WO2023192436A1

WO2023192436A1 - Singleplex or multiplexed assay for complement markers in fresh biological samples

Info

Publication number: WO2023192436A1
Application number: PCT/US2023/016827
Authority: WO
Inventors: Tobin J. CAMMETT
Original assignee: Alexion Pharmaceuticals, Inc.
Priority date: 2022-03-31
Filing date: 2023-03-30
Publication date: 2023-10-05

Abstract

Systems and methods for singleplex or multiplex assays of complement biomarkers in fresh or raw biological fluids are provided.

Description

SINGLEPLEX OR MULTIPLEXED ASSAY FOR COMPLEMENT MARKERS IN FRESH BIOLOGICAL SAMPLES

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63/325,790, filed March 31, 2022, and U.S Provisional Application No. 63/352,762, filed June 16, 2022, the contents of which are incorporated by reference herein in their entirety.

BACKGROUND

FIELD

[0002] The present disclosure generally relates to systems and methods for singleplex or multiplex assays of complement biomarkers in fresh and/or raw biological fluids.

DESCRIPTION OF RELATED ART

[0003] Accurate analysis of complement proteins remains a challenge due to ex- vlvo instability of this complex enzymatic cascade and is confounded by factors including processing time, temperature, coagulation, agitation, freeze/thaw, ambient air, and short-term and long-term storage conditions.

[0004] Complement levels in freshly collected blood and/or urine are not determined routinely in clinical laboratories. Many labs test frozen samples using a wide variety of commercial assays. See, e.g., Jodele et al. Biol Blood Marrow Transplant, 2014 Apr;20(4):518-25); Frazer-Abel etal. {Front Immunol, 2021 Aug 9,12:697313) and Fakhourl etal. {Clin J Am Soc Nephrol, 2019 14(12), 1682-1683). However, these assays are categorized as research use-only (RUO) or lab-developed test (LDT) because they fail in technical and/or clinical validation. Furthermore, these assays cannot be standardized, making commercialization for broad distribution and use challenging. This means that conventional assays and their results alone cannot be used for clinical decision making by healthcare providers (HCP). There exists a need in the art for assays of complement biomarkers in biological fluids. SUMMARY

[0005] In one embodiment, a system for point-of-care rapid assay for complement markers in a freshly collected biological sample can comprise (a) a first module configured for lateral-flow detection of complement markers in a biological sample, said first module comprising a plurality of agents comprising (1) a capture agent located at fixed site in the first module, wherein the capture agent captures the complement marker at the fixed site; and (2) a detection agent for detecting the captured complement marker of (1) at the fixed site; (b) a second module comprising a detector, wherein the second module is compatible with the first module of (a), and wherein the detector is capable of detecting a signal from the detection agent that is specifically associated with the capture agent at the fixed site; and (c) optionally, a dispenser for dispensing a fixed amount of the biological sample into the first module. [0006] In an embodiment, the first module can comprise membrane strip(s) in a cassette.

[0007] In an embodiment, the biological sample can be fresh. The biological sample can be less than 5 hours old. The biological sample can be between about 1 hour and 5 hours old. The biological sample can be between about 1 hour and 5 hours old. The biological sample can be about 1, 2, 3, 4, or 5 hours old.

[0008] In an embodiment, the complement marker can comprise C5b-9 or soluble C5b-9, or a combination thereof. The complement marker can be plasma C5b-9 (sC5b- 9), urine C5b-9 (uC5b-9), or a combination thereof.

[0009] In an embodiment, the capture agent can comprise an antibody that binds specifically to the complement marker. The complement marker can comprise C5b-9, optionally sC5b-9, uC4b-9, or a combination thereof.

[0010] In an embodiment, the detection agent can comprise a labeled second antibody that binds to the capture antibody. The labeled second antibody can be labeled with a radionuclide, fluorescent dye, chemiluminescent agent, microparticle, enzyme, colorimetric label, magnetic label, biotin, or a combination thereof.

[0011] In an embodiment, the biological sample can be urine. The biological sample can be urine, blood, plasma, optionally EDTA plasma, serum, or a combination thereof.

[0012] In an embodiment, the biological sample can be stored at between about -20°C and 25°C. The biological sample can be stored at about -20°C, -4°C, or 25°C. [0013] In an embodiment, the system can be a singleplex system.

[0014] In an embodiment, the system can be a multiplex system.

[0015] In an embodiment, the fixed amount can be between about 1 and 250 mL.

The fixed amount can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 mL. The fixed amount can be about 100 mL.

[0016] In an embodiment, a method for rapidly detecting complement markers in a biological sample at a point-of-care setting can comprise dispensing a fixed amount of the biological sample into a system comprising a first module configured for lateralflow detection of complement markers present in a biological sample, said first module comprising a plurality of agents comprising a capture agent located at fixed site in the cassette, wherein the capture agent captures the complement marker at the fixed site; and (B) incubate the biological sample in the first module for between 1 and 60 minutes;

(C) inserting the first module into a second a second module comprising a detector, wherein the second module is compatible with the first module and wherein the detector is capable of detecting a signal from the detection agent when associated with the capture agent at the fixed site.

[0017] In an embodiment, the first module can comprise membrane strip(s) in a cassette.

[0018] In an embodiment, the biological sample can be fresh. The biological sample can be less than 5 hours old. The biological sample can be between about 1 hour and 5 hours old. The biological sample can be between about 1 hour and 5 hours old. The biological sample can be about 1, 2, 3, 4, or 5 hours old.

[0019] In an embodiment, the complement marker can comprise C5b-9 or soluble C5b-9, or a combination thereof. The complement marker can be plasma C5b-9 (sC5b- 9), urine C5b-9 (uC5b-9), or a combination thereof.

[0020] In an embodiment, the capture agent can comprise an antibody that binds specifically to the complement marker. The complement marker can comprise C5b-9, optionally sC5b-9, uC4b-9, or a combination thereof.

[0021] In an embodiment, the detection agent can comprise a labeled second antibody that binds to the capture antibody. The labeled second antibody can be labeled with a radionuclide, fluorescent dye, chemiluminescent agent, microparticle, enzyme, colorimetric label, magnetic label, biotin, or a combination thereof.

[0022] In an embodiment, the biological sample can be urine. The biological sample can be stored at between about -20°C and 25°C. The biological sample can be stored at about -20°C, -4 °C, or 25°C.

[0023] In an embodiment, the method can be specific for sC5b-9.

[0024] In an embodiment, the method does not cross react with C7, C8, or C9.

[0025] In an embodiment, the method can be specific for fully assembled sC5b-9 complex.

[0026] In an embodiment, the method does not with partial sC5b-9 complexes.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1 depicts levels of soluble complement 5b-9 (sC5b-9; also known as the soluble membrane attack complex, or sMAC) measured in whole blood (WB) samples treated with K2EDTA (WB EDTA), fresh plasma samples treated with K2EDTA (EDTA fresh), frozen plasma samples treated with K2EDTA (EDTA frozen), and plasma samples treated with K2EDTA and then stored for 1 month, 3 months, 6 months, or 12- 13 months (EDTA IM, EDTA 3M, EDTA 6M, and EDTA 12M/13M, respectively). The samples were all BLQ (Below Level of Quantification). FIG. 1 shows that plasma sC5b-9 values increase transiently before falling during increased storage time. Plasma (EDTA) and urine samples were collected from healthy volunteers and kept on ice during transport to the lab. Whole blood (WB) was processed to plasma immediately which was tested fresh then aliquoted and frozen at 80C for retesting after 24-96hr, 1 month (IM), 3 months (3M), 6 months (6M), and 12 months (12M). Consistent with previous published and unpublished results, P-sC5b-9 was significantly elevated after handling and freeze thaw 24-96hr later (p<0.0001). This interval and sample management represents a typical reference laboratory workflow. However, P-sC5b-9 values began to decrease after 1 month in storage falling back to baseline after 3 months (p=0.9842) and below baseline by 6 months (p< 0.0001). Most samples were below limit of quantitation (BLQ) by 12 months. The data on changes in 1 cohort of individual samples and increased variability is consistent with previous reports.

[0028] FIG. 2 depicts fractional change of sC5b-9 levels (versus baseline fresh plasma levels) measured in fresh plasma samples treated with K2EDTA (EDTA fresh), frozen plasma samples treated with K2EDTA (EDTA frozen), and plasma samples treated with K2EDTA and then stored for 1 month, 3 months, 6 months, or 12-13 months (EDTA IM, EDTA 3M, EDTA 6M, and EDTA 12M/13M, respectively). Over time, the levels of sC5b-9 became less quantifiable.

[0029] FIG. 3 depicts levels of sC5b-9 measured in whole blood (WB) samples treated with P100® (WB P100; P100® contains K2EDTA and a protease inhibitor cocktali), fresh plasma samples treated with P100® (P100 fresh), frozen plasma samples treated with P100® (P100 frozen), and plasma samples treated with P100 and then stored for 1 month, 3 months, 6 months, or 12/13 months (P100 IM, P100 3M, P100 6M, and P100 12/13M, respectively). Over time, the levels of sC5b-9 became less quantifiable. The data showed that use of a specialty blood container containing proprietary protease inhibitors and stabilizers delayed the change in sC5b9 levels.

[0030] FIG. 4 depicts fractional change of sC5b-9 levels (versus baseline levels) measured in fresh plasma samples treated with P100® (P100 fresh), frozen plasma samples treated with P100® (P100 frozen), and plasma samples treated with P100® and then stored for 1 month, 3 months, 6 months, or 12-13 months (P100 IM, P100 3M, P100 6M, and P100 12M/13M, respectively). Over time, the levels of sC5b-9 became less quantifiable.

[0031] FIG. 5 depicts nanograms (ng) of urinary complement 5b-9 (uC5b-9) per milligram (mg) creatinine in fresh urine (Urine fresh), frozen urine (Urine frozen), urine stored for 1 month (Urine IM), 3 months (Urine 3M), 6 months (Urine 6M), and 12/13 months (Urine 12M/13M). Overtime, more of the urine samples were BLQ for uC5b-9 (Urine fresh: 2 BLQ; Urine frozen: 3 BLQ; Urine IM: 9 BLQ; Urine 3M: 10 BLQ; Urine 6M: 9 BLQ; Urine 12M/13M: 5 BLQ). Healthy normal donor urine (u-sC5b-9) typically has very low complement activation product levels. Baseline fresh urine values were only 3.59±3.75ng/mg creatinine creating a floor effect. However, an increasing number of samples dropped to BLQ over time.

[0032] FIG. 6 depicts fractional changes in creatinine levels (ng/mg) versus baseline levels in fresh neat urine (Urine fresh), for frozen urine (Urine frozen), 1 month old urine (Urine IM), 3 month old urine (Urine 3M), 6 month old urine (Urine 6M), and 12/13 month old urine (urine 12M/13M).ln all ot the groups (Urine frozen, Urine IM, Urine 3M, Urine 6M, and Urine 12M/13M), creatinine levels were observed above and below the baseline established by Urine fresh. This data depicts that the variability in the measured values from baseline increased significantly over time.

[0033] FIG. 7 depicts sC5b-9 (left) and creatinine (right) measured in fresh urine (Urine fresh; Creatinine fresh), frozen urine (Urine frozen; Creatinine frozen), urine stored for 1 month (Urine IM; Creatinine IM), urine stored for 3 months (Urine 3M; Creatinine 3M), urine stored for 6 months (Urine 6M; Creatinine 6M), and urine stored for 12/13 months (Urine 12M/13M; Creatinine 12M/13M). This data depicts that both sC5b9 and creatinine in urine change over time and to accurately measure or monitor changes in biomarker levels over time, it is important to measure both analytes at the same time. [0034] FIG. 8 depicts Kypha Comp Act® (single analyte system) Urine U-sC5b-9 test is specific for assembled sC5b-9 with a broad linear and dynamic range and full coverage of clinically relevant analytical values. As shown in FIG. 8(A), COMP ACT® (single analyte system) sC5b-9 tests are specific for sC5b-9 and do not cross react with C7, C8, or C9. Proteins and sC5b-9 complex purified from human serum (Comp Tech®). Similarly, FIG. 8(B) shows sC5b-9 tests are specific for fully assembled sC5b-9 complex and do not cross react with partial complexes. sC5b-6 was prepared at 1 pg/ml in 1XPBS and incubated at 37°C for 15 min. Additional components were added and incubated for 30 min each in sequence. Only fully assembled sC5b-9 generated a robust signal. FIG. 8(C) shows data with 40 healthy volunteer urine samples that were collected and analyzed. As expected, normal reference range is very low for complement activation products in urine mean +/- sem. In FIG. 8(D), to evaluate the range and clinical relevance of the assay analytical performance, aHUS patient urine was serially diluted into healthy normal donor urine (n=2, representative patient data shown). In FIG. 8(E), linear range 0 250 ng/ml was established using purified protein in buffer. In FIG. 8(F), dynamic range was evaluated over a broad range 0-10,000 ng/ml. Notably assay quantitation covers broad published values for complement mediated conditions especially aHUS without any additional dilutions or modifications. The broad range is important for aspects of clinical trials like maintaining blinding and reducing user burden by eliminating need for diluting into assay range.

[0035] FIG. 9 depicts Kypha Comp act® (single analyte system) Urine Creatinine Test Performs Comparably to Enzo® Kit and is Suitable for Urinary Complement Analyte Normalization. Creatinine tests are colorimetric enzymatic reaction assays. A creatinine assay was developed to be used on the same Comp act® reader as the C5b-9 test and to normalize urinary uC5b-9 results. The Enzo Colorimetric® detection kit chosen as the predicate assay is calibrated to National Institute of Standards and Technology (NIST) creatinine reference standards. ENZO® Product Data Sheet "Creatinine Colorimetric Detection Kit" (May 31, 2022). Standards prepared per protocol and read on both plate reader and Comp act NC test strip. Time course completed to establish stability of signal on NC solid phase and correlation with Enzo kit for the same standards. The Kypha® urine creatinine test performs comparably to the Enzo® kit.

[0036] FIG. 10 depicts that Urinary C5b-9 (U-sC5b-9) degrades within 5 hours at room temperature (about 25°C) and over three days at 4°C or -20°C. Fresh urine samples were collected from three healthy normal donors and tested immediately or kept at room temperature (about 25°C) for an additional 5 hours or kept at 4°C or -20°C for 72 hours. Testing was performed using the protocol as follows: The lateral flow immunoassay system described here was developed to enable standardized rapid analysis across sites with no complement expertise required (Schramm et al., 2016). Fresh unprocessed urine or whole blood (with one added pipetting step) are applied to each cassette and a quantitative complement activation result is delivered in 30 minutes. A separate urine creatinine test on the same reader was used to normalize urine complement results. Notably, two of these healthy volunteers had moderately elevated values for unknown reasons. (A) Mean U-sC5b-9 dropped by approximately 20% after 5 hours at room temperature (39 ng/ml to 32.67 ng/ml; p<0.05). (B) Samples stored at - 4°C for 72 hours showed a similar reduction approaching significance (p=0.0576). After 72 hr at -20°C, two samples showed a further reduction, but Donor 1 exhibited no change.

DETAILED DESCRIPTION

[0037] It is to be understood that the disclosure is not limited to the particular embodiments of the disclosure described below, as variations of the particular embodiments may be made and still fall within the scope of the appended claims. It is also to be understood that the terminology employed is for the purpose of describing particular embodiments, and is not intended to be limiting. Instead, the scope of the present disclosure will be established by the appended claims.

[0038] The singular forms "a," "an" and "the" include plural reference unless the context clearly dictates otherwise. Likewise, the term "embodiment" includes a single embodiment as well as multiple embodiments. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs.

Detection of Complement Biomarkers in Fresh Biological Fluids

[0039] Systems and methods for singleplex or multiplex assays of complement biomarkers in fresh biological fluids are described herein. It is common practice to collect blood in vessels with Ethylenediaminetetraacetic acid (EDTA) P100® (or EDTA and proteinase inhibitors). However over time, e.g., between 1-6 months, it is nearly impossible to detect complement biomarkers in plasma samples stored with EDTA and/or P100® (EDTA and proteinase inhibitors). The inventors found that the complement biomarkers (e.g., C5b-9) increase in variability over time, e.g., between 1-12 months. This makes it difficult to accurately measure (e.g., BLQ: Below Limit of Quantification) complement biomarkers in biological fluids if the sample is not fresh, e.g., over 1 month post-collection.

[0040] In a singleplex assay, the system is configured to measure a single analyte, e.g., complement biomarkers.

[0041] In a multiplex assay, the system is configured to measure a at least two or more analytes, e.g., complement biomarkers and at least an additional analyte.

[0042] A fresh biological sample can be less than 5 hours old. A fresh biological sample is between about 1 hour and 5 hours old. For example, a fresh biological sample may be about 1, 2, 3, 4, or 5 hours old, as measured from the time of collection (postcollection).

[0043] First, the assays described herein are compatible with freshly collected human blood, plasma, or urine samples. Second, the rapid assay systems and methods described herein can be clinically validated, and scaled up and broadly deployed, to allow clinical sites to collect and complete testing within a short period of time, typically within an hour, with little to no pre-ana lytical manipulation. Third, the assay systems and methods described herein are specifically adapted to monitor the on-set of com pie me nt- mediated disease and progression. As such, the systems and methods of the disclosure provide reliable and on-time information to HCPs on status of complement activity in patients, which allows for more accurate and faster selection and initiation of therapy, thereby drastically improving patient outcomes. [0044] The systems and methods described herein utilize mitigation strategies for improving access to reliable complement testing. In a first aspect, the assay methods described herein systematically eliminate many sources of variability associated with existing clinical testing methods. The methods described herein are designed to offer point-of-care rapid testing using freshly collected samples, which are then assayed within hours of collection by bringing the test to the patient.

[0045] The design and performance characteristics of the systems described herein are compatible with fresh whole blood or raw, unprocessed urine. This is based, partly, on leveraging the features of lateral flow technology to the specific sample matrix (e.g, whole blood or fresh urine) and the conventional reagents (eg., antibodies) for detection of complement markers present in such matrices.

[0046] Application of the systems described herein into the workflow eliminates the need for external fluids, pumps, and/or wash buffers. Furthermore, the calibration curve for each analyte is pre-established and then bar-coded onto each lateral flow cassette, which eliminates pre-analytical variability and allows for rapid and accurate point-of-care quantitation of each analyte of interest. Accordingly, the systems and methods described herein allow for broad deployment as a point-of-care test as they eliminate or reduce the need for specialty trained technicians and/or sophisticated laboratory equipment.

[0047] Furthermore, the assay methods described herein bypass the myriad and unpredictable nature of existing workflows, which are partially caused by inherent limitations associated with the processing, freezing, transportation, reprocessing, and delayed testing of biological samples.

Anti-C5b Antibodies

[0048] Antibodies that bind to C5b as well as methods for making such antibodies are known in the art. Commercially available anti C5b antibodies are available from a number of vendors including, eg., Hycult Biotechnology (catalogue number: HM2080; clone 568) and ABCAM®. (ab46151 or ab46168).

[0049] The biomarkers may be detected using an array. For example, the array may be a protein chip where each address of the array is a well of an assay plate. Each address of the array may be a particle (eg., a bead) having immobilized thereupon a binding agent. [0050] Measuring protein expression levels in a biological sample may be performed by any suitable method. See, e.g., Greenfield (Ed.) (2014) "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. In general, protein levels are determined by contacting a biological sample obtained from a subject with binding agents for the biomarker proteins; detecting, in the sample {e.g., the biological fluid), the levels of one or more of the biomarker proteins that bind to the binding agents; and comparing the levels of one or more of the biomarker proteins in the sample with the levels of the corresponding protein biomarkers in a control sample {e.g., a normal sample). In certain embodiments, a suitable binding agent is a ribosome, with or without a peptide component, an RNA molecule, or a polypeptide {e.g., a polypeptide that comprises a polypeptide sequence of a protein marker, a peptide variant thereof, or a non-peptide mimetic of such a sequence).

[0051] Suitable binding agents also include an antibody specific for a biomarker protein described herein. Suitable antibodies for use in the methods of the present invention include monoclonal and polyclonal antibodies and antigen-binding fragments {e.g., Fab fragments or scFvs) of antibodies. Antibodies, including monoclonal and polyclonal antibodies, fragments and chimeras, may be prepared using methods known in the art. Antibodies to be used in the methods of the invention can be purified by methods well known in the art. Greenfield (Ed.) (2014) "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. Antibodies may also be obtained from commercial sources.

[0052] The binding agent is directly or indirectly labeled with a detectable moiety. The role of a detectable agent is to facilitate the detection step of the diagnostic method by allowing visualization of the complex formed by binding of the binding agent to the protein marker (or fragment thereof). The detectable agent can be selected such that it generates a signal that can be measured and whose intensity is related (preferably proportional) to the amount of protein marker present in the sample being analyzed. Methods for labeling biological molecules such as polypeptides and antibodies are well-known in the art. Any of a wide variety of detectable agents can be used in the practice of the present invention. Suitable detectable agents include, but are not limited to: various ligands, radionuclides, fluorescent dyes, chemiluminescent agents, microparticles {e.g., quantum dots, nanocrystals, phosphors), enzymes {e.g., those used in an ELISA, e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), colorimetric labels, magnetic labels, and biotin, digoxigenin or other haptens and proteins for which antisera or monoclonal antibodies are available. [0053] The binding agents (e.g., antibodies) may be immobilized on a carrier or support (e.g., a bead, a magnetic particle, a latex particle, a microtiter plate well, a cuvette, or other reaction vessel). Examples of suitable carrier or support materials include agarose, cellulose, nitrocellulose, dextran, Sephadex®, Sepharose®, liposomes, carboxymethyl cellulose, polyacrylamides, polystyrene, gabbros, filter paper, magnetite, ion-exchange resin, plastic film, plastic tube, glass, polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, or combinations thereof. Binding agents may be indirectly immobilized using second binding agents specific for the first binding agents (e.g., mouse antibodies specific for the protein markers may be immobilized using sheep anti-mouse IgG Fc fragment specific antibody coated on the carrier or support).

[0054] Protein expression levels in a biological sample may be determined using immunoassays. Examples of such assays are time resolved fluorescence immunoassays (TR-FIA), radioimmunoassays, enzyme immunoassays (e.g., ELISA), immunofluorescence immunoprecipitation, latex agglutination, hemagglutination, Western blot, and histochemical tests, which are conventional methods well-known in the art. Methods of detection and quantification of the signal generated by the complex formed by binding of the binding agent with the protein marker will depend on the nature of the assay and of the detectable moiety (e.g., fluorescent moiety).

[0055] In an example, the presence or amount of protein expression of a gene (e.g., C5b-9) can be determined using a Western blotting technique. For example, a lysate can be prepared from a biological sample, or the biological sample (e.g., biological fluid) itself, can be contacted with Laemmli buffer and subjected to sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE-resolved proteins, separated by size, can then be transferred to a filter membrane (e.g., nitrocellulose) and subjected to immunoblotting techniques using a detectably-labeled antibody specific to the protein of interest. The presence or amount of bound detectably-labeled antibody indicates the presence or amount of protein in the biological sample.

[0056] In an example, an immunoassay can be used for detecting and/or measuring the protein expression of a biomarker protein (e.g., C5b-9). As above, for the purposes of detection, an immunoassay can be performed with an antibody that bears a detection moiety (e.g., a fluorescent agent or enzyme). Proteins from a biological sample can be conjugated directly to a solid-phase matrix (e.g., a multi-well assay plate, nitrocellulose, agarose, Sepharose®, encoded particles, or magnetic beads) or it can be conjugated to a first member of a specific binding pair (e.g., biotin or streptavidin) that attaches to a solid-phase matrix upon binding to a second member of the specific binding pair (e.g., streptavidin or biotin). Such attachment to a solid-phase matrix allows the proteins to be purified away from other interfering or irrelevant components of the biological sample prior to contact with the detection antibody and also allows for subsequent washing of unbound antibody. Here, as above, the presence or amount of bound detectably- labeled antibody indicates the presence or amount of protein in the biological sample. [0057] Alternatively, the protein expression levels may be determined using mass spectrometry based methods or image-based methods known in the art for the detection of proteins. Other suitable methods include 2D-gel electrophoresis, proteomics-based methods such as the identification of individual proteins recovered from the gel (e.g., by mass spectrometry or N-terminal sequencing) and/or bioinformatics.

[0058] Methods for detecting or measuring protein expression can, optionally, be performed in formats that allow for rapid preparation, processing, and analysis of multiple samples. This can be, for example, in multi-well assay plates (e.g., 96 wells or 386 wells) or arrays (e.g., protein chips). Stock solutions for various reagents can be provided manually or robotically, and subsequent sample preparation, pipetting, diluting, mixing, distribution, washing, incubating (e.g., hybridization), sample readout, data collection (optical data) and/or analysis (computer aided image analysis) can be done robotically using commercially available analysis software, robotics, and detection instrumentation capable of detecting the signal generated from the assay. Examples of such detectors include, but are not limited to, spectrophotometers, luminometers, fluorimeters, and devices that measure radioisotope decay.

Diagnostic A ntibodies

[0059] The antibody or antigen-binding fragment thereof may be selected from the group consisting of a humanized antibody, a recombinant antibody, a diabody, a chimerized or chimeric antibody, a monoclonal antibody, a deimmunized antibody, a fully human antibody, a single chain antibody, an Fv fragment, an Fd fragment, an Fab fragment, an Fab' fragment, an F(ab')2 fragment, or a combination thereof.

Monoclonal Antibodies

[0060] The monoclonal antibodies disclosed herein can be of any isotype. The monoclonal antibody can be, for example, an IgM or an IgG antibody, such as IgGl or an lgG2. The class of an antibody that immunospecifically binds C5b-9 can be switched with another (for example, IgG can be switched to IgM), according to well-known procedures. Class switching can also be used to convert one IgG subclass to another, such as from IgGl to lgG2.

[0061] The antibodies of the present invention may be monovalent, bivalent, trivalent or multivalent. For example, monovalent scFvs can be multimerized either chemically or by association with another protein or substance. An scFv that is fused to a hexahistidine tag or a Flag tag can be multimerized using Ni-NTA agarose (Qiagen) or using anti-Flag antibodies (Stratagene, Inc.).

[0062] The antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of C5b-9, or fragment thereof, and a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt et al, J. Immunol. 147:60-69 (1991); U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al. i Immunol. 148:1547-1553 (1992).

Methods for Producing Antibodies

[0063] Antibodies that may be used in the methods described herein (including scFvs and other molecules comprising, or alternatively consisting of antibody fragments or variants of the invention) can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques. Greenfield (Ed.) (2014) "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y.

[0064] Single chain Fvs (scFvs) that immunospecifically bind sC5b-9 or fragment thereof may be generated using phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In particular, DNA sequences encoding VH and VL domains are amplified from animal cDNA libraries e.g., human or murine cDNA libraries of lymphoid tissues) or synthetic cDNA libraries. The DNA encoding the VH and VL domains are joined together by an scFv linker by PCR and cloned into a phagemid vector e.g., p CANT AB 6 or pComb 3 HSS). The vector is electroporated in E. coll an the £ coll is infected with helper phage. Phage used in these methods are typically filamentous phage including fd and M13 and the VH and VL domains are usually recombinantly fused to either the phage gene III or gene VIII. Phage expressing an antigen binding domain that binds to an antigen of interest {e.g., sC5b-9 or fragment thereof) can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Examples of phage display methods that can be used to make the antibodies of the present invention include, but are not limited to, those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al. Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al. Advances in Immunology 57:191-280(1994); WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; W097/13844; and U.S. Patent Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108.

[0065] As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human or humanized antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described below. Techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science 240:1041-1043 (1988).

[0066] To generate whole antibodies, PCR primers including VH or VL nucleotide sequences, a restriction site, and a flanking sequence to protect the restriction site can be used to amplify the VH or VL sequences in scFv clones. Utilizing cloning techniques known to those of skill in the art, the PCR amplified VH domains can be cloned into vectors expressing a VH constant region, e.g., the human gamma 4 constant region, and the PCR amplified VL domains can be cloned into vectors expressing a VL constant region, e.g., human kappa or lambda constant regions. Preferably, the vectors for expressing the VH or VL domains comprise a promoter suitable to direct expression of the heavy and light chains in the chosen expression system, a secretion signal, a cloning site for the immunoglobulin variable domain, immunoglobulin constant domains, and a selection marker such as neomycin. The VH and VL domains may also be cloned into one vector expressing the necessary constant regions. The heavy chain conversion vectors and light chain conversion vectors are then co-transfected into cell lines to generate stable or transient cell lines that express full-length antibodies, e.g., IgG, using techniques known to those of skill in the art.

[0067] Once an antibody that may be used in the methods described herein (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) has been chemically synthesized or recombinantly expressed, it may be purified by any method known in the art for purification of an immunoglobulin molecule, or more generally, a protein molecule, such as, for example, by chromatography e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Further, the antibodies that may be used in the methods described herein may be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification.

[0068] Methods for recombinantly producing antibodies that may be used in the methods described herein are well known to those of ordinary skill in the art. The antibodies may also be produced by constructing, using conventional techniques well known to those of ordinary skill in the art, an expression vector comprising an operon and a DNA sequence encoding the antibodies. Furthermore, the invention relates to vectors, especially plasmids, cosmids, viruses, bacteriophages and other vectors common in genetic engineering, which may comprise the above-mentioned nucleic acid molecules. The nucleic acid molecules contained in the vectors may be linked to regulatory elements that ensure the transcription in prokaryotic and eukaryotic cells. [0069] Vectors comprise elements that facilitate manipulation for the expression of a foreign protein within the target host cell. Conveniently, manipulation of sequences and production of DNA for transformation is first performed in a bacterial host (e.g., £ coli) and usually vectors will include sequences to facilitate such manipulations, including a bacterial origin of replication and appropriate bacterial selection marker. Selection markers encode proteins necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that confer resistance to antibiotics or other toxins, complement auxotrophic deficiencies, or supply critical nutrients not available from complex media. Exemplary vectors and methods for transformation of yeast are described in the art. See, e.g., Burke, et al (2000) Methods in Yeast Genetics Cold Spring Harbor Laboratory Press. [0070] The polynucleotide coding the antibodies may be operably linked to transcriptional and translational regulatory sequences that provide for expression of the polypeptide in yeast cells. These vector components may include, but are not limited to, one or more of the following: an enhancer element, a promoter, and a transcription termination sequence. Sequences for the secretion of the polypeptide may also be included e.g., a signal sequence).

[0071] Nucleic acids are "operably linked" when placed into a functional relationship with another nucleic acid sequence. For example, DNA for a signal sequence is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. Generally, "operably linked" refers broadly to contiguous linked DNA sequences, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous.

[0072] Promoters are untranslated sequences located upstream (5') to the start codon of a structural gene (generally within about 100 to 1000 bp) that control the transcription and translation of particular nucleic acid sequences to which they are operably linked. Such promoters fall into several classes: inducible, constitutive, and repressible promoters (e.g., that increase levels of transcription in response to absence of a repressor). Inducible promoters may initiate increased levels of transcription from DNA under their control in response to some change in culture conditions (eg., the presence or absence of a nutrient or a change in temperature.)

[0073] The expression vectors are transfected into a host cell by convention techniques well known to those of ordinary skill in the art to produce a transfected host cell, said transfected host cell cultured by conventional techniques well known to those of ordinary skill in the art to produce said antibodies.

[0074] The host cells used to express the anti-sC5b-9 antibodies may be either a bacterial cell such as £ coll, yeast {eg., S. cerevisiae), or a eukaryotic cell (eg., a mammalian cell line). A mammalian cell of a well-defined type for this purpose, such as a myeloma cell, 3T3, HeLa, C6A2780, Vero, MOCK II, a Chinese hamster ovary (CHO), Sf9, Sf21, COS, NSO, or HEK293 cell line may be used.

[0075] The general methods by which the vectors may be constructed, transfection methods required to produce the host cell and culturing methods required to produce the antibodies, and fragments thereof, from said host cells all include conventional techniques. Although preferably the cell line used to produce the antibodies is a mammalian cell line, any other suitable cell line, such as a bacterial cell line such as an £ coll-derived bacterial strain, or a yeast cell line, may be used.

[0076] Similarly, once produced the antibodies may be purified according to standard procedures in the art, such as for example cross-flow filtration, ammonium sulphate precipitation, and affinity column chromatography.

[0077] Antibodies binding to biomarkers may be screened using any known methods, eg., binding assays. In a representative method, target biomarkers or antigenic epitope thereof are expressed in standard cells and antibodies are panned using selection techniques known in the art. Antibodies may be ranked, eg., based on binding affinities, for example, a dissociation constant (Kd) of at least IO^-5 M; preferably IO^-8 M; and especially 10 ¹⁰ M. Here, Kd values may be determined using standard binding assays.

[0078] The various embodiments of the present disclosure are described in detail in the following non-limiting and representative examples.

EXAMPLES

EXAMPLE 1

[0079] Plasma (sC5b-9) and urine (uC5b-9) were evaluated for stability in realistic short-term and long-term storage scenarios. Blood (processed to EDTA plasma) or urine were collected and run as described in detail herein.

[0080] Blood is extracted from the Individual, placed in a tube containing EDTA, which centrifuged to remove cells and debris, and the supernatant is the plasma that is tested. This plasma is referred to as EDTA plasma. Briefly, either 20 pl plasma mixed in diluent or 100 pl undiluted urine was applied to a single-use test cassette, developed for 30 min, and analyzed.

[0081] In Experiment 1, urine samples with slightly elevated uC5b-9 were tested immediately and kept at room temp (RT)(about 25°C) for 5 hours (hr) to replicate a common clinical scenario. Mean urine C5b-9 dropped approximately 20% by 5 hours at RT (39 ng/ml to 32.67ng/ml; p<0.05).

[0082] In Experiment 2, EDTA blood samples were collected from 21 healthy donors (HD), and tested immediately or processed to plasma, aliquoted and frozen for retesting after 24-96 hour, 1 month (mo), 3 months, 6 months, and 12 months.

Results

[0083] Compared to sC5b-9 levels in fresh blood (139±57.3 ng/ml), sC5b-9 was significantly elevated in fresh EDTA plasma (173 ±92.9 ng/ml) (values reflecting mean ± SEM; p<0.01 for statistical significance) and further elevated in frozen EDTA plasma (230±90.6 ng/ml) (p<0.01). However, sC5b9 levels decreased after storage at -80°C. For instance, sC5b-9 levels (mean ± SEM) in EDTA plasma after 1 month, 3 months, 6 months, and 12 months of storage at -80 °C were 214±92.5 ng/ml (p<0.01), 180±92.3 ng/ml, 125±50.5 ng/ml (p<0.01), and 98±0 ng/ml, respectively. Multiple samples were below the limit of quantitation (BLQ) by 12 months and excluded.

[0084] These results confirm the pre-analytical instability of complement proteins over time and in two different biological matrices, namely, fresh blood and urine.

Further, these results validate the employability of the systems and methods of the present disclosure in analyzing and/or monitoring complement biomarkers in these freshly collected, complex sample matrices.

EXAMPLE 2

Short- and long-term blood and urine storage: implications for complement C5b-9 research and diagnostic development

[0085] The complement system evolved to respond quickly to invading pathogens, damaged cells, and debris. Unfortunately, similarly rapid in vitro activation or degradation can confound complement analysis. Substantial efforts over almost 40 years have improved best practices among complement laboratories by identifying specific sources of variability: time, temperature, coagulation, agitation, freeze/thaw, ambient air, immunoassay incubation duration, and other factors (Mollnes, et aL, 1988; Schramm et al., 2016). However, three major issues remain. First, scaling standardized methodologies to deliver consistency across multiple laboratories with or without on-site complement expertise has proven difficult and may explain data from the recent International Complement Society External Quality Assessment (Frazer-Abel, et al., 2021; Figure 1). Second, best practices have been derived from healthy normal donor studies, but disease populations may have different preanalytical requirements based on antigen/antibody complexes, genetics, autoantibodies targeting regulators, or other factors likely to impact complement. Finally, complement analytes are unstable at different rates (Mollnes, 1985), making comparisons across analytes and pathways difficult to interpret. Achieving broad utility of complement diagnostics will require ubiquitous access to complement test results, uniformity across sites, and reliability across disease indications.

[0086] Despite the impact of complement-targeted therapeutics, accurate analysis of complement proteins remains a challenge due to ex vivo instability of this complex enzymatic cascade and further confounded by factors including processing time, temperature, coagulation, agitation, freeze/thaw, ambient air, short and long-term storage conditions. Two mitigation strategies can deliver widespread access to reliable complement analysis beyond specialized complement laboratories: (1) standardize the myriad and unpredictable preanalytical factors into a single micromanaged workflow across clinical sites with varying expertise, or (2) systematically eliminate sources of variability with point-of-care rapid testing of a fresh sample within hours of collection. [0087] The inventors evaluated two biological matrices for terminal complement stability in realistic short- and long-term storage scenarios to assess mitigation strategy Option 2. Briefly, healthy donor blood processed to EDTA plasma (P-sC5b-9) or urine (U- sC5b-9) were freshly collected and either 20 pl plasma mixed in diluent or 100 pl undiluted urine was applied to single-use lateral flow test cassettes, developed for 30 minutes, and analyzed. In Experiment 1, urine samples were collected and tested immediately and kept at room temp (RT, e.g., about 25°C) for 5 hours to replicate a common clinical scenario. Mean U-sC5b-9 dropped -20% by 5 hours (39ng/ml to 32.67ng/ml; p<0.05). In Experiment 2, EDTA blood samples were collected and tested immediately or processed to plasma, aliquoted and frozen for retesting after 24-96 hours, 1 months, 3 months, 6 months, and 12 months. Compared to fresh blood (139±57.3ng/ml), P-sC5b-9 was significantly elevated in fresh EDTA plasma (173±92.9)* and further elevated in frozen EDTA plasma (230±90.6)* but decreased after storage time at -80C (Imo 214±92.5*, 3mo 180±92.3, 6mo 125±50.5*, 12mo 98±0). Multiple samples were below the limit of quantitation by 12 months and excluded. These preliminary results quantify the pre-ana lytical instability of complement proteins over time and in two different biological matrices. Efforts to validate and extend this healthy donor fresh vs. frozen assessment in various diseased patient populations are ongoing and will provide important additional insight on defining best practices for accurate assessment of complement activity in clinical samples. *=means±sd, p<0.01 [0088] The methods described herein can be replicated on a larger validation cohort and disease populations, impacting current and future diagnostic applications in multiple ways: (A) Fresh Sample Testing Could Improve Laboratory Standardization: Baseline whole blood and plasma sC5b-9 values were within published normal reference ranges. However, these values fluctuated significantly as a function of time in storage. Because clinical laboratories use different sources of healthy donor samples to set normal reference ranges, time in storage could account for (1) the breadth of the ranges in each lab and (2) differences between labs, either of which could impact clinical interpretation (Bu et al., 2015); (B) Fresh Sample Testing Could Renew and Refine Interest in Certain Clinical Conditions: Ongoing follow up studies will shed light on disease populations, particularly with complement-mediated kidney conditions and urine testing. However, if traditional biobanking methods increase variability and underestimate complement values in old samples, the field could be underestimating the value of complement biomarkers in some conditions; (C) Fresh, unprocessed urine testing is noninvasive and could be done anywhere with little to no additional resources; and (D) Optionally, methods for validating and extending the comparative methods described herein can be employed downstream to the above workflow in samples obtained from various diseased patient populations, which may be used in further development of best practices for accurate testing of clinical samples.

[0089] All references cited in this specification are herein incorporated by reference as though each reference was specifically and individually indicated to be incorporated by reference. The citation of any reference is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such reference by virtue of prior invention. [0090] It will be understood that each of the elements described above, or two or more together may also find a useful application in other types of methods differing from the type described above. Without further analysis, the foregoing will so fully reveal the gist of the present disclosure that others can, by applying current knowledge, readily adapt it for various applications without omitting features that, from the standpoint of prior art, fairly constitute essential characteristics of the generic or specific aspects of this disclosure set forth in the appended claims. The foregoing embodiments are presented by way of example only; the scope of the present disclosure is to be limited only by the following claims.

Claims

CLAIMS:

1. A system for point-of-care rapid assay for complement markers in a freshly collected biological sample comprising:

(a) a first module configured for lateral-flow detection of complement markers in a biological sample, said first module comprising a plurality of agents comprising

(1) a capture agent located at fixed site in the first module, wherein the capture agent captures the complement marker at the fixed site; and

(2) a detection agent for detecting the captured complement marker of (1) at the fixed site;

(b) a second module comprising a detector, wherein the second module is compatible with the first module of (a), and wherein the detector is capable of detecting a signal from the detection agent that is specifically associated with the capture agent at the fixed site; and

(c) optionally, a dispenser for dispensing a fixed amount of the biological sample into the first module.

2. The system of claim 1 wherein the first module comprises membrane strip(s) in a cassette.

3. The system of claim 1 or 2, wherein the biological sample is fresh.

4. The system of claim 3, wherein the biological sample is less than 5 hours old.

5. The system of claim 3 or 4, wherein the biological sample is between about 1 hour and 5 hours old.

6. The system of any one of claims 3-5, wherein the biological sample is between about 1 hour and 5 hours old.

7. The system of any one of claims 3-6, wherein the biological sample is about 1, 2, 3, 4, or 5 hours old.

8. The system of any one of claims 1-7, wherein the complement marker comprises C5b-9 or soluble C5b-9, or a combination thereof.

9. The system of any one of claims 1-8, wherein the complement marker is plasma C5b- 9 (sC5b-9), urine C5b-9 (uC5b-9), or a combination thereof.

10.The system of any one of claims 1-9, wherein the capture agent comprises an antibody that binds specifically to the complement marker. The system of claim 10, wherein the complement marker comprises C5b-9, optionally sC5b-9, uC4b-9, or a combination thereof. The system of any one of claims 1-11, wherein the detection agent comprises a labeled second antibody that binds to the capture antibody. The system of claim 12, wherein the labeled second antibody is labeled with a radionuclide, fluorescent dye, chemiluminescent agent, microparticle, enzyme, colorimetric label, magnetic label, biotin, or a combination thereof. The system of any one of claims 1-13, wherein the biological sample is urine, blood, plasma, optionally EDTA plasma, serum, or a combination thereof. The system of any one of claims 1-14, wherein the biological sample is urine. The system of any one of claims 1-15, wherein the biological sample is stored at between about -20°C and 25°C. The system of any one of claims 1-16, wherein the biological sample is stored at about -20°C, -4°C, or 25°C. The system of any one of claims 1-17, wherein the system is a singleplex system.The system of any one of claims 1-18, wherein the system is a multiplex system.The system of any one of claims 1-19, wherein the fixed amount is between about 1 and 250 ml_. The system of claim 20, wherein the fixed amount is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 mL. The system of claim 20 or 21, wherein the fixed amount is about 100 mL. A method for rapidly detecting complement markers in a biological sample at a point-of-care setting comprising

(A) dispensing a fixed amount of the biological sample into a system comprising a first module configured for lateral-flow detection of complement markers present in a biological sample, said first module comprising a plurality of agents comprising a capture agent located at fixed site in the cassette, wherein the capture agent captures the complement marker at the fixed site; and

(B) incubate the biological sample in the first module for between 1 and 60 minutes; (C) inserting the first module into a second a second module comprising a detector, wherein the second module is compatible with the first module and wherein the detector is capable of detecting a signal from the detection agent when associated with the capture agent at the fixed site. The method of claim 23 wherein the first module comprises membrane strip(s) in a cassette. The method of claim 23 or 24, wherein the biological sample is fresh. The method of claim 25, wherein the biological sample is less than 5 hours old.The method of claim 25 or 26, wherein the biological sample is between about 1 hour and 5 hours old. The method of any one of claims 23-27, wherein the biological sample is between about 1 hour and 5 hours old. The method of any one of claims 23-28, wherein the biological sample is about 1, 2, 3, 4, or 5 hours old. The method of any one of claims 23-29, wherein the complement marker comprises C5b-9 or soluble C5b-9, or a combination thereof. The method of any one of claims 23-30, wherein the complement marker is plasma C5b-9 (sC5b-9), urine C5b-9 (uC5b-9), or a combination thereof. The method of any one of claims 23-31, wherein the capture agent comprises an antibody that binds specifically to the complement marker. The method of claim 32, wherein the complement marker comprises C5b-9, optionally sC5b-9, uC4b-9, or a combination thereof. The method of any one of claims 23-33, wherein the detection agent comprises a labeled second antibody that binds to the capture antibody. The method of claim 34, wherein the labeled second antibody is labeled with a radionuclide, fluorescent dye, chemiluminescent agent, microparticle, enzyme, colorimetric label, magnetic label, biotin, or a combination thereof. The method of any one of claims 23-35, wherein the biological sample is urine, blood, plasma, optionally EDTA plasma, serum, or a combination thereof. The method of any one of claims 23-36, wherein the biological sample is urine.The method of any one of claims 23-37, wherein the biological sample is stored at between about -20°C and 25°C. The method of any one of claims 23-38, wherein the biological sample is stored at about -20°C, -4°C, or 25°C. The method of any one of claims 23-39, wherein the method is specific for sC5b-9.The method of any one of claims 23-40, wherein the method does not cross react with C7, C8, or C9. The method of any one of claims 23-41, wherein the method is specific for fully assembled sC5b-9 complex. The method of any one of claims 23-42, wherein the method does not with partial sC5b-9 complexes. The method of any one of claims 23-43, wherein the fixed amount is between about 1 and 250 mL. The method of claim 44, wherein the fixed amount is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 mL. The method of claim 44 or 45, wherein the fixed amount is about 100 mL.