Methods and kits for measuring antibodies

Abstract

The present invention pertains to a method for assaying a subject-animal antibody contained in a biological sample of a subject-animal. The method is characterized by comprising bringing an antigen, a biological sample of a subject-animal, and anti-subject-animal immunoglobulin antibodies into contact with each other, and assaying a complex formed of the antigen, a subject-animal antibody, and the anti-subject-animal immunoglobulin antibodies, and is characterized in that the anti-subject-animal immunoglobulin antibodies are a plurality of antibodies against a specific class of immunoglobulin, and include antibodies that cross-react with all subclasses of immunoglobulin in the specific class. The present invention also pertains to a kit for assaying a subject-animal antibody contained in a biological sample of a subject-animal. The kit is characterized by including an antigen and anti-subject-animal immunoglobulin antibodies, and is characterized in that the anti-subject-animal immunoglobulin antibodies are a plurality of antibodies against a specific class of immunoglobulin, and include antibodies that cross-react with all subclasses of immunoglobulin in the specific class.

Description

【Technical Field】 【0001】 The present invention relates to a method and a kit for measuring an antibody in a biological sample. 【Background Art】 【0002】 In order to diagnose an infectious disease or an autoimmune disease caused by a virus, a bacterium or a parasite, measurement of an antibody or an autoantibody against a virus, a bacterium or a parasite in a human specimen is performed. Measurement of antibodies against viruses, bacteria and parasites includes hepatitis B virus antibody and chlamydia antibody, etc., and measurement of autoantibodies includes rheumatoid factor and thyroid peroxidase antibody, etc. 【Summary of the Invention】 【Problems to be Solved by the Invention】 【0003】 In the measurement of an antibody for diagnosing an infectious disease or an autoimmune disease caused by a virus, a bacterium or a parasite, it is desirable that false high values and false low values do not occur and that the false positive rate and the false negative rate are low. Further, in an infectious disease, it is desirable that there is a high correlation with a neutralizing antibody. An object of the present invention is to provide an antibody measurement method and an antibody measurement kit in which false low values and false negatives are reduced in the measurement of an antibody contained in a biological sample of a test animal. Furthermore, a more specific object of the present invention is to provide an antibody measurement method and an antibody measurement kit in which the correlation with a neutralizing antibody is improved. 【Means for Solving the Problems】 【0004】 The present invention includes the following inventions in order to solve the above problems. [1] A method for measuring an animal antibody contained in a biological sample of an animal, comprising contacting an antigen with a biological sample of the animal and an anti-animal immunoglobulin antibody, and measuring a complex of the antigen, the animal antibody, and the anti-animal immunoglobulin antibody, wherein the anti-animal immunoglobulin antibody is a plurality of antibodies against a specific class of immunoglobulins and includes antibodies that cross-react with all subclasses of the specific class of immunoglobulins. [2] The method according to [1], wherein the anti-test animal immunoglobulin antibody further comprises an antibody that specifically reacts with an immunoglobulin of a specific subclass of the same class as the specific class. [3] The method according to [1] or [2], wherein the particular class of immunoglobulin is IgG. [4] The method according to any one of [1] to [3], wherein the test animal antibody is an antibody against a non-self or an autoantibody. [5] The method according to [4], wherein the non-self is a pathogenic microorganism, virus, or parasite. [6] The method according to [5], wherein the non-self is a virus. [7] The method according to [6], wherein the virus is SARS-CoV-2. [8] The method according to [1], wherein the plurality of antibodies are anti-test animal immunoglobulin antibodies belonging to different subclasses of the specific class. [9] The method according to any one of [1] to [8], wherein the anti-test animal immunoglobulin antibody or antigen is labeled.

[10] A kit for measuring animal antibodies contained in a biological sample of an animal, comprising an antigen and an anti-animal immunoglobulin antibody, wherein the anti-animal immunoglobulin antibody is a plurality of antibodies against a specific class of immunoglobulins and comprises antibodies that cross-react with all subclasses of the specific class of immunoglobulins.

[11] The kit according to

[10] , wherein the anti-test animal immunoglobulin antibody further comprises an antibody that specifically reacts with an immunoglobulin of a specific subclass of the same class as the specific class.

[12] The kit according to

[10] or

[11] , wherein the particular class of immunoglobulin is IgG.

[13] A kit according to any one of

[10] to

[12] , wherein the antigen is a pathogenic microbial antigen, a viral antigen, a parasitic antigen, or an antigen involved in an autoimmune disease.

[14] The kit according to

[13] , wherein the antigen is a viral antigen.

[15] The kit according to

[14] , wherein the viral antigen is a fragment of the SARS-CoV-2 spike protein.

[16] The kit according to

[10] , wherein the plurality of antibodies are anti-test animal immunoglobulin antibodies belonging to different subclasses of the specific class.

[17] A kit according to any one of

[10] to

[16] , wherein the anti-test animal immunoglobulin antibody or antigen is labeled. [Effects of the Invention] 【0005】 According to the present invention, it is possible to provide an antibody measurement method and an antibody measurement kit that reduce false low values ​​and false negatives in the measurement of antibodies of a test animal contained in a biological sample of the test animal. Furthermore, according to the present invention, more specifically, it is possible to provide an antibody measurement method and an antibody measurement kit with improved correlation to neutralizing antibodies. [Brief explanation of the drawing] 【0006】 [Figure 1] This figure shows the correlation between antibody concentrations against SARS-CoV-2 S-RBD and neutralizing activity against SARS-CoV-2, measured in serum samples from 34 COVID-19 patients. (A) shows the results when using third reagent A, which contains only antibodies that recognize all subclasses of human IgG, as the detection antibody reagent for measuring antibody concentration; (B) shows the results when using third reagent B, which contains only antibodies that specifically recognize human IgG4, as the detection antibody reagent for measuring antibody concentration; and (C) shows the results when using third reagent C, which contains antibodies from both third reagent A and third reagent B. [Figure 2] This figure shows the correlation between antibody concentrations against SARS-CoV-2 S-RBD and neutralizing activity against SARS-CoV-2, measured in serum samples from 34 COVID-19 patients. (A) shows the results when using third reagent A, which contains only antibodies that recognize all subclasses of human IgG, as the detection antibody reagent for measuring antibody concentration; (B) shows the results when using third reagent D, which contains only antibodies that specifically recognize human IgG3, as the detection antibody reagent for measuring antibody concentration; and (C) shows the results when using third reagent E, which contains antibodies from both third reagent A and third reagent D. [Figure 3] This figure shows the correlation between antibody concentrations against SARS-CoV-2 Spike protein and their neutralizing activity against SARS-CoV-2, measured by ELISA using SARS-CoV-2 Spike protein-immobilized plates in serum samples from 34 COVID-19 patients. (A) shows the results when only antibodies that recognize all subclasses of human IgG were used as enzyme-labeled antibodies, and (B) shows the results when a mixture of antibodies that recognize all subclasses of human IgG and antibodies that specifically recognize human IgG3 were used as enzyme-labeled antibodies. [Modes for carrying out the invention] 【0007】 [Antibody measurement method] The present invention provides a method for measuring animal antibodies contained in a biological sample of an animal (hereinafter referred to as "the measurement method of the present invention"). The measurement method of the present invention is a method comprising the steps of contacting an antigen with a biological sample of an animal and an anti-animal immunoglobulin antibody (step 1) and measuring a complex of the antigen, the animal antibody and the anti-animal immunoglobulin antibody (step 2), characterized in that the anti-animal immunoglobulin antibody is a plurality of antibodies against a specific class of immunoglobulin. 【0008】 The test animal can be any animal that possesses an acquired immune system. The test animal can be a mammal, for example, humans, non-human primates, rabbits, guinea pigs, rats, mice, dogs, cats, horses, cattle, pigs, sheep, and goats. Humans are preferred. The biological sample is not particularly limited as long as it is a sample in which antibodies of the test animal may be present, and examples include blood, body fluids, and tissues. Specifically, examples include serum, plasma, whole blood, urine, feces, saliva, ascites, oral mucosa, pharyngeal mucosa, intestinal mucosa, biopsy specimens, and tissue stains of the test animal. Serum or plasma are preferred. Even if the biological sample is collected from the animal being tested, it may have undergone pretreatment such as recovery, concentration, purification, isolation, dilution with buffer solutions, and sterilization by filtration. These pretreatments should be carried out appropriately according to standard procedures. 【0009】 The antibodies of the test animal being measured may be antibodies against non-self or autoantibodies. The target recognized by the antibodies against non-self is not particularly limited as long as it is an immunogenic non-self. The non-self may be pathogenic. Examples include pathogenic microorganisms, viruses, and parasites. Pathogenic microorganisms include fungi, protozoa, bacteria, spirochetes, rickettsiae, chlamydia, mycoplasmas, etc., that cause infectious diseases. Examples include fungi such as Candida and Cryptococcus, malaria parasites, Entamoeba histolytica, Trichomonas vaginalis, Pneumotis carinii pneumoniae, and Echinococcus, as well as bacteria such as Neisseria gonorrhoeae, Neisseria meningitidis, Staphylococcus, Streptococcus, Streptococcus pneumoniae, Shigella, Escherichia coli, Salmonella, Vibrio cholerae, Pseudomonas aeruginosa, Bordetella pertussis, Haemophilus influenzae, Mycobacterium tuberculosis, Neisseria tetanus, Mycobacterium leprae, and Neisseria diphtheriae, spirochetes such as Tryponema pallidum, Mycoplasma pneumoniae, Rickettsia such as Orientia scrub typhus, and Chlamydia such as Chlamydia trachomatis. Examples of viruses include adenoviruses, herpesviruses, papillomaviruses, hepatitis viruses, HIV, measles viruses, influenza viruses, coronaviruses such as SARS-CoV-2, Japanese encephalitis virus, rabies virus, norovirus, and rotavirus. Examples of parasites include lung flukes, liver flukes, liver flukes, Diphyllobothrium mansoni, cysticercosis, filarial worms, roundworms, Anisakis, gnathostoma, Strongyloides, and schistosomiasis. 【0010】 The antibody of the test animal to be measured is preferably an antibody against a virus, and preferably an antibody against SARS-CoV-2. 【0011】 The targets recognized by autoantibodies are not particularly limited as long as they are cells, tissues, or components present in the body's own tissues. The targets recognized by autoantibodies may also be antigens involved in autoimmune diseases. Examples of autoimmune diseases include rheumatoid arthritis, Hashimoto's disease, Graves' disease, antiphospholipid syndrome, insulin autoimmune disease syndrome, pemphigus, bullous pemphigoid, scleroderma, Sjögren's syndrome, Goodpasture syndrome, membranous nephropathy, IgA nephropathy, systemic lupus erythematosus (lupus erythematosus), dilated cardiomyopathy, IgG4-related disease, ANCA-associated vasculitis, myasthenia gravis, Harada's disease, narcolepsy, Burger's disease, type 1 diabetes, multiple sclerosis, neuromyelitis optica, primary biliary cirrhosis, Crohn's disease, ulcerative colitis, mixed connective tissue disease, and Wege. Examples include granulomatosis, vitiligo, polymyositis / dermatitis, thrombocytopenic purpura (ITP), idiopathic Addison's disease, idiopathic autoimmune hepatitis, autoimmune pancreatitis, atrophic gastritis, primary sclerosing cholangitis, aortitis syndrome (Takayasu's arteritis), autoimmune hemolytic anemia, autoimmune inner ear disorders, idiopathic azoospermia, acute disseminated encephalomyelitis, alopecia areata, autoimmune cardiomyopathy, chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, idiopathic pulmonary fibrosis, Guillain-Barré syndrome, lichen sclerosing, microscopic polyangiitis, paroxysmal nocturnal hemoglobinuria, relapsing polychondritis, sarcoidosis, and stiff-person syndrome. 【0012】 Examples of autoantibodies associated with autoimmune diseases include anti-double-stranded DNA antibodies, anti-single-stranded DNA antibodies, anti-histone antibodies, anti-RNA antibodies, anti-RNP antibodies, anti-Sm antibodies, anti-SS-A antibodies, anti-SS-B antibodies, anti-Scl-70 antibodies, anti-PCNA antibodies, anti-ribosomal antibodies, anti-mitochondrial antibodies, anti-centromere antibodies, anti-immunoglobulin antibodies, anti-thyroglobulin antibodies, anti-thyroid peroxidase antibodies, anti-microsomal antibodies, acetylcholine receptor antibodies, anti-erythrocyte antibodies, anti-platelet antibodies, anti-glomerular basement membrane antibodies, anti-islet of Langerhans antibodies, anti-adrenal cortical antibodies, and anti-parietal cell antibodies. 【0013】 In step 1, the antigen, the biological sample of the test animal, and the anti-test animal immunoglobulin antibody are brought into contact. The antigen is the antigen that the test animal antibody to be measured recognizes and binds to. Therefore, the antigen is appropriately selected and used depending on the test animal antibody to be measured. Antigens can be prepared, for example, from cultured cells by known methods. Protein antigens can also be prepared as recombinant proteins by constructing an expression vector containing DNA encoding the desired antigen protein using known genetic engineering techniques, and then introducing the expression vector into suitable host cells and culturing them. The DNA sequence encoding the desired antigen protein can be obtained from known databases (such as NCBI). For example, the accession number of the amino acid sequence of the SARS-CoV-2 spike protein is "QHD43416.1", and the base sequence of the gene encoding it is from positions 21563 to 25384 of accession number "MN908947.3". The antigen may be the full-length protein antigen or a partial fragment (peptide). Examples of protein antigens include the nucleocapsid protein (N protein), spike protein (S protein), and the full-length or partial fragments (peptides) of viral proteins, such as the S1 subunit, S2 subunit, and receptor-binding domain that make up the spike protein. 【0014】 Contact between the antigen, the biological sample of the test animal, and the anti-test animal immunoglobulin antibody can be performed, for example, by bringing the three (antigen, biological sample of the test animal, and anti-test animal immunoglobulin antibody) into contact in the presence of a suitable buffer. Examples of buffers include Tris buffer, phosphate buffer, acetate buffer, borate buffer, citrate buffer, and Veronal buffer. The antigen and biological sample may be brought into contact first, followed by the anti-test animal immunoglobulin antibody; the anti-test animal immunoglobulin antibody and biological sample may be brought into contact first, followed by the antigen; or all three may be brought into contact simultaneously. The anti-test animal immunoglobulin antibody or antigen may be labeled with a labeling substance used in immunoassays. 【0015】 Either a solid-phase antigen or a solid-phase anti-test animal immunoglobulin antibody may be used. The solid-phase support is not particularly limited as long as it is a normal support used in immunoassay. For example, polymer materials such as latex, rubber, polyethylene, polypropylene, polystyrene, styrene-butadiene copolymer, polyvinyl chloride, polyvinyl acetate, polyacrylamide, polymethacrylate, styrene-methacrylate copolymer, polyglycidyl methacrylate, acrylene-ethylene glycol dimethacrylate copolymer, polyvinylidene difluoride (PVDF), silicone, etc., and inorganic materials such as silica gel, glass, inert alumina, magnetic substances, etc. are used. The shape of the support is not particularly limited as long as it is the shape of a normal support used in immunoassay. For example, microplates, spherical, rod-shaped, microparticles (beads), etc. can be mentioned. 【0016】 The ratios of the antigen, the biological sample of the test animal, and the anti-test animal immunoglobulin antibody, the contact time, temperature, pH, and other conditions are not particularly limited and can be appropriately set according to the test animal antibody to be measured, the measurement method to be used, the measuring instrument to be used, etc. For example, the temperature may be 0 to 37 °C, the pH may be pH 6 to 9, and the contact time may be 1 to 120 minutes. 【0017】 In Step 2, the complex of the antigen, the test animal antibody, and the anti-test animal immunoglobulin antibody is measured. The measurement method is not particularly limited as long as it can detect and measure the complex. For example, first, the antigen and the biological sample are contacted to form a complex of the antigen and the test animal antibody, and then an anti-test animal immunoglobulin antibody labeled with the complex of the antigen and the test animal antibody is contacted, and the complex of the antigen, the test animal antibody, and the anti-test animal immunoglobulin antibody is measured by a measurement method according to the labeling substance. Or, first, the anti-test animal immunoglobulin antibody and the biological sample are contacted to form a complex of the anti-test animal immunoglobulin antibody and the test animal antibody, and then an antigen labeled with the complex of the anti-test animal immunoglobulin antibody and the test animal antibody is contacted, and the complex of the antigen, the test animal antibody, and the anti-test animal immunoglobulin antibody is measured by a measurement method according to the labeling substance. 【0018】 The labeling substance is not particularly limited, and can be appropriately selected and used from known labeling substances such as enzymes, fluorescent dyes, fluorescent proteins, radioisotopes, gold colloids, biotin, avidin, etc. Examples of enzymes include, for example, alkaline phosphatase (ALP), peroxidase (POD), microperoxidase, horseradish peroxidase (HRP), β-galactosidase (β-gal), etc. Examples of fluorescent dyes include, for example, fluorescein, fluorescein isothiocyanate (FITC), tetramethylrhodamine (TAMRA), tetramethylrhodamine isothiocyanate (TRICA), Texas red, cyanine 3 (Cy3), cyanine 5 (Cy5), etc. Examples of fluorescent proteins include, for example, allophycocyanin (APC), phycoerythrin (PE), etc. The measurement method of the labeling substance is known. For example, when an enzyme is used as the labeling substance, various measurements can be performed according to the substrate by adding a chromogenic substrate, a fluorescent substrate, a chemiluminescent substrate, etc. as the substrate. 【0019】 The measurement method of the present invention is characterized in that a plurality of antibodies against a specific class of immunoglobulins are used as the anti-test animal immunoglobulin antibody. The correlation between the measurement value obtained by the measurement method of the present invention and the neutralization activity value measured using the same biological sample can be improved. The plurality of antibodies may be used in the form of a mixture. 【0020】 Immunoglobulin antibodies are classified into five classes based on the type of heavy chain within the molecule. For example, in the case of human immunoglobulin antibodies, these are IgG, IgM, IgA, IgD, and IgE. The IgG molecule has a γ chain as its heavy chain, IgM has a μ chain, IgA has an α chain, IgE has an ε chain, and IgD has a δ chain. In the measurement method of the present invention, the test animal antibody to be measured may be any of IgG, IgM, IgA, IgD, or IgE, and it is sufficient to use multiple antibodies against a specific class of test animal immunoglobulin as detection antibodies. The specific class is preferably IgG or IgA with subclasses. In humans, IgG has four subclasses, IgG1 to IgG4, and IgA has two subclasses, IgA1 and IgA2. The specific class is preferably IgG. 【0021】 Multiple antibodies against a specific class of test animal immunoglobulins (hereinafter referred to as "detection antibodies") used as detection antibodies against test animal immunoglobulins may be monoclonal antibodies or polyclonal antibodies. Furthermore, the antibodies may be antibody fragments with antigen-binding ability (e.g., Fab, Fab', F(ab')2, Fv, scFv, diabody, etc.), or low-molecular-weight antibodies to which the variable region of the antibody has been attached. The detection antibodies may be a combination of multiple monoclonal antibodies, a combination of one or more monoclonal antibodies and one or more polyclonal antibodies, or a combination of multiple polyclonal antibodies. 【0022】 Polyclonal antibodies and monoclonal antibodies can be produced by known methods. Polyclonal antibodies can be produced, for example, by immunizing mammals (mice, rats, rabbits, goats, horses, etc.) with an immunogen prepared by dissolving an antigen in PBS and optionally mixing it with an appropriate amount of a common adjuvant (e.g., Freund's complete adjuvant), collecting blood from the immunized animals according to a standard method, separating the serum, and purifying the polyclonal antibody fraction. The immunization method is not particularly limited, but for example, a method of subcutaneous injection or intraperitoneal injection once or multiple times at appropriate intervals is preferred. Monoclonal antibodies can be produced, for example, by fusing immune cells (e.g., spleen cells) obtained from the above-immunized mammals with myeloma cells to obtain a hybridoma, and collecting antibodies from the culture of the hybridoma. Alternatively, antibody genes can be cloned from hybridomas, incorporated into a suitable vector, introduced into host cells, and recombinant monoclonal antibodies can be produced using genetic recombination technology. Furthermore, monoclonal antibodies can also be produced using phage display. Polyclonal antibodies may be used, for example, by removing antibodies that react with a specific subclass through adsorption treatment. 【0023】 The detection antibody preferably contains an antibody that cross-reacts with all subclasses of immunoglobulins of a particular class. That is, if the class of the test animal antibody being measured is IgA, the detection antibody preferably contains a polyclonal or monoclonal antibody that binds to both IgA1 and IgA2, and if the class of the test animal antibody being measured is IgG, the detection antibody preferably contains a polyclonal or monoclonal antibody that binds to all four subclasses of IgG1 to IgG4. The cross-reaction of a polyclonal or monoclonal antibody with both IgA1 and IgA2, or with all four subclasses of IgG1 to IgG4, can be confirmed using known immunoassay methods. 【0024】 Examples of antibodies that cross-react with all subclasses of immunoglobulins include the brand name Mouse Anti-Human IgG Fc-UNLB (Clone: ​​JDC-10, manufactured by Southern Biotech). The recognition sites of antibodies that cross-react with all subclasses of immunoglobulins include the constant region and the Fc region. 【0025】 The detection antibody preferably includes an antibody that cross-reacts with all subclasses of immunoglobulins of the specific class mentioned above, as well as an antibody that specifically reacts with any one subclass of immunoglobulin of that specific class. If the specific class is IgA, one can choose to include a detection antibody containing an antibody that cross-reacts with both IgA1 and IgA2 and an antibody that specifically reacts with IgA1, or a detection antibody containing an antibody that cross-reacts with both IgA1 and IgA2 and an antibody that specifically reacts with IgA2. 【0026】 If a specific class is IgG, you can choose from the following combinations of detection antibodies. (1) Detection antibody containing an antibody that cross-reacts with all subclasses of IgG and an antibody that specifically reacts with IgG1. (2) Detection antibody containing an antibody that cross-reacts with all subclasses of IgG and an antibody that specifically reacts with IgG2. (3) Detection antibody containing an antibody that cross-reacts with all subclasses of IgG and an antibody that specifically reacts with IgG3. (4) Detection antibody containing an antibody that cross-reacts with all subclasses of IgG and an antibody that specifically reacts with IgG4. (5) Detection antibodies including antibodies that cross-react with all subclasses of IgG, antibodies that specifically react with IgG1, and antibodies that specifically react with IgG2. (6) Detection antibodies containing antibodies that cross-react with all subclasses of IgG, antibodies that specifically react with IgG1, and antibodies that specifically react with IgG3. (7) Detection antibodies containing antibodies that cross-react with all subclasses of IgG, antibodies that specifically react with IgG1, and antibodies that specifically react with IgG4. (8) Detection antibodies including antibodies that cross-react with all subclasses of IgG, antibodies that specifically react with IgG2, and antibodies that specifically react with IgG3. (9) Detection antibodies containing antibodies that cross-react with all subclasses of IgG, antibodies that specifically react with IgG2, and antibodies that specifically react with IgG4. (10) Detection antibodies including antibodies that cross-react with all subclasses of IgG, antibodies that specifically react with IgG3, and antibodies that specifically react with IgG4. (11) Detection antibodies including antibodies that cross-react with all subclasses of IgG, antibodies that specifically react with IgG1, antibodies that specifically react with IgG2, and antibodies that specifically react with IgG3. (12) Detection antibodies including antibodies that cross-react with all subclasses of IgG, antibodies that specifically react with IgG1, antibodies that specifically react with IgG2, and antibodies that specifically react with IgG4. (13) Detection antibodies including antibodies that cross-react with all subclasses of IgG, antibodies that specifically react with IgG1, antibodies that specifically react with IgG3, and antibodies that specifically react with IgG4. (14) Detection antibodies including antibodies that cross-react with all subclasses of IgG, antibodies that specifically react with IgG2, antibodies that specifically react with IgG3, and antibodies that specifically react with IgG4. (15) Detection antibodies including antibodies that cross-react with all subclasses of IgG, antibodies that specifically react with IgG1, antibodies that specifically react with IgG2, antibodies that specifically react with IgG3, and antibodies that specifically react with IgG4. 【0027】 Antibodies that specifically react with one subclass of immunoglobulins of a particular class include, for example, antibodies that specifically react with IgG1, such as Mouse anti-Human IgG1 Fc Secondary Antibody (Clone: ​​HP6070, Thermo Fisher Scientific); antibodies that specifically react with IgG2, such as Mouse monoclonal [31-7-4] Anti-Human IgG2 Fc (Clone: ​​31-7-4, Abcam); antibodies that specifically react with IgG3, such as Mouse Anti-Human IgG3 Hinge-UNLB (Clone: ​​HP6050, Southern Biotech); Mouse Anti-Human IgG4 Fc-UNLB (Clone: ​​HP6025, Southern Biotech); Mouse Anti-Human IgG4 pFc'-UNLB (Clone: ​​HP6023, Southern Biotech); and Monoclonal Antibody to Human. Examples include antibodies that specifically react with IgG4, such as IgG4 (Clone: ​​5C3, manufactured by Yamasa Soy Sauce Co., Ltd.). Recognition sites for antibodies that specifically react with one subclass of a particular class of immunoglobulin include the constant region, Fc region, pFc' region, or hinge region. 【0028】 The detection antibody may contain multiple antibodies that specifically react with a particular subclass of immunoglobulin. When the antibody contains multiple antibodies that specifically react with a particular subclass of immunoglobulin, it is preferable that each antibody recognizes a different epitope of that subclass of immunoglobulin. 【0029】 Instead of using an antibody that cross-reacts with all subclasses of immunoglobulins of the specific class mentioned above, the detection antibody may be a combination of antibodies that specifically react with each subclass of immunoglobulin. That is, in the case of IgA, the detection antibody may include an antibody that specifically reacts with IgA1 and an antibody that specifically reacts with IgA2, and in the case of IgG, the detection antibody may include an antibody that specifically reacts with IgG1, an antibody that specifically reacts with IgG2, an antibody that specifically reacts with IgG3, and an antibody that specifically reacts with IgG4. To such a detection antibody, an antibody that specifically reacts with any one or more subclasses of immunoglobulin may be added to make a detection antibody. 【0030】 In step 2, the method for measuring the complex of the antigen, the test animal antibody, and the anti-test animal immunoglobulin antibody includes, specifically, enzyme-linked immunosorbent assay (ELISA), enzyme-enzyme immunoassay (EIA), radioimmunoassay (RIA), fluorescence-enzyme immunoassay (FEIA), fluorescence-enzyme immunoassay (FIA), chemiluminescent enzyme immunoassay (CLEIA), chemiluminescent immunoassay (CLIA), electrochemiluminescent immunoassay (ECLIA), immunocomplex transfer assay, immunochromatography (ICA), luminescent oxygen channeling immunoassay (LOCI), and liquid-phase binding assay-electrokinetic assay. This can be performed using known immunoassay methods such as Analyte (LBA-EATA method), capillary electrophoresis, Western blotting, latex immunohistochemistry (NIA method), latex turbidiometry (TIA method), microparticle counting immunoaggregation assay (PCIA method), surface plasmon resonance (SPR method), AlphaLISA method, and assays that detect the presence of the target molecule using fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET). 【0031】 By using the measurement method of the present invention, the correlation between the measured value of the test animal antibody and the neutralizing antibody can be improved compared to the measurement method using a single detection antibody (anti-test animal immunoglobulin antibody). When using the measurement method of the present invention, the correlation coefficients between the antibody measurement value and the neutralizing antibody are preferably R=0.7 or higher, R=0.75 or higher, R=0.8 or higher, R=0.85 or higher, and R=0.9 or higher. The method for evaluating the activity (neutralizing activity) of the neutralizing antibody varies depending on the antigen recognized by the test animal antibody, and can be measured using a known method for measuring neutralizing activity depending on the antigen recognized by the test animal antibody. 【0032】 〔kit〕 The present invention provides a kit (hereinafter referred to as "the kit of the present invention") for measuring animal antibodies contained in a biological sample of an animal. By using the kit of the present invention, the measurement method of the present invention can be carried out simply and quickly. The kit of the present invention is characterized in that the reagent (hereinafter referred to as "detection antibody reagent") for detecting the animal antibody to be measured is a plurality of antibodies against a specific class of immunoglobulin. 【0033】 The detection antibody reagent provides the detection antibody described above as a reagent. The detection antibody reagent is a combination of multiple antibodies as described above, and may be provided as a reagent in the form of a mixture of multiple antibodies in one container, or as a reagent in the form of multiple antibodies provided in separate containers. The antibodies contained in the detection antibody reagent may be labeled. The detection antibody reagent can be prepared, for example, by dissolving the antibody in a buffer suitable for the antibody solution. If the reagent is in the form of a mixture, it can be prepared by mixing each antibody solution. As the buffer, for example, Tris buffer, phosphate buffer, acetate buffer, borate buffer, citrate buffer, veronal buffer, etc., with a pH of 6 to 9, preferably pH 7 to 8, can be used. The detection antibody reagent may be lyophilized. 【0034】 The kit of the present invention may include, in addition to the detection antibody reagent, a reagent containing an antigen recognized by the antibody of the test animal to be measured (hereinafter referred to as "antigen reagent"). Since the antigen differs depending on the antibody of the test animal to be measured, a kit equipped with the desired antigen reagent is provided individually. The antigen may be labeled. The antigen reagent can be prepared, for example, by dissolving the antigen in a buffer suitable for the antigen solution. Suitable buffers for the antigen solution are the same as those suitable for the detection antibody reagent. The antigen reagent may be lyophilized. 【0035】 For example, when conducting a test kit to measure animal antibodies that recognize SARS-CoV-2, the full-length or fragment of the SARS-CoV-2 spike protein can be used as the antigen. The full-length or fragment of the SARS-CoV-2 spike protein can be produced as a recombinant protein using known genetic engineering techniques. 【0036】 In addition to the above, the kit of the present invention may also include a buffer solution for sample dilution, reagents for measuring the labeled substance, a multiwell plate or tube, instructions for use, etc. [Examples] 【0037】 The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. 【0038】 [Example 1: Measurement of IgG antibodies against SARS-CoV-2 S-RBD in COVID-19 patients] Using serum samples from 34 COVID-19 patients who visited Fujita Health University, the amount of IgG antibodies against the SARS-CoV-2 Spike protein receptor binding domain (S-RBD) in the serum was measured, and the neutralizing activity values ​​against COVID-19 in the serum of these patients were compared. 【0039】 1-1 Preparation of Reagents The reagents necessary for the measurement were prepared using the following reagent raw materials: MES (2-morpholinoethanesulfonic acid, manufactured by Dojin Chemical Laboratories), sodium chloride (manufactured by Fujifilm Wako Pure Chemical Corporation), BSA (manufactured by Sigma-Aldrich Japan Co., Ltd.), boric acid (manufactured by Fujifilm Wako Pure Chemical Corporation), sodium luminol salt (manufactured by Sigma-Aldrich Japan Co., Ltd.), phosphoric acid (manufactured by Fujifilm Wako Pure Chemical Corporation), and hydrogen peroxide (manufactured by Fujifilm Wako Pure Chemical Corporation). 【0040】 (1) First reagent (antigen-solid-phase magnetic particle reagent) Magnetic silica particles (product name: Magrapid, manufactured by Sanyo Chemical Industries, Ltd.) were successively reacted with γ-aminopropyltriethoxysilane (manufactured by Shin-Etsu Chemical Co., Ltd.) and succinic anhydride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). The mixture was magnetized using a neodymium magnet, and the supernatant was removed to obtain magnetic particles containing carboxyl groups. Subsequently, SARS-CoV-2 S-RBD (manufactured by ACRO Biosystems) was reacted with N-hydroxysuccinimide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and WSC (water-soluble carbodiimide, manufactured by Dojin Chemical Laboratories) at 26°C for 12-16 hours. The mixture was magnetized using a neodymium magnet, and the supernatant was removed to prepare antigen-solid-phase magnetic particles, and the first reagent consisting of the following composition was prepared. ·S-RBD antigen solid phase magnetic particles 0.25mg / mL 50mM MES (pH 5.5) 500 mM sodium chloride 【0041】 (2) Second reagent (reaction buffer) A buffer solution with the following composition was prepared. 50mM MES (pH 6.0) 300 mM sodium chloride 【0042】 (3) Third reagent (detection antibody reagent) Three types of reagents containing the following peroxidase-labeled antibodies were prepared as a third reagent to measure the amount of IgG antibodies in patient serum. Reagent A: Antibody that recognizes all subclasses of human IgG (hereinafter referred to as "anti-human IgG antibody") Reagent B: Antibody that specifically recognizes human IgG4 (hereinafter referred to as "anti-human IgG4 antibody") Reagent C: Mixture of anti-human IgG antibody and anti-human IgG4 antibody 【0043】 For the enzyme-labeled anti-human IgG antibodies in Reagent 3A and Reagent 3C, we used diluted enzyme-labeled antibodies included in the COVID-19 ELISA reagent (product name: COVID-19 IgG Test ELISA Kit Wako (S-RBD), manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). The enzyme-labeled antibodies included in this kit recognize all subclasses of human IgG. 【0044】 (3-1) Preparation of the third reagent A The composition of the third reagent A is shown below. 53.3% enzyme-labeled anti-human IgG antibody solution 50mM MES (pH 6.5) 150 mM sodium chloride 2.0% BSA 【0045】 (3-2) Preparation of the third reagent B A commercially available peroxidase-labeled anti-human IgG4 antibody (Clone: ​​5C3, manufactured by Yamasa Shoyu Co., Ltd.) was used as the anti-human IgG4 antibody. Using this antibody, a third reagent B consisting of the following composition was prepared. • 1000 ppm POD-labeled anti-human IgG4 antibody (1000-fold dilution) 50mM MES (pH 6.5) 150 mM sodium chloride 2.0% BSA 【0046】 (3-3) Preparation of the third reagent C Reagent C, consisting of the following composition, was prepared by mixing the antibodies used in the third reagent A and the third reagent B. • 53.3% enzyme-labeled anti-human IgG antibody solution • 1000 ppm POD-labeled anti-human IgG4 antibody (1000-fold dilution) 50mM MES (pH 6.5) 150 mM sodium chloride 2.0% BSA 【0047】 (4) Fourth reagent A fourth reagent was prepared, consisting of the following composition. • 200 mM boric acid (pH 8.55) 150 mM sodium chloride • 1g / L luminol sodium salt 【0048】 (5) Fifth reagent A fifth reagent was prepared, consisting of the following composition. • 68 μL / L Phosphate 670 μL / L hydrogen peroxide 【0049】 (6) Other reagents "AcuraSeed B / F Separation Solution" (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was used as the washing solution. For calibration curve samples, 400 ng / mL of antibody against SARS-CoV-2 S-RBD recombinant with human IgG1 (manufactured by ACRO Biosystems) was defined as 1 U / mL, and diluted samples of 1.4 U / mL, 14.3 U / mL, 28.6 U / mL, 52.4 U / mL, 76.1 U / mL, and 104.7 U / mL were prepared. 【0050】 1-2 Measurement of antibody concentration against SARS-CoV-2 S-RBD The concentration of IgG antibodies against SARS-CoV-2 S-RBD in human serum was measured using the Accuraseed automated chemiluminescent enzyme immunoassay analyzer (registered trademark, manufactured by Fujifilm Techno Products Co., Ltd.) according to the following procedure using reagents 1 through 5 and the washing solution. 【0051】 50 μL of the first reagent was added to the reaction cuvette, magnetized using a neodymium magnet, and the supernatant was removed. Subsequently, 140 μL of the second reagent and 10 μL of the measurement sample (COVID-19 patient serum or calibration curve sample) were added and mixed, and the mixture was heated at 37°C for 3 minutes. After heating, the mixture was magnetized using a neodymium magnet to remove reagents other than magnetic particles, and washed three times with washing solution. Next, 50 μL of the third reagent was added, and the mixture was heated at 37°C for 3 minutes. After heating, the mixture was magnetized using a neodymium magnet to remove reagents other than magnetic particles, and washed three times with washing solution. After washing, 100 μL of the fourth reagent and 100 μL of the fifth reagent were added, and after a reaction at 37°C for 20 seconds, the luminescence was measured. A calibration curve showing the relationship between the IgG antibody content against SARS-CoV-2 S-RBD and the luminescence was created from the luminescence measured for each calibration curve sample, and the IgG antibody content against SARS-CoV-2 S-RBD in the serum of COVID-19 patients was determined. Since a calibration curve could not be created using the calibration sample for the third reagent B, the calibration curve created with the third reagent A was used for the analysis. 【0052】 1-3 Neutralizing activity values ​​against SARS-CoV-2 The neutralizing activity against SARS-CoV-2 was measured according to the neutralization test method described in the "COVID-19 Serological Testing Manual" published by the National Institute of Infectious Diseases. 【0053】 1-4 Results Figure 1 shows the correlation between antibody concentration and neutralizing activity. (A) shows the results when using third reagent A, (B) shows the results when using third reagent B, and (C) shows the results when using third reagent C. Compared to when using third reagent A or third reagent B, an improvement in correlation was observed when using third reagent C. 【0054】 Similarly, when Clone:JDC-10 (Southern Biotech) was used as the enzyme-labeled anti-human IgG antibody solution in the third reagent, instead of the enzyme-labeled antibody included in the COVID-19 IgG ELISA Kit Wako (S-RBD), it was confirmed that the correlation improved when using third reagent C compared to when using third reagent A or third reagent B. 【0055】 Furthermore, when Clone:HP6025 (Southern Biotech) or Clone:HP6023 (Southern Biotech) were used instead of the peroxidase-labeled anti-human IgG4 antibody (Clone: ​​5C3) in the third reagent, it was confirmed that the correlation improved when using third reagent C compared to when using third reagent A or third reagent B. 【0056】 Furthermore, when Clone:JDC-10 (Southern Biotech) was used instead of the enzyme-labeled antibody included in the COVID-19 IgG ELISA Kit Wako (S-RBD) as the enzyme-labeled anti-human IgG antibody solution, and when Clone:HP6025 (Southern Biotech) or Clone:HP6023 (Southern Biotech) was used instead of the peroxidase-labeled anti-human IgG4 antibody (Clone: ​​5C3) as the POD-labeled anti-human IgG4 antibody, it was confirmed that the correlation improved when using third reagent C compared to when using third reagent A or third reagent B. 【0057】 [Example 2: Measurement of IgG antibodies against SARS-CoV-2 S-RBD in COVID-19 patients] 2-1 Reagents and Methods In place of the anti-human IgG4 antibody used in the third reagents B and C of Example 1, a commercially available POD-labeled anti-human IgG3 antibody (Clone: ​​HP6050, Southern Biotech) that specifically recognizes human IgG3 (hereinafter referred to as "anti-human IgG3 antibody") was used to prepare third reagent D, which contains only the anti-human IgG3 antibody, and third reagent E, which is a mixture of the anti-human IgG antibody and the anti-human IgG3 antibody. 【0058】 The composition of the third reagent D is as follows: • 100 ppm POD-labeled anti-human IgG3 antibody (10,000-fold dilution) 50mM MES (pH 6.5) 150 mM sodium chloride 2.0% BSA The composition of the third reagent E is as follows: • 53.3% enzyme-labeled anti-human IgG antibody solution • 100 ppm POD-labeled anti-human IgG3 antibody (10,000-fold dilution) 50mM MES (pH 6.5) 150 mM sodium chloride 2.0% BSA 【0059】 The antibody concentration against SARS-CoV-2 S-RBD and the neutralizing activity value against SARS-CoV-2 were measured in the serum of 34 COVID-19 patients using the same method as in Example 1, except that reagent D was used instead of reagent B, and reagent E was used instead of reagent C. Since a calibration curve could not be created using the calibration sample for reagent D, the calibration curve created with reagent A was used for the analysis. 【0060】 2-2 Results Figure 2 shows the correlation between antibody concentration and neutralizing activity. (A) shows the results when using third reagent A, (B) shows the results when using third reagent D, and (C) shows the results when using third reagent E. Compared to when using third reagent A or third reagent D, an improvement in correlation was observed when using third reagent E. 【0061】 [Example 3: Measurement of IgG antibodies against SARS-CoV-2 Spike protein in COVID-19 patients] 3-1 Reagents and Methods Using SARS-CoV-2 Spike protein immobilized plates (hereinafter referred to as "S-full immobilized plates") and COVID-19 ELISA reagent (product name: COVID-19 IgG test ELISA kit Wako (S-RBD), manufactured by Fujifilm Wako Pure Chemical Corporation), the amount of IgG antibodies against SARS-CoV-2 Spike protein in the serum of 34 COVID-19 patients who visited Fujita Health University was measured and compared with the neutralizing activity value against COVID-19 in the serum of the same patients. The enzyme-labeled antibody included in this kit recognizes all subclasses of human IgG. 【0062】 (1) Fabrication of S-full immobilization plate S-full immobilized plates were prepared by reacting SARS-CoV-2 Spike protein (CerTest) dissolved in 50 mM carbonate buffer with a 96-well plate (Thermo Fisher Scientific, Inc.), and then blocking with phosphate buffer containing 1.0% Block Ace (K.A.C. Co., Ltd.). 【0063】 (2) Preparation of enzyme-labeled antibodies For the evaluation of the third reagent A, the enzyme-labeled antibody included in the kit was used as is. For the evaluation of the third reagent E, the enzyme-labeled antibody solution included in the kit was used with the POD-labeled anti-human IgG3 antibody used in Example 2 added to a concentration of 1000 ppm. (3) Preparation of washing solution The concentrated washing solution (10x) included in the kit was diluted 10 times with purified water produced using a Milli-Q water production system (Merck Millipore K.K.) to prepare the washing solution. 【0064】 (4)Measurement method Samples of serum from the same 34 COVID-19 patients as in Examples 1 and 2, diluted 201-fold with the sample diluent provided in the kit, or the standard provided in the kit, were added to an S-full immobilized plate and reacted at room temperature for 1 hour. Next, the plates were washed three times with 300 μL of washing solution, and then 50 μL each of enzyme-labeled antibody solution for evaluating the third reagent A or the third reagent E was added, and the plates were reacted at room temperature for 1 hour. The plates were washed three times with 300 μL of washing solution, 100 μL of chromogenic solution was added, and after 10 minutes, 100 μL of stop solution was added, and the absorbance at the primary wavelength of 450 nm and the secondary wavelength of 620 nm was measured. The amount of IgG antibody against S-full in the serum of COVID-19 infected patients was calculated from the calibration curve obtained from the standard. 【0065】 3-2 Results Figure 3 shows the correlation between the amount of IgG antibody against S-full calculated above and the neutralization activity value measured in Example 1. (A) shows the results of evaluating the third reagent A, and (B) shows the results of evaluating the third reagent E. Compared to using an antibody that recognizes all subclasses of anti-human IgG alone (third reagent A), an improvement in correlation was observed when using both an antibody that recognizes all subclasses of anti-human IgG and an anti-human IgG3 antibody (third reagent E). 【0066】 It should be noted that the present invention is not limited to the embodiments and examples described above, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included within the technical scope of the present invention. Furthermore, all academic and patent documents mentioned herein are incorporated herein by reference.

Claims

[Claim 1] A method for measuring antibodies of a test animal contained in a biological sample of a test animal, comprising contacting an antigen with a biological sample of the test animal and an anti-test animal immunoglobulin antibody, and measuring a complex of the antigen, the test animal antibody, and the anti-test animal immunoglobulin antibody, wherein the anti-test animal immunoglobulin antibody is a plurality of antibodies against a specific class of immunoglobulins. The plurality of antibodies include a combination of antibodies that cross-react with all subclasses of the particular class of immunoglobulins and antibodies that specifically react with a particular subclass of immunoglobulin of the same class as the particular class, The above combination is one of the following (1) to (17): (1) Antibodies that cross-react with all subclasses of IgG and antibodies that specifically react with IgG1; (2) Antibodies that cross-react with all subclasses of IgG and antibodies that specifically react with IgG2; (3) Antibodies that cross-react with all subclasses of IgG and antibodies that specifically react with IgG3; (4) Antibodies that cross-react with all subclasses of IgG and antibodies that specifically react with IgG4; (5) Antibodies that cross-react with all subclasses of IgG, antibodies that specifically react with IgG1, and antibodies that specifically react with IgG2; (6) Antibodies that cross-react with all subclasses of IgG, antibodies that specifically react with IgG1, and antibodies that specifically react with IgG3; (7) Antibodies that cross-react with all subclasses of IgG, antibodies that specifically react with IgG1, and antibodies that specifically react with IgG4; (8) Antibodies that cross-react with all subclasses of IgG, antibodies that specifically react with IgG2, and antibodies that specifically react with IgG3; (9) Antibodies that cross-react with all subclasses of IgG, antibodies that specifically react with IgG2, and antibodies that specifically react with IgG4; (10) Antibodies that cross-react with all subclasses of IgG, antibodies that specifically react with IgG3, and antibodies that specifically react with IgG4; (11) Antibodies that cross-react with all subclasses of IgG, antibodies that specifically react with IgG1, antibodies that specifically react with IgG2, and antibodies that specifically react with IgG3; (12) Antibodies that cross-react with all subclasses of IgG, antibodies that specifically react with IgG1, antibodies that specifically react with IgG2, and antibodies that specifically react with IgG4; (13) Antibodies that cross-react with all subclasses of IgG, antibodies that specifically react with IgG1, antibodies that specifically react with IgG3, and antibodies that specifically react with IgG4; (14) Antibodies that cross-react with all subclasses of IgG, antibodies that specifically react with IgG2, antibodies that specifically react with IgG3, and antibodies that specifically react with IgG4; (15) Antibodies that cross-react with all subclasses of IgG, antibodies that specifically react with IgG1, antibodies that specifically react with IgG2, antibodies that specifically react with IgG3, and antibodies that specifically react with IgG4; (16) an antibody that cross-reacts with all subclasses of IgA and an antibody that specifically reacts with IgA1; or (17) An antibody that cross-reacts with all subclasses of IgA and an antibody that specifically reacts with IgA2. [Claim 2] The method according to claim 1, wherein the immunoglobulin of the specific class is IgG. [Claim 3] The method according to claim 1 or 2, wherein the test animal antibody is an antibody against a non-self or an autoantibody. [Claim 4] The method according to claim 3, wherein the non-self is a pathogenic microorganism, a virus, or a parasite. [Claim 5] The method according to any one of claims 1 to 4, wherein the anti-test animal immunoglobulin antibody or antigen is labeled. [Claim 6] A kit for measuring antibodies of a test animal contained in a biological sample of a test animal, comprising an antigen and an anti-test animal immunoglobulin antibody, wherein the anti-test animal immunoglobulin antibody is a plurality of antibodies against a specific class of immunoglobulins. The plurality of antibodies include a combination of antibodies that cross-react with all subclasses of immunoglobulins of the specific class and antibodies that specifically react with a specific subclass of immunoglobulin of the same class as the specific class, The above combination is one of the following (1) to (17): (1) Antibodies that cross-react with all subclasses of IgG and antibodies that specifically react with IgG1; (2) Antibodies that cross-react with all subclasses of IgG and antibodies that specifically react with IgG2; (3) Antibodies that cross-react with all subclasses of IgG and antibodies that specifically react with IgG3; (4) Antibodies that cross-react with all subclasses of IgG and antibodies that specifically react with IgG4; (5) Antibodies that cross-react with all subclasses of IgG, antibodies that specifically react with IgG1, and antibodies that specifically react with IgG2; (6) Antibodies that cross-react with all subclasses of IgG, antibodies that specifically react with IgG1, and antibodies that specifically react with IgG3; (7) Antibodies that cross-react with all subclasses of IgG, antibodies that specifically react with IgG1, and antibodies that specifically react with IgG4; (8) Antibodies that cross-react with all subclasses of IgG, antibodies that specifically react with IgG2, and antibodies that specifically react with IgG3; (9) Antibodies that cross-react with all subclasses of IgG, antibodies that specifically react with IgG2, and antibodies that specifically react with IgG4; (10) Antibodies that cross-react with all subclasses of IgG, antibodies that specifically react with IgG3, and antibodies that specifically react with IgG4; (11) Antibodies that cross-react with all subclasses of IgG, antibodies that specifically react with IgG1, antibodies that specifically react with IgG2, and antibodies that specifically react with IgG3; (12) Antibodies that cross-react with all subclasses of IgG, antibodies that specifically react with IgG1, antibodies that specifically react with IgG2, and antibodies that specifically react with IgG4; (13) Antibodies that cross-react with all subclasses of IgG, antibodies that specifically react with IgG1, antibodies that specifically react with IgG3, and antibodies that specifically react with IgG4; (14) Antibodies that cross-react with all subclasses of IgG, antibodies that specifically react with IgG2, antibodies that specifically react with IgG3, and antibodies that specifically react with IgG4; (15) Antibodies that cross-react with all subclasses of IgG, antibodies that specifically react with IgG1, antibodies that specifically react with IgG2, antibodies that specifically react with IgG3, and antibodies that specifically react with IgG4; (16) an antibody that cross-reacts with all subclasses of IgA and an antibody that specifically reacts with IgA1; or (17) An antibody that cross-reacts with all subclasses of IgA and an antibody that specifically reacts with IgA2. [Claim 7] The kit according to claim 6, wherein the immunoglobulin of the specific class is IgG. [Claim 8] The kit according to claim 6 or 7, wherein the antigen is a pathogenic microorganism antigen, a viral antigen, a parasite, or an antigen involved in an autoimmune disease. [Claim 9] The kit according to any one of claims 6 to 8, wherein the anti-test animal immunoglobulin antibody or antigen is labeled.

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