Detection of antibodies using antigens

Abstract

The invention relates to detection of antibodies using antigens. Example complexes include a solid support and at least two different types of antigens attached to the solid support. The complexes include a carrier protein attached to a solid support. At least one different type of antigen is attached to the carrier protein. At least one different type of antigen is attached to the solid support by a carrier protein.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 538,042, entitled “Detecting Antibodies Using Antigens,” filed on September 12, 2023. U.S. Provisional Application No. 63 / 538,042 is incorporated herein by reference. Technical Field

[0003] This specification refers to the use of antigens to detect antibodies. Background of the Invention

[0005] Antinuclear antibodies (ANA) are autoantibodies that bind to the contents of the cell nucleus and are a prominent feature of autoimmune connective tissue diseases. An example of an ANA test is the detection of ANA in a subject's biological fluids such as blood or body tissue. In some cases, autoantibodies targeting cytoplasmic antigens such as Jo-1 or ribosomal P protein are detected and reported when performing an indirect immunofluorescence (IIF) assay using HEp-2 cells. Therefore, it is recommended that the term be changed to anticellular antibodies or "ACA". However, the term ANA is still used in the literature, so ANA and ACA are used interchangeably in this article.

[0006] A positive ANA test result means that there are autoantibodies in the subject, which may be a sign of disease or other conditions. Multi-analyte assays / detection systems that detect multiple types of ANA use a single antigen immobilized on a single solid support (such as particles or microbeads). Antibodies in the sample bind to the antigen, and the results are based on detection such as fluorescence of the bound antigen / antibody. The amount of fluorescence detected is proportional to the amount of antibody contained in the sample. A relatively large amount of antibodies indicates a higher probability of a disease associated with the target antigen.

[0007] Compared to IIF assays on cell substrates (e.g., HEp-2 cells), some multi-analyte assays / detection systems of the above type do not include enough antigen to capture enough antibodies, making detection of diseases and other conditions challenging in some cases. Additionally, depending on the disease or condition, multiple beads need to be used in these assays, which can lead to inefficient workflows because each bead needs to be detected and the combined signal analyzed to determine the disease or condition. Calibration using multiple beads can also be time-consuming, and analysis can require complex computational assistance and high technical skills. SUMMARY OF THE INVENTION

[0009] An exemplary complex includes a solid support and at least two different types of antigens attached to the solid support. The complex may include one or more of the following features, alone or in combination.

[0010] The complex may include a carrier attached to a solid support. At least one of the different types of antigens may be attached to the carrier. At least one of the different types of antigens may be attached to the solid support via the carrier. The complex may include a linker for attaching at least one of the different types of antigens to the carrier. The linker may include a polyethylene glycol (PEG) spacer or biotin.

[0011] The carrier may include at least one of streptavidin, polymeric protein, polymeric streptavidin and biotin, bovine serum albumin (BSA), human serum albumin (HSA), polyethylene glycol (PEG) spacer attachment, or adipic acid dihydrazide (ADH).

[0012] The composite material may include a plurality of carriers. Each carrier may be attached to a solid support. Each different type of antigen may be attached to a carrier of the plurality of carriers. Each different type of antigen may be attached to the solid support via a respective one of the plurality of carriers.

[0013] The complex may include a carrier attached to a solid support. Each different type of antigen may be attached to the carrier. Each different type of antigen may be attached to the solid support via the carrier.

[0014] At least two different types of antigens may include markers for a group of subject diseases or conditions. At least one different type of antigen may be part of a cell extract attached to a solid support. The first different type of antigen may include a purified antigen and the second different type of antigen may be part of a cell extract.

[0015] The at least two different types of antigens may include at least two of the following antigens: Scl-70, centromere, dsDNA, Ro52, Ro60, SS-B, Ribo-P, DFS-70, EJ, Sp100, Gp210, actin, AMA-M2, AMA-M2 / MIT3, TIF1-γ, NXP-2, SAE, PL-7, PL-12, OJ, HMGCR, SRP, Mi-2, MDA-5, RNA helicase, Ki / SL, Sm / RNP, SS-A, PM / Scl, RNA Pol III, Th / To, fibronectin, Sm, Ku, Jo-1, RNP, BICD2, HK, KL, LC1, LKM-1, SLA, SS-A / Ro52, Scl-70, Ku, centromere protein, U11 / U12 RNP, hexokinase-1 or Kelch-12.

[0016] At least one different type of antigen may bind to the antinuclear antibody (ANA). The complex may include a label associated with a solid support. The label may be associated with a disease, condition, or analyte that can be detected by at least two different antigens.

[0017] An example kit includes: a first solid support including a first antigen attached thereto, wherein the first antigen includes at least two different types of antigens; a second solid support including at least one second antigen attached thereto, wherein at least one second antigen is different from each first antigen. One or more first antigens or at least one second antigen may include a portion of a purified antigen or cell extract. An example kit may include one or more of the following features alone or in combination.

[0018] The first solid support may include a first marker and the second solid support may include a second marker. The first marker may be different from the second marker. The first antigen may include a marker for a first disease or condition. The first marker may be associated with the first disease or condition. The at least one second antigen may include a marker for a second disease or condition. The second marker may be associated with a second disease or condition.

[0019] The first antigen may include a first label that generates a signal in response to binding with a corresponding antibody. The at least one second antigen may include at least one second label that generates a signal in response to binding with at least one corresponding antibody. The first label and the at least one second label may be of the same type or of different types.

[0020] An exemplary method includes contacting a biological sample from a subject with a complex, the complex including a solid support and at least two different types of antigens attached to the solid support; detecting the presence of an antibody bound to the complex in the biological sample, wherein the presence of the antibody bound to the complex indicates the presence of a disease. The method may include one or more of the following features, alone or in combination.

[0021] The method may include detecting a solid support based on a label on the solid support. The label may be associated with a disease. Detection may include detecting signals from labels on at least two different types of antigens.

[0022] The complex may include one of a plurality of complexes contacted with the biological sample. Each complex may have a different configuration associated with a different disease. Each of the plurality of complexes may include a respective solid support; and one or more antigens attached to the respective solid support.

[0023] At least a portion of the systems and techniques (including determinations) described in this specification may be controlled by executing instructions stored on one or more non-transitory machine-readable storage media on one or more processing devices. Examples of non-transitory machine-readable storage media include read-only memory, optical disk drives, storage disk drives, and random access memory. At least a portion of the systems and techniques (including determinations) described in this specification may be controlled using a computing system comprising one or more processing devices and a memory storing instructions that may be executed by one or more processing devices to perform various control operations. The compositions, devices, systems, and techniques (including determinations) described in this specification may be configured by design, construction, formulation, arrangement, placement, programming, operation, activation, deactivation, and / or control.

[0024] Two or more features described in this specification (including this abstract) may be combined to form embodiments not specifically described in this specification.

[0025] The details of one or more embodiments are set forth in the accompanying drawings and the description below. Other features and advantages will be apparent from the description, drawings, and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1A , 1B and 1C show a table of antigens and their accession numbers.

[0027] Figure 2 is a diagram conceptually showing an example of a solid support-antigen complex comprising one carrier and ten antigens.

[0028] Figure 3 is a diagram conceptually showing an example of a solid support-antigen complex comprising an antigen attached to the support via a linker.

[0029] Figure 4 is a diagram conceptually showing four example combinations of antigens on four different solid supports.

[0030] Figure 5 is a diagram conceptually showing four example combinations of antigens on four different solid supports.

[0031] Figure 6 is a diagram conceptually showing four example combinations of antigens on four different solid supports.

[0032] Figure 7 Photographs showing three examples of autoantibodies that can produce positive indirect immunofluorescence results but are not commonly used in the differential diagnosis of connective tissue diseases (CTDs).

[0033] Figure 8 is a flow chart showing an example of operations involved in a process (assay) of determining the likelihood that a subject has a certain disease, condition, or analyte using a solid support-antigen complex.

[0034] Fig. 9 is a graph showing an example of a receptor operating characteristic curve (ROC) analysis depicting the ability to detect autoantibodies to individual autoantigens (Ro52, Ro60, RNP, La / SS-B) using a mixture of antigens coupled to a single bead.

[0035] Fig.10 is a bar graph showing the reactivity of individual antigens relative to the reactivity of individual antigens coupled to beads.

[0036] Fig.11 is a graph showing the results of a clinical study using ten antigens on a solid support and using an FDA (Food and Drug Administration) approved device to screen biological samples for diseases.

[0037] Fig.12 is a graph showing how the results of a clinical study using ten antigens to screen for disease compare to test results obtained using previous technology and thousands of samples.

[0038] Fig.13 is a graph showing the results of a clinical study using ten antigens on a solid support to screen for a disease and how these results compare to those obtained using previous technology and thousands of samples.

[0039] Like reference numerals refer to like elements. DETAILED DESCRIPTION OF THE INVENTION

[0041] Described herein are examples of multi-analyte assays for screening for antibodies such as antinuclear antibodies (ANA) / anticellular antibodies (ACA) in biological samples. In some embodiments, the multi-analyte assay is configured to cover more analytes than the ANA solid phase assay for screening ANA and approximate the broader antibody coverage detected by the ANA HEp-2 cell assay.

[0042] Example antigens are molecules, substances, foreign particulate matter, or allergens that can bind to specific antibodies. Antigens can be chemically synthesized, recombinantly synthesized, or isolated from natural sources.

[0043] Antibodies include binding agents configured to specifically bind to an antigen. Example antibodies include polypeptide products of B cells or their recombinant equivalents, which can bind to specific molecular antigens and include two heavy chains and two light chains. Each amino terminal portion of each chain includes a variable region that confers binding specificity. Antibodies can include full-length antibodies and functional fragments such as these. When used for antibodies, example functional fragments include a portion of an antibody, including, for example, a heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment was derived. These functional fragments may include Fd, Fv, Fab, F(ab'), F(ab)2, F(ab')2, single-chain Fv (scFv), diabodies, triabodies, tetrabodies and minibodies, etc., as described in, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989); Myers (ed.), Molec. Biology and Biotechnology: A Comprehensive Desk Reference, New York: VCH Publisher, Inc.; Huston et al, Cell Biophysics, 22: 189-224 (1993); Pluckthun and Skerra, Meth. Enzymol., 178: 497-515 (1989); and Day, ED, Advanced Immunochemistry, Second Edition. Ed., Wiley-Liss, Inc., New York, NY (1990) () () (). Functional fragments of antibodies such as these can be used in the assays described herein.

[0044] ANA is a type of autoantibody. Autoantibodies include antibodies produced by the subject's own immune system against one or more of the subject's own polypeptides or antigens. Autoantibodies are produced by the subject's immune system when the immune system is unable to fully or partially distinguish between self-tissue components and non-self-tissue components. Examples of autoantibodies include immunoglobulins (Ig) against tissue components of the subject that produced the autoantibodies.

[0045] When used against ANA, exemplary anti-X antibodies include ANAs specific for antigen X or ANAs pan-specific for one or more antigens X. For example, an anti-centromere antibody is an ANA that is specific for centromere or pan-specific. The anti-X antibody may include any antibody class (IgG, IgM, IgD, IgA, and IgE) and subclass (IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or a functional fragment thereof. In some embodiments, the anti-X antibody includes a human ANA against a human antigen X.

[0046] The exemplary assays described herein immobilize multiple antigens on a solid support as part of a detection scheme. For example, two or more (e.g., multiple) types of antigens can be immobilized on a single solid support. The antigens immobilized on a single solid support form a solid support-antigen complex capable of detecting two or more types of antibodies, such as two or more types of ANA.

[0047] In some embodiments, the solid support can be pre-prepared and coupled to the antigen using one or more binding techniques. Binding techniques may include, but are not limited to, covalent binding via carboxyl groups, covalent binding by click chemistry, biotin-streptavidin, adsorption, His capture, or tags (such as tags identified below). Covalent binding agents may include any chemical or biological reagent that can be used to covalently fix the antigen directly to the surface of the solid support. Covalent binding agents may include carboxyl-amine reactive groups, such as carbodiimides, such as EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) or DCC (N,N′-dicyclohexylcarbodiimide), amine reactive groups such as N-hydroxysuccinimide (NHS) esters or iminoesters, sulfhydryl reactive crosslinkers such as maleimides, haloacetyl or pyridyl disulfides; carbonyl reactive crosslinkers such as hydrazines or alkoxyamines; photoreactive crosslinkers such as aryl azides or diazines; or chemical selective linking groups such as Staudinger reaction pairs. Click chemistry combinations can include photoclick reactions of alkenes and tetrazoles, [3+2] cycloadditions of alkenes and azides, reverse-demand Diels-Alder reactions of alkenes and tetrazines, copper(I)-catalyzed azide-alkyne cycloadditions (CuAAC), strain-promoted azide-alkyne cycloadditions (SPAAC), strain-promoted alkyne-nitrone cycloadditions (SPANC), and reactions of strained alkenes.

[0048] In some cases, combining two or more antigens onto a single solid support using covalent binding can be challenging because different antigens may compete with each other for binding to the same site on the solid support. In some cases, if one or more antigens cannot bind to the solid support, the detection using these antigens will not exist. Sometimes, different antigen ratios and different antigen-to-solid support coupling strategies can be used to address these issues. For example, antigens with lower binding affinity can be added at higher concentrations compared to antigens with higher binding affinity to increase the amount bound to the solid support. This control over the ratio of antibodies can also be used to account for differences in binding affinity between various antibodies and their corresponding antigens. For example, if the first antigen binds weakly to its corresponding antibody, the beads may require a higher amount of the first antibody on the beads than the second antibody that binds more strongly to its corresponding antibody.

[0049] Non-covalent binding can use any chemical or biological reagent that non-covalently immobilizes the antigen on the surface of the solid support, such as affinity tags including biotin or capture agents such as streptavidin or anti-tag antibodies, such as anti-His6 or anti-Myc antibodies.

[0050] In some embodiments, the example assay uses a carrier to bind to an antigen. For example, the carrier is bound to a solid support and the antigen is bound to the carrier. Therefore, in these examples, the antigen is indirectly attached to the solid support. For example, by providing a uniform binding / reaction surface for the antibody, this configuration can reduce the possibility that individual antigens compete with each other for binding to the solid support in an unpredictable manner. In addition, the type and / or amount of antigen bound to the carrier can be selected as described above to guide the binding so that different antigens are bound to the carrier in a controlled ratio. In some embodiments, the molar ratio between the first antigen and the second antigen can be in the range of 10:1 to 1:10.

[0051] Examples of carriers that can be used include, but are not limited to, proteins such as streptavidin, bovine serum albumin (BSA), human serum albumin (HSA), and protein conjugates such as polymeric streptavidin. The carrier may include a cross-linking agent such as glutaraldehyde, a diisocyanate, a carbodiimide (e.g., 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide and N-hydroxysuccinimide), a polyepoxide (e.g., 1,4-butanediol diglycidyl ether and phenyl glycidyl ether), an acyl azide, dithiobis succinimidyl propionate, disuccinimidyl suberate (DGS), ethylene glycol bis(succinimidyl succinate) (EGS), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxy-(6-amidinohexanoate) (LC-SMCC), succinimidyl 4-(p-maleimidophenyl) butyrate (SMPB), formaldehyde, nitrogen mustard, and adipic acid dihydrazide (ADH). Other carriers may include synthetic or naturally derived polymers such as dendrimers and hyperbranched polymers. Examples of dendrimers and hyperbranched polymers include polyester-8-hydroxy-1-ethynyl bis-MPA dendrimer generation 3 (Sigma Product No. 686646), polyester-16-hydroxy-1-ethynyl bis-MPA dendrimer, 4th generation (Sigma Product No. 686638), polyester-32-hydroxy-1-ethynyl bis-MPA dendrimer, 5th generation (Sigma Product No. 686611), polyester-8-hydroxy-1-carboxybis-MPA dendrimer, 3rd generation (Sigma Product No. 686670), polyester-16-hydroxy-1-carboxybis-MPA dendrimer, 4th generation (Sigma Product No. 686662), polyester-32-hydroxy-1-carboxybis-MPA dendrimer, 5th generation (Sigma Product No. 686654), hyperbranched bis-MPA polyester-16-hydroxy, 2nd generation (Sigma Product No. 686603), hyperbranched bis-MPA polyester-32-hydroxy, 3rd generation (Sigma Product No. 686581) and hyperbranched bis-MPA polyester-6,4-hydroxy, 4th generation (Sigma Product No. 686573). Examples of naturally derived polymers include polysaccharides such as cellulose, chitin, chitosan, alginate, hyaluronic acid, proteins and polymer conjugates. In some embodiments, carriers that can be used include streptavidin, bovine serum albumin (BSA), human serum albumin (HSA) and protein conjugates such as polymeric streptavidin.

[0052] In some embodiments, two or more different antigens can be bound to the same carrier, thereby increasing the number of antigens (indirectly) attached to the solid support substrate. Different antigens can be bound to the carrier using a linker, such as biotin, polyethylene glycol (PEG), his-tag, SpyTag using SpyCatcher technology or other small molecule synthetic proteins or peptides.

[0053] Antigens that can be simultaneously attached to a single solid support directly or indirectly via a carrier include, but are not limited to, the following antigens: Scl-70, centromere, dsDNA, Ro52, Ro60, SS-B, Ribo-P, DFS-70, EJ, Sp100, Gp210, actin, AMA-M2, AMA-M2 / MIT3, TIF1-γ, NXP-2, SAE, PL-7, PL-12, OJ, HMGCR, SRP, Mi-2, MDA-5, RNA helicase, Ki / SL, Sm / RNP, SS-A, PM / Scl, RNA Pol III, Th / To, fibronectin, Sm, Ku, Jo-1, RNP, BICD2, HK, KL, LC1, LKM-1, SLA, SS-A / Ro52, Scl-70, Ku, centromere protein, U11 / U12 RNP, hexokinase-1 and Kelch- 12. In some embodiments, the antigen is a native antigen, a recombinant antigen, or a small peptide sequence targeting a specific antibody epitope of the entire protein.

[0054] Cross Figure 1A , 1B Table 10 shown in 1C and 1C shows examples of antigens 11 (proteins) that can be simultaneously attached to a single solid support directly or indirectly via a carrier and their accession numbers. Figure 1A , 1B Two or more antigens as shown in 1C and / or any combination of the antigens listed above can be simultaneously attached to the same solid support. Figure 1A , 1B Two or more antigens as shown in 1C and / or any combination of the antigens listed above can be simultaneously attached to the same carrier (attached to a solid support).

[0055] In some embodiments, the antigen can be part of a cell extract or fragment. Exemplary cell extracts or fragments include intact parts of cells that have not been distilled or purified into a single antigen. Exemplary cell extracts can contain multiple antigens. Thus, the cell extract itself can be attached to a solid support using any of the techniques described herein - for example, directly attached or attached to a carrier directly or through a linker. The cell extract can be attached to the same solid support as the purified antigen, or it can be attached to a different solid support. The cell extract attached to the solid support can be detected using the assays described herein in the same manner as the purified antigen attached to the solid support.

[0056] In one example, a multi-analyte assay can include individual and purified antigens and cell extracts (e.g., HEp-2 cell extracts) attached to different solid supports but analyzed together to improve the performance of ANA / ACA assays. In one example, cell extracts and purified antigens are used in combination, but on different solid supports. By combining the cell extract with the solid support, the assay can detect all antigens in the cell extract.

[0057] Figure 2 An example of a solid support 14 is shown, in this case beads, to which ten antigens 16 are attached via respective carriers 18. In this example, except for the RNP / SM which is a complex of two antigens, the other antigens are attached to a respective carrier. The following table shows the relative molar ratios of the antigens in the process of preparing to attach the antigens.

[0058]

[0059]

[0060] Figure 3 An example of a solid support 20 is shown, in this example a bead, to which 15 antigens 22 are attached via respective linkers and carriers. In this example, the 15 antigens 22 are each attached to the same carrier 24 via respective linkers 26 (here biotin). Figure 3 In the embodiment, only one carrier 24 is attached to the solid support 20, but a plurality of carriers may be attached to the solid support 3, each carrier having a plurality of antigens attached thereto.

[0061] Any number of antigens can be attached directly or indirectly (e.g., via a carrier with or without a linker) to a single solid support. For example, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more antigens can be attached directly or indirectly to a single solid support. In an example, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more antigens can be attached to each carrier.

[0062] Examples of solid supports, solid surfaces, or other grammatical equivalents include any material that is suitable or modifiable to be suitable for attaching an antigen to which an autoantibody can bind. Example materials include, for example, glass and modified or functionalized glass, plastics (including acrylic resins, polystyrene, methyl styrene, polyurethane, Teflon, etc.). TM etc.), paramagnetic materials, thoria sol, carbon graphite, titanium oxide, latex or cross-linked dextran (such as agarose), cellulose polysaccharide, nylon or nitrocellulose, ceramics, resins, silicon or silicon-based materials (including silicon and modified silicon), carbon metal, inorganic glass, optical fiber bundles and various other polymers.

[0063] In some embodiments, the solid support can be or include holes or arrays of holes or depressions on the surface. These can be made using techniques such as photolithography, stamping, molding, and microetching. In some embodiments, the solid support can include a multi-well plate, such as a 96-well plate, a 384-well plate, or a 1536-well plate. In some embodiments, the solid support can be located in a flow cell or flow cell device (e.g., Biacore TM chip or protein chip).

[0064] An example solid support can be or include a bead, a sphere, a particle, a membrane, a chip, a slide, or a test tube. The bead can include a sphere or a particle. A sphere, a particle, or its grammatical equivalent can include discrete non-planar particles, for example, micron or nanometer sized particles. A sphere can include, for example, a microsphere. The particle can include, for example, a nanoparticle. In some embodiments, the shape of the bead can be irregular. Alternatively or additionally, the bead can also be porous. Example bead sizes can range from nanometers to millimeters, and in some embodiments, the beads used range from about 0.2 microns to about 200 microns. In some embodiments, the size range of the bead can range from about 0.5 to about 5 microns. In some embodiments, microbeads less than 0.2 microns and greater than 200 microns can be used.

[0065] The solid support can be or include a patterned surface suitable for fixing purified proteins (such as antigens) in an ordered pattern (such as on a protein chip). Example patterned surfaces include arrangements of different regions in or on an exposed layer of a solid support. For example, one or more regions can be features where one or more purified proteins are present. These features can be separated by gap regions where purified proteins are not present. In some embodiments, the pattern can be a feature in an xy format with rows and columns as units. In some embodiments, the pattern can be a repeated arrangement of features and / or gap regions. In some embodiments, the pattern can be a random arrangement of features and / or gap regions. Examples of patterned surfaces that can be used for example assays and complexes are described in U.S. Patent Application Publication No. 2008 / 0280785, U.S. Patent Application Publication No. 2003 / 0153013, and PCT Publication No. WO2009 / 039170, each of which is incorporated herein by reference.

[0066] The exemplary solid supports described herein can also be used in the kits and techniques (eg, methods / assays) described herein.

[0067] Different types of antigens can be attached to the same solid support using the same or different immobilization techniques. In this regard, "immobilization" and "attachment" are used interchangeably herein, and both terms are intended to include, but are not limited to, covalent attachment or binding and non-covalent attachment or binding, unless otherwise expressly stated or the context indicates otherwise.

[0068] The antigen type on a single solid support and / or the antigen ratio on a single solid support can vary according to the antibody to be detected. Antigen type and ratio can vary because of disease state or antigen group. In some embodiments, antigens are combined on a single solid support by disease area (such as SjS / MCTD spectrum, SSc / myositis, SLE and liver disease). In addition, the ratio of antigens also can be adjusted according to the analyte to be detected or the disease.

[0069] For example, the combination of antigens on the solid support may be associated with a particular disease or condition. In the example, Figure 2 The combination of antigens on the solid support 14 is related to connective tissue diseases. Figure 2 The complex 15 in the present invention can be used to bind to antibodies in a biological sample of a subject, which antibodies indicate a connective tissue disease in the subject. In another example, the complex described herein can use antigens associated with autoimmune liver disease, because some antibodies in such diseases will produce positive ANA. Antigens associated with autoimmune liver disease can be bound to a solid support, because some antibodies in such diseases will produce positive ANA. Attaching these antigens can improve the differential diagnosis of systemic autoimmune diseases.

[0070] The set of antigens and / or cell extracts attached to a single solid support may include antigens and / or cell extracts grouped by their disease areas, such as but not limited to

[0071] 1. A solid support (30, Figure 4 ), including antigens Sm / RNP, SS-A, Ro52, Ro60 and SS-B.

[0072] 2. A solid support for measuring SSc / myositis (31, Figure 4 ), including PM / Scl, RNA Pol III, Th / To, fibronectin, and Mi-2.

[0073] 3. A solid support for measuring SLE (32, Figure 4 ), including dsDNA, Sm, Ribo-P and Ku.

[0074] 4. A solid support for determining liver autoimmune diseases (33, Figure 4 ), including gp210, AMA-M2 / MIT3 and sp100.

[0075] The set of antigens and / or cell extracts attached to a single solid support may include antigens and / or cell extracts grouped according to their importance / recognition and sensitivity, e.g.

[0076] 5. A solid support (ENA 4) (36, Figure 5 ), including Sm, RNP, SS-A / Ro52, Ro60 and SS-B+dsDNA.

[0077] 6. A solid support (ENA 7) (37, Figure 5 ), including +Scl-70 and Jo-1.

[0078] 7. A solid support (CTD Screen Plus) (38, Figure 5 ), including +Ku, PM / Scl, RNA Pol III, Th / To, fibronectin, and Mi-2.

[0079] 8. A solid support (liver) (39, Figure 5 ), including gp210, AMA-M2 / MIT3 and sp100.

[0080] 9. A solid support (40, Figure 6 ), containing Sm, RNP, SS-A / Ro52, Ro60, SS-B, SCL-70, Jo-1+dsDNA.

[0081] 10. A solid support (41, Figure 6 ), containing +Ku, PM / Scl, RNA Pol III, Th / To, fibrin, Mi-2, Ki / SL, and possibly one or more other substances.

[0082] 11. A solid support (43, Figure 6 ), containing HEp-2 cell extract.

[0083] 12. A solid support (44, Figure 6 ), containing gp210, AMA-M2 / MIT3 and sp100.

[0084] 13. A solid support for measuring myositis-specific antibodies, comprising EJ, HMGCR, MDA5, Mi-2, NXP2, OJ, PL-7, PL-12, SAE, SRP, and TIF1γ.

[0085] 14. A solid support for measuring liver autoimmune diseases, comprising gp210, hexokinase-1, Kelch-12, LC1, LKM-1, AMA-M2 / MIT3, SLA, and sp100.

[0086] In some embodiments, a combination of antigens and / or cell extracts can be used to detect an analyte rather than determining whether a subject has a disease or condition. The analyte can be an antibody that binds to an antigen on a solid support.

[0087] In some embodiments, complexes for detecting different diseases or conditions can be combined in a single assay. For example, complexes for detecting different diseases can have different labels, such as colorimetric labels, dyes, or other labels described herein. These labels make it possible to distinguish complexes in the same assay, thereby identifying which disease complex is positive for a particular disease. Therefore, a single assay can be used to detect multiple diseases, saving time and money. Analysis can also be simplified compared to methods involving multiple beads, because the latter must interpret multiple signals to determine whether a disease or condition is present. In some embodiments, complexes for detecting analytes and complexes for detecting one or more diseases or conditions can be used in combination in a single assay.

[0088] Diseases or conditions that can be detected or diagnosed using the assays and complexes described herein include, but are not limited to, systemic lupus erythematosus (SLE), Sjögren's syndrome (SjS), systemic sclerosis (SSc), mixed connective tissue disease (MCTD), idiopathic inflammatory myopathy (IIM), unidentified / undifferentiated CTD, rheumatoid arthritis (RA), autoimmune hepatitis (AIH), antiphospholipid syndrome (APS), dense-speckled or nucleolar IFA, autoimmune gastritis (AIG), infectious HIV, HCV, syphilis, primary biliary cholangitis (PBC), ANCA-associated vasculitis (AAV), and idiopathic inflammatory myopathy (IIM).

[0089] The label can be conjugated to any of the antigens described herein. The conjugation can include non-covalent or covalent cross-linking. For example, the label can include a molecular entity that can emit a signal that can be used as a readout or measurement for detecting an analyte. There are many types of labels. Examples of these classes include fluorophores, enzymes, chemiluminescent moieties, radioactive moieties, organic dyes, small molecules, polypeptides or functional fragments thereof.

[0090] Examples of fluorophores include fluorescent dyes such as phycoerythrin (PE), fluorescein isothiocyanate (FITC), tetramethylrhodamine (TRITC), BODIPY, and Dyes. Fluorescent dyes also include fluorescence resonance energy transfer (FRET) dyes or time-resolved (TR)-FRET dyes. Fluorophore labels also include fluorescent proteins, such as green fluorescent protein (GFP) and cyan fluorescent protein (CFP). Examples of enzyme labels include alkaline phosphatase (AP) or horseradish peroxidase (HRP). "When any of the substrates S^'S^'-tetramethylbenzidine (TMB), 3,3'-diaminobenzidine (DAB) or 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) is applied to HRP, a color (color development) or light (chemiluminescence) signal is generated. Examples of radioactive labels include, for example, carbon-14 or tritium. Examples of small molecule labels include biotin, resins such as agarose beads, and fluorescently labeled magnetic beads or nanoparticles such as colloidal gold. Examples of polypeptide or functional fragment labels include avidin, streptavidin or neutravidin that have affinity for biotin. Examples of polypeptide or functional fragment tags also include hemagglutinin (HA), glutathione-S-transferase (GST) or c-myc.

[0091] In some configurations, the label conjugated to the antigen includes an additional substrate or binding agent. For example, the HRP label conjugated to the antigen includes a substrate to identify the antigen. A variety of other label configurations are known. Any label can be used in the assays described herein. In some embodiments, the label configuration can include PE, antigen X: anti-X antibody complex binding agent, anti-X antibody binding agent or Ig binding agent conjugated to antigen X. The labels described herein can be used in the assays described herein to detect antibodies bound to the antigen. "Antigen X" used herein includes but is not limited to any antigen listed herein.

[0092] The ratio of antigens attached to a single solid support can be selected to achieve the desired sensitivity to one or more antibodies. For example, the ratio of antigens in the reaction mixture used to add the antigen to the solid support can be controlled to achieve the desired detection sensitivity. This can include adding more of one antigen than one or more other types of antigens on a single solid support. For example, in order to detect lupus, an additional amount of dsDNA can be added to the reaction mixture so that it is attached to the solid support to enhance the lupus signal. In some embodiments, beads containing cell extracts (such as HEp-2 cells) can have a higher weight ratio of cell extracts than single antigens and cell extracts. For example, the weight ratio of cell extracts to single antigens can be greater than 1:1, such as between 10:1 and 1:1.

[0093] The example assays described herein can utilize antigens to improve the differential diagnosis of systemic autoimmune disorders. Examples of these assays can detect antibodies to cellular antigens by (i) combining different antigens on a single solid support with the same unique identifier in a multi-analyte assay, and / or (ii) providing antigens bound to autoantibodies to cellular antigens on a single solid support, but these antigens are not typically used for CTD diagnosis. Examples of such antigens include, but are not limited to, those listed in Table 1 below.

[0094]

[0095] Table 1: Intracellular autoantigens targeted by antibodies not associated with connective tissue disease but that are ANA positive using the HEp-2 IIF method

[0096] Figure 7 Also shown are examples of autoantibodies 45 that can produce positive indirect immunofluorescence but are not typically used to diagnose CTD.

[0097] Figure 8is a flow chart showing operations included in an example process (assay) 50 for detecting or diagnosing a disease using a solid support-antigen complex of the type described herein. The operations include contacting a biological sample of a subject with one or more complexes of the type described herein (50a). This can be achieved by contacting the biological sample with a fluid containing the complexes. Each complex can include a solid support and at least two different types of antigens attached to the solid support. Different complexes can be configured to detect antibodies associated with different diseases, conditions, or analytes. The operations include detecting (50b) the presence of one or more antibodies bound to the complexes in the biological sample.

[0098] The presence of one or more antibodies bound to the complex indicates a disease associated with the complex. Detection can be performed using a diagnostic test instrument or detector to identify a signal from the complex, such as fluorescence or chemiluminescence, and to identify a label associated with the complex that emits the signal. The level (e.g., intensity) of the signal can be compared (50c) to a predetermined benchmark to determine (50d) the likelihood that the subject suffers from a disease condition associated with the antibody bound to the complex.

[0099] For example, a signal (e.g., a fluorescent or chemiluminescent signal) may be measured in advance for a subject known to have a disease associated with each complex and used to create a curve that correlates the signal level with the likelihood that the subject has a particular disease. The curve may be retrieved in operation 50c. The measured signal level may be compared to the curve to determine the likelihood that the subject has a disease associated with each complex. These operations may be performed for complexes associated with different diseases and for complexes associated with a particular analyte. In the case of an analyte, a curve of signal levels associated with different analytes present in a test sample may be generated in advance and used to determine the likelihood that the analyte is present in a biological sample.

[0100] Fig. 9 Shown is a receptor operating characteristic curve (ROC) analysis used to differentiate antigen reactivity with a mixture of the four antigens shown (i.e., RNP, Ro52, Ro60, and SSB). Two formats are compared, in the first format, the antigens are all coupled to a single bead, and in the second format, each antigen is coupled to a different bead as a homogenous population. Two different preparations 52 and 53 are shown, and the two formats are compared separately. The sensitivity of the two preparations is plotted on the Y axis and plotted relative to the specificity on the X axis. The figure shows that the specificity and sensitivity of the two formats are comparable.

[0101] Fig.10is a bar graph showing the reactivity of a single antigen relative to the reactivity / signal of the same antigen contained on a solid support (beads) containing RNP, Ro52, Ro60, and SSB. This demonstrates that the reactivity of the antigens coupled to a single support is within the same order of magnitude as the reactivity of only one antigen coupled to the beads. It is noteworthy that the reactivity / signal can be improved by including more antibody surface density.

[0102] Fig.11 is a graph showing the results of a clinical study using ten antigens on a solid support and using an FDA (Food and Drug Administration) approved device (here system, it is Inova The graph shows the false positive rate and true positive rate for the two technologies. Curve 60 plotted using ten antigens on a solid support is similar to curve 61 plotted using the FDA-approved device. As shown in text box 64, the two assays have similar sensitivity and specificity, where sensitivity represents the percentage of subjects who are accurately identified as having a disease and specificity represents the percentage of subjects who are identified as having the disease.

[0103] Fig.12 is a graph showing how the results of a clinical study using ten antigens to screen for disease compare to test results obtained using prior art and thousands of samples. Prior art includes Quanta in SA The results are shown in Figure 64. The results are shown in Figure 65. The results are shown in Figure 66. The results are shown in Figure 67. The results are shown in Figure 68. The results are shown in Figure 69. The results are shown in Figure 70. The results are shown in Figure 71.

[0104] Fig.13 is a graph showing the results of a clinical study using ten antigens on a solid support to screen for disease and how these results compare to those obtained using previous technologies and thousands of samples. The graph relates the false positive rate to the true positive rate. Previous technologies include Quanta System, Nova Digital IFA Reader and The assay was performed on the Quanta system. Curve 71 is from system, curve 72 comes from System; Curve 73 from Nova The curve 74 is from an assay described herein using ten antigens on a solid support. As shown, the curve 74 is from an assay described herein using ten antigens on a solid support, and its sensitivity 75 is close to 90%, which is consistent with The sensitivity of the systems is comparable. Note that the shading associated with each curve (eg, 76) corresponds to the 95% confidence level for the data. The overlap of shading between the curves indicates that the accuracy of the assay described herein using ten antigens on one solid support is comparable to that of the prior art.

[0105] The exemplary assays described herein can be used in conjunction with a multi-analyte detection system such as In an exemplary embodiment, two or more solid supports (e.g., two, three, four, five, six, etc.), each having a combination of two or more antigens attached directly or indirectly through the use of a carrier or a carrier and a linker, are mixed together and then loaded into a tube, which can be part of the kit. The remaining reactions are performed as described above. The plan is underway.

[0106] In an example protocol, antigens are attached to paramagnetic particles with unique tags or signatures and incubated with diluted patient samples, which can be body fluids such as blood or saliva. After incubation at 37°C for 9.5 minutes, the particles are washed and incubated for another 9.5 minutes. The particles are then incubated with anti-human IgG conjugated to phycoerythrin (PE) at 37°C to label the bound autoantibodies. After the final wash cycle, the median fluorescence intensity (MFI) on the particles is captured using a digital imager and the captured detection results are analyzed to extract meaningful information for each analyte. Detection can also be performed using an indirect immunofluorescence assay (IFA).

[0107] Cassette systems include multiple antigens attached to a single solid support, allowing for the use of fewer solid supports compared to techniques that use one antigen on a single solid support. This may reduce the number of solid supports and, therefore, the number of cassettes required to achieve the same number of tests. However, multiple types of antigens can capture more types of target antibodies, allowing for a comprehensive analysis of the test sample. In addition, by selecting the type and ratio of antigens per solid support, test sensitivity can be improved relative to techniques that use one antigen per solid support.

[0108] In some embodiments, a single cartridge as described herein may include one or more (e.g., four) solid supports containing the above-mentioned antigen groups 1 to 4. In some embodiments, a single cartridge (e.g., a cartridge as described herein) may include one or more (e.g., four) solid supports containing the above-mentioned antigen groups 5 to 8. In some embodiments, a single cartridge (e.g., a cartridge as described herein) may include two or more (e.g., four) solid supports containing the above-mentioned antigen groups 5 to 12.

[0109] Currently, the most commonly used technology for detecting ANA as part of the diagnosis of ANA-associated rheumatic diseases is the HEp-2 IIF test, which has high sensitivity but low specificity. Solid-phase bead-based systems may be less sensitive than such systems. Based on previous studies, the sensitivity of such solid-phase assays is approximately 75%, depending on the composition of the cohort. In this case, a sensitivity of 75% means that in a group of test subjects known to have the disease, the assay for a certain disease can identify 75% of the test subjects as having the disease. Including multiple antigens per solid support has the potential to increase assay sensitivity by approximately 10%. Including multiple antigens per solid support may also improve assay performance, especially in the case of systemic sclerosis and myositis.

[0110] Therefore, the example assay described in this article can be extended to The capabilities of multi-analyte detection systems such as ANA and IIF on HEp-2 cells allow these systems to provide results comparable to those of IIF on HEp-2 cells for the detection of ANA.

[0111] Software that processes test results in diagnostic tests, such as the software described herein, systems, and / or process 50 above, can be configured to combine multiple results (from multiple antigens) to provide new indications or more accurate indications. Such software can implement machine learning algorithms to provide these new indications or more accurate indications.

[0112] Also described herein are example embodiments of kits for detecting, diagnosing, and / or monitoring a disease or condition. Example kits may include one or more of the example solid support-antigen complexes described herein. For example, a kit may include a solid support-antigen complex for detecting a first disease, a solid support-antigen complex for detecting a second disease different from the first disease, a solid support-antigen complex for detecting a third disease different from the first and second diseases, and the like. For example, a kit may include a solid support-antigen complex for detecting a plurality of different analytes. A kit may include both solid support-antigen complexes for detecting one or more different diseases and solid support-antigen complexes for detecting one or more different analytes.

[0113] Examples of kits described herein may include anti-X antibody specific detection probes. The detection probe may include an antigen, such as an antigen or an antibody specific binding polypeptide as described herein. Thus, in certain examples, the anti-X antibody specific detection probe may include, for example, an antigen X fragment, an antigen X: anti-X antibody complex binder, an anti-X antibody binder, or an Ig binder or protein. Examples of antibody specific binding polypeptides include any polypeptide that can specifically recognize an antibody and its functional fragments. Examples of antibody specific binding polypeptides include IgG binding proteins, receptors, chimeric receptors, and binding polypeptides identified by random or combinatorial library screening.

[0114] Examples of IgG binding proteins include protein A and protein G. Antibody-specific binding polypeptides can be chemically synthesized or purified from natural sources, or recombinantly produced. Antibody-specific binding polypeptides described herein can be mammalian, including mice, rabbits, goats, chickens, donkeys, etc.

[0115] When referring to binding to a target such as an antibody, the detection probe may bind to the target directly or indirectly. For example, direct binding agents include antibodies or other antibody-specific binding polypeptides that specifically recognize antigen X: anti-X antibody complexes, and antibodies or other antibody-specific binding polypeptides that specifically bind to anti-X antibodies. Detection probes may be made specific to a target using anti-Ig or other antibody-specific binding polypeptides that bind to Ig. Such antibodies and antibody-specific binding polypeptides may be made specific to anti-X antibodies by, for example, capturing anti-X antibodies with antigen X and washing away non-anti-X Ig before adding anti-Ig or other antibody-specific binding polypeptides that bind to Ig. Other configurations that separate or partition such binding complexes to achieve specific binding to a target may be used as indirect mechanisms to render the detection probe specific to a target. Thus, an "anti-X antibody-specific detection probe" may include, for example, antigen X, antigen X: anti-X antibody complex binding agents, anti-X antibody binding agents, and Ig binding agents.

[0116] The detection probe may include a marker or reporter label. For example, the marker or reporter label includes one or more molecules that are configured to generate a detection signal indicative of an anti-X antibody to which the detection probe binds. The marker or reporter label may be attached or conjugated to the detection probe by non-covalent or covalent cross-linking. Non-covalent and covalent fixation of the reporter label to the detection probe may be described, for example, in Dennler et al, "Antibody conjugates: from heterogeneous populations to defined reagents,"

[0117] The marker or reporter tag can generate a variety of signals, depending on the type of reporter tag.

[0118] An exemplary kit may include a solid support, examples of which are described herein. The kit may also include a control. Exemplary controls include positive or negative standards for comparison with a test sample in which the presence or amount of anti-X antibodies is unknown. The kit may include a positive control. In some embodiments, the positive control includes a sample containing a detectable amount of anti-X antibodies or functional fragments thereof or a level above a threshold. In some embodiments, the positive control may be obtained from a diseased subject with an anti-X antibody level above a threshold. Alternatively or additionally, the positive control may also include an antibody or functional fragment thereof specific for the antigen. The antibody or functional fragment thereof may be selected from a monoclonal or polyclonal antibody. In other embodiments, the kit may include a negative control. The negative control may include a sample that does not contain detectable anti-X antibodies or functional fragments thereof or a level below a threshold. In some embodiments, the negative control may be obtained from a healthy control individual or may be synthesized in vitro. For example, the negative control may include water or a buffer.

[0119] Exemplary kits may include vectors, examples of which are described herein.Exemplary kits may include linkers, examples of which are described herein.

[0120] For example, the kit may include one or more auxiliary reagents. Example auxiliary reagents include substances, mixtures, materials or components useful for performing the assay or composition. Example auxiliary reagents include conjugated reagents, buffers, instructions, instruments, etc.

[0121] In some embodiments, the kit may include reagents, and the reagents used in the kit may include any conjugation reagents known in the art, including covalent and non-covalent conjugation reagents. Covalent conjugation reagents may include any chemical or biological reagents that can be used to covalently fix a polypeptide on a surface. Covalent conjugation reagents may include carboxyl-amine reactive groups, such as carbodiimides, such as EDC or DCC; amine reactive groups, such as N-hydroxysuccinimide (NHS) esters or imidoesters; sulfhydryl reactive crosslinkers, such as maleimide, haloacetyl or pyridyl disulfide; carbonyl reactive crosslinkers such as hydrazine or alkoxyamine; photoreactive crosslinkers such as aryl azide or diazines; or chemical selective linking groups such as Staudinger reaction pairs. Non-covalent fixation reagents may include any chemical or biological reagents that can be used to non-covalently fix a polypeptide on a surface, such as affinity tags such as biotin or capture reagents such as streptavidin or anti-tag antibodies, such as anti-His6 or anti-Myc antibodies.

[0122] Exemplary reagents may include combinations of conjugation reagents. Such combinations include, for example, EDC and NHS, which can be used to immobilize proteins on surfaces such as carboxylated dextran matrices (e.g., BIAcore TM CM5 chip or dextran-based beads). The conjugation reagent combination can be stored as a pre-mixed reagent combination, or one or more conjugation reagents in the combination can be stored separately from other conjugation reagents.

[0123] In some embodiments, the reagents used in the kit may include a coating buffer. The coating buffer may include sodium carbonate-sodium hydroxide or phosphate. In some embodiments, the coating buffer may be 0.1M NaHCCb (e.g., about pH 9.6).

[0124] In some embodiments, the reagent may include a wash buffer. The wash buffer may include a tris (hydroxymethyl) aminomethane (Tris) based buffer, such as Tris buffered saline (TBS) or a phosphate buffer, such as phosphate buffered saline (PBS). The wash buffer may include a detergent, such as an ionic or non-ionic detergent. In some embodiments, the wash buffer may be a PBS buffer having a pH of about 7.4, including a concentration of about 0.05% 20.

[0125] In some embodiments, the reagents may include a dilution buffer. Any dilution buffer known in the art may be included in the kit. Examples of dilution buffers include proteins, such as bovine serum albumin (BSA), and detergents, such as 20. In some embodiments, the dilution buffer may be PBS having a pH of about 7.4, comprising about 1% BSA and about 0.05% 20.

[0126] In some embodiments, the reagents may include a detection or assay buffer.Any detection or assay buffer known in the art may be included in the kit.

[0127] The detection or assay buffer may be a colorimetric detection or assay buffer, a fluorescent detection or assay buffer, or a chemiluminescent detection or assay buffer. The colorimetric detection or assay buffer includes, but is not limited to, PNPP (p-nitrophenyl phosphate), ABTS (2,2'-azo-bis(3-ethylbenzothiazoline-6-sulfonic acid)) or OPD (o-phenylenediamine). The fluorescent detection or assay buffer includes QuantaBlu TM or QuantaRed TM (Thermo Waltham, MA). Chemiluminescent detection or assay buffers may include luminol or luciferin. The detection or assay buffer may also include a trigger such as H2O2 and a tracer such as isoluminol-conjugate.

[0128] In some embodiments, reagents may include solutions for calibration or testing. In some embodiments, reagents may include stop solutions. Any stop solutions known in the art may be included in the test kit. Example stop solutions terminate or delay the further development of detection reagents and corresponding determination signals. Stop solutions may include low pH buffers (such as glycine buffer, pH value is 2.0), chaotropic agents (such as guanidinium chloride, sodium dodecyl sulfate (SDS)) or reducing agents (such as dithiothreitol, b-mecaptoethanol) etc.

[0129] In some embodiments, the reagent may include a cleaning reagent. The cleaning reagent may include any cleaning reagent known in the art. In some embodiments, the cleaning reagent may be a cleaning reagent recommended by the manufacturer of the automated assay system.

[0130] In some embodiments, kits of the type described herein can be used to perform assays on a detection instrument, such as a detection instrument or system as described herein. In some embodiments, the detection instrument is configured to detect and measure labels. To this end, the detection instrument can be configured to detect or measure fluorescence, luminescence, chemiluminescence or absorption, reflection, transmission, birefringence or refractive index. In some embodiments, the detection instrument can use confocal and non-confocal microscopes, microplate readers, flow cytometers, etc.

[0131] In some embodiments, the kit can include components suitable for collecting biological samples, as described below. The components can include collection tubes, columns, syringes, needles, etc.

[0132] In some embodiments, the kit may include instructions for use of the kit components. The instructions may be in any form and may be within or outside the kit. The instructions provide detailed information about the protocol and analytical techniques.

[0133] The components of the kit can be in different physical states. For example, some or all of the components can be lyophilized, in aqueous solution or frozen. These components can include two or more antigens to be attached to a solid support as described herein, a solid support, a detection probe, and auxiliary reagents.

[0134] The kit can be customized according to a specific assay technology. For example, in some embodiments, the kit can be a FIA kit, a CIA kit, a RIA kit, a multiplex immunoassay kit, a protein / peptide array immunoassay kit, a SPRIA kit, an IIF kit, an ELISA, a particle-based multi-analyte technology (PMAT kit) or a dot blot kit. In some embodiments, the ELISA kit may include a wash buffer, a sample diluent, a secondary antibody-enzyme conjugate, a detection reagent, and a stop solution. In some embodiments, the dot blot kit may include a wash buffer, a sample diluent, a secondary antibody-enzyme conjugate, a detection reagent, and a stop solution. In some embodiments, the CIA kit may include a wash buffer, a sample diluent, a tracer (such as an isoluminol-conjugate) and a trigger (such as H2O2). In some embodiments, a multiplex kit may include a wash buffer, a sample diluent, and a secondary antibody-enzyme conjugate. In some embodiments, the kit can be customized according to the Luminex platform, for example, including Beads.

[0135] The kits can be used to diagnose diseases, such as but not limited to CTD or autoimmune diseases, by providing a mechanism to detect anti-X antibodies bound to antigen X. The kits can be used to detect anti-X antibodies by any of the techniques disclosed herein. The antigen X: anti-X antibody complex or antigenic fragment thereof can have a stoichiometry of anti-X antibodies of one to one or more. In some embodiments, there can be one anti-X antibody per antigen X in the complex; in some embodiments, there can be two anti-X antibodies per antigen X in the complex; in some embodiments, there can be more than two anti-X antibodies per antigen X in the complex. Techniques for measuring the binding stoichiometry of two antigens are well known in the art and include, for example, isothermal titration calorimetry (ITC) and ultracentrifugation.

[0136] In some embodiments, the antigen X: anti-X antibody complex or antigenic fragment thereof can include a plurality of complexes having the same stoichiometry. For example, all of the complexes in the plurality of complexes have one anti-X antibody for each antigen X. In some embodiments, the antigen X: anti-X antibody complex or antigenic fragment thereof can be a plurality of complexes having different stoichiometries. For example, some of the complexes in the plurality of complexes can have one anti-X antibody per antigen X, while other complexes in the plurality of complexes can have more than one anti-X antibody per antigen X.

[0137] In some embodiments, antigen X can bind to an anti-X antibody with high affinity. In some embodiments, the binding of an anti-X antibody binding site by an anti-X antibody can have a greater than 2-fold, greater than 3-fold, greater than 4-fold, greater than 5-fold, greater than 8-fold, greater than 10-fold, greater than 15-fold, greater than 20-fold, greater than 25-fold, greater than 50-fold, greater than 100-fold, greater than 300-fold, greater than 1000-fold, greater than 3000-fold, greater than 10000-fold, greater than 30000-fold, or greater than 100000-fold greater binding affinity.

[0138] A lower dissociation constant (KDS) for the antigen X: anti-X antibody complex or a higher association constant (KAS) for each anti-X antibody and antigen X demonstrates a higher binding affinity. In some embodiments, the dissociation constant (KDS) for the X: anti-X antibody complex may be less than 1 mM (millimolar), less than 300 nM (nanomolar), less than 100 nM, less than 30 nM, less than 10 nM, less than 3 nM, less than 1 nM, less than 300 pM, less than 100 pM, less than 30 pM, less than 10 pM, less than 3 pM, or less than 1 pM. Techniques for measuring antibody-antigen binding affinity are known and include ELISA, isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR).

[0139] The presence of an increase in anti-X antibodies in a subject compared to a healthy control group can indicate the presence of a disease or a risk of developing a disease. Thus, a measurable increase in anti-X antibodies to antigen X can be used to diagnose a disease associated with anti-X antibodies. Example techniques for detecting and comparing anti-X antibody levels to a control (e.g., a predetermined baseline) are provided below.

[0140] In some embodiments, the level of anti-X antibodies is detected. In some embodiments, antigen X:anti-X antibody complexes can be formed using the compositions and techniques described herein, and anti-X antibodies in the complexes can be detected.

[0141] In some embodiments, the detection of elevated levels of anti-X antibodies compared to healthy control individuals indicates that the subject suffers from a disease associated with anti-X antibodies. In some embodiments, after the anti-X antibody-associated disease is diagnosed using the support antigen complexes and techniques described herein, the presence of the anti-X antibody-associated disease can be further confirmed based on various symptoms associated with the onset or presence of the anti-X antibody-associated disease. For example, clinical symptoms associated with systemic sclerosis and myositis.

[0142] In some embodiments, detection of elevated anti-X antibody levels compared to healthy control individuals indicates that the subject is at risk for developing clinical symptoms of an anti-X antibody-associated disease. In some embodiments, the subject may be at risk for developing clinical symptoms of an anti-X antibody-associated disease within less than 3 months, less than 6 months, less than 9 months, less than 12 months, less than 18 months, less than 2 years, less than 3 years, less than 4 years, less than 5 years, less than 6 years, less than 7 years, less than 8 years, less than 9 years, less than 10 years, less than 12 years, less than 14 years, or less than 16 years after the elevated anti-X antibody levels are determined.

[0143] In some embodiments, the presence of elevated anti-X antibody levels, as compared to healthy control individuals, indicates a greater than 5%, greater than 10%, greater than 15%, greater than 20%, greater than 25%, greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90% likelihood that the subject will develop clinical symptoms of an anti-X antibody-related disease within 5 years after the elevated anti-X antibody levels are determined. In some embodiments, the presence of elevated anti-X antibody levels may indicate a greater than 2 times, greater than 3 times, greater than 4 times, greater than 5 times, greater than 6 times, greater than 7 times, greater than 8 times, greater than 9 times, or greater than 10 times likelihood that the subject will develop clinical symptoms of an anti-X antibody-related disease within 5 years after the elevated anti-X antibody levels are determined, as compared to healthy control individuals.

[0144] The terms "subject", "organism", "individual" or "patient" are used as synonyms and interchangeably and may refer to a vertebrate mammal. For example, the subject of the present disclosure may include a healthy subject, an asymptomatic subject, and a diseased subject.

[0145] A subject may be "suspected of having a disease associated with an anti-X antibody," such as determined by the presence of certain risk factors known in the art. Example risk factors include, for example, genetic susceptibility, personal disease history, lifestyle factors, environmental factors, diagnostic indicators, etc.

[0146] A subject at risk for having or developing an anti-X antibody-associated disease may have a genetic predisposition to develop an anti-X antibody-associated disease, or have a family history of an anti-X antibody-associated disease or other autoimmune disease. A subject may be exposed to certain lifestyle factors (e.g., smoking) or environmental factors that promote the development of an anti-X antibody-associated disease. A subject may exhibit clinical disease manifestations of an anti-X antibody-associated disease. A subject may be a patient undergoing a clinical examination to diagnose an anti-X antibody-associated disease or to assess the risk of developing an anti-X antibody-associated disease.

[0147] In some embodiments, the subject may have anti-X antibodies in their body fluids (such as blood) or tissues. In some embodiments, the subject may have elevated anti-X antibodies in their body fluids or tissues compared to a normal healthy subject. In some embodiments, the subject does not have elevated anti-X antibodies in their body fluids or tissues compared to a normal healthy subject.

[0148] In some embodiments, the subject may be a naive patient. In some embodiments, the subject may be receiving treatment for an anti-X antibody-related disease (e.g., drug therapy). In some embodiments, the subject may be in remission. In some embodiments, remission may be drug-induced. In some embodiments, remission may be drug-free remission.

[0149] In some embodiments, the subject can be an animal model of an anti-X antibody-associated disease. In some embodiments, the animal model can be a mouse, rabbit, or primate model of an anti-X antibody-associated disease. In some embodiments, the animal model can involve inducing an anti-X antibody response by immunizing or inoculating an animal with antigen X.

[0150] The subject may be "seronegative for known anti-X antibodies associated with the disease," i.e., a blood test fails to detect known anti-X antibodies associated with the disease.

[0151] In some embodiments, the known anti-X antibody associated with the disease may be anti-mitochondrial antibody (AMA), ANA, anti-multinuclear dots (MND) autoantibody, anti-nuclear body (NB) autoantibody, anti-hexokinase 1 (HK1) antibody, and anti-KLHL12 antibody.

[0152] In some embodiments, the subject may be asymptomatic. Asymptomatic subjects include healthy subjects with substantially no risk or only a low risk of developing an anti-X antibody-related disease (e.g., the probability of an asymptomatic patient developing an anti-X antibody-related disease during the next five years is less than 10%, less than 5%, less than 3%, or less than 1%). Asymptomatic subjects also include healthy subjects with a higher risk of developing an anti-X antibody-related disease (e.g., the probability of an asymptomatic patient developing an anti-X antibody-related disease during the next five years is greater than 50%, greater than 70%, greater than 90%, or greater than 95%). Asymptomatic subjects also include diseased subjects who may show mild early diagnostic indicators of an anti-X antibody-related disease, but otherwise have no disease or discomfort.

[0153] It should be noted that the terms "healthy control individuals," "healthy subjects," and grammatical equivalents thereof are used interchangeably and refer to subjects who do not have elevated anti-X antibody levels above a baseline or known or established standard value, which represent subjects who do not have a disease associated with an anti-X antibody.

[0154] In some embodiments, healthy subjects may never have suffered from a certain disease. In some embodiments, healthy subjects may have been ill before. In some embodiments, healthy subjects may receive a routine physical examination. In some embodiments, healthy subjects may be members of control groups such as clinical trials. In some embodiments, healthy subjects may have a risk of infectious diseases, which is determined by the presence of certain risk factors well known in the art. These risk factors include but are not limited to genetic susceptibility, personal disease history, family disease history, lifestyle factors, environmental factors, diagnostic indicators, etc.

[0155] The baseline or standard for determining or defining a subject as a subject who does not suffer from a disease associated with anti-X antibodies is a reference interval. In diagnostic or health-related fields, a reference interval is a range of values ​​observed in a reference subject (which may be a healthy control individual) expressed as a specific percentile. The reference interval can be any range of values ​​determined by a person skilled in the art. See CLSI, "How to define and determine reference intervals in the clinical laboratory: approved guideline," C28: A2 (2000).

[0156] In some cases, the reference interval can be strict or less strict, depending on the specific analyte being measured or the disease being studied. In some embodiments, the reference interval can be set to the 95th percentile. To increase specificity and reduce sensitivity, for example, to increase stringency, a higher cutoff value can be used, such as the 96th percentile or the 97th percentile or the 98th percentile or the 99th percentile.

[0157] If the anti-X antibody level is at least above the 95th percentile relative to the anti-X antibody level of healthy control subjects, the subject may be considered to have elevated anti-X antibodies. In other embodiments, if the anti-X antibody level is above the 96th, 97th, 98th, or 99th percentile, the subject may be considered to have elevated anti-X antibodies. If the subject's anti-X antibody level meets or exceeds any of the disclosed reference intervals, the subject may be considered to have a disease associated with anti-X antibodies.

[0158] In some embodiments, the presence of anti-X antibodies can be based on a comparison of the signal to the background in a healthy subject. In some embodiments, the presence of anti-X antibodies can be increased or decreased relative to the average or median anti-X antibody level observed in a population of healthy subjects. In some embodiments, anti-X antibodies may not be present in healthy subjects. In some embodiments, the detection of anti-X antibody levels does not exceed the noise of the corresponding detection method used to determine the anti-X antibody level. In some embodiments, if the detection of anti-X antibody levels exceeds the noise of the corresponding assay used to determine the anti-X antibody level, then anti-X antibodies can be considered to be present in the sample. In some embodiments, if the signal in the anti-X antibody detection assay is at least two standard deviations higher than the noise (such as the average or mean signal of the control sample), then anti-X antibodies can be considered to be increased in the sample. In some embodiments, if the level of anti-X antibodies exceeds a predetermined threshold level, then anti-X antibodies can be considered to be present in the sample. The anti-X antibody threshold level can be determined by a skilled artisan (such as a clinician) based on various factors, such as the specific goals of the clinical trial or the diagnostic and prognostic significance of a certain anti-X antibody level or the results of another diagnostic test for a disease associated with anti-X antibodies that does not involve the detection of anti-X antibody levels.

[0159] Anti-X antibodies can be detected in a variety of different biological samples obtained from a subject. Example "biological samples" include any specimen from an organism that can be used for analysis or diagnosis. For example, a biological sample can include a liquid sample, such as whole blood, plasma, serum, synovial fluid, amniotic fluid, sputum, pleural effusion, peritoneal effusion, cerebrospinal fluid, urine, bile, saliva, or tears. Biological samples can also include solid tissue samples, such as liver biopsy samples, bone marrow samples, oral samples, or other solid or semi-solid cell aggregate samples. For purposes of the present disclosure, a biological sample obtained from a subject can be any sample that contains or is suspected of containing autoantibodies, and also includes any material from a subject in which anti-X antibodies can be detected.

[0160] The biological sample can be or include, for example, whole blood, serum, plasma, or sputum. In some embodiments, the biological sample of the present disclosure can be a tissue biopsy, such as a liver biopsy. The length of the liver biopsy can be less than 10 mm, less than 11 mm, less than 12 mm, less than 13 mm, less than 14 mm, less than 15 mm, less than 16 mm, less than 17 mm, less than 18 mm, less than 19 mm, less than 20 mm, less than 21 mm, less than 22 mm, less than 21 mm, less than 23 mm, less than 24 mm, or less than 25 mm. In addition to the length disclosed herein, the liver biopsy can also include at least 4 portal triads, at least 5 portal triads, at least 6 portal triads, at least 7 portal triads, at least 8 portal triads, at least 9 portal triads, at least 10 portal triads, or at least 11 portal triads.

[0161] The biological sample can be obtained from any subject that has or is suspected of having anti-X antibodies. The biological sample can also be obtained from any subject that does not have or is suspected of not having anti-X antibodies. Thus, in some embodiments, the biological sample can be obtained from symptomatic subjects, asymptomatic subjects, and anti-X antibody negative subjects. In some embodiments, the biological sample can be obtained from a mammal.

[0162] Biological samples can be collected and processed immediately, or collected, frozen and subsequently processed. Biological samples can also include multiple samples from one of the subjects described herein. In some embodiments, multiple biological samples can be regularly collected in the time of more than 12 hours, more than 1 day, more than 2 days, more than 3 days, more than 4 days, more than 5 days, more than 6 days, more than 7 days, more than 10 days, more than 14 days, more than 3 weeks, more than 1 month, more than 2 months, more than 3 months, more than 4 months, more than 5 months, more than 6 months, more than 9 months, more than 12 months, more than 18 months, more than 24 months, more than 30 months, more than 3 years, more than 4 years or more than 5 years.

[0163] In some embodiments, the exemplary techniques described herein can be performed by contacting a biological sample with 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more support-antigen complexes described herein. Two or more antigens can be bound to a single solid support as described herein.

[0164] Detecting the presence of bound anti-X antibody can include contacting the anti-X antibody with an anti-X antibody-specific detection probe (eg, an antigen) and detecting specific binding of the antibody to the detection probe.

[0165] The reporter label can be used to detect the antibody. The function of the reporter label is to generate a signal for detecting the biomarker. In some embodiments, the reporter label comprises a ligand or a microparticle. In some embodiments, the ligand is biotin. In some embodiments, the microparticle comprises a nanoparticle. The reporter label can be attached to any detection probe used herein, for example, by non-covalent or covalent cross-linking. The reporter label may include a label of the type described herein. For example, the label may include a fluorophore, an enzyme, a chemiluminescent moiety, a radioactive moiety, an organic dye, a small molecule, a polypeptide or a functional fragment thereof. In some embodiments, the label includes a fluorescent label. In some embodiments, the fluorescent label is PE, horseradish peroxidase or alkaline phosphatase. In some embodiments, the label is conjugated to the detection probe of the present disclosure, as described above.

[0166] As described herein, a disease associated with an anti-X antibody can be determined in a subject by detecting the presence of bound anti-X antibodies. Techniques for detecting, measuring and / or quantifying signals generated by the markers of the present disclosure are well known in the art and include detection of fluorescence, luminescence, chemiluminescence or absorbance, reflection, transmission, birefringence or refractive index. Optical techniques include imaging techniques (such as confocal and non-confocal microscopy) and non-imaging techniques (such as microplate readers). In some embodiments, techniques for detecting anti-X antibodies in biological samples may include visualization, quantification, or both of fluorescent, colorimetric or absorbance signals in biological samples.

[0167] Elements of the different embodiments described may be combined into other embodiments not specifically described above. Elements may not be included in the system described above without adversely affecting its operation or the operation of the entire system. In addition, various independent elements may be combined into one or more separate elements to perform the functions described in this specification.

[0168] Other implementations not specifically described in this specification are also within the scope of the following claims.

Claims

1. A compound comprising: a solid support; and At least two different types of antigens are attached to the solid support.

2. The composite of claim 1, further comprising: a carrier, which is attached to the solid support; At least one of the different types of antigens is attached to the carrier, and at least one of the different types of antigens is attached to the solid support via the carrier.

3. The composite of claim 2, further comprising: A linker is provided to attach at least one of the different types of antigens to the carrier. The complex of claim 3 , wherein the linker comprises a polyethylene glycol (PEG) spacer or biotin.

5. The complex of claim 2, wherein the carrier comprises at least one of streptavidin, polymeric protein, polymeric streptavidin with biotin, bovine serum albumin (BSA), human serum albumin (HSA), a polyethylene glycol (PEG) spacer attachment, or adipic acid dihydrazide (ADH).

6. The composite of claim 1, further comprising: a plurality of carriers, each carrier being attached to the solid support; Each of the different types of antigens is attached to one of the multiple carriers, respectively, and each of the different types of antigens is attached to the solid support via one of the multiple carriers.

7. The composite of claim 1, further comprising: a carrier, which is attached to the solid support; Each of the different types of antigens is attached to the carrier, and each of the different types of antigens is attached to the solid support via the carrier.

8. The complex of claim 1, wherein the at least two different types of antigens comprise a panel of markers for a disease or condition in the subject.

9. The complex of claim 1, wherein at least one of said different types of antigens is part of a cell extract attached to said solid support.

10. The complex of claim 1, wherein a first of said different types of antigens comprises a purified antigen and a second of said different types of antigens is part of a cellular extract.

11. The complex of claim 1, wherein the at least two different types of antigens comprise at least two of the following: Scl-70, centromere, dsDNA, Ro52, Ro60, SS-B, Ribo-P, DFS-70, EJ, Sp100, Gp210, actin, AMA-M2, AMA-M2 / MIT3, TIF1-γ, NXP-2, SAE, PL-7, PL-12, OJ, HMGCR, SRP, Mi-2, MDA-5, RNA helicase, Ki / SL, Sm / RNP, SS-A, PM / Scl, RNA Pol III, Th / To, fibronectin, Sm, Ku, Jo-1, RNP, BICD2, HK, KL, LC1, LKM-1, SLA, SS-A / Ro52, Scl-70, Ku, centromere protein, U11 / U12 RNP, hexokinase-1 or Kelch-12.

12. The complex of claim 1, wherein at least one of the different types of antigens is bound to an antinuclear antibody (ANA).

13. The composite of claim 1, further comprising: Associated with the solid support is a label associated with a disease, disorder or analyte detectable using the at least two different antigens.

14. A kit comprising a first solid support comprising first antigens attached thereto, wherein the first antigens comprise at least two different types of antigens; and a second solid support comprising at least one second antigen attached thereto, the at least one second antigen being different from the first antigen; Wherein one or more of the first antigen or the at least one second antigen comprises a purified antigen or is part of a cell extract.

15. The kit of claim 14, wherein the first solid support has a first label, the second solid support has a second label, and the first label is different from the second label.

16. The kit of claim 14, wherein the first antigen comprises a marker for a first disease or condition, the first marker being associated with the first disease or condition; and Wherein the at least one second antigen comprises a marker for a second disease or condition, and the second marker is associated with the second disease or condition.

17. The kit of claim 14, wherein the first antigen comprises a first label that generates a signal in response to binding to a corresponding antibody; and wherein the at least one second antigen comprises at least one second marker that produces a signal in response to binding to at least one corresponding antibody; The first marker and the at least one second marker are of the same type or of different types.

18. A method comprising: contacting a biological sample from a subject with a complex comprising: Solid support; as well as at least two different types of antigens attached to the solid support; and detecting the presence of antibodies bound to the complex in the biological sample, wherein the presence of the antibodies bound to the complex is indicative of a disease.

19. The method of claim 18, further comprising: detecting the solid support based on a label on the solid support, wherein the label is associated with the disease; Wherein detecting comprises detecting signals from labels on the at least two different types of antigens.

20. The method of claim 18, wherein the complex is one of a plurality of complexes contacted with the biological sample, each of the plurality of complexes having a different configuration associated with a different disease, each of the plurality of complexes comprising: a respective solid support; and One or more antigens are attached to said respective solid supports.

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