises, prior to step (a), mixing a linking agent comiected to a targeting agent complement with a binding reagent comprising a supplemental linking agent, thereby forming the coating solution comprising the binding reagent bound to the linking agent. In embodiments, the method comprises forming about 200 pL to about 1000 pL, or about 300 pL to about 800 pL, or about 400 pL to about 600 pL of the coating solution. In embodiments, step (a) comprises incubating the linking agent and the binding reagent at about 15 °C to about 30 °C, about 18 °C to about 28 °C, about 20 °C to about 26 °C, or about 22 °C to about 24 °C. In embodiments, the method comprises forming about 500 pL of the coating solution by incubating about 300 pL of a solution comprising the linking agent and about 200 pL of a solution comprising the binding reagent, at about room temperature (e.g., about 22 °C to about 28 °C) for about 30 minutes. In embodiments, the incubating is performed without shaking. In embodiments, the assay further comprises contacting the coating solution with a stop solution (e.g.. about 100 pL to about 500 pL, or about 150 pL to about 300 pL. or about 200 pL of a stop solution) to stop the binding reaction betw een the linking agent and supplemental linking agent. In embodiments, the coating solution and the stop solution are incubated for about 10 minutes to about 1 hour, about 20 minutes to about 40 minutes, or about 30 minutes. In embodiments, the coating solution and the stop solution are incubated at about 15 °C to about 30 °C, about 18 °C to about 28 °C, about 20 °C to about 26 °C. or about 22 °C to about 24 °C. In embodiments, the method further comprises, following incubation of the coating solution with the stop solution, diluting the coating solution using the stop solution, e.g., by 2-fold, 5-fold, 10-fold, or 20-fold, to a working concentration as described herein. In embodiments, the targeting agent and targeting agent complement comprise complementary oligonucleotides. In embodiments, the linking agent comprises avidin or streptavidin, and the supplemental linking agent comprises biotin.

[00213] In embodiments, about 10 pL to about 200 pL, about 5 pL to about 100 pL, about 10 pL to about 90 pL, about 15 pL to about 80 pL, about 20 pL to about 70 pL, about 30 pL to about 60 pL, or about 50 pL of the coating solution or a solution containing the biotinylated binding reagent are added to each well of the plate. In embodiments, the plate is incubated at about 15 °C to about 30 °C. about 18 °C to about 28 °C, about 20 °C to about 26 °C, or about 22 °C to about 24 °C. In embodiments, the plate is incubated for about 10 minutes to about 6 hours, or about 30 minutes to about 4 horns, or about 45 minutes to about 2 horns, or about 1 hour. In embodiments, the plate is incubated at about room temperature (e.g., about 22 °C to about 28 °C) for at least 30 minutes. In embodiments, the plate is incubated at about room temperature (e.g., about 22 °C to about 28 °C) for about 1 horn. In embodiments, the plate is incubated without shaking. In embodiments, the plate is incubated with shaking, e.g., at about 500 to 1000 rpm. In embodiments, the plate is incubated with shaking at about 700 rpm.

[00214] In embodiments, about 5 LIL to about 50 pL. about 10 pL to about 40 pL, about 20 pL to about 30 pL, about 15 pL, about 25 pL, about 35 pL, or about 50 pL of the sample, calibration reagent, or control reagent are added to each well of the plate. In embodiments, the plate is incubated at about 15 °C to about 30 °C, about 18 °C to about 28 °C, about 20 °C to about 26 °C, or about 22 °C to about 24 °C. In embodiments, the plate is incubated for about 10 minutes to about 6 hours, or about 30 minutes to about 4 hours, or about 45 minutes to about 2 hours, or about 1 hour. In embodiments, the plate is incubated at about room temperature (e.g., about 22 °C to about 28 °C) for at least 30 minutes. In embodiments, the plate is incubated at about room temperature (e.g., about 22 °C to about 28 °C) for about 1 hour. In embodiments, the plate is incubated without shaking. In embodiments, the plate is incubated with shaking, e.g., at about 500 to 1000 rpm. In embodiments, the plate is incubated with shaking at about 700 rpm.

[00215] In embodiments where the assay is a classical or bridging serology assay, about 5 pL to about 50 pL, about 10 pL to about 40 pL, about 10 pL to about 20 pL, about 20 pL to about 30 pL, about 15 pL. about 25 pL, about 35 pL, or about 50 pL of the mixture comprising the binding reagent and the detection reagent to each well of the plate. In embodiments, the plate is incubated at about 15 °C to about 30 °C, about 18 °C to about 28 °C. about 20 °C to about 26 °C, or about 22 °C to about 24 °C. In embodiments, the plate is incubated for about 10 minutes to about 6 hours, or about 30 minutes to about 4 horns, or about 45 minutes to about 2 hours, or about 1 horn. In embodiments, the plate is incubated at about room temperature (e.g.. about 22 °C to about 28 °C) for at least 30 minutes. In embodiments, the plate is incubated at about room temperature (e.g., about 22 °C to about 28 °C) for about 1 horn. In embodiments, the plate is incubated without shaking. In embodiments, the plate is incubated with shaking, e.g., at about 500 to 1000 rpm. In embodiments, the plate is incubated with shaking at about 700 rpm.

[00216] In embodiments where the assay is a competitive serology assay, about 5 pL to about 50 pL, about 10 pL to about 40 pL. about 10 pL to about 20 pL, about 20 pL to about 30 pL, about 15 pL, about 25 pL. about 35 pL, or about 50 pL of a solution comprising the ACE2 detection reagent are added to each well of the plate. In embodiments, tire plate is incubated at about 15 °C to about 30 °C, about 18 °C to about 28 °C. about 20 °C to about 26 °C, or about 22 °C to about 24 °C. In embodiments, the plate is incubated for about 10 minutes to about 6 hours, or about 30 minutes to about 4 horns, or about 45 minutes to about 2 hours, or about 1 hour. In embodiments, the plate is incubated at about room temperature (e.g., about 22 °C to about 28 °C) for at least 30 minutes. In embodiments, the plate is incubated at about room temperature (e.g., about 22 °C to about 28 °C) for about 1 hour. In embodiments, the plate is incubated without shaking. In embodiments, the plate is incubated with shaking, e.g.. at about 500 to 1000 rpm. In embodiments, the plate is incubated with shaking at about 700 rpm.

[00217] In embodiments, step (d) comprises adding a read buffer to each well of the plate. Read buffers are further described herein. In embodiments, about 5 pL to about 200 pL, about 5 pL to about 150 pL, about 5 pL to about 100 pL. about 10 pL to about 80 pL, about 20 pL to about 60 pL, about 40 pL, about 50 pL. about 100 pL, or about 150 pL of the read buffer is added to each well. In embodiments, the measuring comprises reading the plate, e.g., on a plate reader as described herein. In embodiments, the assay comprises reading the plate immediately following addition of the read buffer.

Quantitation Embodiments

[00218] In embodiments, the invention further provides a method of determining viral exposure in a subject, (a) comprising conducting an immunoassay method described herein on a biological sample of the subject; (b) detecting the virus, viral component, and/or biomarker (e.g., antibody biomarker or inflammatory or tissue damage biomarker) as described herein; (c) determining if the amount of detected virus, viral component, and/or biomarker is higher or lower relative to a control: and (d) determining the viral exposure of the subject based on the determination of (c). In embodiments, the method comprises normalizing the detected amount of biomarker (e.g., antibody biomarker) to a control and determining whether the subject is exposed to, infected by, and/or immune to the virus. In embodiments, the control is a biological sample containing a known amount of biomarkers (e.g., antibody biomarkers or inflammatory or tissue damage biomarkers). In embodiments, the control is a biological sample obtained from a subject know n to have never been exposed to the virus. In embodiments, the control is a biological sample obtained from a subject known to have recovered from an infection by the virus. In embodiments, the virus is a coronavirus, hr embodiments, tire virus is SARS-CoV-2.

[00219] In embodiments comprising a multiplexed immunoassay for quantifying the amount of a biomarker (e.g., an antibody biomarker or an inflammatory or tissue damage biomarker), the method further comprises determining a threshold value of the biomarker in a healthy subject, hr embodiments, tire threshold value is detennined from the aggregate results of measured biomarker amounts in multiple healthy subjects. For example, tire aggregate results from a certain number of samples can determine the percentile, e.g., 99^th percentile or greater, of biomarker levels in a healthy subject. Various statistical models and algorithms can be utilized to calculate the extent of viral infection and/or degree of immunity in a subject by comparing and/or normalizing the subject's measured biomarker amount to the threshold value of that biomarker.

[00220] In embodiments, the multiplexed immunoassay for quantifying the amount of an antibody biomarker (e.g., a serology assay described herein) that specifically binds to a viral antigen is performed substantially with an assay that measures inhibition of binding between a viral protein and its associated host receptor, e.g., the binding of the coronavirus spike protein to the ACE2 receptor or the NRP1 receptor. In embodiments, the antibody biomarker inhibits binding betw een the viral protein and its associated host receptor. In embodiments, the inhibition assay indirectly detects the antibody biomarker. In embodiments. simultaneous direct detection (e.g., utilizing a viral antigen as a binding reagent) and indirect detection (e.g., measuring inhibition between a viral protein and its receptor) of the antibody biomarker improves specificity of the antibody biomarker detection.

[00221] In embodiments, the invention provides methods of assessing the affinity of an antibody biomarker to a viral antigen described herein, e.g., a SARS-CoV-2 antigen. As used in the context of antibodies, "affinity" refers to the strength of interaction between an epitope (e.g., on a viral antigen) and an antibody's antigen-binding site. In embodiments, the invention provides methods of assessing the binding kinetics between an antibody biomarker and viral antigen described herein. Methods of measuring antibody affinity and/or binding kinetics include, e.g., surface plasmon resonance (SPR) and bio-layer interference (BLI). Antibody affinity measurement is further described in, e.g., Underwood, Advances in Virus Research 34:283-309 (1988); Azimzadeh et al., J Mol Recognition 3(3): 108-116 (1990); Hearty et al., Methods Mol Biol 907:411-442 (2012); and Singhal et al., Anal Chem 82(20): 8671-8679 (2010).

[00222] In embodiments, the invention provides methods of assessing the affinity of a neutralizing antibody to a viral antigen described herein, e.g., a SARS-CoV-2 antigen. In embodiments, the affinity determination of a neutralizing antibody in a serum or plasma sample for SARS-CoV-2 comprises: a) titrating a labeled competitor to a surface comprising a known amount of SARS-CoV-2 S protein to determine the K,j, between the labeled ACE2 competitor and S protein; b) titrating: (i) a plasma sample known to contain neutralizing antibody for SARS-CoV-2 S while maintaining a constant ACE2 concentration, as described by equation 1(a); and (ii) ACE2 while maintaining a constant sample concentration, as described by equation 1(b); and c) solving the system of equations 1(a) and 1(b) to determine the average antibody concentration in sample [A ] and average affinity K_di. As described in equation 1(a), if the binding of fixed labeled ACE2, [£], is plotted against the log concentration of the unlabeled competitor antibody, [A], the resulting inhibition curve will be shifted by a factor of log(l+|£|/^).

[00223] Affinity’ measurements are further described, e.g., in Huhne et al., British Journal of Pharmacology 161: 1219-1237 (2010).

Assays for Viral Nucleic Acids

Amplification and Detection of Viral Nucleic Acid

[00224] In embodiments, the invention provides a method for detecting a coronavirus in a biological sample, comprising: a) contacting the biological sample with a binding reagent that specifically binds a nucleic acid of the coronavirus; b) forming a binding complex comprising the binding reagent and the coronavirus nucleic acid; and c) detecting the binding complex, thereby detecting the coronavirus in the biological sample. In embodiments, the coronavirus is SARS-CoV-2. In embodiments, the coronavirus nucleic acid is RNA. In embodiments, the binding reagent comprises a single stranded oligonucleotide. [00225] In embodiments, the sample comprises a coronavirus nucleic acid. In embodiments, the method further comprises amplifying the coronavirus nucleic acid to fonn one or more additional copies of the coronavirus nucleic acid sequence, forming a plurality of binding complexes, each binding complex comprising a copy of the coronavirus nucleic acid sequence, and detecting the plurality of binding complexes, thereby detecting the coronavirus in the biological sample. In embodiments, the coronavirus nucleic acid is RNA, and the amplifying comprises reverse transcribing the RNA to form a cDNA, and amplifying the cDNA using polymerase chain reaction (PCR) to form a PCR product comprising a copy of the coronavirus nucleic acid sequence. In embodiments, tire reverse transcription to form a cDNA and the PCR to amplify the cDNA are performed in a single reaction mixture. In embodiments, the reaction mixture further comprises a glycosylase enzyme. In embodiments, the glycosylase removes non-specific products from the reaction mixture. In embodiments, the glycosylase is uracil-N-glycosylase. In embodiments, the sample comprises an RT-PCR product, e.g., cDNA. In embodiments, the cDNA is generated from a coronavirus nucleic acid. In embodiments, the method comprises amplify ing the cDNA using PCR to form a PCR product comprising a copy of tire coronavirus nucleic acid sequence. In embodiments, the PCR is performed for about 10 to about 60 cycles, about 20 to about 50 cycles, or about 30 to about 40 cycles. In embodiments, the cDNA is amplified with a first primer that comprises a binding partner of the binding reagent and a second primer that comprises a detectable label or binding partner thereof, to fonn the PCR product. In embodiments, the first primer is a PCR forw ard primer and comprises the binding partner of the binding reagent at a 5' end. In embodiments, the second primer is a PCR reverse primer and comprises the detectable label or binding partner thereof at a 3' end. In embodiments, the PCR product comprises, in 5' to 3' order: the binding partner of the binding reagent, a copy of the coronavirus nucleic acid sequence, and the detectable label or binding partner thereof.

[00226] In embodiments, the first and second primers amplify a coronavirus nucleic acid sequence that encodes a protein, e.g.. any of the coronavirus proteins described herein such as S, E, M, N, or a nonstructural protein. In embodiments, the first and second primers amplify a non-coding coronavirus nucleic acid sequence, i.e., that does not encode a gene. In embodiments, the first and second primers amplify a coronavirus nucleic acid sequence capable of identifying a coronavirus species. In embodiments, the coronavirus nucleic acid is SARS-CoV-2 RNA.

[00227] In embodiments, the method is a multiplexed method. In embodiments, the cDNA is amplified using multiple sets of primers, wherein each set of primers comprises a PCR forw ard primer and a PCR reverse primer as described herein. In embodiments, the PCR forw ard primer in each set of primers comprises a binding partner of the same binding reagent. In embodiments, the PCR forward primer in each set of primers comprises a binding partner of different binding reagents. In embodiments, each set of primers amplifies a different region of the cDNA to generate a plurality of PCR products, each having a different coronavirus nucleic acid sequence. For example, a first set of primers amplifies a region that encodes for the S protein, a second set of primers amplifies a region that encodes for the N protein, a third set of primers amplifies a region for a noncoding region, etc. In embodiments, each coronavirus nucleic acid sequence corresponds to a different binding reagent. In embodiments, the coronavirus nucleic acid sequence of the PCR product is identified by determining the binding reagent that binds the PCR product. In embodiments, the coronavirus nucleic acid is SARS-CoV-2 RNA. In embodiments, the primers for amplifying a region that encodes for the S protein are described in the accompanying Sequence Lising. In embodiments, the primer comprises a modified nucleotide, e.g., a locked nucleic acid (LNA).

[00228] In embodiments, the binding reagent comprises a single-stranded oligonucleotide, and the binding partner of the binding reagent comprises a complementary oligonucleotide of the binding reagent. In embodiments, the binding reagent further comprises a targeting agent complement. In embodiments, the targeting agent complement comprises an oligonucleotide that is complementary to a targeting agent on a surface, as described herein. In embodiments, the binding reagent is immobilized to the surface via the targeting agent - targeting agent complement interaction. In embodiments, each PCR product binds to a binding reagent to form one or more binding complexes on the surface. In embodiments comprising different binding reagents corresponding to different coronavirus nucleic acid sequences, each binding reagent is located at a distinct binding domain on die surface, and the detected coronavirus nucleic acid sequence is identified by the location of the binding complex on the surface.

[00229] In embodiments, tire method comprises detecting the binding complex(es). In embodiments, the PCR product comprises a detectable label. In embodiments, the PCR product comprises a binding partner of a detectable label. Detectable labels are described herein. In embodiments, the detectable label is an electrochemiluminescence (ECL) label. In embodiments, the PCR product comprises biotin, and the detectable label comprises an ECL label linked to avidin or streptavidin. In embodiments, the PCR product comprises avidin or streptavidin, and the detectable label comprises an ECL label linked to biotin. Additional non-limiting examples of binding partners that can be on the detection probe and detectable label are provided herein.

[00230] In embodiments, RNA is extracted from a sample containing an RNA virus (e.g., SARS-CoV-2). and the extracted RNA is converted to cDNA. A "Master Mix" is prepared by combining a forward primer comprising a 5' binding reagent complement sequence and a cDNA complement sequence, a reverse primer comprising a cDNA reverse complement sequence and a 3' binding partner of a detectable label, and other PCR components such as dNTPs and DNA polymerase (e.g.. Taq polymerase). The cDNA and Master Mix are combined, and PCR is performed for 30 to 40 cycles to form a plurality of PCR products, each PCR product comprising the 5' binding reagent complement sequence and 3' binding partner of a detectable label. Each PCR product hybridizes to a binding reagent on a surface. The surface is then contacted with a detectable label, which binds to the PCR product. The PCR product bound to the detectable label is then subjected to detection as described herein.

[00231] In an alternative embodiment, the Master Mix comprises the components for performing the reverse transcription reaction and the PCR reaction, e.g., reverse transcriptase, DNA polymerase, forward and reverse primers, nucleotides, magnesium, ribonuclease inhibitor, and glycosylase, and the RNA extracted from die sample is added to the Master Mix, such that the reverse transcription reaction and the PCR reaction are performed with a single reaction mixture to form the PCR product. In embodiments, the single reaction mixture is: (1) incubated at a first temperature sufficient to activate the glycosylase; (2) incubated at a second temperature sufficient to perform the reverse transcription; and (3) incubated at temperature sufficient to perform PCR. In embodiments, the PCR product is bound to the surface and detected as described herein.

Detection of Viral Nucleic Acids and Single Nucleotide Polymorphisms (SNPs)

[00232] In embodiments, the invention provides a method for detecting a coronavirus nucleic acid in a biological sample. In embodiments, the invention provides a method of identifying the circulating strains of SARS-CoV-2 without sequencing a large number of SARS-CoV-2 isolates. Certain strains of SARS-CoV-2 are associated with increased transmissibility (e.g., the B.l.1.7, 501Y.V2, and P.l strains) and diminished efficacy against currently available vaccines. In embodiments, the invention provides a method of real-time monitoring and assessing transmission patterns of SARS-CoV-2. In embodiments, the invention provides a method for determining a SARS-CoV-2 strain (e.g., the L strain or S strain, or the S-D614 or S-D614G strain, or the variants in FIG. 1 A as described herein) in a biological sample.

[00233] In embodiments, the invention provides method for detecting a single nucleotide polymorphism (SNP) in a target nucleic acid, wherein the target nucleic acid is a SARS-CoV-2 nucleic acid, comprising: (a) contacting a sample comprising the target nucleic acid with (i) a targeting probe, wherein the targeting probe comprises a first region complementary to a polymorphic site of the target nucleic acid that comprises the SNP, and wherein the targeting probe comprises an oligonucleotide tag; and (ii) a detection probe, wherein the detection probe comprises a second region complementary to an adjacent region of the target nucleic acid comprising tire polymorphic site, and wherein the detection probe comprises a detectable label; (b) hybridizing tire targeting and detection probes to tire target nucleic acid; (c) ligating the targeting and detection probes that hybridize with perfect complementarity at the polymorphic site to form a ligated target complement comprising the oligonucleotide tag and the detectable label; (d) contacting the product of (c) with a surface comprising an immobilized binding reagent, wherein tire binding reagent comprises an oligonucleotide complementary to the oligonucleotide tag; (e) forming a binding complex on the surface, wherein the binding complex comprises the binding reagent and the ligated target complement; and (f) detecting the binding complex, thereby detecting the SNP at the polymorphic site.

[00234] In embodiments, the method comprises an oligonucleotide ligation assay (OLA). OLA and other nucleic acid detection methods are described, e.g., in WO 2020/227016. In embodiments, the OLA method is used to detect, identify, and/or quantify a coronavirus nucleic acid (e.g., RNA). In embodiments, the coronavirus nucleic acid encodes the N gene. In embodiments, the coronavirus nucleic acid is the N1 region, N2 region, or N3 region of the N gene, as described herein. In embodiments, the OLA method is used to detect, identify, and/or quantify a single nucleotide polymorphism (SNP) at a polymorphic site in a coronavirus nucleic acid (e.g., RNA). In embodiments, the coronavirus is SARS-CoV-2. hr embodiments, the method detects any of the SNPs as shown in FIGS. 1A and 1C.

[00235] In embodiments, the OLA method for detecting a coronavirus nucleic acid or for detecting an SNP comprises: (a) contacting the biological sample with: (i) a targeting probe, wherein, when the method is for detecting the coronavirus nucleic acid, the targeting probe is complementary to a first region of a target nucleic acid; or wherein, when the method is for detecting the SNP. the targeting probe is complementary to a polymorphic site of a target nucleic acid, wherein the target nucleic acid is, e.g., the coronavirus nucleic acid or an RT-PCR product described herein, and wherein the targeting probe comprises an oligonucleotide tag; and (ii) a detection probe, wherein, when the method is for detecting the coronavirus nucleic acid, the detection probe is complementary to a second region that is adjacent to the first region of the target nucleic acid; or wherein, when the method is for detecting the coronavirus nucleic acid, the detection probe is complementary to an adjacent region of the target nucleic acid containing the distinct SNP; (b) hybridizing the targeting and detection probes to the target nucleic acid; (c) ligating the targeting and detection probes that hybridize with perfect complementarity to the first and second regions of the target nucleic acid when the method is for detecting the coronavirus nucleic acid; or ligating the targeting and detection probes that hybridize with perfect complementarity at the polymorphic site when the method is for detecting the SNP, to form a ligated target complement comprising the oligonucleotide tag and the detectable label; (d) contacting the product of (c) with a surface comprising a binding reagent immobilized in one or more binding domains, wherein the binding reagent comprises an oligonucleotide complementary to the oligonucleotide tag; (c) forming a binding complex on the surface betw een the binding reagent and the ligated target complement; and (f) detecting the binding complex, thereby detecting, identifying, and/or quantifying the coronavirus nucleic acid or the SNP at the polymorphic site of the coronavirus nucleic acid. In embodiments, die coronavirus is SARS-CoV-2. In embodiments, the coronavirus nucleic acid is RNA. In embodiments, the sample comprises the coronavirus nucleic acid. In embodiments, the sample comprises an RT-PCR product, e.g.. cDNA that is generated from the coronavirus nucleic acid.

[00236] In embodiments, the ligating of the oligonucleotide probes is dependent on three events: (1) the targeting and detection probes must hybridize to complementary sequences within the target nucleic acid; (2) the targeting and detection probes must be adjacent to one another in a 5'- to 3'- orientation with no intervening nucleotides; and (3) the targeting and detection probes must have perfect base-pair complementarity with the target nucleic acid at the ligation site. A single nucleotide mismatch between the primers and target may inhibit ligation. In embodiments, the melting temperature (T_M) of the oligonucleotide probes is about 55°C to about 70°C. about 58°C to about 68°C, about 60°C to about 67°C. or about 62°C to about 66°C. In embodiments, the ligation is performed at about 60°C to about 70°C. about 61°C to about 69°C, or about 62°C to about 68°C. In embodiments, the ligation is performed at about 60°C. about 61 °C, about 62°C, about 63°C, about 64°C. about 65°C, about 66°C, about 67°C, about 68°C, about 69°C, or about 70°C.

[00237] In embodiments, the targeting probe comprises, in 5'- to 3'- order: the oligonucleotide tag, and a sequence that is complementary to a first region of the target nucleic acid. In embodiments where the method detects a polymorphic site (SNP), the first region of the target nucleic acid comprises the polymorphic site. In embodiments, the oligonucleotide tag comprises a single-stranded oligonucleotide. In embodiments, the oligonucleotide tag does not hybridize with the target nucleic acid. In embodiments, the detection probe comprises, in 5'- to 3'- order: a sequence that is complementary to a second region of the target nucleic acid that is adjacent to the first region, and a detectable label or binding partner thereof. In embodiments, the 5' end of the targeting probe is phosphorylated and is adjacent to the 3' hydroxyl of the detection probe when the targeting and detection probes are hybridized to the target nucleic acid, such that the ends of the targeting and detection probes are ligated by formation of a phosphodiester bond. In embodiments, the 5' end of the detection probe is phosphorylated and is adjacent to the 3' hydroxyl of the targeting probe when the targeting and detection probes are hybridized to the target nucleic acid, such that the ends of the targeting and detection probes are ligated by formation of a phosphodiester bond. Tn embodiments, the targeting probe and/or the detection probe comprises a modified nucleotide, e.g., a locked nucleic acid (LN A).

[00238] In embodiments, the targeting and detection probes are ligated using a template-dependent ligase, for example, a DNA ligase such as E. coli DNA ligase, T4 DNA ligase, T. aquaticus (Taq) ligase, T. Thermophilus DNA ligase, or Pyrococcus DNA ligase. In embodiments, the ligase is a thermostable ligase. In embodiments, the targeting and detection probes are ligated by chemical ligation. In embodiments, the hybridization and ligation are performed in a combined step, for example, using multiple thermocycles and a thermostable ligase.

[00239] In embodiments where the method detects a coronavirus nucleic acid (e.g., the SARS-CoV-2 N gene or the Nl, N2, and/or N3 regions thereof), the targeting probe hybridizes to the target nucleic acid such that tire terminal 5' nucleotide of the targeting probe hybridizes with the first region in the target nucleic acid, and the detection probe hybridizes to tire second region in the target nucleic acid that is adjacent to the first region and provides a 3' end for the ligation of the targeting and detection probes. In embodiments, the detection probe hybridizes to die target nucleic acid such that the terminal 5' nucleotide of the detection probe hybridizes with the first region in the target nucleic acid, and the targeting probe hybridizes to the second region in the target nucleic acid that is adjacent to the first region and provides a 3' end for the ligation of the targeting and detection probes. In embodiments, the detection probe hybridizes to the target nucleic acid such that the terminal 3' nucleotide of the detection probe hybridizes with the first region in the target nucleic acid, and the targeting probe hybridizes to the second region of the target nucleic acid that is adjacent to the first region and provides a 5' end for the ligation of the targeting and detection probes.

[00240] In embodiments where the method detects an SNP, the targeting probe hybridizes to the target nucleic acid such that the terminal 5' nucleotide of the targeting probe hybridizes with the SNP in the target nucleic acid, and the detection probe hybridizes to the target nucleic acid adjacent to the SNP and provides a 3' end for the ligation of the targeting and detection probes. In embodiments, the detection probe hybridizes to the target nucleic acid such that tire terminal 5' nucleotide of the detection probe hybridizes with the SNP in the target nucleic acid, and the targeting probe hybridizes to the target nucleic acid adjacent to tire SNP and provides a 3' end for the ligation of the targeting and detection probes. In embodiments, the detection probe hybridizes to the target nucleic acid such that the terminal 3' nucleotide of the detection probe hybridizes with the SNP in the target nucleic acid, and the targeting probe hybridizes to the target nucleic acid adjacent to the SNP and provides a 5' end for tire ligation of the targeting and detection probes. [00241] In embodiments, the method further comprises providing a blocking probe during the ligating of the targeting and detection probes. In embodiments, a blocking probe reduces non-specific bridging background during the ligation reaction. In embodiments, the blocking probe comprises a single stranded oligonucleotide that is complementary to die target nucleic acid and straddles the ligation site but does not comprise an oligonucleotide tag or a detectable label or binding partner thereof. In embodiments, the blocking probe comprises a single stranded oligonucleotide that is complementary to a probe designed to hybridize to the target nucleic acid. Without being bound by theory, it is believed that the presence of a blocking probe can reduce formation of complexes in which die target nucleic acid functions as a "bridge" for probes that are annealed to the target nucleic acid, but not ligated to one another, such that the complex can generate a false signal. In embodiments, a pair of blocking probes is provided during the ligating. In embodiments, one or more blocking probes is provided during the ligating in excess over the corresponding targeting and/or detection probes.

[00242] In embodiments, the detection probe comprises a detectable label. In embodiments, the detection probe comprises a binding partner of a detectable label. Detectable labels arc described herein. In embodiments, the detectable label is an electrochemiluminescence (ECL) label. In embodiments, the detection probe comprises biotin, and the detectable label comprises an ECL label linked to avidin or streptavidin. In embodiments, the detection probe comprises avidin or streptavidin, and the detectable label comprises an ECL label linked to biotin. Additional non-limiting examples of binding partners that can be on the detection probe and detectable label are provided herein.

[00243] In embodiments, the target nucleic acid in the sample comprises a coronavirus nucleic acid. In embodiments, the target nucleic acid in the sample comprises an RT-PCR product, e.g., cDNA generated from the coronavirus nucleic acid. In embodiments, the method further comprises amplifying the target nucleic acid prior to contacting with the oligonucleotide probes. In embodiments, the method does not comprise amplifying the target nucleic acid. In embodiments, the nucleic acid is coronavirus RNA. and the method comprises reverse transcribing the coronavirus RNA into cDNA prior to step (a). In embodiments, the targeting probe and/or detection probe hybridize to the cDNA strand comprising the SNP of interest. In embodiments where the SNP of interest is in a protein coding sequence, the targeting probe and/or detection probe hybridize to the cDNA strand comprising the protein coding sequence. In embodiments, the targeting probe and/or detection probe hybridize to the cDNA strand comprising a complement of the SNP of interest. Tn embodiments wherein the SNP of interest is in a protein coding sequence, the targeting probe and/or detection probe hybridize to the cDNA strand comprising the complementary strand of the protein coding sequence. In embodiments, the coronavirus is SARS-CoV-2.

[00244] In embodiments, a region of the SARS-CoV-2 genome surrounding the SNP of interest is reverse transcribed prior to step (a). In embodiments, the cDNA formed by the reverse transcription is amplified by PCR. Exemplary PCR primers for amplification are in the accompanying Sequence Listing and described in Lu et al., Emerg Infect Dis 26(8): 1654-1665 (2020). In embodiments, the method comprises detecting an SNP in a synthetic oligonucleotide template. In embodiments, die region surrounding a SARS-CoV-2 genome location described in FIG. 1A and/or FIG. 1C is reverse transcribed prior to step (a). [00245] An embodiment of the OLA method for detecting an SNP described herein is represented schematically in FIGS. 4A-4D. In FIGS. 4A-4C. a target nucleic acid (1) that comprises an SNP (2) is contacted with: a targeting probe (3) that comprises an oligonucleotide tag (4) and a sequence that is complementary to the SNP, and a detection probe (5) that comprises detectable label (6). The targeting and detection probes (3. 5) hybridize to the target nucleic acid, and the targeting and detection probes that hybridize with perfect complementarity at the SNP are ligated to form a ligated target complement (11) comprising the oligonucleotide tag and detectable label. The reaction mixture containing the ligated target complement is contacted with a surface comprising one or more binding reagents (7) immobilized in one or more binding domains (9). A signal (10) is detected if the ligated target complement is immobilized on the surface via hybridization of the complementary oligonucleotides in the oligonucleotide tag and the binding reagent. In FIG. 4D, the targeting probe has a mismatch with the SNP in the target nucleic acid, and thus, hybridization and ligation do not occur.

[00246] In embodiments, the method is a multiplexed OLA method. In embodiments where the method detects a coronavirus nucleic acid, the biological sample is contacted with one or more targeting probes and one or more detection probes to different regions of the coronavirus nucleic acid to form a plurality of ligated target complements. In embodiments, targeting probes for individual coronavirus nucleic acid regions comprise oligonucleotide tags corresponding to the individual coronavirus nucleic acid regions. In embodiments where tire method detects an SNP, the biological sample is contacted with one or more SNP- specific targeting probes and one or more detection probes to form a plurality of ligated target complements. In embodiments, the detection probes comprise identical sequences. In embodiments, each of the one or more SNP-specific targeting probes hybridizes to a different SNP at the target nucleic acid (e.g., any of the SARS-CoV-2 genome locations and SNPs in FIGS. 1A and 1C herein). In embodiments, targeting probes for different SNPs comprise different oligonucleotide tags. In embodiments, the targeting probes for different coronavirus nucleic acid regions or for different SNPs have substantially the same melting temperatures (T_M), e.g.. within about 5°C, within about 4°C. within about 3°C, within about 2°C. or within about 1°C. In embodiments, the surface comprises a plurality of binding reagents capable of hybridizing to the different oligonucleotide tags. In embodiments, a plurality of binding complexes, each comprising a ligated target complement and its corresponding binding reagent, are formed on the surface, and the binding complexes are detected, thereby detecting, identify ing, and/or quantifying each of the different coronavirus nucleic acid regions or each of the SNPs at the polymorphic site of the coronavirus nucleic acid. In embodiments, tire coronavirus is SARS-CoV-2. In embodiments, the coronavirus nucleic acid is RNA. In embodiments, the different coronavirus regions comprise the Nl, N2, and N3 regions of SARS-CoV-2.

[00247] In embodiments, the multiplexed OLA method simultaneously detects at least 2. at least 3, at least 4, at least 5. at least 6, at least 7, at least 8. at least 9, or at least 10 coronavirus nucleic acids as described herein, hr embodiments, tire multiplexed OLA method simultaneously detects at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 SNPs. In embodiments, the multiplexed OLA method detects any combination of the SNPs in FIG. 1 A. In embodiments, the multiplexed OLA method simultaneously detects the reference SARS-CoV-2 strain and one or more variants, e.g., by detecting both the wild-type nucleotide and the variant SNP at a genome location. As used herein, "variant" refers to a strain that has one or more mutations relative to the SARS-CoV-2 reference strain NC 045512. In embodiments, the multiplexed OLA method comprises contacting the biological sample with a surface comprising at least 2. at least 3, at least 4, at least 5. at least 6, at least 7, at least 8. at least 9, or at least 10 distinct binding domains, wherein each binding domain comprises a unique binding reagent, each unique binding reagent capable of recognizing a different oligonucleotide tag as described herein.

[00248] In embodiments, the method further comprises detecting a control gene. In embodiments, the control gene comprises an endogenous gene of the subject from which the biological sample was obtained. In embodiments, the control gene comprises the human RPP30 gene. In embodiments, the targeting probe for the human RPP30 gene comprises SEQ ID NO:35 or 37. In embodiments, the detection probe for the human RPP30 gene comprises SEQ ID NO:36 or 38. In embodiments, the blocking probe for the human RPP30 gene comprises any one of SEQ ID NOs:51-54.

[00249] In embodiments, the invention provides a method for detecting a coronavirus nucleic acid in a biological sample. In embodiments, the method comprises: (a) contacting the biological sample with (i) a polymerase; (ii) a forward primer, wherein the forw ard primer binds to a first region of a target nucleic acid (e.g., tire coronavirus nucleic acid or an RT-PCR product described herein), and wherein tire forward primer comprises an oligonucleotide tag; and (iii) a reverse primer, wherein the reverse primer binds to a second region of the target nucleic acid; (b) amplifying the target nucleic acid using the polymerase to form an amplified target nucleic acid comprising the oligonucleotide tag; (c) hybridizing the amplified target nucleic acid with an internal detection probe that is complementary to at least a portion of the amplified target nucleic acid, thereby forming a hybridized target; (d) contacting the hybridized target with a surface comprising a binding reagent immobilized in one or more binding domains, wherein the binding reagent comprises an oligonucleotide complementary to the oligonucleotide tag; (e) fonning a binding complex on the surface between the binding reagent and the hybridized target; and (f) detecting the binding complex, thereby detecting, identifying, and/or quantifying the coronavirus nucleic acid. In embodiments, the coronavirus is SARS-CoV-2. In embodiments, the coronavirus nucleic acid is RNA. In embodiments, the target nucleic acid is the Nl. N2. and/or N3 regions of SARS-CoV-2. In embodiments, the sample comprises the coronavirus nucleic acid. In embodiments, the sample comprises an RT-PCR product, e g., cDNA that is generated from the coronavirus nucleic acid.

[00250] In embodiments, the method further comprises amplifying the target nucleic acid prior to contacting with the oligonucleotide probes. In embodiments, the nucleic acid is coronavirus RNA, and the method comprises reverse transcribing the coronavirus RNA into cDNA prior to step (a). In embodiments, the coronavirus is SARS-CoV-2

[00251] In embodiments, the internal detection probe comprises a detectable label. In embodiments, the internal detection probe comprises a binding partner of a detectable label. Detectable labels are described herein. In embodiments, the detectable label is an electrochemiluminescence (ECL) label. In embodiments, the internal detection probe comprises biotin, and the detectable label comprises an ECL label linked to avidin or streptavidin. In embodiments, the internal detection probe comprises avidin or streptavidin, and the detectable label comprises an ECL label linked to biotin. Additional non-limiting examples of binding partners that can be on the internal detection probe and detectable label are provided herein.

[00252] In embodiments, the method further comprises detecting a control gene. In embodiments, the control gene comprises an endogenous gene of the subject from which the biological sample was obtained. Suitable control genes are known to those of skill in the art. In embodiments, the control gene comprises the human RPP30 gene. In embodiments, the forw ard primer for the human RPP30 gene comprises SEQ ID NO: 64. In embodiments, the reverse primer for tire human RPP30 gene comprises SEQ ID NO: 65. In embodiments, the internal detection probe for the human RPP30 gene comprises SEQ ID NO:66.

Manual and Automated Embodiments

[00253] The methods herein can be performed manually, using automated technology, or both. Automated technology may be partially automated, e.g.. one or more modular instruments, or a fully integrated, automated instrument. Exemplary automated systems and apparatuses are described in WO 2018/017156, WO 2017/015636, and WO 2016/164477. In embodiments, the methods herein are performed in an automated cartridge reader as described herein. Manual and automated systems for use with the methods and kits described herein are known in the art and described, e.g., in U.S. Publication No. 2022/0003766 and U.S. Publication No. 2021/0349104.

Antibody and Composition

[00254] In embodiments, the invention provides an antibody or antigen-binding fragment thereof that specifically binds a viral antigen described herein, e.g., a SARS-CoV-2 protein. In embodiments, the invention provides an antibody or antigen-binding fragment thereof that specifically binds a SARS-CoV-2 N protein or a SARS-CoV-2 S protein. In embodiments, the invention provides an antibody or antigenbinding fragment thereof that specifically binds SARS-CoV-2 SI, S2, S-ECD, S-NTD, or S-RBD. In embodiments, the invention provides an antibody or antigen-binding fragment thereof that specifically binds a SARS-CoV-2 S protein or submit or fragment thereof that comprises any’ of the mutations in FIGS. 1A and IB. In embodiments, the invention provides an antibody or antigen-binding fragment thereof that specifically binds an S protein from SARS-CoV-2, an S protein from SARS-CoV, an S protein from MERS-CoV, an S protein from HCoV-HKUl, an S protein from HCoV-OC43, an S protein from HCoV- NL63, an S protein from HCoV-229E, an N protein from SARS-CoV-2, an N protein from SARS-CoV, an N protein from MERS-CoV, an N protein from HCoV-HKUl, an N protein from HCoV-OC43, an N protein from HCoV-NL63, an N protein from HCoV-229E, an HA from influenza B, an HA from influenza A Hl, an HA from influenza A H3, an HA from influenza A H7, an F protein from RSV, or a combmation thereof.

[00255] In embodiments, the antibody or antigen-binding fragment thereof is a binding reagent, e.g., as disclosed herein, for an assay described herein, e.g., for detecting a viral component in a sample. In embodiments, the antibody or antigen-binding fragment thereof is capable of being immobilized onto a surface, e.g.. as disclosed herein. In embodiments, the invention provides a composition comprising: (i) the antibody or antigen-binding fragment thereof; and (ii) a surface. In embodiments, the antibody or antigenbinding fragment thereof is immobilized onto the surface.

[00256] In embodiments, the antibody or antigen-binding fragment thereof is a detection reagent, e.g.. as disclosed herein, for an assay described herein, e.g.. for detecting a viral component in a sample. In embodiments, the antibody or antigen-binding fragment thereof comprises a detectable label. In embodiments, the antibody or antigen-binding fragment is capable of being conjugated with a detectable label. In embodiments, the invention provides a composition comprising: (i) the antibody or antigenbinding fragment thereof; (ii) a detectable label, e.g., as disclosed herein; and (iii) a reagent for conjugating the detectable label to the antibody or antigen-binding fragment thereof. In embodiments, the detectable label is an ECL label. In embodiments, the antibody or antigen-binding fragment thereof comprises a nucleic acid probe. In embodiments, tire antibody or antigen-binding fragment is capable of being conjugated with a nucleic acid probe. In embodiments, the invention provides a composition comprising: (i) die antibody or antigen-binding fragment thereof; (ii) a nucleic acid probe, e.g., as disclosed herein; and (iii) a reagent for conjugating the nucleic acid probe to the antibody or antigen-binding fragment thereof.

[00257] In embodiments, die antibody or antigen-binding fragment thereof is a calibration reagent, e.g., as disclosed herein, for a serology assay, e.g., a classical, bridging, or competitive serology assay, e.g., as disclosed herein. In embodiments, the antibody or antigen-binding fragment tiiereof is a competitor for a competitive serology assay.

[00258] In embodiments, the invention provides a tiierapeutic composition comprising the antibody or antigen-binding fragment thereof. In embodiments, the therapeutic composition is capable of treating or preventing infection by a virus described herein, e.g., SARS-CoV-2 and/or a variant thereof.

[00259] In embodiments, the invention provides a composition comprising (i) the antibody or antigenbinding fragment thereof and (ii) a viral antigen that specifically binds the antibody or antigen-binding fragment.

[00260] In general, an antibody (used interchangeably with the term "immunoglobulin") comprises at least the variable domain of a heavy chain: typically . an antibody comprises the variable domains of a heavy chain and a light chain. Both the heavy and light chains are divided into regions of structural and functional homology. Generally, the variable domain of a heavy chain (V_H) or light chain (V_L) determines antigen recognition and specificity, and the constant domain of a heavy chain (CHI, CHS, or CHS) or light chain (CL) confers biological properties such as secretion, receptor binding, complement binding, and the like. Generally, the N-terminal portion of an antibody chain is a variable portion, and the C-terminal portion is a constant region; the CHS and CL domains ty pically comprise the C-terminus of the heavy⁷ chain and light chain, respectively.

[00261] In general, antibodies are encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immuno globulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. [00262] In general, the variable region allows tire antibody to selectively recognize and specifically bind epitopes on antigens. Thus, the V_L domain and VH domain, or a subset of the complementarity determining regions (CDR) within these variable domains, of an antibody combine to form the variable region that forms an antigen binding domain. The antigen binding domain is typically defined by three CDRs on each of the V_L and VH domains. The six "complementarity determining regions" or "CDRs" typically present in each antigen binding domain are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen binding domain. The antigen binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the antigen. This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope.

[00263] In embodiments, the antibody or antigen-binding fragment thereof comprises a constant region comprising an IgA. IgD, IgE, IgG. or IgM domain. In embodiments, the antibody or antigen-binding fragment thereof comprises an IgG domain. In embodiments, the antibody or antigen-binding fragment thereof is an IgGl, IgG2, IgG3, or IgG4 iso type antibody or antigen-binding fragment thereof. In embodiments, the antibody or antigen-binding fragment thereof is IgG2a, IgG2b, or IgG2c subclass antibody or antigen-binding fragment thereof.

[00264] In embodiments, die antibody or antigen-binding fragment thereof is derived from a mouse, rat, goat, rabbit, chicken, guinea pig, hamster, horse, sheep, ferret, minx, or bat. In embodiments, the antibody or antigen-binding fragment thereof is humanized. In embodiments, the antibody or antigen-binding fragment thereof is capable of being administered to a human or an animal subject described herein, e.g., mouse, rat, ferret, minx, bat, a domestic animal, or an NHP. In embodiments, the antibody or antigenbinding fragment thereof is non-immunogcnic to a human or an animal subject described herein, e.g.. mouse, rat, ferret, minx, bat, a domestic animal, or an NHP.

Kits

[00265] In embodiments, the invention provides a kit comprising, in one or more vials, containers, or compartments: (a) a viral antigen that specifically binds a biomarker, e g., an antibody biomarker; and (b) a detection reagent that specifically binds die biomarker, e.g., the antibody biomarker. In embodiments, the kit further comprises a surface. Antibody biomarkers and their binding partners, e.g., viral antigens, are described herein. In embodiments, die detection reagent is an antibody or antigen-binding fragment thereof. In embodiments, the detection reagent is a second copy of the viral antigen.

[00266] In embodiments, the viral antigen is a respiratory virus antigen. In embodiments, the respiratory virus is a coronavirus, an influenza virus, a paramyxovirus, an adenovirus, a bocavirus, a pneumovirus, an enterovirus, a rhinovirus, or a combination thereof. In embodiments, the viral antigen is a coronavirus S protein or fragment tiiereof. In embodiments, the coronavirus is SARS-CoV, MERs-CoV. SARS-CoV-2. HCov-OC43, HCoV-229E. HCoV-NL63, HCoV-HKUl. or a combination thereof. In embodiments, the viral antigen is SARS-CoV-2 S protein, SI subunit. S2 subunit, S-RBD, M protein. E protein, N protein, or a combination thereof.

[00267] In embodiments, the invention provides a kit comprising, in one or more vials, containers, or compartments: (a) a binding reagent that specifically binds a biomarker, e.g., an inflammatory or tissue damage response biomarker; and (b) a detection reagent that specifically binds the biomarker, e.g.. the inflammatory or tissue damage response biomarker. In embodiments, the kit further comprises a surface. Inflammatory and tissue damage response biomarkers and binding and detection reagents therefor are described herein. In embodiments, die binding reagent is an antibody or antigen-binding fragment. In embodiments, the detection reagent is an antibody or antigen-binding fragment thereof.

[00268] In embodiments, the invention provides a kit comprising: (a) one or more binding reagents that specifically bind one or more antibody biomarkers, wherein each antibody biomarker specifically binds to a SARS-CoV-2 antigen; and (b) one or more detection reagents. In embodiments, the one or more antibody biomarkers comprises IgG, IgA. IgM, or a combination thereof. In embodiments, the IgG, IgA. and/or IgM is from a human, mouse, rat, ferret, minx, bat, or combination thereof. In embodiments, the one or more binding reagents comprises the SARS-CoV-2 antigen(s) to which the antibody biomarkers specifically bind. In embodiments, the one or more detection reagents specifically binds to the antibody biomarker. In embodiments, the one or more detection reagents specifically binds IgA, IgG, or IgM. In embodiments, the one or more detection reagents comprises the SARS-CoV-2 antigcn(s) to which the antibody biomarkers specifically bind. In embodiments, the one or more detection reagents comprises ACE2. In embodiments, die kit further comprises a surface. In embodiments, the surface comprises a single assay plate. In embodiments, the surface comprises a multi-well assay plate, wherein each well comprises four distinct binding domains, e.g., as shown in FIG. 20A. In embodiments, the surface comprises a multi-well assay plate, wherein each well comprises ten distinct binding domains, e.g., as shown in FIG. 20B. In embodiments, the assay plate is a 96-well assay plate. In embodiments, the assay plate is a 384-well assayplate.

[00269] In embodiments, the kit described herein comprises a surface comprising one or more binding domains, wherein each binding domain comprises a binding reagent, e.g., a viral antigen immobilized thereon or capable of being immobilized thereon. In embodiments, each binding domain comprises an immobilized antigen of a panel of antigens. In embodiments, the surface comprises a well of an assay plate. In embodiments, each well of the assay plate comprises ten distinct binding domains, wherein each binding domain comprises an immobilized viral (e.g.. a respiratory virus such as SARS-CoV-2) antigen of a panel of viral antigens as described herein. In embodiments, the panel of viral antigens, e.g., SARS-CoV-2 antigens, are immobilized or capable of being immobilized on a surface comprising Spots 1-10 as shown in FIG. 20B, wherein the antigens are arranged as shown in Tables 2A-2I. In embodiments, the S protein mutations from the SARS-CoV-2 strains of Tables 2A-2I are described in Table 1A. In embodiments, the S-RBD mutations from the SARS-CoV-2 strains of Tables 2A-2I are described in Table IB. In embodiments, the detection reagents of the kit comprise the same antigens as those immobilized on the surface. In embodiments, the detection reagents of the kit comprise one or more antibodies that specifically bind IgA, IgG, or IgM. In embodiments, the detection reagents of the kit comprise ACE2.

[00270] In embodiments, the surface of the kits described herein comprises a multi-well assay plate. In embodiments, the surface comprises avidin or streptavidin. In embodiments, each binding reagent comprises biotin. In embodiments, the surface comprises a targeting agent. In embodiments, the kit further comprises a linking agent connected to a targeting agent complement. In embodiments, each binding reagent comprises a supplemental linking agent. In embodiments, the targeting agent and targeting agent complement comprise complementary oligonucleotides. In embodiments, the linking agent comprises avidin or streptavidin, and the supplemental linking agent comprises biotin. Targeting agents, targeting agent complements, linking agents, and supplemental linking agents are further described herein.

[00271] In embodiments, the invention provides a kit comprising: (a) one or more binding reagents, each binding reagent binding specifically to: (1) a viral component; (2) a host antibody biomarker; or (3) a host inflammatory and/or tissue damage response biomarker; and (b) one or more detection reagents, each detection reagent binding specifically to the viral component, host antibody biomarker, or the host inflammatory and/or tissue damage response biomarker. In embodiments, the detection reagent that binds to the host antibody biomarker binds IgA, IgG, or IgM. In embodiments, the detection reagent is ACE2. In embodiments, the viral component is a viral protein. In embodiments, the viral component is a viral nucleic acid. In embodiments, the virus is a coronavirus. In embodiments, the virus is SARS-CoV-2. In embodiments, the kit further comprises a surface.

[00272] In embodiments, die invention provides a combmation of any of tire kits described herein. In embodiments, the combination of kits is provided as a single kit, comprising the components of each of the individual kits.

[00273] In embodiments, the binding reagent is an antibody or antigen-binding fragment thereof. In embodiments, the detection reagent is an antibody or antigen-binding fragment thereof. In embodiments, any of the detection reagents described herein comprises a detectable label as described herein. In embodiments, the detection reagent comprises a nucleic acid probe as described herein. In embodiments, the kit comprises first and second detection reagents, and the first and second detection reagents respectively comprise first and second nucleic acid probes as described herein. In embodiments, the kit further comprises a reagent for conjugating tire detection reagent to a detectable label or a nucleic acid probe.

[00274] In embodiments, the detection reagent is lyophilized. In embodiments, the detection reagent is provided in solution. In embodiments, the binding reagent is immobilized on the binding domain. In embodiments, the binding reagent is provided in solution. In embodiments, the reagents and other components of the kit are provided separately. In embodiments, they are provided separately according to their optimal shipping or storage temperatures.

[00275] Reagents and methods for immobilizing binding reagents to surfaces, e.g.. via targeting agents/targeting agent complements, linking agents/supplemental linking agents, and bridging agents are described herein. In embodiments, tire surface is a plate. In embodiments, the surface is a multi-well plate. Non-limiting examples of plates include the MSD® SECTOR™ and MSD QUICKPLEX® assay plates, e.g.. MSD® GOLD™ 96-well Small Spot Streptavidin plate. In embodiments, the surface is a particle. In some embodiments, the particle comprises a microsphere. In embodiments, tire particle comprises a paramagnetic bead. In embodiments, the surface is a cartridge. In embodiments, the surface comprises an electrode. In embodiments, the electrode is a carbon ink electrode. [00276] In embodiments, the kit further comprises a calibration reagent. In embodiments, the calibration reagent comprises a known quantity of tire virus, viral component, or biomarker as described herein. In embodiments, multiple calibration reagents comprise a range of concentrations of the virus, viral component, or biomarker. In embodiments, the multiple calibration reagents comprise concentrations of the virus, viral component, or biomarker near the upper and lower limits of quantitation for the immunoassay. In embodiments, the multiple concentrations of the calibration reagent span the entire dynamic range of the immunoassay. In embodiments, the calibration reagent comprises an antibody biomarker. In embodiments, the antibody biomarker is a neutralizing antibody as described herein. In embodiments, the neutralizing antibody is a monoclonal antibody. In embodiments, the calibration reagent comprises a neutralizing antibody that specifically binds the SARS-CoV S protein, the SARS-CoV-2 S protein, or both. In embodiments, the calibration reagent is derived from human serum known to contain one or more antibodies that specifically bind to one or more viral antigens described herein. In embodiments, the one or more antibodies is human IgG, human IgM, or a combination thereof. In embodiments, the calibration reagent comprises an antibody that specifically binds the SARS-CoV S protein, an antibody that specifically binds SARS-CoV-2 S-NTD, an antibody that specifically binds SARS-CoV-2 S protein, an antibody that specifically binds SARS-CoV-2 S-RBD, an antibody that specifically binds SARS-CoV-2 N protein, an antibody that specifically binds HCoV-OC43 S protein, an antibody that specifically binds HCoV-HKUl S protein, an antibody that specifically binds MERS-CoV S protein, an antibody that specifically binds HCoV-NL63 S protein, an antibody that specifically binds HCoV-229E S protein, an antibody that specifically binds influenza A/Hong Kong H3 HA protein, an antibody that specifically binds influenza B/Brisbane HA protein, an antibody that specifically binds influenza A/Shanghai H7 HA protein, an antibody that specifically binds influenza A/Micliigan Hl HA protein, an antibody that specifically binds influenza B/Phuket HA protein, and an antibody that specifically binds RSV pre-fusion F protein. In embodiments, the calibration reagent comprises an IgG that specifically binds to SARS-CoV-2 S protein, an IgG that specifically binds to SARS-CoV-2 N protein, an IgG drat specifically binds to SARS-CoV-2 S- RBD. an IgM that specifically binds to SARS-CoV-2 S protein, an IgM that specifically binds to SARS- CoV-2 N protein, an IgM that specifically binds to SARS-CoV-2 S-RBD, an IgA that specifically binds to SARS-CoV-2 S protein, an IgA that specifically binds to SARS-CoV-2 N protein, and an IgA that specifically binds to SARS-CoV-2 S-RBD.

[00277] In embodiments, the calibration reagents are provided in the kit at the following concentrations: about 1 to about 10 BAU/mL of an IgG that specifically binds to SARS-CoV-2 S protein, about 0.1 to about 5 BAU/mL of an IgG that specifically binds to SARS-CoV-2 N protein, about 5 to about 20 BAU/mL of an IgG that specifically binds to SARS-CoV-2 S-RBD, about 0.1 to about 2 BAU/mL of an IgM that specifically binds to SARS-CoV-2 S protein, about 1 to about 5 BAU/mL of an IgM that specifically binds to SARS-CoV-2 N protein, about 0.1 to about 2 BAU/mL of an IgM that specifically binds to SARS-CoV- 2 S-RBD, about 1 to about 5 BAU/mL of an IgA that specifically binds to SARS-CoV-2 S protein, about 1 to about 10 BAU/mL of an IgA that specifically binds to SARS-CoV-2 N protein, and about 0.1 to about 5 BAU/mL of an IgA that specifically binds to SARS-CoV-2 S-RBD. Concentrations of the calibration reagents provided herein are defined according to the "First WHO International Standard for anti-SARS- CoV-2 immunoglobulin" (NIBSC code: 20/136).

[00278] In embodiments, the calibration reagents are provided in tire kit at the following concentrations: about 6.31 BAU/mL of an IgG that specifically binds to SARS-CoV-2 S protein, about 1.89 BAU/mL of an IgG that specifically binds to SARS-CoV-2 N protein, about 8.16 BAU/mL of an IgG that specifically binds to SARS-CoV-2 S-RBD, about 0.867 BAU/mL of an IgM that specifically binds to SARS-CoV-2 S protein, about 2.64 BAU/mL of an IgM that specifically binds to SARS-CoV-2 N protein, about 0.466 BAU/mL of an IgM that specifically binds to SARS-CoV-2 S-RBD, about 3.09 BAU/mL of an IgA that specifically binds to SARS-CoV-2 S protein, about 5.57 BAU/mL of an IgA that specifically binds to SARS-CoV-2 N protein, and about 1.56 BAU/mL of an IgA that specifically binds to SARS-CoV-2 S- RBD.

[00279] In embodiments, the calibration reagent is a positive control reagent. In embodiments, the calibration reagent is a negative control reagent. In embodiments, the positive or negative control reagent is used to provide a basis of comparison for the biological sample to be tested with the methods of the present invention. In embodiments, the positive control reagent comprises multiple concentrations of the virus, viral component, or biomarker. In embodiments, the positive control reagent comprises an antibody. In embodiments, the positive control reagent comprises human IgG, IgM, IgA, or a combination thereof. In embodiments, the positive control reagent comprises an antibody that specifically binds the SARS-CoV-2 S protein, SARS-CoV-2 N protein, SARS-CoV-2 S-RBD, or a combination thereof, hi embodiments, the positive control reagent comprises an IgG that specifically binds to SARS-CoV-2 S protein, an IgG that specifically binds to SARS-CoV-2 N protein, an IgG that specifically binds to SARS-CoV-2 S-RBD. an IgM that specifically binds to SARS-CoV-2 S protein, an IgM that specifically binds to SARS-CoV-2 N protein, an IgM that specifically binds to SARS-CoV-2 S-RBD, an IgA that specifically binds to SARS- CoV-2 S protein, an IgA that specifically binds to SARS-CoV-2 N protein, and an IgA that specifically binds to SARS-CoV-2 S-RBD.

[00280] In embodiments, the positive control reagent is provided in the kit at the following concentrations: about 0.005 to about 1 BAU/mL of an IgG that specifically binds to SARS-CoV-2 S protein; about 0.001 to about 0.1 BAU/mL of an IgG that specifically binds to SARS-CoV-2 N protein; about 0.005 to about 1 BAU/mL of an IgG that specifically binds to SARS-CoV-2 S-RBD; about 0.001 to about 0. 1 BAU/mL of an IgM that specifically binds to SARS-CoV-2 S protein; about 0.01 to about 0.1 BAU/mL of an IgM that specifically binds to SARS-CoV-2 N protein; about 0.001 to about 0.1 BAU/mL of an IgM that specifically binds to SARS-CoV-2 S-RBD; about 0.005 to about 0.5 BAU/mL of an IgA that specifically binds to SARS-CoV-2 S protein; about 0.005 to about 0.5 BAU/mL of an IgA that specifically binds to SARS-CoV-2 N protein; and about 0.001 to about 0.1 BAU/mL of an IgA that specifically binds to SARS-CoV-2 S-RBD. In embodiments, the positive control reagents are provided in the kit at the following concentrations: about 0.1504, about 0.0372, and about 0.0133 BAU/mL of an IgG that specifically binds to SARS-CoV-2 S protein; about 0.0457, about 0.0078, and about 0.0025 BAU/mL of an IgGthat specifically binds to SARS-CoV-2 N protein; about 0.1952, about 0.0576, and about 0.0148 BAU/mL of an IgG that specifically binds to SARS-CoV-2 S-RBD; about 0.0187. about 0.0054, and about 0.0077 BAU/mL of an IgM that specifically binds to SARS-CoV-2 S protein; about 0.061, about 0.030, and about 0.0285 BAU/mL of an IgM that specifically binds to SARS-CoV-2 N protein; about 0.011, about 0.0047, and about 0.0068 BAU/mL of an IgM that specifically binds to SARS-CoV-2 S-RBD; about 0.0768, about 0.023, and about 0.0103 BAU/mL of an IgA that specifically binds to SARS-CoV-2 S protein; about 0.1414, about 0.0237. and about 0.0379 BAU/mL of an IgA that specifically binds to SARS-CoV-2 N protein; and about 0.0394. about 0.0131, and about 0.0085 BAU/mL of an IgA that specifically binds to SARS-CoV-2 S-RBD.

[00281] In embodiments, the calibration reagent is lyophilized. In embodiments, the calibration reagent is provided in solution. In embodiments, the calibration reagent is provided as a stock concentration that is 5X, 10X, 20X, 30X, 40X, 50X, 60X, 70X, 80X, 90X, 100X. 125X, 150X or higher fold concentrations of tire highest working concentration of the calibration reagent. In embodiments, the kit further comprises a diluent for preparing multiple concentrations of the calibration reagent. In embodiments, tire calibration reagent provided in the kit is diluted 1: 10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:140, 1:160, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:1500, 1:2000, 1:2500, 1:3000, 1:3500, 1:4000, 1:4500, 1:5000, 1:5500, 1:6000, 1:6500, 1:7000, 1:7500, 1:8000, 1:8500, 1:9000, 1:9500, 1: 10000, 1:20000, 1:30000, 1:40000, or 1:50000 to provide multiple concentrations of the calibration reagent. In embodiments, the kit comprises multiple calibration reagents at multiple concentrations, e.g., two or more, three or more, four or more, or five or more concentrations. In embodiments, the multiple concentrations of calibration reagents are used to calculate a standard curve. In embodiments, the multiple concentrations of calibration reagents provide thresholds indicating low, medium, or high levels of the virus, viral component, or biomarker being measured.

[00282] In embodiments, the kit further comprises a sample collection device. In embodiments, the sample collection device is an applicator stick. In embodiments, the sample collection device is a swab. In embodiments, the sample collection device is a tissue scraper. In embodiments, the sample collection device is a vial or container for collecting a liquid sample.

[00283] In embodiments, the kit further comprises one or more of a buffer, e.g., assay buffer, reconstitution buffer, storage buffer, read buffer, wash buffer and the like; a diluent; a blocking solution; an assay consumable, e.g., assay modules, vials, tubes, liquid handling and transfer devices such as pipette tips, covers and seals, racks, labels, and the like; an assay instrument; and/or instructions for carrying out the assay.

[00284] In embodiments, the kit comprises lyophilized reagents, e.g.. detection reagent and/or calibration reagent. In embodiments, the kit comprises one or more solutions to reconstitute the lyophilized reagents. [00285] In embodiments, a kit comprising the components above include stock concentrations of the components that are 5X, 10X, 20X, 30X, 40X, 50X, 60X, 70X, 80X, 90X, 100X, 125X, 150X or higher fold concentrations of the working concentrations of the immunoassays herein.

[00286] In embodiments, the invention provides a kit for collecting a biological sample. In embodiments, die kit for collecting a biological sample can be provided to a subject for collecting the subject's own sample, e.g.. saliva sample. The collected sample can then be provided by the subject, e.g., delivered in person or via postal service, to a laboratory for analysis. In embodiments, the kit further comprises an assay instrument, e.g., an assay cartridge and/or a cartridge reader, for the subject to analyze the collected sample. In embodiments, the kit comprises a sample collection device, e.g.. an applicator stick, a swab, a tissue scraper, or a vial or container for collecting a liquid sample. In embodiments, the sample collection device comprises a straw for collecting a saliva sample. In embodiments, the sample collection device comprises a storage solution that stabilizes the sample. In embodiments, the sample collection device comprises a unique sample identifier, e.g., a barcode. In embodiments, the kit further comprises instructions for collecting the sample and/or for analyzing the sample in an assay instrument. In embodiments, the kit farther comprises an absorbent material, e.g., a tissue. In embodiments, the kit further comprises a secondary container (e.g., a bag) to secure the sample collection device. In embodiments, the kit further comprises a pre-paid postage label or a pre-paid envelope or box for mailing the collected sample.

[00287] All references cited herein, including patents, patent applications, papers, textbooks and the like, and fac references cited therein, to tire extent that they arc not already, arc hereby incorporated herein by reference in their entirety.

EXEMPLARY EMBODIMENTS

[00288] The following are exemplary embodiments of the inventions disclosed herein.

[00289] Embodiment (E) 1 is a kit for detecting one or more antibody biomarkers of interest in a sample, the kit comprising, in one or more vials, containers, or compartments:

(a) a surface comprising one or more binding domains, wherein each binding domain comprises an immobilized antigen of a panel of antigens, and wherein the panel of antigens comprises:

(1) an S protein from the following SARS-CoV-2 strains: wild-type, BA. l, XBB.1.16.1, XBB.1.5, BQ.1.1, XBB.1.16, XBB.2.3, XBB. l, and BA.5; and an N protein from wild-ty pe SARS-CoV-2; or

(2) an S protein from the following SARS-CoV-2 strains: wild-ty pe FL.1.5.1, BA.2.86, XBB.1.5, EG.5.1, XBB.1.16, XBB.2.3, XBB.1.16.6, and BA.5; and an N protein from wild-type SARS- CoV-2; or

(3) an S protein from the following SARS-CoV-2 strains: wild-ty pe, FL.1.5.1, BA.2.86, XBB.1.5, EG.5.1, JN.l. JD.1.1, HV. l, and BA.5; and an N protein from wild-type SARS-CoV-2, and

(b) one or more detection reagents, wherein each detection reagent comprises a detection antibody, a detection antigen, or an ACE detection reagent.

[00290] E2 includes the kit of E 1. wherein the detection reagent comprises an electrochemiluminescent (ECL) label.

[00291] E3 includes the kit of El or E2, wherein the surface comprises an electrode.

[00292] E4 includes the kit of any one of El to E3, wherein the surface comprises a well of a multi -well plate, and wherein each well comprises 1 to 10 binding domains.

[00293] E5 includes the kit of any one of El to E4, wherein the panel of antigen comprises (1). [00294] E6 includes the kit of any one of El to E4, wherein the panel of antigen comprises (2).

[00295] E7 includes the kit of any one of El to E4. wherein the panel of antigen comprises (3).

[00296] E8 is a method of detecting one or more antibody biomarkers of interest in a sample, comprising:

(a) contacting the sample with a surface comprising one or more binding domains, wherein each binding domain comprises an immobilized antigen of a panel of antigens, and wherein the panel of antigens comprises:

(1) an S protein from the following SARS-CoV-2 strains: wild-type, BA. l, XBB.1.16.1, XBB.1.5, BQ.1.1, XBB.1.1 , XBB.2.3, XBB. l. and BA.5; and an N protein from wild-type SARS-CoV-2; or

(2) an S protein from the following SARS-CoV-2 strains: wild-type FL.1.5.1, BA.2.86, XBB.1.5. EG.5.1, XBB. l.16, XBB.2.3, XBB.l.16.6, and BA.5; and anN protein from wild-type SARS- CoV-2; or

(3) an S protein from the following SARS-CoV-2 strains: wild-type, FL.1.5.1, BA.2.86, XBB.1.5, EG.5.1, JN.l, JD.1.1, HV. l, and BA.5; and an N protein from wild-ty pe SARS-CoV-2;

(b) forming a binding complex in each binding domain, wherein the binding complex comprises die immobilized antigen and an antibody biomarker that binds to the immobilized antigen;

(c) contacting the binding complex in each binding domain widi a detection reagent; and;

(d) detecting the binding complex in each binding domain, thereby detecting the one or more antibody biomarkers in the sample.

[00297] E9 includes the method of E8. wherein the detection reagent comprises a detection antibody, a detection antigen, or an ACE detection reagent

[00298] E10 includes the method of E8 or E9, wherein the detection reagent comprises an ECL label. [00299] El l includes the method of any one of E8 to E10, wherein die surface comprises an electrode. |00300] E12 includes the method of any one of E8 to El 1, wherein die surface comprises a well of a multi-well plate, and wherein each well comprises 1 to 10 binding domains.

[00301] E13 includes the method of any one of E8 to E12, wherein the detection reagent comprises an ECL label, the surface comprises an electrode, and the detecting comprises applying a voltage to the surface and measuring an ECL signal generated from the ECL label on the detection reagent.

[00302] E14 includes the method of any one of E8 to E13, wherein the panel of antigen comprises (1).

[00303] E15 includes the method of any one of E8 to E13, wherein the panel of antigen comprises (2).

[00304] E16 includes the method of any one of E8 to E13, wherein the panel of antigen comprises (3).

EXAMPLES

Example 1. Bridging Serology Assay for SARS-CoV-2 on Patient Samples

[00305] A bridging serology' assay to detect SARS-CoV-2 antibodies was performed using the SARS- CoV-2 S-RBD antigen. Hie bridging serology assay used the simultaneous binding of antibodies to immobilized viral antigen and detection tag-labeled viral antigen, leading to a highly specific isotypeindependent measurement of immune reactivity. The tested samples were from the same COVID-19 positive and normal patients as described in Example 4. except the samples were diluted 10-fold or 100- fold.

[00306] Results are shown in FIG. 2. For both 10-fold and 100-fold sample dilutions, clear separation of signal was observed between the COVID- 19 patient and normal samples. High signals detected in the COVID- 19 sera indicates the presence of anti-S-RBD antibodies.

Example 2. Neutralization Serology Assay for SARS-CoV-2 on Patient Samples

[00307] As described herein, neutralization serology assays (also termed "competitive serology assay") can allow assessment of the potential protective serological response present in the patient. A neutralization serology assay was performed to test SARS-CoV-2 antibody binding to immobilized SARS-CoV-2 S protein in the presence of the host cell protein receptor, ACE2, as competitor for the antigen. The ACE2 is labeled with a detection label. The tested samples were from tire same CO VID-19 positive and nonnal patients as described in Example 4, except the samples were diluted 10-fold or 100-fold.

[00308] Results are shown in FIG. 3. For both the 10-fold and 100-fold sample dilutions, clear separation of signal was observed between the COVID-19 patients and normal samples. Lower signal detected in the COVID-19 sera indicates inhibition of the interaction between the SARS-CoV-2 S protein and its cognate receptor. ACE2, in COVID-19 patient samples.

Example 3. Correlation of Different Serology Assay Formats

[00309] Results of a bridging serology assay and a neutralization serology assay were plotted against results of a classical serology assay to determine the correlation betw een the different serology assay formats. The plot is shown in FIG. 6, indicating the results between different assay formats are well- correlated.

Example 4. Evaluation of Assay Performance

[00310] The sensitivity and specificity of serology assays were detennined for IgG and IgM using SARS- CoV-2 S, S-RBD, and N antigens, and ACE2 competitor (neutralization) serology assays using SARS- CoV-2 S and S-RBD antigens. ROC analysis was used to identify the threshold for each combination of format and antigen that maximized the sum of the specificity and sensitivity for separating the late infections from the nonnal controls. This threshold was then applied to both the early infection and late infection data sets.

[00311] Using the optimal thresholds, the different assays shown in FIG. 7 provided similar sensitivity and specificity values, with largely overlapping 95% confidence limits. All the assays provided point estimates for specificity that were greater than or equal to 95%, except for the measurement of IgM against SARS-CoV-2 S protein. Measurements of IgG using the indirect serology format provided the highest point estimates for sensitivity' in classifying late infections (point estimates of sensitivity for S. RBD and N ranged from 93% to 94%). Point estimates for measurement of IgM using the indirect serology format or inhibitory antibodies using the ACE2 competition format w ere slightly low er ranging from 85% to 91%, with the best sensitivity in both formats provided by the SARS-CoV-2 S antigen (91% sensitivity in both the IgM and ACE2 formats). All the assays provided lower sensitivity for measuring early infection with point estimates for sensitivity ranging from 26% to 47%. There was no evidence that measurement of IgM provided statistically higher sensitivity for classification of the early infection samples, relative to measurement of IgG.

Example 5. SARS-CoV-2 Single Nucleotide Polymorphism (SNP) Assay

[00312] Singleplex and multiplex oligonucleotide ligation assays (OLA) were performed to detect single nucleotide polymorphisms (SNPs) at SARS-CoV-2 genome locations 8782 (OT mutation). 11083 (G>T mutation). 23403 (A>G mutation), and 28144 (T>C mutation). A pair of targeting probes and detection probes for each polymorphic site were designed to allow for single-base discrimination at the SNP site, as described in embodiments herein. The targeting probes have unique 5' oligonucleotide tag sequences that are complementary to binding reagents on specific binding domains on a multi-well plate. The detection probes have a 5' phosphate group for ligation and a 3' biotin. Taq DNA ligase was used to join the targeting probe and detection probe that aligned correctly on the sample. Fragments of unmodified template complements were added to prevent bridging of unligated probes. The OLA cycling conditions were: 2 minutes at 95°C, then 30 cycles of 30 seconds at 95°C and 2 minutes at 65°C. The plates were blocked with a blocking solution for 30 minutes at 37°C during the OLA cycling.

[00313] The plate was washed, and the ligated probes were hybridized to a binding reagent on a plate. In the singleplex format, each well only contained the binding reagents specific for one SNP. In the multiplex format, each well of contained ten binding domains ("spots"), wherein a unique binding reagents was immobilized in each spot, allowing for detection of up to five SNPs per well. The hybridization was performed in hybridization buffer (50 |iL per well), with a one-hour incubation at 37°C.

[00314] The hybridized ligated probes were then detected by streptavidin- SULFO-T AG™. Briefly, following the hybridization, tire plate was washed, and a detection solution was added (50 pL per well) and incubated for 30 minutes at 37°C. The plate was washed, and read buffer was added (150 pL), and the plate was then read using a plate reader.

[00315] Ten (10) nasal swabs from COVID- 19-positive patients obtained from BOCA Biolistics (Deerfield Beach, FL) were tested using both the singleplex and multiplex OLA assay formats. RNA was extracted using the MAGMAX™ Viral/Pathogen Ultra Nucleic Acid Isolation Kit (Applied Biosystems). RT-PCR was conducted using site-specific primers using TAQPATH™ 1-step RT-qPCR Master Mix (Applied Biosystems). Reference strain RNAs were obtained from BEI Resources, NIAID, NIH: Genomic RNA from SARS-Related Coronavirus 2, Isolate USA-WA1/2020, NR-52285, GenBankMN985325, deposited by the Centers for Disease Control and Prevention; Genomic RNA from SARS-Related Coronavirus 2. Isolate Hong Kong/VM20001061/2020. NR-52388, GenBankMT547814, deposited by the University of Hong Kong.

[00316] Results from a test assay using synthetic oligonucleotide template containing the SNPs of interest are shown FIG. 8. The synthetic oligonucleotide templates showed high specificity for the appropriate allele using ligation temperature of 65°C.

[00317] Results from the singleplex assay format with patient samples are shown in FIG. 9. There was a high prevalence of the L strain type (8782C and 28144 T) that also have the D614G mutation. Only one strain had the "asymptomatic" allele (11083T). Results were also compared to a fully sequenced reference strain, with all results matching the published sequences for these strains. Results from the multiplex assay format with patient samples are shown in FIG. 10 and were consistent with the results from the singleplex assays shown in FIG. 9.

[00318] Reproducibility of the multiplex assay was also assessed. All assays had good allele frequency reproducibility, and the SNP determinations for all ten samples and the reference strain were consistent between the runs for all three experiments.

Example 6. Detection of SARS-CoV-2 Nucleocapsid Protein in Human Samples

[00319] A detection assay was used to test for SARS-CoV-2 N protein in the following samples: nasopharyngeal swabs from 12 patients who tested positive for CO VID-19, nasopharyngeal swabs from 6 patients who tested negative for COVID-19, and normal (CO VID-19 negative) human saliva, serum, and EDTA plasma. The detection assay was performed as follows: To each well of a 96-well plate containing immobilized anti-Nucleocapsid capture antibody, add 25 pL labeled anti-Nucleocapsid detection antibody labeled with nucleic acid probe, and 25 pL sample. Incubate for 1 hour at room temperature with shaking. Wash plate and add extension solution. Incubate for 15 minutes at room temperature with shaking. Wash plate and add detection solution. Incubate for 45 minutes at room temperature with shaking. Wash plate, add 150 pL ECL Read Buffer, and read plate. Total protocol time is approximately 2 hours. The samples were all tested without dilution. Assay results of the SARS-CoV-2 N protein concentration are shown in FIG. 11. The results for the negative nasopharyngeal swab and normal saliva, serum, and EDTA plasma were comparable, while the positive nasopharyngeal swab samples had significantly higher concentration of SARS-CoV-2 N protein.

[00320] The dilution linearity and spike recovery of the assay were also tested to determine whether the assay is affected by component(s) that may be present in the biological sample matrix (also known as the "sample matrix effect"). For dilution linearity', the normal human serum, EDTA plasma, saliva, and CO VID-19 negative human nasopharyngeal swab samples were spiked with calibrator and tested at different dilutions. Percent recovery at each dilution level was normalized to tire dilution-adjusted, neat concentration, and shown in FIG. 12.

[00321] For spike recovery, nonnal human serum, EDTA plasma, saliva, and COVID-19 negative human nasopharyngeal swab samples were spiked with calibrator at three levels. Spiked samples were tested neat. Percent recovery is shown in FIG. 13. Based on die results in FIGS. 12 and 13, samples may be diluted to reduce sample matrix effects.

Example 7. SARS-CoV-2 Serology Assays with Variant Panels

[00322] Samples from SARS-CoV-2-infected individuals in die United States in early 2020 (known to be infected widi wild-type SARS-CoV-2 ("Wuhan")); SARS-CoV-2-infected individuals in die United Kingdom (dominating strain: SARS-CoV-2 strain B.1.1.7); or SARS-CoV-2 -infected individuals in South Africa (dominating strain: SARS-CoV-2 strain 501Y.V2, also known as B.1.351) were tested using a viral antigen panel that included the wild-type S protein from SARS-CoV-2, S-RBD from SARS-CoV-2 strain 501Y.V2, N protein from SARS-CoV-2. S-RBD from SARS-CoV-2 strain P. l. S-RBD from SARS-CoV-2 strain B.l.1.7, S protein from SARS-CoV-2 strain P. l, S protein from SARS-CoV-2 strain B.1.1.7. S protein from SARS-CoV-2 strain 501Y.V2, and wild-type S-RBD from SARS-CoV-2. The viral antigens were immobilized in a 96-well plate or within an assay cartridge. Detection was performed using an anti- IgG antibody labeled with an electrochemiluminescence (ECL) label.

[00323] The results are shown in FIG. 14. The measured ratios of antibodies against wild-type SARS- CoV-2 versus SARS-CoV-2 strain B.l .1.7 were plotted on the x-axis, and the measured ratios of antibodies against wild-type SARS-CoV-2 versus SARS-CoV-2 strain 501Y.V2 were plotted on the y-axis. As shown in FIG. 14. samples from the wild-type SARS-CoV-2-mfected patients clustered in the top right quadrant; samples from the UK clustered in the top quadrant; and samples from South Africa clustered in the lower left quadrant. Thus, the samples can be differentiated by geographical region based on binding to the S protein or S-RBD in the serology panel. This approach can be used in epidemiology studies to determine tire circulating strain in a population or geographical region.

Example 8. Matched Fingerstick Blood and Saliva Sample Testing

[00324] A study was performed to demonstrate that self-collected saliva provides information similar to finger-stick blood even after the saliva is at ambient temperature for hours to days without preservatives. Details of the study are described in Campbell et al., Scientific Reports 12:6560 (2022). Consistent with prior studies that report a correlation between antibody levels in serum and plasma, very high concordance was observed between self-collected saliva and finger-stick blood in the identification of individuals with IgG reactivity to the RBD and full-length forms of CoV-2 spike protein.

[00325] The degree of correlation between finger-stick blood and saliva measurements (FIG. 15) depends on variations among individuals in the rate of antibody transit into the mouth, the rate of saliva flow diluting tire antibody, and possibly other factors associated with the degree of compliance with instructions for sample collection such as delaying collection after eating or drinking. The almost perfect agreement we observed in the classification of serostatus using saliva and finger-stick blood suggests that the difference in die observed antibody activity in positive subjects vs. negative controls is large enough to compensate for the increased variability in saliva samples. The ratio of anti-N antibodies to anti-S antibodies in saliva and finger-stick blood (FIG. 16) correlates more strongly than the ratio of tire absolute concentrations (FIG. 15). showing that die effect of variations among donors in salivary flow rates and rates of antibody transit can be reduced through normalization approaches.

Example 9. SARS-CoV-2 Strain Identification

[00326] Samples from ~200 individuals in the United States infected with wild-type SARS-CoV-2 ("Wuhan") and 32 individuals in Soutii Africa infected with SARS-CoV-2 strain 501 Y.V2, also known as B.1.351, were tested using a 10-spot variant SARS-CoV-2 S-RBD panel as shown in FIG. 17, including the following mutations: (1) L452R; (2) K417N, E484K, and N501Y; (3) E484K; (4) K417T, E484K, and N501Y; (5) S477N; (6) N501Y and A570D; (7) E484K andN501Y; (8) L452R and E484Q; (9) Q414K and N450K mutations; and (10) wild-type SARS-CoV-2 S-RBD, wherein all mutations are relative to wild-type S-RBD from SARS-CoV-2. The panels were used in indirect IgG Serology and ACE2 competition assay formats as described in the previous Examples. [00327] The results in FIG. 17 show the signals for each of the antigens after normalization to the signal from the wild-type SARS-CoV-2 S-RBD. Each set of connected dots shows the normalized signals from each antigen for one study subject. The upper figure shows results of the ACE-2 competition assay; the lower figure shows the serology results. The results show that the sample reactivity towards the wild-type Wuhan strain circulating in the United States at the time of sample collection can be clearly separated from the circulating B.1 .351 strain in South Africa.

[00328] FIG. 18 shows a subset of the data in a different format: for each sample, assay signals for the wild-type SARS-CoV-2 S-RBD (x-axis) were plotted against the signals for the SARS-CoV-2 S-RBD from the B.1.351 strain. While absolute serology signals may vary from subject to subject, the ratio of the signals on the two spots remained remarkably consistent between subjects in one region (and presumably exposed to the same strain), as shown by the data points from each region falling on two different lines with tw o different slopes. The results in FIG. 18 show that while the signal from one spot may not be sufficient to distinguish between the wild type and B.1.351 SARS-CoV-2 infections, combining the results from two spots provided almost complete separation.

Example 10. Host Biomarker Detection in Serum and Cerebrospinal Fluid of COVID-19 Patients [00329] Cerebrospinal fluid (CSF) and serum from acute CO VID-19 patients were obtained and measured for the following panel of cytokines: IL-6, IL-10, IL-12p70, IL-4. TNF-a, IL-2, IL-1 , IFN-y. and IL-17A. The cytokines w ere measured using the Format 2 immunoassay described in Example 7. i.e.. each cytokine was detected using a detection reagent linked to a nucleic acid probe, wherein upon binding of the detection reagent to the cytokine, the nucleic acid probe is extended to form an extended sequence, and the extended sequence is contacted with a probe comprising a detectable label for detection.

[00330] The measured cytokine levels from COVID-19 patients were compared against cytokine levels from non-COVID-19 control subjects. Results are shown in FIG. 19, with CO VID-19 patients in light grey- on the left side of each CSF or serum result panel, and non-COVID-19 control subjects in dark grey on the right side of each CSF or serum result panel. The results showed that IL-2, IL-6, IL-10, IFN-y, and TNF-a levels in serum and CSF were generally higher in the acute CO VID-19 patient group than the control group. In general, the cytokine concentrations in CSF were approximately an order of magnitude lower than in serum, except for IL-6, for which the concentrations in CSF and serum were comparable.

Claims

WHAT IS CLAIMED IS:

1. A kit for detecting one or more antibody biomarkers of interest in a sample, the kit comprising, in one or more vials, containers, or compartments:

(1) an S protein from the following SARS-CoV-2 strains: wild-type, BA.l, XBB.1.16.1, XBB.1.5, BQ.1.1, XBB.1.16, XBB.2.3, XBB. l, and BA.5; and an N protein from wild-type SARS-CoV-2; or

(2) an S protein from the following SARS-CoV-2 strains: wild-type, FL.1.5.1, BA.2.86, XBB.1.5, EG.5.1, XBB.1.16, XBB.2.3, XBB.1.16.6, and BA.5; and an N protein from wild-type SARS-CoV-2; or

(3) an S protein from the following SARS-CoV-2 strains: wild-type, FL.1.5.1, BA.2.86, XBB.1.5. EG.5.1. JN.l, JD.1.1, HV. l, and BA.5; and an N protein from wild-type SARS-CoV-2. and

2. The kit of claim 1, wherein the detection reagent comprises an electrochemiluminescent (ECL) label.

3. The kit of claim 1 or 2, wherein the surface comprises an electrode.

4. The kit of any one of claims 1 to 3, wherein the surface comprises a well of a multi-well plate, and wherein each well comprises 1 to 10 binding domains.

5. The kit of any one of claims 1 to 4. wherein the panel of antigen comprises (1).

6. The kit of any one of claims 1 to 4, wherein die panel of antigen comprises (2).

7. The kit of any one of claims 1 to 4, wherein the panel of antigen comprises (3).

8. A method of detecting one or more antibody biomarkers of interest in a sample, comprising:

(1) an S protein from the following SARS-CoV-2 strains: wild-type, BA. l, XBB.1.16.1, XBB.1.5, BQ.1.1, XBB.1.1 , XBB.2.3, XBB. L and BA.5; and an N protein from wild-type SARS-CoV-2; or

(2) an S protein from the following SARS-CoV-2 strains: wild-type FL.1.5.1, BA.2.86, XBB.1.5. EG.5.1, XBB.1.16, XBB.2.3, XBB.1.16.6, and BA.5; and an N protein from wild-type SARS- CoV-2; or

(3) an S protein from the following SARS-CoV-2 strains: wild-type, FL.1.5.1, BA.2.86, XBB.1.5, EG.5.1, JN.l, JD.1.1, HV. l, and BA.5; and an N protein from wild-type SARS-CoV-2, (b) forming a binding complex in each binding domain, wherein the binding complex comprises the immobilized antigen and an antibody biomarker that binds to the immobilized antigen;

(c) contacting the binding complex in each binding domain with a detection reagent; and

9. The method of claim 8. wherein the detection reagent comprises a detection antibody, a detection antigen, or an ACE detection reagent.

10. The method of claim 8 or 9, wherein tire detection reagent comprises an ECL label.

11. The method of any one of claims 8 to 10. wherein tire surface comprises an electrode.

12. The method of any one of claims 8 to 10, wherein die surface comprises a well of a multi-well plate, and wherein each well comprises 1 to 10 binding domains.

13. The method of any one of claims 8 to 12, wherein the detection reagent comprises an ECL label, the surface comprises an electrode, and the detecting comprises applying a voltage to the surface and measuring an ECL signal generated from the ECL label on the detection reagent.

14. The method of any one of claims 8 to 13. wherein the panel of antigen comprises (1).

15. The method of any one of claims 8 to 13. wherein the panel of antigen comprises (2).

16. The method of any one of claims 8 to 13, wherein the panel of antigen comprises (3).