CN117178190A - Assay for the detection of SARS-COV-2 - Google Patents

Abstract

The present disclosure relates to detecting a protein from a SARS-CoV-2 virus or a fragment thereof in a sample obtained from a subject using a first antibody or antigen-binding fragment thereof that binds to a protein from a SARS-CoV-2 virus or a fragment thereof and a second antibody or antigen-binding fragment thereof that binds to a protein from a SARS-CoV-2 virus or a fragment thereof.

Description

Assay for the detection of SARS-COV-2

Cross Reference to Related Applications

The present application requires U.S. provisional application No. 63/060,975, filed 8/4 in 2020; 63/065,898 submitted on day 8, 14 of 2020; 63/067,051 submitted on 8/18/2020; and 63/126,336 filed on 12/16/2020, the contents of each of which are hereby incorporated by reference in their entirety.

Statement of sequence Listing

The text of the computer readable sequence listing entitled "38691-601_sequence_list_st25," filed herewith, was created at month 8, 4 of 2021, with a file size of 13,107 bytes, which is hereby incorporated by reference in its entirety.

Technical Field

The present disclosure relates to components and methods for detecting SARS-CoV-2 antigen.

Background

At the end of 2019, a new type of coronavirus (SARS-CoV-2 (2019-nCoV)) appears as a human pathogen, causing fever, severe respiratory disease and pneumonia. A disease associated with SARS-CoV-2 (2019-nCoV) is designated as COVID-19. The novel coronavirus is a member of the genus beta coronavirus, closely related to several bat coronaviruses and severe acute respiratory syndrome coronaviruses (SARS-CoV). However, unlike SARS-CoV, SARS-CoV-2 (2019-nCoV) spreads rapidly from person to person.

By the end of month 7 of 2021, over 1.9 million cases of COVID-19 were diagnosed in over 200 countries, and the complications of COVID-19 were considered to be the cause of death in over 400 thousands of people. Because of the health risks associated with SARS-CoV-2 transmission, there is a need for methods and kits for assessing coronavirus transmission in humans, including methods for determining the presence and/or detecting the amount of SARS-CoV-2 protein in one or more samples obtained from a subject.

Disclosure of Invention

The present disclosure provides methods, devices, and kits for detecting the presence or determining the amount of SARS-CoV-2 in a sample from a subject.

In some embodiments, the method comprises: contacting a sample obtained from the subject with a first antibody or antigen-binding fragment thereof that specifically binds to a protein or fragment thereof from the SARS-CoV-2 virus under conditions that allow the protein or fragment thereof from the SARS-CoV-2 virus (if present in the sample) to bind to the first antibody or antigen-binding fragment thereof; contacting the sample with a conjugate comprising a second antibody that specifically binds to a protein from the SARS-CoV-2 virus or a fragment thereof and a detectable label; and assessing the presence of the signal from the detectable label, wherein the presence of the signal from the detectable label indicates the presence of a protein from the SARS-CoV-2 virus or fragment thereof in the sample.

In some embodiments, the protein from SARS-CoV-2 virus or fragment thereof is a nucleocapsid (N) protein. The first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof may recognize different epitopes of a protein from the SARS-CoV-2 virus (e.g., the SARS-CoV-2 virus nucleocapsid (N) protein).

The first antibody or antigen-binding fragment thereof may comprise: (i) A heavy chain variable region comprising a complementarity determining region 1 (CDR) amino acid sequence having at least 70% identity to SEQ ID No. 1, a CDR2 amino acid sequence having at least 70% identity to SEQ ID No. 2, and a CDR3 amino acid sequence having at least 70% identity to SEQ ID No. 3, and (ii) a light chain variable region comprising a CDR1 amino acid sequence having at least 70% identity to SEQ ID No. 4, a CDR2 amino acid sequence having at least 70% identity to SEQ ID No. 5, and a CDR3 amino acid sequence having at least 70% identity to SEQ ID No. 6. In some embodiments, the first antibody or antigen binding fragment thereof comprises a heavy chain variable region amino acid sequence having at least 70% identity to SEQ ID No. 7 and a light chain variable region amino acid sequence having at least 70% identity to SEQ ID No. 8. In some embodiments, the first antibody or antigen binding fragment thereof comprises a heavy chain variable region amino acid sequence having at least 70% identity to SEQ ID NO. 17 and a light chain variable region amino acid sequence having at least 70% identity to SEQ ID NO. 18.

The second antibody or antigen-binding fragment thereof may comprise: (i) A heavy chain variable region comprising a complementarity determining region 1 (CDR) amino acid sequence having at least 70% identity to SEQ ID No. 9, a CDR2 amino acid sequence having at least 70% identity to SEQ ID No. 10, and a CDR3 amino acid sequence having at least 70% identity to SEQ ID No. 11, and (ii) a light chain variable region comprising a CDR1 amino acid sequence having at least 70% identity to SEQ ID No. 12, a CDR2 amino acid sequence having at least 70% identity to SEQ ID No. 13, and a CDR3 amino acid sequence having at least 70% identity to SEQ ID No. 14. In some embodiments, the second antibody or antigen binding fragment thereof comprises a heavy chain variable region amino acid sequence having at least 70% identity to SEQ ID NO. 15 and a light chain variable region amino acid sequence having at least 70% identity to SEQ ID NO. 16.

In some embodiments, the method is performed using an immunoassay. The immunoassay may be an enzyme-linked immunosorbent assay (ELISA) or a lateral flow immunoassay (LFA).

The sample may be any sample from a subject comprising or suspected of comprising SARS-CoV 02. In some embodiments, the sample comprises a nasal or nasopharyngeal swab or brush, saliva, mucus, blood, serum, or plasma.

Also disclosed herein is a lateral flow device comprising: a first antibody or antigen-binding fragment thereof that specifically binds to a protein from the SARS-CoV-2 virus or fragment thereof; and a second antibody or antigen-binding fragment thereof that specifically binds to a protein from SARS-CoV-2 or a fragment thereof. In some embodiments, the first antibody or antigen-binding fragment thereof is immobilized. In some embodiments, the test line comprises a first antibody or antigen binding fragment thereof. In some embodiments, the second antibody or antigen binding fragment thereof comprises a detectable label. In some embodiments, the sample pad comprises a second antibody or antigen-binding fragment thereof.

In some embodiments, the protein from SARS-CoV-2 virus or fragment thereof is a nucleocapsid (N) protein. The first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof may recognize different epitopes of a protein from the SARS-CoV-2 virus (e.g., the SARS-CoV-2 virus nucleocapsid (N) protein).

The first antibody or antigen-binding fragment thereof may comprise: (i) A heavy chain variable region comprising a complementarity determining region 1 (CDR) amino acid sequence having at least 70% identity to SEQ ID No. 1, a CDR2 amino acid sequence having at least 70% identity to SEQ ID No. 2, and a CDR3 amino acid sequence having at least 70% identity to SEQ ID No. 3, and (ii) a light chain variable region comprising a CDR1 amino acid sequence having at least 70% identity to SEQ ID No. 4, a CDR2 amino acid sequence having at least 70% identity to SEQ ID No. 5, and a CDR3 amino acid sequence having at least 70% identity to SEQ ID No. 6. In some embodiments, the first antibody or antigen binding fragment thereof comprises a heavy chain variable region amino acid sequence having at least 70% identity to SEQ ID No. 7 and a light chain variable region amino acid sequence having at least 70% identity to SEQ ID No. 8. In some embodiments, the first antibody or antigen binding fragment thereof comprises a heavy chain variable region amino acid sequence having at least 70% identity to SEQ ID NO. 17 and a light chain variable region amino acid sequence having at least 70% identity to SEQ ID NO. 18.

The second antibody or antigen-binding fragment thereof may comprise: (i) A heavy chain variable region comprising a complementarity determining region 1 (CDR) amino acid sequence having at least 70% identity to SEQ ID No. 9, a CDR2 amino acid sequence having at least 70% identity to SEQ ID No. 10, and a CDR3 amino acid sequence having at least 70% identity to SEQ ID No. 11, and (ii) a light chain variable region comprising a CDR1 amino acid sequence having at least 70% identity to SEQ ID No. 12, a CDR2 amino acid sequence having at least 70% identity to SEQ ID No. 13, and a CDR3 amino acid sequence having at least 70% identity to SEQ ID No. 14. In some embodiments, the second antibody or antigen binding fragment thereof comprises a heavy chain variable region amino acid sequence having at least 70% identity to SEQ ID NO. 15 and a light chain variable region amino acid sequence having at least 70% identity to SEQ ID NO. 16.

Kits comprising the lateral flow devices described herein are also disclosed. The kit may also include at least one or both of an extraction buffer and a sampling device (e.g., a nasal swab).

Other aspects and embodiments of the disclosure will be apparent from the following detailed description and the accompanying drawings.

Drawings

FIG. 1 is a graph of dose response for an exemplary lateral flow assay to test for hook effect.

Detailed Description

The present disclosure is based, at least in part, on the development of methods using a pair of antibodies that allow for the detection of SARS-CoV-2 with increased specificity and lower detection limits.

As used herein, the terms "comprise", "include", "having", "has", "can", "contain" and variants thereof are intended to be open transitional phrases, terms or words that do not exclude the possibility of additional acts or structures. The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments "comprising," "consisting of," and "consisting essentially of," whether or not explicitly stated.

For recitation of ranges of values herein, each intervening number is explicitly contemplated to be of the same accuracy. For example, for the range of 6 to 9, the numbers 7 and 8 are considered in addition to 6 and 9, and for the range of 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0 are explicitly considered.

As used herein, the term "immunoglobulin" or "antibody" refers to a protein found in the blood or other body fluids of vertebrates that is used by the immune system to identify and neutralize foreign bodies, such as bacteria and viruses. Typically, an immunoglobulin or antibody is a protein comprising at least one Complementarity Determining Region (CDR). CDRs form the "hypervariable regions" of the antibody, which are responsible for antigen binding (discussed further below). Intact immunoglobulins generally consist of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each heavy chain contains an N-terminal variable (V _H ) The region and three C-termini are constant (C _H1 、C _H2 And C _H3 ) Regions, and each light chain contains an N-terminal variable (V _L ) The region and one C-terminal constant (C _L ) A zone. Based on the amino acid sequence of the constant domain, the light chain of an antibody can be designated as one of two different types (kappa) or lambda (lambda)). In a typical immunoglobulin, each light chain is linked to a heavy chain by disulfide bonds, and the two heavy chains are linked to each other by disulfide bonds. Light chain variable regionAligned with the variable region of the heavy chain, and the light chain constant region is aligned with the first constant region of the heavy chain. The remaining constant regions of the heavy chain are aligned with each other.

The variable regions of each pair of light and heavy chains form the antigen binding site of the antibody. V (V) _H And V _L The regions have the same general structure, and each region contains four framework (FW or FR) regions. As used herein, the term "framework region" refers to a relatively conserved amino acid sequence located between CDRs within a variable region. There are four framework regions in each variable domain, designated FR1, FR2, FR3 and FR4. The framework regions form beta sheets of the structural framework that provides the variable region (see, e.g., c.a. janeway et al (edit), immunobiology, 5 th edition, garland Publ ishing, new York, n.y. (2001)).

The framework regions are connected by three CDRs. As discussed above, the three CDRs, termed CDR1, CDR2 and CDR3, form the "hypervariable region" of the antibody, which is responsible for antigen binding. The CDRs form loops that connect and in some cases comprise a portion of the beta-sheet structure formed by the framework regions. Although the constant regions of the light and heavy chains are not directly involved in binding of the antibody to the antigen, the constant regions may affect the orientation of the variable regions. The constant region also exhibits various effector functions, such as participation in antibody-dependent complement-mediated lysis or antibody-dependent cytotoxicity via interactions with effector molecules and cells.

As used herein, when an antibody or other entity (e.g., an antigen binding domain) "specifically recognizes" or "specifically binds" an antigen or epitope, it preferentially recognizes the antigen in a complex mixture of proteins and/or macromolecules, and binds the antigen or epitope with a significantly higher affinity than other entities that do not display the antigen or epitope. In this regard, "significantly higher affinity" means that the affinity is sufficiently high to enable detection of an antigen or epitope that is distinguishable from an entity using the desired assay or measurement device. Typically, this means having at least 10 ⁷ M ^-1 (e.g.,>10 ⁷ M ^-1 、>10 ⁸ M ^-1 、>10 ⁹ M ^-1 、>10 ¹⁰ M ^-1 、>10 ¹¹ M ^-1 、>10 ¹² M ^-1 、>10 ¹³ M ^-1 etc.) binding constant (K _a ) Is used for the binding affinity of (a) to the substrate. In certain such embodiments, the antibody is capable of binding to a different antigen, so long as the different antigen comprises the particular epitope. In some cases, for example, homologous proteins from different species may comprise the same epitope.

As used herein, "antibody" refers to: a monoclonal antibody; monospecific antibodies (e.g., may be monoclonal or may be produced by means other than production of monospecific antibodies by normal germ cells); a multispecific antibody; a human antibody; humanized antibodies (fully or partially humanized); animal antibodies such as, but not limited to, birds (e.g., ducks or geese), sharks, whales, and mammals including non-primates (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, etc.) or non-human primates (e.g., monkeys, chimpanzees, etc.); recombinant antibodies, chimeric antibodies, single chain variable fragments ("scFv"), single chain antibodies, single domain antibodies, fab fragments, F (ab') ₂ Fragments, disulfide-linked Fvs ("sdFv") and anti-idiotype ("anti-Id") antibodies, dual domain antibodies, dual Variable Domain (DVD) or Triple Variable Domain (TVD) antibodies (dual variable domain immunoglobulins and methods for making them are described in Wu, c.et al Nature Biotechnology,25 (11): 1290-1297 (2007) and PCT international application WO 2001/058956, each of which is incorporated herein by reference) or domain antibodies (dabs) (e.g., as described in Holt et al (2014) Trends in Biotechnology 21:484-490), and include (e.g., as described in cartilaginous fish and camelids) naturally occurring single domain antibodies sdabs or synthetic single domain antibodies sdabs (e.g., nanobody h), or other domain structures) and functionally active epitope-binding fragments of any of the foregoing. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an analyte binding site. Immunoglobulin molecules may be of any type (e.g., igG, igE, igM, igD, igA and IgY), class (e.g., igG1, ig)G2, igG3, igG4, igA1, and IgA 2) or subclasses. For simplicity, an anti-analyte antibody is often referred to herein as an "anti-analyte antibody" or simply an "analyte antibody".

The terms "fragment of an antibody", "antibody fragment" and "antigen-binding fragment" are used interchangeably herein to refer to one or more fragments of an antibody that retain the ability to specifically bind an antigen (see generally Holliger et al, nat. Biotech.,23 (9): 1126-1129 (2005)). Antigen binding fragments of any of the antibodies described herein are within the scope of the invention. An antibody fragment desirably comprises, for example, one or more CDRs, a variable region (or portion thereof), a constant region (or portion thereof), or a combination thereof. In some embodiments, the portion does not include the constant heavy chain domain of the Fc region of the intact antibody (i.e., CH2, CH3, or CH4, depending on the antibody isotype). Examples of antibody fragments include, but are not limited to: (i) Fab fragment, which is composed of V _L 、V _H 、C _L And C _H1 A monovalent fragment of composition, (ii) F (ab') ₂ A fragment which is a bivalent fragment comprising two Fab fragments linked at the hinge region by a disulfide bridge, (iii) an Fv fragment consisting of V of the antibody single arm _L And V _H Domain composition, (iv) Fab 'fragments, which are disrupted by using mild reducing conditions F (ab') ₂ The disulfide bridge of the fragment results, (V) disulfide-stabilized Fv fragment (dsFv), (vi) domain antibody (dAb), which is an antibody single variable region domain (V) that specifically binds antigen _H Or V _L ) A polypeptide, (vii) a Fab' -SH fragment, (viii) an Fd fragment, (ix) a diabody, (x) a single chain Fv (scFv) molecule, (xi) a single chain polypeptide comprising only one light chain variable domain, (xii) a single chain polypeptide comprising three CDRs of a light chain variable domain, (xii) a single chain polypeptide comprising only one heavy chain variable region, and (xiii) a single chain polypeptide comprising three CDRs of a heavy chain variable region.

Antibody fragments that recognize specific epitopes can be generated by known techniques. For example, fab and F (ab') ₂ Fragments can be produced by using enzymes such as papain (to produce two identical Fab fragments) or pepsin (to produce F (ab') ₂ Fragment)) proteolytic cleavage of immunoglobulin moleculesTo produce. F (ab') of IgG molecules ₂ The fragment retains two antigen binding sites of a larger ("parent") IgG molecule, including two light chains (containing variable light chain and constant light chain regions), the CH1 domain of the heavy chain, and the disulfide-bond forming hinge region of the parent IgG molecule. Thus, F (ab') ₂ The fragment is still able to cross-link the antigen molecule as the parent IgG molecule.

As used herein, a "fragment antigen binding fragment" or "Fab fragment" refers to an antibody fragment that binds an antigen and contains one antigen binding site, one complete light chain, and a portion of one heavy chain. Fab is composed of V _L 、V _H 、C _L And C _H1 A monovalent fragment consisting of a domain. Fab is made up of one constant domain and one variable domain of each of the heavy and light chains. The variable domain contains a paratope (antigen binding site) at the amino terminus of the monomer, which contains a set of complementarity determining regions. Each arm of Y thus binds an epitope on the antigen. Fab fragments may be generated as described in the art (e.g., using papain, which may be used to cleave immunoglobulin monomers into two Fab fragments and one Fc fragment), or may be produced recombinantly.

"F (ab') ₂ Fragment "refers to an antibody produced by pepsin digestion of an entire IgG antibody to remove most of the Fc region while leaving some of the hinge region intact. F (ab') ₂ Fragments have two antigen-binding F (ab) moieties linked together by disulfide bonds and are therefore bivalent, with a molecular weight of about 110kDa. Bivalent antibody fragment (F (ab') ₂ Fragments) are smaller than the intact IgG molecules and are better able to penetrate into the tissue, thereby promoting better antigen recognition in immunohistochemistry. Use of F (ab') ₂ Fragments also avoid non-specific binding to Fc receptors on living cells or to protein A/G. F (ab') ₂ Fragments can bind and precipitate antigens.

As used herein, "framework" (FR) or "framework sequence" may mean the variable region minus the remaining sequence of CDRs. Because the exact definition of CDR sequences can be determined by different systems, the meaning of framework sequences correspondingly tends to be interpreted differently. The six CDRs (CDR-L1, -L2 and-L3 for the light chain and CDR-H1, -H2 and-H3 for the heavy chain) also divide the framework regions on the light and heavy chains into four subregions (FR 1, FR2, FR3 and FR 4) on each chain, with CDR1 located between FR1 and FR2, CDR2 located between FR2 and FR3, and CDR3 located between FR3 and FR 4. Without designating a particular subregion as FR1, FR2, FR3 or FR4, the framework region (as referred to by others) represents the combined FR within the variable region of a single naturally occurring immunoglobulin chain. As used herein, one FR means one of four subregions, and a plurality of FR means two or more of the four subregions constituting the framework region.

Human heavy and light chain FR sequences are known in the art and can be used as heavy and light chain "acceptor" framework sequences (or simply "acceptor" sequences) to humanize non-human antibodies using techniques known in the art. In one embodiment, the human heavy and light chain acceptor sequences are selected from publicly available databases such as V-base (HyperText transfer protocol: vbase (dot) mrc-cpe (dot) cam (dot) ac (dot) uk)) or International The framework sequences listed in the information systems (HyperText transfer protocol: imgt cis fr/text/IMGTrepertoire/Locus genes /).

"recombinant antibody" refers to an antibody prepared by one or more steps comprising cloning all or part of a nucleic acid sequence encoding one or more monoclonal antibodies into an appropriate expression vector by recombinant techniques, and subsequently expressing the antibody in an appropriate host cell. The term includes, but is not limited to, recombinantly produced monoclonal antibodies, chimeric antibodies, humanized antibodies (fully or partially humanized), multispecific or multivalent structures formed from antibody fragments, bifunctional antibodies, heteroconjugate antibodies,As described in (i) hereinIs a variant of the antibody. (double variable domain immunoglobulins and methods for preparing them are described in Wu, c. Et al, nature Biotechnology,25:1290-1297 (2007).

The terms "nucleic acid", "polynucleotide", "nucleotide sequence" and "oligonucleotide" are used interchangeably herein and refer to polymers or oligomers of pyrimidine and/or purine bases, preferably cytosine, thymine and uracil, respectively, and adenine and guanine (see Albert l. Lehninger, principles of Biochemistry, at 793-800 (Worth pub.1982)). The term encompasses any deoxyribonucleotide, ribonucleotide or peptide nucleic acid component and any chemical variant thereof, such as methylated, methylolated or glycosylated forms of these bases. The polymers or oligomers may be heterogeneous or homogeneous in composition, may be isolated from naturally occurring sources, or may be artificially or synthetically produced. Furthermore, the nucleic acid may be DNA or RNA or a mixture thereof, and may exist permanently or temporarily in single-stranded or double-stranded form (including homoduplex, heteroduplex, and hybridized states). In some embodiments, the nucleic acid or nucleic acid sequence comprises other types of nucleic acid structures such as, for example, DNA/RNA helices, peptide Nucleic Acids (PNAs), morpholino nucleic acids (see, e.g., braasch and Corey, biochemistry,41 (14): 4503-4510 (2002) and U.S. Pat. No. 5,034,506), locked nucleic acids (LNA; see wahlestdt et al, proc.Natl. Acad.Sci.U.S. A.,97:5633-5638 (2000)), cyclohexenyl nucleic acids (see Wang, j.am.chem.soc.,122:8595-8602 (2000)), and/or ribozymes. The terms "nucleic acid" and "nucleic acid sequence" may also encompass a strand comprising non-natural nucleotides, modified nucleotides and/or non-nucleotide building blocks (e.g., "nucleotide analogs") that may exhibit the same function as a natural nucleotide.

The terms "peptide," "polypeptide," and "protein" are used interchangeably herein and refer to polymeric forms of amino acids of any length, which may include encoded and non-encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

The terms "immunogen" and "antigen" are used interchangeably herein and refer to any molecule, compound, or substance that induces an immune response in an animal (e.g., a mammal). An "immune response" may require, for example, antibody production and/or activation of immune effector cells. An antigen in the context of the present disclosure may include any subunit, fragment or epitope of any protein or non-protein (e.g., carbohydrate or lipid) molecule that elicits an immune response in a mammal. An "epitope" refers to an antigenic sequence recognized by an antibody or antigen receptor. Epitopes are also known in the art as "antigenic determinants". In certain embodiments, an epitope is an antigenic region that is specifically bound by an antibody. In certain embodiments, an epitope may include a chemically active surface group of a molecule, such as an amino acid, a sugar side chain, a phosphoryl group, or a sulfonyl group. In certain embodiments, an epitope may have a particular three-dimensional structural feature (e.g., a "conformational" epitope) and/or a particular charge feature. The antigen may be a protein or peptide of viral, bacterial, parasitic, fungal, protozoan, prion, cellular or extracellular origin, which elicits an immune response in a mammal, preferably resulting in protective immunity.

As used herein, the terms "detectable label" and "label" refer to a moiety that can produce a signal that is detectable visually or instrumentally. In some embodiments, the label is a direct label, e.g., an entity that is readily visible to the naked eye in its natural state or by means of a filter and/or an applied stimulus (e.g., ultraviolet light that promotes fluorescence). For example, tiny colored particles such as dye sols, metal sols (e.g., gold) and colored latex particles are very suitable. In some embodiments, the label is an indirect label, such as an enzyme, e.g., alkaline phosphatase and horseradish peroxidase. Indirect labeling typically requires the addition of one or more developing reagents (such as substrates) to detect the visible signal.

As used herein, the terms "present" or "absent" (or, alternatively, "present" or "absent") are used in a relative sense to describe the amount or level of a particular entity (e.g., analyte). For example, when an analyte is said to be "present" in a test sample, this means that the level or amount of this analyte is above a predetermined threshold; conversely, when an analyte is said to be "absent" in a test sample, this means that the level or amount of this analyte is below a predetermined threshold. The predetermined threshold may be a threshold for detectability associated with a particular test for detecting an analyte or any other threshold. An analyte is "present" in a sample when it is "detected" in the sample; when an analyte is "undetected," it is "absent" in the sample. In addition, a sample in which the analyte is "detected" or in which the analyte is "present" is a sample that is "positive" for the analyte. Samples in which the analyte is "undetected" or in which the analyte is "absent" are samples that are "negative" for the analyte.

As used herein, the term "analyte" refers to a compound or composition that is detected and/or measured by specific binding of a ligand, receptor, or enzyme (e.g., an antibody or antigen). In some embodiments, the analyte is a protein or a nucleic acid. In some embodiments, the analyte is an antigen. In some embodiments, the analyte is a fragment of an antigen. In some embodiments, the analyte is an analyte analog or analyte derivative (e.g., an analyte that is altered by chemical or biological means). In some embodiments, the analyte is an epitope. In some embodiments, the term "analyte" refers to a protein and/or nucleic acid from the SARS-CoV-2 virus. In some embodiments, the analyte is a fragment and/or epitope of a protein and/or nucleic acid from SARS-CoV-2 virus. In some embodiments, the analyte is SARS-CoV-2 spike protein (the "S" protein provided by UniProtKB accession number P0DTC 2) or a spike protein receptor binding domain (see, e.g., wrapp (2020) "Cryo-EM Structure of the 2019-nCoV spike in the prefusion conformation" Science 367:1260-63; walls (2020) "Structure, function, and Antigenicity of the SARSCoV-2Spike Glycoprotein"Cell 180:1-12, each of which is incorporated herein by reference). In some embodiments, the analyte is a viral transcription and/or replication protein (e.g., a replicase polyprotein 1a (R1 a) as provided by UniProtKB accession number P0DTC1 or a replicase polyprotein 1ab (R1 ab) as provided by UniProtKB accession number P0DTD 1. In some embodiments, the analyte is a viral budding protein (e.g., protein 3a as provided by UniProtKB accession number P0DTC3 or an envelope small membrane protein (E) as provided by P0DTC 4)). In some embodiments, the analyte is a viral morphogenic protein (e.g., a membrane protein (M) as provided by UniProtKB accession number P0DTC 5.) in some embodiments, the analyte is a nonstructural protein 6 (e.g., as provided by UniProtKB accession number P0DTC 6), a protein 7A (NS 7A) (e.g., as provided by UniProtKB accession number P0DTC 7), a protein 7B (NS 7B) (e.g., as provided by UniProtKB accession number P0DTC 8), a protein (e.g., protein 3, or a protein (e.g., protein 3 d) as provided by UniProtKB accession number P0DTC 8) in some embodiments, the analyte is a non-structural protein (e.g., protein 3, such as provided by UniProtKB accession number P0DTC 8) or a protein (e.g., protein B) as provided by UniProtKB accession number P0DTC 9).

As used herein, "system" refers to a plurality of real and/or abstract components that operate together for common purposes. In some embodiments, a "system" is an integrated assembly of hardware and/or software components. In some embodiments, each component of the system interacts with and/or is associated with one or more other components. In some embodiments, the system refers to a combination of components and software for controlling and directing the method.

As used herein, the term "sample" refers to any sample comprising SARS-CoV-2 or a portion or component thereof, or possibly comprising SARS-CoV-2 or a portion or component thereof. Thus, the term "sample" refers to a material that is to be tested for the presence or amount of an analyte (e.g., SARS-CoV-2) or a portion or component thereof. Preferably, the sample is a fluid sample, preferably a liquid sample. For example, the sample may be a bodily fluid such as blood (including, for example, capillary blood, venous blood, dried blood spots, etc.), serum, plasma, ocular fluid, urine, mucus, semen, nasal or nasopharyngeal swab, pharyngeal swab, tears, sweat, or saliva. Viscous liquid, semi-solid, or solid specimens can be used to produce liquid solutions, eluents, suspensions, or extracts that can be samples. For example, a pharyngeal swab or a genital swab may be suspended in a liquid solution to prepare the sample.

"point-of-care device" refers to a device for providing medical diagnostic testing at or near a point-of-care (i.e., outside of a laboratory), at the time and place of patient care, such as at a hospital, doctor's office, emergency or other medical care facility, patient's home, nursing home, and/or long-term care and/or terminal care facility. Examples of point of care devices include those produced by Abbott Laboratories (Abbott Park, IL) (e.g., i-STAT and i-STAT affinity, universal Biosensors (Rowville, australia) (see US 2006/0134713), axis-Shield PoC AS (Oslo, norway) and Clinical Lab Products (Los Angeles, USA).

As used herein, "sensitivity" of an assay refers to the proportion of subjects that are positive in result that are correctly identified as positive (e.g., those subjects with a disease or medical condition they are undergoing testing). For example, this may include correctly identifying a subject infected with a coronavirus (such as a beta coronavirus) from a subject that has not been or has not been infected with a coronavirus (such as a beta coronavirus). In some aspects, the sensitivity of an assay may be determined by evaluating the change in signal to noise (S/N) ratio of the assay. For example, in some aspects, an increase in the S/N ratio may indicate increased assay sensitivity for a particular analyte (e.g., SARS-CoV-2 nucleocapsid protein).

As used herein, "specificity" of an assay refers to the proportion of subjects that are negative in result that are correctly identified as negative (e.g., those subjects that do not have the disease or medical condition they are being tested for). For example, this may include correctly identifying a subject infected with a coronavirus (such as a beta coronavirus) from a subject that has not been infected with a coronavirus (such as a beta coronavirus).

Unless defined otherwise herein, scientific and technical terms used in connection with the present disclosure shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of the terms should be clear; if any potential ambiguity exists, the definitions provided herein take precedence over any dictionary or external definitions. Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular.

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

1. Antibodies to

According to the disclosed methods, a sample is contacted with a first antibody or antigen-binding fragment thereof (e.g., fab) that specifically binds to a protein or fragment thereof from the SARS-CoV-2 virus and a second antibody or antigen-binding fragment thereof (e.g., fab) that specifically binds to a protein or fragment thereof from the SARS-CoV-2 virus. The first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof recognize different epitopes of a protein from SARS-CoV-2 virus or fragment thereof. In some embodiments, the protein from the SARS-CoV-2 virus is a viral genome packaging protein (e.g., a nucleocapsid (N) protein, e.g., as provided by UniProtKB accession number P0DTC 9).

The first antibody or antigen-binding fragment thereof comprises: (i) A heavy chain variable region comprising a CDR1 amino acid sequence having at least 70% (e.g., 75%, 80%, 85%, 90%, 95%, 98%) identity to SEQ ID No. 1, a CDR2 amino acid sequence having at least 70% identity to SEQ ID No. 2, and a CDR3 amino acid sequence having at least 70% identity to SEQ ID No. 3, and (ii) a light chain variable region comprising a CDR1 amino acid sequence having at least 70% identity to SEQ ID No. 4, a CDR2 amino acid sequence having at least 70% identity to SEQ ID No. 5, and a CDR3 amino acid sequence having at least 70% identity to SEQ ID No. 6.

Alternatively, the first antibody or antigen binding fragment thereof may comprise heavy chain variable region CDR1, CDR2 and CDR3 amino acid sequences having at least 90% identity to SEQ ID No. 1, SEQ ID No. 2 and/or SEQ ID No. 3, respectively, and/or light chain variable region CDR1, CDR2 and CDR3 amino acid sequences having at least 90% identity to SEQ ID No. 4, SEQ ID No. 5 and/or SEQ ID No. 6, respectively.

In one embodiment, (i) each of the heavy chain variable region CDR1, CDR2 and/or CDR3 amino acid sequences comprises, consists essentially of or consists of SEQ ID No. 1, SEQ ID No. 2 and/or SEQ ID No. 3, respectively, and (ii) each of the light chain variable region CDR1, CDR2 and/or CDR3 amino acid sequences comprises, consists essentially of or consists of SEQ ID No. 4, SEQ ID No. 5 and/or SEQ ID No. 6, respectively. When the heavy and/or light chain CDRs 1, CDR2, and CDR3 of the disclosed antibodies consist essentially of the amino acid sequences described above, additional components (e.g., protein portions that facilitate purification or isolation, such as biotin) that do not substantially affect the antibody or antigen binding fragment thereof can be included in the CDRs. When the heavy and/or light chain CDRs 1, CDR2, and CDR3 of the disclosed antibodies consist of the amino acid sequences described above, each CDR does not comprise any additional components (e.g., components that are not endogenous to the CDR).

In some embodiments, the first antibody or antigen binding fragment thereof comprises: heavy chain variable region (V) _H ) An amino acid sequence comprising, consisting essentially of, or consisting of SEQ ID No. 7; and a light chain variable region (V _L ) An amino acid sequence comprising, consisting essentially of, or consisting of SEQ ID No. 8. When V is _H The amino acid sequence consists essentially of SEQ ID NO. 7 and V _L When the amino acid sequence consists essentially of SEQ ID NO. 8, additional components (e.g., protein portions that facilitate purification or isolation, such as biotin or His tags) that do not substantially affect the antibody or antigen binding fragment thereof may be included in the heavy or light chain variable region. When V is _H The amino acid sequence consists of SEQ ID NO. 7 and V _L The amino acid sequence is represented by SEQ ID NO. 8 groupIn turn, the heavy and light chain variable regions do not comprise any additional components (e.g., components that are not endogenous to the heavy or light chain variable regions).

In other embodiments, the first antibody or antigen binding fragment thereof may comprise a heavy chain variable region amino acid sequence having at least 70% identity to SEQ ID No. 7 and a light chain variable region amino acid sequence having at least 70% identity to SEQ ID No. 8.

In some embodiments, the first antibody or antigen binding fragment thereof comprises: heavy chain variable region (V) _H ) An amino acid sequence comprising, consisting essentially of, or consisting of SEQ ID No. 17; and a light chain variable region (V _L ) An amino acid sequence comprising, consisting essentially of, or consisting of SEQ ID No. 18. When V is _H The amino acid sequence consists essentially of SEQ ID NO. 17 and V _L When the amino acid sequence consists essentially of SEQ ID NO. 18, additional components (e.g., protein portions that facilitate purification or isolation, such as biotin or His tags) that do not substantially affect the antibody or antigen binding fragment thereof may be included in the heavy or light chain variable region. When V is _H The amino acid sequence consists of SEQ ID NO. 17 and V _L When the amino acid sequence consists of SEQ ID NO. 18, the heavy and light chain variable regions do not comprise any additional components (e.g., components that are not endogenous to the heavy or light chain variable regions).

In other embodiments, the first antibody or antigen binding fragment thereof may comprise a heavy chain variable region amino acid sequence having at least 70% identity to SEQ ID NO. 17 and a light chain variable region amino acid sequence having at least 70% identity to SEQ ID NO. 18.

The second antibody or antigen-binding fragment thereof comprises: (i) A heavy chain variable region comprising a CDR1 amino acid sequence having at least 70% (e.g., 75%, 80%, 85%, 90%, 95%, 98%) identity to SEQ ID No. 9, a CDR2 amino acid sequence having at least 70% identity to SEQ ID No. 10, and a CDR3 amino acid sequence having at least 70% identity to SEQ ID No. 11, and (ii) a light chain variable region comprising a CDR1 amino acid sequence having at least 70% identity to SEQ ID No. 12, a CDR2 amino acid sequence having at least 70% identity to SEQ ID No. 13, and a CDR3 amino acid sequence having at least 70% identity to SEQ ID No. 14.

Alternatively, the second antibody or antigen binding fragment thereof may comprise heavy chain variable region CDR1, CDR2 and CDR3 amino acid sequences having at least 90% identity to SEQ ID NO. 9, SEQ ID NO. 10 and/or SEQ ID NO. 11, respectively, and/or light chain variable region CDR1, CDR2 and CDR3 amino acid sequences having at least 90% identity to SEQ ID NO. 12, SEQ ID NO. 13 and/or SEQ ID NO. 14, respectively.

In one embodiment, (i) each of the heavy chain variable region CDR1, CDR2 and/or CDR3 amino acid sequences comprises, consists essentially of or consists of SEQ ID NO 9, SEQ ID NO 10 and/or SEQ ID NO 11, respectively, and (ii) each of the light chain variable region CDR1, CDR2 and/or CDR3 amino acid sequences comprises, consists essentially of or consists of SEQ ID NO 12, SEQ ID NO 13 and/or SEQ ID NO 14, respectively. When the heavy and/or light chain CDRs 1, CDR2, and CDR3 of the disclosed antibodies consist essentially of the amino acid sequences described above, additional components (e.g., protein portions that facilitate purification or isolation, such as biotin) that do not substantially affect the antibody or antigen binding fragment thereof can be included in the CDRs. When the heavy and/or light chain CDRs 1, CDR2, and CDR3 of the disclosed antibodies consist of the amino acid sequences described above, each CDR does not comprise any additional components (e.g., components that are not endogenous to the CDR).

In some embodiments, the second antibody or antigen binding fragment thereof comprises: heavy chain variable region (V) _H ) An amino acid sequence comprising SEQ ID NO:15. consists essentially of, or consists of; and a light chain variable region (V _L ) An amino acid sequence comprising, consisting essentially of, or consisting of SEQ ID No. 16. When V is _H The amino acid sequence essentially consists of SEQ ID NO. 15 and V _L When the amino acid sequence consists essentially of SEQ ID NO. 16, additional components (e.g., protein portions that facilitate purification or isolation, such as biotin or His tags) that do not substantially affect the antibody or antigen binding fragment thereof may be included in the heavy or light chain variable region. When V is _H The amino acid sequence consists of SEQ ID NO. 15 and V _L When the amino acid sequence consists of SEQ ID NO. 16, the heavy and light chain variable regions do not comprise any additional components (e.g., components that are not endogenous to the heavy or light chain variable regions).

In other embodiments, the second antibody or antigen binding fragment thereof may comprise a heavy chain variable region amino acid sequence having at least 70% identity to SEQ ID NO. 15 and a light chain variable region amino acid sequence having at least 70% identity to SEQ ID NO. 16.

As described herein, nucleic acid or amino acid sequence "identity" may be determined by comparing a nucleic acid or amino acid sequence of interest to a reference nucleic acid or amino acid sequence. The percent identity is the number of identical (e.g., identical) nucleotide or amino acid residues between the sequence of interest and the reference sequence divided by the length of the longest sequence (e.g., the length of the sequence of interest or the reference sequence, whichever is longer). Many mathematical algorithms for obtaining optimal alignments and calculating identities between two or more sequences are known and incorporated into many available software programs. Examples of such programs include CLUSTAL-W, T-Coffee and ALIGN (for alignment of nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST 2.1, BL2SEQ and updated versions thereof), and FASTA programs (e.g., FASTA3x, FAS) ^TM And SSEARCH) (for sequence alignment and sequence similarity search). Sequence alignment algorithms are also disclosed in, for example, altschul et al, J.molecular biol.,215 (3): 403-410 (1990), et al, proc. Natl. Acad. Sci. USA,106 (10): 3770-3775 (2009), durbin et al, editions, biological Sequence Analysis: probabilistic Models of Proteins and Nucleic Acids, cambridge University Press, cambridge, UK (2009), soning, bioinformatics,21 (7): 951-960 (2005), altschul et al, nucleic Acids Res.,25 (17): 3389-3402 (1997) and Gusfield, algorithms on Strings, trees and Sequences, cambridge Univers ity Press, cambridge UK (1997)).

One or more amino acids of the above-mentioned antibodies or antigen fragments thereof may be replaced or substituted with different amino acids. An amino acid "substitution" or "substitution" refers to the replacement of one amino acid at a given position or residue with another amino acid at the same position or residue within the polypeptide sequence.

Furthermore, one or more amino acids may be inserted into the antibody or antigen binding fragment thereof (e.g., into the heavy and/or light chain variable region amino acid sequences). Any number of any suitable amino acids may be inserted into the amino acid sequence of an antibody or antigen binding fragment thereof. In this regard, at least one amino acid (e.g., 2 or more, 5 or more, or 10 or more amino acids), but no more than 20 amino acids (e.g., 18 or less, 15 or less, or 12 or less amino acids) may be inserted into the amino acid sequence of an antibody or antigen binding fragment thereof. For example, 1-10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) can be inserted into the amino acid sequence of an antibody or antigen binding fragment thereof. In this regard, amino acids may be inserted into an antibody or antigen-binding fragment thereof at any suitable position. Preferably, the amino acid is inserted into a CDR (e.g., CDR1, CDR2, or CDR 3) of an antibody or antigen binding fragment thereof.

The antibodies or antigen-binding fragments thereof employed in the methods of the invention are not limited to polypeptides comprising the specific amino acid sequences described herein. In fact, the antibody or antigen-binding fragment thereof may comprise any heavy or light chain polypeptide that competes with the antibody or antigen-binding fragment thereof of the invention for binding to tenofovir (tenofovir) or a tenofovir derivative. Antibody competition can be determined using conventional peptide competition assays, such as, for example, ELISA, western blot, or immunohistochemical methods (see, e.g., U.S. Pat. nos. 4,828,981 and 8,568,992; and Braitbard et al, proteome sci, 4:12 (2006)).

Antibodies may be produced by any of a number of techniques known in the art. For example, by a host cell into which expression vectors encoding the heavy and light chains are transfected by standard techniques. The various forms of the term "transfection" are intended to encompass the variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Although antibodies can be expressed in prokaryotic or eukaryotic host cells, it is preferred to express antibodies in eukaryotic cells, and most preferred to express antibodies in mammalian host cells, since such eukaryotic cells (particularly mammalian cells) are more likely than prokaryotic cells to assemble and secrete correctly folded and immunocompetent antibodies.

Exemplary mammalian host cells for expression of recombinant antibodies include chinese hamster ovary (CHO cells) (including DHFR-CHO cells, which are described in Urlaub and Chas in, proc.Natl. Acad.Sci.USA,77:4216-4220 (1980), for use with DHFR selectable markers, e.g., as described in Kaufman and Sharp, J.mol.biol.,159:601-621 (1982)), NS0 myeloma cells, COS cells, and SP2 cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow expression of the antibody in the host cell, or more preferably, secretion of the antibody into the medium in which the host cell is grown. Antibodies can be recovered from the culture medium using standard protein purification methods. In some aspects, antibodies can be purified in CHO and/or HEK cells using conventional techniques known in the art.

Host cells can also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It will be appreciated that variations of the above procedure may be performed. For example, it may be desirable to transfect a host cell with DNA encoding a functional fragment of an antibody light chain and/or heavy chain. Recombinant DNA techniques may also be used to remove some or all of the DNA encoding one or both of the light and heavy chains that are not necessary for binding to the antigen of interest. Molecules expressed from such truncated DNA molecules are also encompassed within antibodies.

In a preferred system for recombinant expression of an antibody or antigen-binding portion thereof, a recombinant expression vector encoding both an antibody heavy chain and an antibody light chain is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are each operably linked to CMV enhancer/AdMLP promoter regulatory elements to drive high level transcription of the genes. The recombinant expression vector also carries a DHFR gene that allows selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification. The selected transformant host cells are cultured to allow expression of the antibody heavy and light chains, and the whole antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare recombinant expression vectors, transfect host cells, select transformants, culture the host cells, and recover antibodies from the culture medium. Still further, the present disclosure provides a method of synthesizing a recombinant antibody by culturing a host cell in a suitable medium until the recombinant antibody is synthesized. The method may further comprise isolating the recombinant antibody from the culture medium.

The humanized antibody may be an antibody or variant, derivative, analog or fragment or portion thereof that specifically binds to an antigen of interest and that comprises a Framework (FR) region having substantially the amino acid sequence of a human antibody and a Complementarity Determining Region (CDR) having substantially the amino acid sequence of a non-human antibody. Humanized antibodies may be derived from non-human species antibodies that bind to a desired antigen, having one or more Complementarity Determining Regions (CDRs) from a non-human species and framework regions from a human immunoglobulin molecule.

As used herein, the term "substantially" in the context of CDRs refers to CDRs having amino acid sequences that are at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequences of CDRs of a non-human antibody. Humanized antibody packageComprising substantially all or at least one and usually two variable domains (Fab, fab ', F (ab') ₂ FabC, fv), wherein all or substantially all CDR regions correspond to those of a non-human immunoglobulin (e.g., a donor antibody), and all or substantially all framework regions are those of a human immunoglobulin consensus sequence. According to one aspect, the humanized antibody further comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. In some embodiments, the humanized antibody comprises at least a variable domain of a light chain and a heavy chain. Antibodies may also include CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, the humanized antibody contains only humanized light chains. In some embodiments, the humanized antibody contains only humanized heavy chains. In particular embodiments, the humanized antibody comprises only a humanized variable domain of a light chain and/or a humanized variable domain of a heavy chain.

The humanized antibody may be selected from any class of immunoglobulins, including IgM, igG, igD, igA and IgE, and any isotype, including but not limited to IgG1, igG2, igG3, and IgG4. Humanized antibodies may comprise sequences from more than one class or isotype and specific constant domains may be selected to optimize desired effector functions using techniques well known in the art.

The Framework (FR) and CDR regions of a humanized antibody need not correspond exactly to the parent sequence, e.g., the donor antibody CDR or consensus framework may be mutagenized by substitution, insertion, and/or deletion of at least one amino acid residue such that the CDR or framework residue at that site does not correspond to the donor antibody or consensus framework. However, in one embodiment, such mutations will not be extensive. Typically, at least 90%, at least 95%, at least 98%, or at least 99% of the humanized antibody residues will correspond to residues of the parent FR and CDR sequences. As used herein, the term "consensus framework" refers to a framework region in a consensus immunoglobulin sequence. As used herein, the term "consensus immunoglobulin sequence" refers to a sequence formed by the most frequently occurring amino acids (or nucleotides) in the family of related immunoglobulin sequences (see, e.g., winnaker, from Genes to Clones (Verlagsgesel lschaft, weinheim, germany 1987)). In the immunoglobulin family, each position in the consensus sequence is occupied by the most frequently occurring amino acid at that position in the family. If the frequency of occurrence of the two amino acids is the same, either one may be included in the consensus sequence.

Humanized antibodies can be designed to minimize unwanted immune responses to rodent anti-human antibodies that limit the duration and effectiveness of therapeutic applications of those moieties in human recipients. The humanized antibody may have one or more amino acid residues introduced into the humanized antibody from a non-human source. These non-human residues are often referred to as "input" residues, which are typically taken from the variable domain. Humanization may be performed by replacing the corresponding sequence of a human antibody with a hypervariable region sequence. Thus, such "humanized" antibodies are chimeric antibodies in which significantly less than the entire human variable domain has been replaced with a corresponding sequence from a non-human species. See, for example, U.S. Pat. No. 4,816,567, the contents of which are incorporated herein by reference. The humanized antibody may be a human antibody in which some hypervariable region residues and possibly some FR residues are replaced with residues from similar sites in a rodent antibody. Humanization or engineering of the antibodies of the present disclosure may be performed using any known method, such as, but not limited to, U.S. Pat. nos. 5,723,323;5,976,862;5,824,514;5,817,483;5,814,476;5,763,192;5,723,323;5,766,886;5,714,352;6,204,023;6,180,370;5,693,762;5,530,101;5,585,089;5,225,539; and those described in 4,816,567.

Humanized antibodies can retain high affinity for SARS-CoV-2 antigen and other advantageous biological properties. Humanized antibodies can be prepared by a process of analyzing a parent sequence and various conceptual humanized products using a three-dimensional model of the parent and humanized sequences. Three-dimensional immunoglobulin models are commonly available. Computer programs are available that illustrate and display the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Examination of these shows that it is possible to analyze the possible role of residues in the function of the candidate immunoglobulin sequence, e.g. to analyze residues affecting the ability of the candidate immunoglobulin to bind its antigen. In this way, framework residues can be selected and combined from the receptor and input sequences such that desired antibody characteristics, such as increased affinity for SARS-CoV-2, are achieved. Generally, hypervariable region residues may be directly and most substantially involved in influencing antigen binding.

As an alternative to humanization, human antibodies (also referred to herein as "fully human antibodies") may be generated. For example, human antibodies can be isolated from libraries via PROfus ion and/or yeast-related techniques. Transgenic animals (e.g., mice, which are capable of producing a complete human antibody repertoire in the absence of endogenous immunoglobulin production after immunization, e.g., chimeric and germ-line mutant mice, in which the heavy chain linker region of the antibody (J _H ) Homozygous deletion of the gene results in complete inhibition of endogenous antibody production. Transferring an array of human germline immunoglobulin genes in such germline mutant mice will result in the production of human antibodies following antigen challenge. Humanized or fully human antibodies can be according to U.S. patent No. 5,770,429;5,833,985;5,837,243;5,922,845;6,017,517;6,096,311;6,111,166;6,270,765;6,303,755;6,365,116;6,410,690;6,682,928; and 6,984,720, the contents of each of which are incorporated herein by reference.

2. Method of

The disclosed methods comprise contacting a sample obtained from a subject with a first antibody or antigen-binding fragment thereof comprising a detectable label and specifically binding to a protein or fragment or epitope thereof from SARS-CoV-2 virus as described herein under conditions that allow the protein or fragment or epitope thereof from SARS-CoV-2 virus, if present in the sample, to bind to the first antibody or antigen-binding fragment thereof to form a first complex. In some embodiments, the protein from the SARS-CoV-2 virus is a viral genome packaging protein (e.g., a nucleocapsid (N) protein, e.g., as provided by UniProtKB accession number P0DTC 9).

The sample may be subjected to one or more treatment steps prior to contact with the primary antibody or antibody-binding fragment thereof. In some embodiments, such processing steps include the addition of one or more preservatives or stabilizers to facilitate storage of the sample or transport of the sample from the collection location to the testing location. In some embodiments, such processing steps include a purification step (e.g., using a filter, centrifugation, etc.) that removes one or more components from the sample to enrich the sample for the analyte of interest.

The primary antibody or antigen-binding fragment thereof may be contacted with the sample using any suitable method known in the art. As used herein, the term "contacting" refers to any type of combined action that brings an antibody, particularly an antibody immobilized on a solid support, into sufficient proximity to an analyte of interest (e.g., a protein from the SARS-CoV-2 virus) in a sample such that a binding interaction occurs if the analyte of interest is present in the sample that is specific for the antibody. Contacting can be accomplished in a number of different ways including directly combining the sample with the antibody or antigen binding fragment thereof, or exposing the sample to a solid support comprising the antibody or antigen binding fragment thereof by introducing the solid support in close proximity to the sample. The contacting may be repeated as many times as necessary or for a requisite amount of time so that binding interactions occur.

The methods described herein are desirably performed using an immunoassay. As used herein, the term "immunoassay" refers to a biochemical test that measures the presence or concentration of a large or small molecule in a solution by using an antibody or antigen. Any suitable immunoassay may be used, and a variety of immunoassay types, configurations, and formats are known in the art and are within the scope of the present disclosure. Suitable types of immunoassays include, but are not limited to, enzyme-linked immunosorbent assays (ELISA), lateral flow assays, competitive inhibition immunoassays (e.g., forward and reverse), radioimmunoassays (RIA), fluorescent Immunoassays (FIA), chemiluminescent immunoassays (CLIA), counting Immunoassays (CIA), enzyme-multiplied immunoassay technology (EMIT), one-step antibody detection assays, homogeneous assays, heterogeneous assays, in-flight capture assays (capture on the fly assay), single molecule detection assays, and the like. Such methods are disclosed, for example, in U.S. patent 6,143,576;6,113,855;6,019,944;5,985,579;5,947,124;5,939,272;5,922,615;5,885,527;5,851,776;5,824,799;5,679,526;5,525,524;5,480,792;11,022,598; international patent application publication WO 2016/161400; and Adamczyk et al, anal.Chim. Acta,579 (1): 61-67 (2006).

The immunoassay format may be "direct" or "indirect" or "sandwich". Sandwich formats involve the use of capture and detection antigens to immobilize and detect antigens in a sample. Specifically, the surface of a solid support (e.g., ELISA plate, bead, etc.) is coated with a capture antibody or antigen-binding fragment thereof that binds and immobilizes a target antigen present in a sample applied thereto. The detection antibody is then added to the complex or brought into contact with the complex. The detection antibody may be directly labeled with an antibody ("direct sandwich immunoassay") to allow detection and quantification of the antigen. Alternatively, if the detection antibody is unlabeled, a second enzyme-conjugated detection antibody may be used ("indirect sandwich assay").

Thus, the disclosed methods can further comprise contacting the sample with a conjugate comprising a second antibody, wherein the second antibody or antigen-binding fragment thereof (part of the conjugate) specifically binds to a target antigen (e.g., a protein from SARS-CoV-2 virus or a fragment or epitope thereof), which results in the binding of the conjugate to the captured analyte and the formation of an immune sandwich (also referred to herein as an "immune sandwich complex"). It will be appreciated that in a sandwich immunoassay format, the first and second antibodies recognize two different non-overlapping epitopes on the target analyte/antigen.

In certain embodiments, the primary antibody or antigen-binding fragment thereof may be attached to or immobilized on a solid support. The terms "solid phase" and "solid support" are used interchangeably herein and refer to any material that can be used to attach and/or attract and immobilize one or more antibodies. Any solid support known in the art may be used in the methods described herein. Examples of suitable solid supports include electrodes, test tubes, beads, microparticles, nanoparticles, wells of microwell plates or plates, gels, colloids, biological cells, sheets, strips and chips.

In one embodiment, the solid support desirably comprises a plurality (e.g., 2 or more, 50 or more, 100 or more, 1,000 or more, or 5,000 or more) of antibodies or antigen binding fragments thereof immobilized on its surface that bind to a protein from SARS-CoV-2 virus or a fragment or epitope thereof. As used herein, the term "immobilized" refers to a stable association of a binding member with a solid support surface. As discussed herein, after incubation between the solid support and the sample for a sufficient time, the protein from the SARS-CoV-2 virus, or a fragment or epitope thereof (if present in the sample), is desirably captured on the surface of the solid support via the immobilized antibody.

The antibody or antibody fragment may be attached to the solid support via a linkage, which may include any portion, functionalization, or modification that facilitates attachment of the antibody to the support and/or antibody. The linkage between the antibody and the support may include one or more chemical or physical bonds (e.g., non-specific attachment via van der waals forces, hydrogen bonding, electrostatic interactions, hydrophobic/hydrophilic interactions, etc.) and/or chemical spacers that provide such bonds. Antibodies can be attached to a variety of solid supports using any number of techniques (see, e.g., U.S. patent 5,620,850; and heller, acc. Chem. Res.,23:128 (1990)).

In some embodiments, the binding affinity between the protein from SARS-CoV-2 virus or a fragment or epitope thereof and the first or second antibody or antibody fragment should be sufficient to maintain binding under assay conditions that include a washing step to remove non-specifically bound molecules or particles. It is desirable to maintain contact (e.g., incubation) for a time sufficient to allow binding interaction between the protein from the SARS-CoV-2 virus, or fragment or epitope thereof, and the first or second antibody or antibody fragment. In addition, incubation may be performed in a binding buffer that promotes specific binding interactions, such as, for example, albumin (e.g., BSA), a non-ionic detergent (Tween-20, triton X-100), and/or a protease inhibitor (e.g., PMSF). The binding affinity and/or specificity of the first or second antibody or antibody fragment may be manipulated or altered in the assay by altering the binding buffer.

Any unbound antibody, antibody fragment or conjugate component may be separated from the immunocompetent by any suitable means, such as, for example, droplet drive, electrophoresis, electrowetting, dielectrophoresis, electrostatic drive, electric field mediation, electrode mediation, capillary force, chromatography, centrifugation, aspiration or Surface Acoustic Wave (SAW) based washing methods.

The method further comprises assessing the presence of a signal from the detectable label conjugated to the second antibody, wherein the presence of the signal from the detectable label is indicative of the presence of a protein from the SARS-CoV-2 virus or a fragment or epitope thereof in the sample.

Suitable detectable labels include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials (see, e.g., zola, monoclonal Antibodies: A Manual of Techniques, CRC Press, inc. (1987)). For example, the detectable label may be a radioisotope (e.g ³ H、 ¹⁴ C、 ³² P、 ³⁵ S or ¹²⁵ I) A fluorescent or chemiluminescent compound (e.g., fluorescein isothiocyanate, rhodamine, or luciferin) or an enzyme (e.g., alkaline phosphatase, beta-galactosidase, or horseradish peroxidase). Any method known in the art for conjugating antibodies separately to a detectable label may be employed in the context of the present disclosure (see, e.g., hunter et al, nature,144:945 (1962), david et al, biochemistry,13:1014 (1974), pain et al, j. Immunol. Meth.,40:219 (1981), and Nygren, j. Histochem. And cytochem.,30:407 (1982)). The signal generated from the detectable label attached to the antibody may be measured according to the spectral properties.

It is to be understood that different constellations of the antigen capture and immunocompetent formation methods described above are within the scope of the present disclosure. In fact, the various components of the solid support, conjugates, and detectable labels described above may be arranged or utilized in any suitable combination, conformation, or form. For example, the disclosed methods can be performed in one, delayed one, or two step formats, wherein the sample is incubated with a first antibody and then with a second antibody, and vice versa. The assay reagents (e.g., microparticles, conjugates, fluorophores, etc.) may be pre-mixed or added sequentially as appropriate.

In one embodiment, a lateral flow assay is used. Lateral flow assays provide techniques for the definitive detection and/or quantitative measurement of analytes in a short period of time using antigen-antibody interactions (e.g., using immunochromatography). These tests typically use an assay device in the form of an assay test strip or a device in which the assay strip is mounted within a plastic cartridge. See, for example, international patent application publication No. WO2011102563A1; U.S. patent No. 8,828,739, each of which is incorporated herein by reference.

Lateral flow assays are typically provided in devices comprising: a lateral flow test strip (e.g., nitrocellulose or filter paper), a sample application area (e.g., a sample pad), a test result area (e.g., a test line), an optional control result area (e.g., a control line), and an analyte-specific binding agent that binds to a detectable label (e.g., a colored particle or an enzyme detection system). See, for example, U.S. patent No. 6,485,982;6,187,598;5,622,871;6,565,808;6,809,687; and 10,717,082, each of which is incorporated herein by reference. In some embodiments, the technology relates to a test device that includes a reagent-impregnated test strip to provide a specific binding assay (e.g., an immunoassay).

In some embodiments, the present disclosure relates to a lateral flow device that is suitable for use in a home, clinic, or hospital and is intended to quickly give analysis results with minimal skill and user involvement. In some embodiments, the use of the devices described herein involves a method for a user to perform a series of operations to provide observable test results.

In some of these methods, the lateral flow device comprises: a first antibody that specifically binds to a protein from the SARS-CoV-2 virus or a fragment thereof; and a second antibody that specifically binds to a protein from SARS-CoV-2 virus or a fragment thereof. The description of antibodies provided above is related to the lateral flow devices described herein. In some embodiments, the test line of the device comprises a first antibody. In some embodiments, the sample pad comprises a second antibody.

In some embodiments, the sample is applied to a portion of the test strip and allowed to permeate through the strip material, typically by means of an eluting solvent (such as water) and/or a suitable extraction buffer (e.g., optionally containing a detergent). In so doing, the sample enters or passes through a detection zone in the test strip, in which a specific binding reagent (e.g., an antibody) for an analyte suspected of being present in the sample (e.g., a protein from the SARS-CoV-2 virus or a fragment or epitope thereof) is immobilized. Analytes present in the sample may thus bind within the detection zone. The extent to which the analyte is bound in this region can be determined by means of a labelled reagent which can also be subsequently incorporated into or applied to the test strip.

In some embodiments, the analytical test device includes a hollow housing constructed of a moisture impermeable solid material containing a dry porous carrier in direct or indirect communication with the exterior of the housing so that a liquid test sample can be applied to the porous carrier. In some embodiments, the device further comprises a labeled specific binding reagent for the analyte, and the labeled specific binding reagent is free to move within the porous carrier when in a wet state. In some embodiments, the device comprises unlabeled specific binding reagents for the same analyte, and the unlabeled reagents are permanently immobilized in a detection zone on the carrier material, and therefore do not move in a wet state. The relative positioning of the labelled reagent and the detection zone is such that a liquid sample applied to the device can obtain the labelled reagent and subsequently penetrate into the detection zone, and the device provides the extent (if any) to which the labelled reagent enters the detection zone to be observed.

In some embodiments, the device comprises a porous solid phase material carrying a labeled reagent in a first region, the labeled reagent remaining in the first region when the porous material is in a dry state, but the labeled reagent freely migrating through the porous material when the porous material is wet (e.g., by application of an aqueous liquid sample suspected of containing an analyte). In some embodiments, the porous material includes unlabeled specific binding reagents in a second region that is spatially distinct from the first region, the reagents being specific for the analyte and capable of participating in a "sandwich" or "competition" reaction with the labeled reagents. The unlabeled specific binding reagent is firmly immobilized on the porous material such that the unlabeled specific binding reagent does not migrate freely when the porous material is in a wet state.

In some embodiments, a device as described herein is contacted with an aqueous liquid sample suspected of containing an analyte such that the sample permeates through the porous solid phase material by capillary action, through the first zone into the second zone, and the labeled reagent subsequently migrates from the first zone to the second zone, the presence of the analyte in the sample being determined by observing the extent, if any, of binding of the labeled reagent in the second zone.

In some embodiments, the labeled reagent is a specific binding partner for the analyte. The labeled reagent, analyte (if present), and immobilized unlabeled specific binding reagent act synergistically in a "sandwich" reaction. If the analyte is present in the sample, this will result in binding of the labelled reagent in the second zone. In sandwich format, the two binding reagents are specific for different epitopes on the analyte. In some embodiments, the first antibody is immobilized. In some embodiments, the second antibody comprises a detectable label.

In some embodiments, the test strip (e.g., carrier material) comprises nitrocellulose. This is a considerable advantage over some other strip materials, such as paper, because of its natural ability to bind proteins without prior sensitization. Specific binding reagents, such as immunoglobulins, can be applied directly to the nitrocellulose and immobilized thereon. No chemical treatment is required that might interfere with the basic specific binding activity of the reagent. Simple materials (such as polyvinyl alcohol) can then be used to block unused binding sites on nitrocellulose. In addition, nitrocellulose of various pore sizes is readily available and this aids in the selection of carrier materials that are particularly suited to requirements such as sample flow rates.

In some embodiments, a porous solid phase material is coupled to a porous receiving member to which a liquid sample may be applied and from which the sample may permeate into the porous solid phase material. In some embodiments, the porous solid phase material is contained within a moisture impermeable outer shell (cas) or shell (hous) and a porous receiving member attached to the porous solid phase material extends out of the shell and may be used as a means to allow a liquid sample to enter the shell and permeate the porous solid phase material. The shell should be provided with a second zone that enables visualization of the porous solid phase material (carrying immobilized unlabeled specific binding reagent) from outside the shell, so that the means of measuring the result (e.g. a well placed in place) can be visualized. If desired, the shell may also be provided with other means which enable another region of porous solid phase material to be observed from outside the shell and which incorporate a control reagent which enables an indication to be given as to whether the assay procedure has been completed. In some embodiments, the shell is provided with a removable cap or shield that can protect the protruding porous receiving member during storage prior to use. If desired, the cap or shield may be replaced onto the protruding porous receiving member after sample application while the assay procedure is performed. Optionally, the labeled reagent may be incorporated elsewhere within the device, such as into a water-absorbent sample collection member, but this is not preferred.

In some embodiments, the device is configured as a kit suitable for use in a hospital, clinic, or home. In some embodiments, the kit comprises a plurality (e.g., two) of devices, individually wrapped in a moisture impermeable package, and packaged with appropriate instructions to the user.

In some embodiments, the device includes an optional "control zone". The "control" zone, if present, may be designed to convey to the user an extraneous signal that the device has been operated. For example, the control zone may be loaded with an antibody (e.g., goat anti-rabbit IgG) that will bind to the labeled antibody (e.g., labeled rabbit IgG) from the first zone to confirm that the sample has permeated the test strip. In some embodiments, the first zone comprises an antigen and/or antibody that is independent of the analyte and that is specifically captured at the control zone. In some embodiments, the control zone may contain an anhydrous reagent that produces a color change or color formation when wetted, such as anhydrous copper sulfate that will turn blue when wetted by an aqueous sample. As a further alternative, the control zone may contain immobilized analyte that reacts with excess labeled reagent from the first zone. Since the purpose of the control zone is to indicate to the user that the test has been completed, the control zone should be located downstream of the second zone where the desired test results are recorded. Thus, the positive control indicator tells the user that the sample has penetrated the desired distance through the test device.

Ideally, the results of the assay should be distinguishable by eye, and in order to facilitate this, it is necessary that the direct labelling is concentrated at the detection zone. In some embodiments, the detectable label is a direct label, e.g., an entity that is readily visible to the naked eye in its natural state or by means of a filter and/or an applied stimulus (e.g., ultraviolet light that promotes fluorescence). For example, tiny colored particles such as dye sols, metal sols (e.g., gold) and colored latex particles are very suitable. Concentration of the labels into small areas or volumes (e.g., test lines) can produce readily detectable signals, e.g., strongly colored areas. If desired, this can be assessed by eye or by instrument.

In some embodiments, the techniques include the use of indirect labeling. Indirect labels such as enzymes, e.g., alkaline phosphatase and horseradish peroxidase, may be used, but these typically require the addition of one or more developers (such as substrates) to detect the visible signal. Such additional reagents may be incorporated into the porous solid phase material or the sample receiving member (if present) such that they are dissolved or dispersed in the aqueous liquid sample. Alternatively, the developer may be added to the sample prior to contact with the porous material, or the porous material may be exposed to the developer after the binding reaction has occurred.

In some embodiments, the sample flow is caused to continue beyond the detection zone and sufficient sample is applied to the porous material such that this may occur, and any excess labeled reagent from the first zone that does not participate in any binding reaction in the second zone is flushed out of the detection zone by such continued flow. If desired, an absorbent "slot" may be provided at the distal end of the carrier material. The absorption cell may consist of, for example, whatman 3MM chromatographic paper and should provide sufficient absorption capacity to allow any unbound conjugate to be washed away from the detection zone. As an alternative to such a well, it is sufficient to have a length of porous solid phase material extending beyond the detection zone.

In some embodiments, the carrier material is in the form of a strip or sheet, the reagents are applied to the carrier material in spatially distinct regions, and the liquid sample is allowed to permeate through the sheet or strip from one side or end to the other side or the other.

In some embodiments, the material comprising the porous solid phase is nitrocellulose. This has the advantage that the antibodies in the second region are firmly immobilized without prior chemical treatment. For example, if the porous solid phase material comprises paper, antibody immobilization in the second zone needs to be performed by chemical coupling using, for example, CNBr, carbonyldiimidazole or triphenol chloride.

After the antibody is applied to the detection zone, the remainder of the porous solid phase material is treated to block any remaining binding sites elsewhere. For example, blocking may be achieved by treatment with a protein (e.g., bovine serum albumin or milk protein) or with polyvinyl alcohol or ethanolamine or any combination of these agents. The labelled reagent for the first zone may then be dispensed onto a dry carrier and the labelled reagent will move in the carrier when in a wet state. Between each of these different processing steps (sensitization, application of unlabeled reagents, blocking and application of labeled reagents), the porous solid phase material is dried.

The disclosed methods can include a quality control component. "quality control components" in the context of immunoassays and kits described herein include, but are not limited to, calibrators, controls, and sensitivity groups. A "calibrator" or "standard" (e.g., one or more, such as a plurality) may be used in order to establish an interpolated calibration (standard) curve of analyte (such as antigen) concentration. Alternatively, a single calibrator near a reference or control level (e.g., a "low," "medium," or "high" level) may be used. Multiple calibrants (e.g., more than one calibrant or different amounts of calibrants) can be used in combination to make up a "sensitivity group". The calibrator is optionally part of a series of calibrators, where each calibrator is different from the other calibrators in the series, such as, for example, concentration or detection method (e.g., colorimetric or fluorescent detection).

In addition to SARS-CoV-2, the disclosed methods can further comprise detecting one or more pathogens or antigens thereof. The pathogen may be an infectious disease of interest, or any infectious organism that may cause symptoms of a disease that require differential diagnosis for proper treatment. Infectious organisms of interest include bacteria (including but not limited to Escherichia species, streptococcus species, haemophilus species, staphylococcus species, neisseria species), viruses (including but not limited to adenoviruses, enteroviruses, echoviruses, human herpesviruses, mumps virus Ag), influenza viruses, parainfluenza viruses, respiratory Syncytial Viruses (RSV), other human coronaviruses, rhinoviruses, human metapneumoviruses) or eukaryotic pathogens (including fungi and protozoa). In some embodiments, the method further comprises detecting influenza virus, rhinovirus, RSV and/or adenovirus.

Detection of other pathogens may use the same types of methods used to detect SARS-CoV-2 antigen, e.g., immunoassays as described above. Alternatively, detection of other pathogens may use non-immunoassays for detection, including, but not limited to, nucleic acid detection (e.g., microarrays), nucleic acid amplification (e.g., RT-PCR) and/or sequencing (e.g., next generation sequencing), immunofluorescence assays, serological assays, and cell culture-based assays.

Detection of other pathogens may be performed simultaneously with, before or after detection of the SARS-Cov-2 antigen. For example, if the method of detecting other pathogens is the same type of immunoassay, the sample may be incubated with the detection reagents for the other pathogens, while simultaneously incubating with the reagents for detecting SARS-Cov-2 antigen (e.g., a single sample pad for multiple test strips in a lateral flow assay).

3. Kit and instrument

Kits for performing the above methods are also provided herein. The instructions included in the kit may be affixed to the packaging material or may be included as a package insert. The instructions may be written or printed materials, but are not limited thereto. The present disclosure contemplates any medium capable of storing and conveying such instructions to an end user. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term "description" may include a web site address of an internet site providing the description.

The kit may comprise a lateral flow device as described herein. In some embodiments, the lateral flow device is in a sealed package (e.g., wrapped in a moisture impermeable package). The lateral flow device may be disposable. In some embodiments, the device is configured as a kit suitable for use in a hospital, clinic, or home. In some embodiments, the kit comprises a plurality (e.g., two) of devices, individually wrapped in a moisture impermeable package, and packaged with appropriate instructions to the user.

The kit may comprise a sample container. The sample container may be an absorption cell. The sample holder may be a cartridge comprising a microfluidic module. The sample holder may be an ELISA plate. The sample containers can include one or more reagents (e.g., binding buffers and antibodies) that can be used to perform the methods disclosed herein. The sample holder may also include other materials that may be desired from a user's perspective, such as buffers, diluents, standards (e.g., calibrators and controls), and/or any other material that may be used for sample processing, washing, or any other step in performing an assay.

The kit may also include a reference standard for detecting a protein from the SARS-CoV-2 virus or fragment thereof in the sample. The reference standard may be used to establish an interpolated and/or extrapolated standard curve for the concentration of a protein or fragment thereof from the SARS-CoV-2 virus. The kit may include reference standards at different concentration levels. For example, the kit may include one or more reference standards having a high concentration level, a medium concentration level, or a low concentration level. This can be optimized according to the assay in terms of the concentration range of the reference standard.

The kit may also include quality control components (e.g., a sensitive group, a calibrator, and a positive control). The preparation of quality control reagents is well known in the art and is described on the inserts of a variety of immunodiagnostic products. Sensitive panel members are optionally used to establish assay performance characteristics and are useful indicators of kit reagent integrity and assay standardization.

The kit may optionally also include other reagents necessary to detect additional pathogens in the sample, as well as any reference standards or quality control components thereof.

The kit may optionally also include other reagents required to perform the assay or to facilitate quality control evaluation, such as buffers, salts, enzymes, enzyme cofactors, substrates, detection reagents, and the like. Other components, such as buffers and solutions for separation and/or processing of the test sample (e.g., pretreatment reagents or extraction buffers) may also be included in the kit. The kit may additionally include one or more other controls. One or more of the components of the kit may be lyophilized, in which case the kit may further comprise reagents suitable for reconstitution of the lyophilized components. One or more of the components may be in liquid form.

The various components of the kit are optionally provided in suitable containers as desired. The kit may also include a container for holding or storing the sample (e.g., a container or cartridge for the sample). The kit may optionally contain reaction vessels, mixing vessels, and other components that aid in preparing reagents or testing samples, where appropriate. The kit may also include one or more sample collection/retrieval instruments for aiding in obtaining a test sample (e.g., microsampling devices, microneedles, or other minimally invasive painless blood collection methods for obtaining, storing, or aspirating tissue samples; blood collection tubes; lancets; capillary blood collection tubes; other single-fingertip piercing blood collection methods; oral swabs, nasal/pharyngeal swabs; 16 gauge or other size needles, scalpels, or lasers (e.g., particularly hand-held), syringes, sterile containers, or cannulas).

The concepts, kits and methods as described herein can be implemented on any system or instrument, including any manual, automated or semi-automated system for conducting immunoassays. In certain embodiments, the assays, kits, and kit components described herein can be performed in a hospital, home, or clinic.

In certain embodiments, the assays, kits, and kit components described herein can be performed on a high throughput immunoassay laboratory system, such as, for example, in Abbott ^TM ARCHITECT ^TM (Abbott Laboratories) performed on an immunoassay analyzer. Such devices and their components are described in, for example, U.S. patent 5,468,646;5,543,524;5,545,739;5,565,570;5,669,819; and 5,783,699.

In certain embodiments, the assays, kits, and kit components described herein can be implemented on an electrochemical or other hand-held or point-of-care assay system, such AS, for example, abbott Point of Care in performing sandwich assays and Axi s-Shield POC ASAbbott Laboratories) are implemented on electrochemical assay systems. Immunosensors and methods of making and operating in disposable testing devices are described, for example, in U.S. patent 5,063,081;7,419,821;7,682,833; and 7,723,099 and U.S. patent application publication No. 2004/0018577.

4. Examples

Example 1

Lateral flow assay

An exemplary lateral flow assay for detecting SARS-CoV-2 using a sandwich assay is provided. In particular, the device includes a first region (e.g., a reagent region) that includes a labeled antibody specific for a protein from the SARS-CoV-2 virus or fragment thereof, such as a monoclonal antibody labeled with a detectable label. The device includes a second antibody immobilized in a second region (e.g., a detection region) that is specific for a different epitope of a protein from SARS-CoV-2 virus or a fragment thereof. The presence of a visible line at the detection zone (e.g., at the test line) is indicative of a positive test.

Initial testing of the device was performed on heat-inactivated SARS-CoV-2 virus diluted onto a foam swab (applied to the reagent zone). LOD was found to be 22.5% Tissue Culture Infection Dose (TCID) ₅₀ )(TCID ₅₀ ) Test dilution or 1125TCID ₅₀ /mL。

No cross-reactivity was observed with many other viruses tested, including adenovirus (types 1, 5 and 7), enterovirus (types EV68 and D68), echovirus (types 1 and 2), human herpesvirus (types 1 and 2), mumps virus, influenza A virus (H1N 1 strain (A/Virginia/ATCC 1/2009), H1N1 strain (A/WS/33) and H3N2 strain (A/Hongkong/8/68)), influenza B virus (strain (B/Lee/40)), parainfluenza virus (types 1, 2, 3 and 4A), respiratory syncytial virus or RSV (types A and B), human coronavirus (HKU 1, NL63, OC43 and E), rhinovirus (type A16), MERs-CoV and human metapneumovirus (16A 1). In addition, co-infection with a non-SARS-CoV-2 virus (influenza A, rhinovirus, RSV or adenovirus) does not affect SARS CoV-2 detection near the limit of detection.

However, when using 25ng/mL to 25. Mu.g/mL of human SARS coronavirus nucleoprotein, cross-reactivity with human SARS coronavirus was observed, probably due to 79.6% similarity between the genome of SARS-CoV and the genome of SARS-CoV-2. Samples containing 2.5ng/mL human SARS coronavirus nucleoprotein did not cause cross-reaction.

Cross-reactivity with other organisms including Candida albicans (Chlamydia pneumoniae), streptococcus pyogenes (Streptococcus pyogenes) (group a 19615), staphylococcus aureus (Staphylococcus aureus), staphylococcus saprophyticus (Staphylococcus saprophyticus), neisseria species (neisseria lactose (Neisseria lactamica)), escherichia coli, staphylococcus hemolyticus (Staphylococcus haemolyticus), streptococcus salivarius (Streptococcus salivarius), haemophilus parahaemolyticus (Hemophilus parahaemolyticus), proteus vulgaris, moraxella catarrhalis (Moraxella catarrhalis), klebsiella pneumoniae (Klebsiella pneumoniae), clostridium necrobacter (Fusobacterium necrophorum) and mycobacterium tuberculosis (Mycobacterium tuberculosis) was not observed. Furthermore, no cross-reactivity was observed with pooled human nasal wash samples.

Tests were also performed that included a variety of known interfering substances, including: endogenous substances such as mucins, hemoglobin, triglycerides, jaundice (bilirubin), rheumatoid factors, antinuclear antibodies, pregnancy and whole blood, and expectorants such as guaifenesin; bronchodilators such as albuterol and ephedrine; antihistamines such as chlorpheniramine (chlorpheniramine) and diphenhydramine (diphenhydramine); nasal decongestants such as phenylephrine hydrochloride (phenylephrine hydrochloride) and oxymetazoline hydrochloride (oxymetazoline hydrochloride); antiviral drugs such as ribavirin (ribavirin), oseltamivir (oseltamivir), and zanamivir (zanamivir); antibiotic drugs such as amoxicillin (amoxicillin); commonly used drugs such as acetylsalicylic acid and ibuprofen; antihypertensive agents such as chlorothiazide (chlorthiazide) and indapamide (indapamide); antidiabetic agents such as glimepiride (sulfonylureas) and indapamide; and putative covd-19 drugs such as ivermectin (ivermectin), lopinavir (lopinavir), ritonavir (ritonavir), and chloroquine phosphate. Solutions of interfering substances are incorporated onto foam swabs or sample libraries and the test is completed as described above. No interference was observed at the concentrations tested.

Example 2

Lateral flow assay kit

An exemplary lateral flow assay kit includes at least one or all of the following, either alone or within a lateral flow device: extracting the buffer solution; a lateral flow device; a positive control swab comprising recombinant SARS-CoV-2N protein in a 1X XTBE and 1% bsa solution; a nitrocellulose test strip having a test line and a control line; patient swabs (e.g., foam head applicators); and at least one or both of the first and second antibodies or fragments thereof. A lateral flow device comprising a test strip assembly comprising: bridge pads (e.g., ahlstrom 1281-Ahlstrom Filtration inc., mt. Holly Springs, pa.), conjugate pads (e.g., PN PK 002123), sample pads (e.g., ahlstrom 1281-Ahlstrom Filtration inc., mt. Holly Springs, pa.), antibody pads (e.g., ahlstrom 904-Ahlstrom Filtration inc., mt. Holly Springs, pa.), and shells. The extraction buffer contained 200mM Tricine, 1.2% NaCl, 0.75% Zwi ttergent, 0.5% Tween 20 and 0.0125% azide, pH 8.8. Embedding or treating the test wire with a solution comprising: 2mg/mL BSA-Fab, 1.5% trehalose, 50mM Tris, 0.1% azide and 0.02% Intrawhite (UV). The control line is embedded in or treated with a solution comprising: 1mg/mL chicken IgY, 1% trehalose, 50mM Tris, 0.1% azide, and 0.05% FD & C blue dye.

The second antibody or conjugate antibody or fragment thereof is provided with the following buffers: resuspension buffers (e.g., 5mM borate, 0.1% casein, 0.01% PEG compound, and 0.01% azide, pH 7.4) and drying buffers (e.g., 5mM borate, 2% enzymatic casein (N-Z Case), 0.1% Triton X-100, 2% Tween 20, 6% sucrose, and 0.02% azide, pH 8). The second antibody or conjugate antibody or fragment thereof may comprise EQ ID NO. 15 and EQ ID NO. 16 or derivatives thereof, and donkey anti-chicken without BSA.

Example 3

Lateral flow assay kit

An exemplary lateral flow device containing the first and second antibodies as disclosed herein was tested using 243 nasopharyngeal specimens (60 PCR positive and 183 PCR negative) collected from individuals suspected of being exposed to covd-19 or developing symptoms of covd-19 over the past 7 days. The results showed an overall percent identity of 97.9% (table 1).

Table 1:

example 4

Lateral flow assay comparison

The clinical sensitivity and specificity of the exemplary lateral flow device was tested against a reference method comprising real-time PCR (RT-PCR) of SARS-CoV-2 in a sample from a nasal swab of a patient suspected of having a COVID-19 infection. One exemplary lateral flow device comprises full length versions of the first and second antibodies described herein, while the second lateral flow device comprises Fab antibody fragments of the first and second antibodies. Two nasal swabs per patient were collected; one was used for direct testing using a lateral flow assay, while the other was placed in a virus transfer medium and cooled to 2-8 ℃ for subsequent RT-PCR analysis. The order of nasal swab collection was random.

An exemplary lateral flow device comprising full length antibodies was found to have a positive percentage of identity of approximately 68% for all samples (symptomatic patients) compared to RT-PCR analysis (table 2). The percent positive identity was approximately 90% compared to the culturable virus measurement (CT cutoff = 23), although the percent negative identity was slightly reduced (table 3). Most patients (100) developed symptoms less than 7 days and the results were similar to the summary results for all patients as shown in table 2. However, a smaller sample size for patients with symptoms occurring for 8-10 days, 11-14 days, or greater than 15 days results in a larger 95% confidence interval.

Table 2:

table 3:

exemplary lateral flow devices comprising Fab antibody fragments produced significantly higher percent positive identity than devices using full length antibodies. The percent positive identity for all patient samples (symptomatic and asymptomatic patients) was approximately 90%; for symptomatic patients, the percent positive identity was 91.5% (table 4). Similar results were found by comparing Fab side-stream devices with culturable virus (CT cutoff = 23). Due to the low sample size, there was a large error in the percent positive consistency for asymptomatic patients, as indicated by the 95% confidence interval. (Table 4).

Table 4:

most symptomatic patients (102/126) develop symptoms less than 7 days. In those samples, the percent positive and overall percent identity were all increased to 97.1% and 98.0%, respectively; the percent negative identity remained essentially unchanged. However, similar to the total number of asymptomatic patients, low sample volumes for symptomatic patients lasting 8-10 days (5 patients), 11-14 days (13 patients) and over 15 days (6 patients) resulted in a large number of errors in the percent positive consistency measurement of 100.0% (29.2, 100.0), 62.5% (24.5, 91.5) and 100.0% (2.5, 100.0), respectively. As shown in table 5, samples from symptomatic patients lasting 10 days or less resulted in an overall percent identity of about 98%.

Table 5:

the use of Fab antibody fragments instead of full length antibodies in the lateral flow device increased the percent positive identity from about 68% to about 90% and the percent overall identity from about 88% to about 95% for all patient samples tested, and increased the percent positive, percent negative and percent overall identity for samples from patients with symptoms lasting 10 days or less.

Example 5

Lateral flow assay

Additional tests were performed on heat-inactivated SARS-CoV-2 isolated from positive patients using a full length version of the lateral flow device comprising the first and second antibodies as described herein. LOD was found to be 79% of the Tissue Culture Infection Dose (TCID) ₅₀ ) Ml ((TCID) ₅₀ )/mL)。

The samples were also used to determine if a hook effect (also known as high dose hook effect) was present. The hook effect is caused by an excess of target protein that reacts simultaneously and transiently with the immobilized and labeled antibody. Thus, the hook effect refers to a false negative result that can be seen when very high levels of target are present in the test sample. To overcome the hook effect, the specimen may have to be diluted. As shown in fig. 1, up to and including 1.0x10 ^5.8 (630,957)(TCID ₅₀ ) No significant hooking effect was seen at the concentration of/mL.

Example 6

Clinical evaluation of nasopharyngeal and nasal specimens Using lateral flow devices

Clinical assessment of sensitivity and specificity was performed using an exemplary lateral flow device against a reference method comprising real-time PCR (RT-PCR) of SARS-CoV-2 in a sample from a nasopharyngeal specimen of a patient suspected of having a COVID-19 infection. Exemplary lateral flow devices include full length versions of the first and second antibodies described herein.

An exemplary lateral flow device comprising full length antibodies was found to have a sensitivity (also referred to as percent positive identity above) of about 91% for all samples compared to RT-PCR analysis (table 6). Specificity (also referred to above as percent negative identity) exceeds 99%.

Samples were also classified based on the days after onset of symptoms. As shown in table 7, the sensitivity of all positive subjects was greater than 90% from 0-7 days after onset of symptoms. In addition, the specificity of negative subjects 0-3 days after onset of symptoms was 100%, and the specificity of negative subjects 4-7 days after onset of symptoms was greater than 99%.

Table 6:

table 7:

an exemplary lateral flow device was used to test clinical sensitivity and specificity for a reference method including real-time PCR (RT-PCR) of SARS-CoV-2 in a sample from a nasal swab of a patient suspected of having a COVID-19 infection. Exemplary lateral flow devices include full length versions of the first and second antibodies described herein.

An exemplary lateral flow device comprising full length antibodies was found to have a sensitivity of about 91% for all samples compared to RT-PCR analysis (table 8). The specificity is greater than 99%.

Samples were also classified based on the days after onset of symptoms. As shown in table 9, the sensitivity of all positive subjects was greater than 90% from 0-7 days after onset of symptoms. In addition, the specificity of negative subjects 4-7 days after onset of symptoms was 100%, and the specificity of negative subjects 4-7 days after onset of symptoms was greater than 99%.

Table 8:

table 9:

furthermore, as described in example 4 and used above, after using a portion or a majority of the sample in a lateral flow assay, the remaining sample is used to complete RT-PCR, rather than using a second sample.

An exemplary lateral flow device comprising full length antibodies was found to have a sensitivity of about 98% for all samples compared to RT-PCR analysis of the remaining samples (table 10). The specificity is greater than 99%. The overall consistency is greater than 99%.

Samples were also grouped by days after onset of symptoms. As shown in table 11, the sensitivity of all positive subjects 0-3 days after onset of symptoms was 100%, and the sensitivity of all positive subjects 4-7 days after onset of symptoms was greater than 96%. In addition, the specificity of negative subjects 4-7 days after onset of symptoms was 100%, and the specificity of negative subjects 4-7 days after onset of symptoms was greater than 99%.

Table 10:

table 11:

example 7

SARS-CoV-2 variants

To evaluate the effects of nucleocapsid mutations found in circulating SARS-CoV-2 strains (including delta and lambda strains), recombinant proteins carrying mutations identified in clinical specimens were prepared for testing. Mutations tested alone or in combination include: d63G, R203K, R203M, G204R, R209I, A V, Q229H, M234I, S235F, D348Y, P365S, E367Q, A376T, D377Y and Wild Type (WT) reference controls. These mutations represent a unique spectrum of nucleocapsid sequences of several circulatory lineages: b.1.1.7, b.1.617.1, b.1.617.2, b.1.617.3, b.1.618, ay.1, ay.2, p.2, b.1.526, b.1.526.1, b.1.526.2 and a group of strains from italy. Western blot and high throughput immunoassays using antibodies as disclosed herein confirm that all mutants and WT recombinant antigens (rags) are detected with sensitivity comparable to WT controls.

It is to be understood that the foregoing detailed description and accompanying examples are only illustrative and should not be taken as limiting the scope of the disclosure, which is defined only by the appended claims and equivalents thereof.

Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art and may be made without departing from the spirit and scope of the disclosed embodiments.

Sequence listing

<110> Yapei rapid diagnosis International Infinite responsibility corporation (Abbott Rapid Diagnostics International Unlimited Company)

<120> assay for detecting SARS-COV-2

<130> ALERE-38691.601

<150> US 63/126,336

<151> 2020-12-16

<150> US 63/067,051

<151> 2020-08-18

<150> US 63/065,898

<151> 2020-08-14

<150> US 63/060,975

<151> 2020-08-04

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<170> PatentIn version 3.5

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Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Met His Trp

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Met Ile Asp Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Arg Phe Lys

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Asp Lys

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Cys Ala Arg Ser Leu Leu Arg Gly Val Tyr Ala Met Asp Tyr Trp

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Cys Lys Ala Ser Gln Ser Val Ser Asn Asp Val Ala Trp

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Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg

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Cys Gln Gln Asp Tyr Ser Ser Pro Tyr Thr Phe

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Gln Val Gln Leu Gln Gln Ser Gly Pro Gln Leu Val Arg Pro Gly Ala

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Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr

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Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

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Gly Met Ile Asp Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Arg Phe

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Lys Asp Lys Ala Thr Leu Thr Val Asp Arg Ser Ser Ser Thr Ala Tyr

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Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

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Ala Arg Ser Leu Leu Arg Gly Val Tyr Ala Met Asp Tyr Trp Gly Gln

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Gly Thr Ser Val Thr Val Ser Ser

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Ser Ile Val Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly

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Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp

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Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

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Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

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Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Gln Ala

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Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro Tyr

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Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

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Ser Tyr Ala Ile Ser

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Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln

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Gly

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Gly Tyr Trp Gly Ser Gly Tyr His Tyr Tyr Gly Met Asp Val

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Gly Gly Asn Asn Ile Gly Ser Lys Ser Val His

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Tyr Asp Ser Asp Arg Pro Ser

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Gln Val Trp Asp Arg Ser Ser Asp Leu Val Val

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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Lys Lys Pro Gly Ser

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

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Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

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Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

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Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

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Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

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Ala Arg Gly Tyr Trp Gly Ser Gly Tyr His Tyr Tyr Gly Met Asp Val

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Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly

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Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

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Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

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Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

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Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

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Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

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Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

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Cys

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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Ser Val Ala Pro Gly Lys

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Thr Ala Arg Ile Pro Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val

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His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

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Tyr Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

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Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

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Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Arg Ser Ser Asp Leu

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Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys

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Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln

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Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly

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Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly

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Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala

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Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser

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Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val

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Ala Pro Thr Glu Ser Ser

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Gln Val Gln Leu Gln Gln Ser Gly Pro Gln Leu Val Arg Pro Gly Ala

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Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr

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Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

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Gly Met Ile Asp Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Arg Phe

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Lys Asp Lys Ala Thr Leu Thr Val Asp Arg Ser Ser Ser Thr Ala Tyr

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Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

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Ala Arg Ser Leu Leu Arg Gly Val Tyr Ala Met Asp Tyr Trp Gly Gln

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Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val

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Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr

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Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr

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Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val

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Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser

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Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala

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Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys

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Ser Ile Val Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly

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Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp

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Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

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Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

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Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Gln Ala

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Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro Tyr

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Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala

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Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly

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Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile

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Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu

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Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser

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Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr

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Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser

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Phe Asn Arg Asn Glu Ser

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Claims (19)

1. A method of detecting SARS-CoV-2 virus in a sample obtained from a subject, the method comprising:

contacting a sample obtained from a subject with a first antibody or antigen-binding fragment thereof that specifically binds to a protein or fragment thereof from the SARS-CoV-2 virus under conditions that allow the protein or fragment thereof from the SARS-CoV-2 virus (if present in the sample) to bind to the first antibody or antigen-binding fragment thereof, wherein the first antibody or antigen-binding fragment thereof comprises:

(i) A heavy chain variable region comprising a complementarity determining region 1 (CDR) amino acid sequence having at least 70% identity to SEQ ID NO. 1, a CDR2 amino acid sequence having at least 70% identity to SEQ ID NO. 2 and a CDR3 amino acid sequence having at least 70% identity to SEQ ID NO. 3,

(ii) A light chain variable region comprising a CDR1 amino acid sequence having at least 70% identity to SEQ ID No. 4, a CDR2 amino acid sequence having at least 70% identity to SEQ ID No. 5, and a CDR3 amino acid sequence having at least 70% identity to SEQ ID No. 6;

contacting the sample with a conjugate comprising a second antibody that specifically binds to the protein from SARS-CoV-2 virus or a fragment thereof and a detectable label, wherein the second antibody or antigen binding fragment thereof comprises:

(i) A heavy chain variable region comprising a complementarity determining region 1 (CDR) amino acid sequence having at least 70% identity to SEQ ID NO. 9, a CDR2 amino acid sequence having at least 70% identity to SEQ ID NO. 10 and a CDR3 amino acid sequence having at least 70% identity to SEQ ID NO. 11,

(ii) A light chain variable region comprising a CDR1 amino acid sequence having at least 70% identity to SEQ ID No. 12, a CDR2 amino acid sequence having at least 70% identity to SEQ ID No. 13, and a CDR3 amino acid sequence having at least 70% identity to SEQ ID No. 14; and

Assessing the presence of a signal from the detectable label, wherein the presence of a signal from the detectable label indicates the presence of the protein from SARS-CoV-2 virus or fragment thereof in the sample.

2. The method of claim 1, performed using an immunoassay.

3. The method of claim 1 or claim 2, wherein the immunoassay is an enzyme-linked immunosorbent assay (ELISA) or a lateral flow immunoassay (LFA).

4. A method according to any one of claims 1 to 3, wherein the sample comprises a nasal swab or brush, saliva, mucus, blood, serum or plasma.

5. The method of any one of claims 1-4, wherein the first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof recognize different epitopes of the protein from SARS-CoV-2 virus.

6. The method of any one of claims 1-5, wherein the protein from SARS-CoV-2 virus or fragment thereof is a nucleocapsid (N) protein.

7. The method of any one of claims 1-6, wherein the first antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence having at least 70% identity to SEQ ID No. 7 and a light chain variable region amino acid sequence having at least 70% identity to SEQ ID No. 8.

8. The method of any one of claims 1-7, wherein the second antibody or antigen binding fragment thereof comprises a heavy chain variable region amino acid sequence having at least 70% identity to SEQ ID No. 15 and a light chain variable region amino acid sequence having at least 70% identity to SEQ ID No. 16.

9. The method of any one of claims 1-8, further comprising detecting at least one or more additional pathogens or antigens thereof.

10. A lateral flow device, comprising:

a first antibody or antigen-binding fragment thereof that specifically binds to a protein from SARS-CoV-2 virus or fragment thereof, wherein the first antibody or antigen-binding fragment thereof comprises:

(i) A heavy chain variable region comprising a complementarity determining region 1 (CDR) amino acid sequence having at least 70% identity to SEQ ID NO. 1, a CDR2 amino acid sequence having at least 70% identity to SEQ ID NO. 2 and a CDR3 amino acid sequence having at least 70% identity to SEQ ID NO. 3,

(ii) A light chain variable region comprising a CDR1 amino acid sequence having at least 70% identity to SEQ ID No. 4, a CDR2 amino acid sequence having at least 70% identity to SEQ ID No. 5, and a CDR3 amino acid sequence having at least 70% identity to SEQ ID No. 6; and

A second antibody or antigen-binding fragment thereof that specifically binds to a protein from SARS-CoV-2 virus or fragment thereof, wherein the second antibody or antigen-binding fragment thereof comprises:

(i) A heavy chain variable region comprising a complementarity determining region 1 (CDR) amino acid sequence having at least 70% identity to SEQ ID NO. 9, a CDR2 amino acid sequence having at least 70% identity to SEQ ID NO. 10 and a CDR3 amino acid sequence having at least 70% identity to SEQ ID NO. 11,

(ii) A light chain variable region comprising a CDR1 amino acid sequence having at least 70% identity to SEQ ID No. 12, a CDR2 amino acid sequence having at least 70% identity to SEQ ID No. 13, and a CDR3 amino acid sequence having at least 70% identity to SEQ ID No. 14.

11. The lateral flow device of claim 10, wherein the first antibody or antigen-binding fragment thereof is immobilized.

12. The lateral flow device of claim 10 or 11, wherein the second antibody or antigen-binding fragment thereof comprises a detectable label.

13. The lateral flow device of any one of claims 10-12, wherein the first antibody or antigen-binding fragment thereof and the second antibody or fragment thereof recognize different epitopes of the protein from SARS-CoV-2 virus.

14. The lateral flow device of any one of claims 10-13, wherein a sample pad comprises the second antibody or antigen-binding fragment thereof.

15. The lateral flow device of any one of claims 10-14, wherein a test line comprises the first antibody or antigen-binding fragment thereof.

16. A kit comprising the lateral flow device of any one of claims 10-15 in a sealed package.

17. The kit of claim 16, further comprising an extraction buffer.

18. The kit of any one of claims 16 or 17, further comprising a sampling device.

19. The kit of claim 18, wherein the sampling device comprises a nasal swab.