WO2022118947A1 - Method and kit for assaying antibody - Google Patents

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

Methods and kits for measuring antibodies

The present invention relates to a method and a kit for measuring an antibody in a biological sample.

In order to diagnose infectious diseases or autoimmune diseases caused by viruses, bacteria and parasites, antibodies against viruses, bacteria and parasites in human specimens or autoimmune diseases are measured. Hepatitis B virus antibody and chlamydia antibody are known for measuring antibodies against viruses, bacteria and parasites, and rheumatoid factor and thyroid peroxidase antibody are known for measuring autoantibodies.

In the measurement of antibodies for diagnosing infectious diseases or autoimmune diseases caused by viruses, bacteria and parasites, it is desirable that false highs and false lows do not occur and false positive rates and false negative rates are low. Furthermore, in infectious diseases, it is desirable that the correlation with neutralizing antibodies is high.
An object of the present invention is to provide an antibody measuring method and an antibody measuring kit in which false low values and false negatives are reduced in the measurement of a test animal antibody contained in a biological sample of a test animal.
Further, more specifically, it is an object of the present invention to provide an antibody measuring method and an antibody measuring kit having an improved correlation with a neutralizing antibody.

The present invention includes the following inventions in order to solve the above problems.
[1] A method for measuring a test animal antibody contained in a biological sample of a test animal, in which the antigen and the biological sample of the test animal are brought into contact with an anti-test animal immunoglobulin antibody, and the antigen and the test are tested. The anti-test animal immunoglobulin antibody comprises measuring a complex of an animal antibody and an anti-test animal immunoglobulin antibody, wherein the anti-test animal immunoglobulin antibody is a plurality of antibodies against a particular class of immunoglobulin, said particular class. A method comprising an antibody that cross-reacts with immunoglobulins of all subclasses of.
[2] The method according to the above [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] above, wherein the particular class of immunoglobulin is IgG.
[4] The method according to any one of [1] to [3] above, wherein the test animal antibody is an antibody against non-self or an autoantibody.
[5] The method according to [4] above, wherein the non-self is a pathogenic microorganism, a virus or a parasite.
[6] The method according to [5] above, wherein the non-self is a virus.
[7] The method according to [6] above, wherein the virus is SARS-CoV-2.
[8] The method according to [1] above, 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] above, wherein the anti-test animal immunoglobulin antibody or antigen is labeled.
[10] A kit for measuring a test animal antibody contained in a biological sample of a test animal, which comprises an antigen and an anti-test animal immunoglobulin antibody, and the anti-test animal immunoglobulin antibody is a specific class. A kit comprising a plurality of antibodies against an immunoglobulin of the above, comprising an antibody that cross-reacts with all subclasses of the immunoglobulin of the particular class.
[11] The kit according to the above [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] above, wherein the particular class of immunoglobulin is IgG.
[13] The kit according to any one of the above [10] to [12], wherein the antigen is a pathogenic microbial antigen, a viral antigen, a parasite, or an antigen involved in an autoimmune disease.
[14] The kit according to the above [13], wherein the antigen is a viral antigen.
[15] The kit according to the above [14], wherein the viral antigen is a fragment of a peaplomer of SARS-CoV-2.
[16] The kit according to the above [10], wherein the plurality of antibodies are anti-test animal immunoglobulin antibodies belonging to different subclasses of the specific class.
[17] The kit according to any one of [10] to [16] above, wherein the anti-test animal immunoglobulin antibody or antigen is labeled.

According to the present invention, it is possible to provide an antibody measuring method and an antibody measuring kit in which false low values and false negatives are reduced in the measurement of a test animal antibody contained in a biological sample of a test animal.
Further, according to the present invention, more specifically, it is possible to provide an antibody measuring method and an antibody measuring kit having an improved correlation with a neutralizing antibody.

The antibody concentration against SARS-CoV-2 S-RBD and the neutralization activity value against SARS-CoV-2 were measured for the sera of 34 cases collected from COVID-19 patients, respectively, and the correlation between the two was shown. As a result of using the third reagent A containing only the antibody recognizing all subclasses of human IgG as the detection antibody reagent used in (A) for measuring the antibody concentration, (B) is for detection used in measuring the antibody concentration. As a result of using the third reagent B containing only the antibody specifically recognizing human IgG4 as the antibody reagent, (C) the third reagent C containing both the antibody of the third reagent A and the antibody of the third reagent B. It is a result when. The antibody concentration against SARS-CoV-2 S-RBD and the neutralization activity value against SARS-CoV-2 were measured for the sera of 34 cases collected from COVID-19 patients, respectively, and the correlation between the two was shown. As a result of using the third reagent A containing only the antibody recognizing all subclasses of human IgG as the detection antibody reagent used in (A) for measuring the antibody concentration, (B) is for detection used in measuring the antibody concentration. As a result of using the third reagent D containing only the antibody specifically recognizing human IgG3 as the antibody reagent, (C) the third reagent E containing both the antibody of the third reagent A and the antibody of the third reagent D. It is a result when. Antibodies to SARS-CoV-2 Spike protein measured by ELISA using a SARS-CoV-2 Spike protein-immobilized plate and neutralization to SARS-CoV-2 in 34 sera collected from COVID-19 patients. It is a figure showing the correlation with the activity value, and as a result of the case where (A) uses only an antibody that recognizes all subclasses of human IgG as an enzyme-labeled antibody, (B) is all subclasses of human IgG as an enzyme-labeled antibody. This is the result when a mixture of an antibody that recognizes human IgG3 and an antibody that specifically recognizes human IgG3 is used.

[Antibody measurement method]
The present invention provides a method for measuring a test animal antibody contained in a biological sample of a test animal (hereinafter referred to as "the measuring method of the present invention"). The measuring method of the present invention comprises contacting the antigen with a biological sample of the test animal and the anti-test animal immunoglobulin antibody (step 1), and the antigen, the test animal antibody, and the anti-test animal immunoglobulin antibody. A method comprising the step of measuring a complex (step 2), characterized in that the anti-test animal immunoglobulin antibody is a plurality of antibodies against a particular class of immunoglobulin.

The test animal may be an animal having an adaptive immune system. The test animal may be a mammal, and examples thereof include humans, non-human primates, rabbits, guinea pigs, rats, mice, dogs, cats, horses, cows, pigs, sheep, and goats. It is preferably human. The biological sample is not particularly limited as long as it is a sample in which the antibody of the test animal can be present, and examples thereof include blood, body fluid, and tissue. Specific examples thereof include serum, plasma, whole blood, urine, stool, saliva, ascites, oral mucosa, pharyngeal mucosa, intestinal mucosa, biopsy sample, and tissue staining of a test animal. Preferred is serum or plasma.
The biological sample may be a sample collected from a test animal or may have been subjected to pretreatment such as recovery, concentration, purification, isolation, dilution with a buffer solution, or filtration sterilization. These pretreatments may be appropriately performed according to a conventional method.

The test animal antibody to be measured may be an antibody against non-self or an autoantibody. The target recognized by the antibody against the non-self is not particularly limited as long as it is a non-self having immunogenicity. The non-self may be pathogenic. For example, pathogenic microorganisms, viruses, parasites and the like can be mentioned. Pathogenic microorganisms include fungi, protozoans, bacteria, spirochete, rickettsia, chlamydia, mycoplasma, etc. that cause infectious diseases. For example, fungi such as Candida and Cryptococcus, malaria protozoa, diarrhea amoeba, vaginal trichomonas, pneumotiscarini pneumonia, protozoa such as Echinocox, gonorrhea, epidemic meningitis, staphylococci, streptococci, pneumonia diplococci, erythema. , Escherichia coli, Salmonella, Cholera, C. Chlamydia such as Riquetcia and Chlamydia trachomatis. Examples of viruses include adenovirus, herpesvirus, papillomavirus, hepatitis virus, AIDS virus, hemp virus, influenza virus, coronavirus such as SARS-CoV-2, Japanese encephalitis virus, mad dog disease virus, norovirus, and rotavirus. Can be mentioned. Examples of parasites include lung flukes, liver flukes, fasciola hepatica, Spirometra erinaceus, scab, filamentous worms, roundworms, Anisakis, jaw worms, fecal nematodes, and blood-sucking flukes.

The test animal antibody to be measured is preferably an antibody against a virus, and preferably an antibody against SARS-CoV-2.

The target recognized by the autoantibody is not particularly limited as long as it is a cell, tissue, or component existing in the living body of the autoantibody. The target recognized by the autoantibody may be an antigen involved in an autoimmune disease. Examples of autoimmune diseases include rheumatoid arthritis, Hashimoto's disease, Based's disease (Graves' disease), antiphospholipid antibody syndrome, insulin autoimmune disease syndrome, scab, scab, scleroderma, Sjogren's syndrome, and Good Pasture's syndrome. (Goodpasture syndrome), membranous nephropathy, IgA nephropathy, systemic erythematosus (Lupus erythematosus), dilated myocardial disease, IgG4-related disease, ANCA-related vasculitis, severe myasthenia, Harada disease, Narcolepsy, Burger Disease, type I diabetes, polysclerosis, optic neuromyelitis, primary biliary cirrhosis, Crohn's disease, ulcerative colitis, mixed connective tissue disease, Wegener's granulomatosis, leukoplakia vulgaris, polymyositis / dermatitis, Thrombocytopenic purpura (ITP), idiopathic azison disease, idiopathic autoimmune hepatitis, autoimmune pancreatitis, atrophic gastric inflammation, primary sclerosing cholangitis, aortitis syndrome (hyperan arteritis), autoimmune hemolysis Sexual anemia, autoimmune internal ear disorders, idiopathic asperemia, acute diffuse encephalomyelitis, alopecia alopecia, autoimmune myocardial disease, chronic inflammatory demyelinating polyneuritis, Churg-Strauss syndrome, idiopathic lung Fibrosis, Gillan Valley syndrome, sclerosing lichen, microscopic polyangiitis, paroxysmal nocturnal hemochromatosis, recurrent polychondritis, sarcoidosis, Stiffperson syndrome and the like.

Examples of autoantibodies related to autoimmune diseases include anti-double-stranded DNA antibodies, anti-single-stranded DNA antibodies, anti-histon antibodies, anti-RNA antibodies, anti-RNP antibodies, anti-Sm antibodies, anti-SS-A antibodies, and anti-antibodies. SS-B antibody, anti-Scl-70 antibody, anti-PCNA antibody, anti-ribosome antibody, anti-mitochontic antibody, anti-centromea antibody, anti-immunoglobulin antibody, anti-tyroglobulin antibody, anti-thyroid peroxidase antibody, anti-microsome antibody, acetylcholine receptor antibody , Anti-erythrocyte antibody, anti-platelet antibody, anti-globulous basal membrane antibody, anti-Langerhans islet antibody, anti-adrenal cortex antibody, anti-gastric wall cell antibody and the like.

In step 1, the antigen, the biological sample of the test animal, and the anti-test animal immunoglobulin antibody are brought into contact with each other. The antigen is an antigen that is recognized and bound by the test animal antibody to be measured. Therefore, the antigen is appropriately selected and used according to the test animal antibody to be measured. The antigen can be prepared, for example, from cultured cells by a known method. Further, the protein antigen can be used as a recombinant protein by constructing an expression vector containing a DNA encoding a desired antigen protein using a known gene recombination technique, introducing the expression vector into an appropriate host cell, and culturing the vector. Can be prepared. The DNA sequence encoding the desired antigenic protein can be obtained from a known database (such as NCBI). For example, the amino acid sequence of SARS CoV-2 spike protein has an accession number of "QHD43416.1", and the base sequence of the gene encoding this is the accession number "MN908947.3" at positions 21563 to 25384. ..
The antigen may be the full length of the protein antigen or a partial fragment (peptide).
Examples of protein antigens include full-length or partial fragments of viral proteins such as viral nucleocapsid protein (N protein), spike protein (S protein), S1 subunit, S2 subunit, and receptor-binding domain that make up the spike protein. Peptide).

Contact between the antigen and the biological sample of the test animal and the anti-test animal immunoglobulin antibody is performed, for example, in the presence of an appropriate buffer (antigen, biological sample of the test animal and anti-test animal immunoglobulin antibody). There is a method of contacting the antibody. Examples of the buffer solution include Tris buffer solution, phosphate buffer solution, acetate buffer solution, borate buffer solution, citrate buffer solution, veronal buffer solution and the like. The antigen and the biological sample may be contacted first, and then the anti-test animal immunoglobulin antibody may be contacted, or the anti-test animal immunoglobulin antibody and the biological sample may be contacted first, and then the antigen is contacted. Alternatively, the three parties may be brought into contact with each other at the same time. The anti-test animal immunoglobulin antibody or antigen may be labeled with a labeling substance used for immunoassay.

An immobilized antigen may be used, or an immobilized anti-test animal immunoglobulin antibody may be used. The immobilized support is not particularly limited as long as it is a normal support used for immunoassay, and is, for example, latex, rubber, polyethylene, polypropylene, polystyrene, styrene-butadiene copolymer, polyvinyl chloride, polyvinyl acetate, and the like. Polymer materials such as polyacrylamide, polymethacrylate, styrene-methacrylate copolymer, polyglycidyl methacrylate, achlorine-ethylene glycol dimethacrylate copolymer, polyvinylidene difluoride (PVDF), silicone, silica gel, glass, inert alumina. , Inorganic materials such as magnetic materials are used. The shape of the support is not particularly limited as long as it is the shape of a normal support used for immunoassay, and examples thereof include microplates, spheres, rods, and fine particles (beads).

Conditions such as the ratio of the antigen, the biological sample of the test animal to the anti-test animal immunoglobulin antibody, the contact time, the temperature, the pH, etc. are not particularly limited, and the test animal antibody to be measured, the measurement method to be used, and the use are used. It can be set as appropriate according to the measuring device and the like. For example, the temperature may be 0-37 ° C., the pH may be pH 6-9, and the contact time may be 1-120 minutes.

In step 2, the complex of the antigen, the test animal antibody, and the anti-test animal immunoglobulin antibody is measured. The measuring method is not particularly limited as long as it can detect and measure the complex. For example, the antigen and the biological sample are first contacted to form a complex of the antigen and the test animal antibody, and the anti-test animal immunoglobulin antibody labeled on the complex of the antigen and the test animal antibody is contacted and labeled. Examples thereof include a method of measuring a complex of an antigen, a test animal antibody, and an anti-test animal immunoglobulin antibody by a measurement method according to a substance. Alternatively, first, the anti-test animal immunoglobulin antibody and the biological sample are brought into contact with each other to form a complex of the anti-test animal immunoglobulin antibody and the test animal antibody, and then the anti-test animal immunoglobulin antibody and the test animal antibody are combined. Examples thereof include a method in which a labeled antigen is brought into contact with the complex and a 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.

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, radioactive isotopes, colloidal gold, biotin, and avidin. Examples of the enzyme include alkaline phosphatase (ALP), peroxidase (POD), microperoxidase, horse radish peroxidase (HRP), β-galactosidase (β-gal) and the like, and examples of the fluorescent dye include fluorosane. Examples thereof include fluorescein isothiocyanate (FITC), tetramethyllodamine (TAMRA), tetramethyllodamine isothiocyanate (TRICA), Texas red, cyanine 3 (Cy3), cyanine 5 (Cy5), and the like. Examples include allophicocyanine (APC) and phycoerythrin (PE). A method for measuring a labeling substance is known. For example, when an enzyme is used as a labeling substance, various measurements are performed depending on the substrate by adding a color-developing substrate, a fluorescent substrate, a chemically luminescent substrate, or the like as a substrate. be able to.

The measuring method of the present invention is characterized in that the anti-test animal immunoglobulin antibody uses a plurality of antibodies against a specific class of immunoglobulin. It is possible to improve the correlation between the measured value obtained by the measuring method of the present invention and the neutralizing activity value measured using the same biological sample. Multiple antibodies may be used in the form of a mixture.

Immunoglobulin antibodies are classified into five classes, IgG, IgM, IgA, IgD and IgE, depending on the type of heavy chain in the molecule, for example, in the case of human immunoglobulin antibodies. The IgG molecule has a γ chain as a 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 and IgE, and for detecting a plurality of antibodies against immunoglobulins of a specific class of test animals. It may be used as an antibody. The particular class is preferably IgG or IgA with subclasses. In humans, IgG has four subclasses of IgG1 to IgG4, and IgA has two subclasses of IgA1 and IgA2. IgG is preferred for a particular class.

Multiple antibodies against a particular class of test animal immunoglobulin (hereinafter referred to as "detection antibody") used as anti-test animal immunoglobulin antibodies for detection may be monoclonal antibodies or polyclonal antibodies. It may be an antibody. The antibody is a low-molecular-weight antibody to which an antibody fragment having an antigen-binding ability (for example, Fab, Fab', F (ab') ₂ , Fv, scFv, diabody, etc.) and a variable portion of the antibody are bound. You may. The detection antibody may be a combination of a plurality of monoclonal antibodies, a combination of one or more monoclonal antibodies and one or more polyclonal antibodies, or a combination of a plurality of polyclonal antibodies.

Polyclonal antibody and monoclonal antibody can be produced by known methods. Polyclonal antibody immunizes mammals (mouse, rat, rabbit, goat, horse, etc.) using, for example, an antigen dissolved in PBS and optionally mixed with an appropriate amount of a usual adjuvant (for example, Freund's complete adjuvant) as an immunogen. It can be prepared by collecting blood from an immunized animal according to a conventional 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 preferable. The monoclonal antibody can be produced, for example, by fusing immune cells (for example, splenocytes) obtained from the immunized mammal and myeloma cells to obtain a hybridoma, and collecting the antibody from the culture of the hybridoma. can. It is also possible to clone an antibody gene from a hybridoma, incorporate it into an appropriate vector, introduce it into a host cell, and use gene recombination technology to produce a recombinant monoclonal antibody. Furthermore, monoclonal antibodies can also be prepared using the phage display method. As the polyclonal antibody, for example, a polyclonal antibody excluding an antibody that reacts with a specific subclass by an adsorption treatment or the like may be used.

The detection antibody preferably contains an antibody that cross-reacts with immunoglobulins of all subclasses of a specific class. That is, when the class of the test animal antibody to be measured is IgA, the detection antibody preferably contains a polyclonal antibody or a monoclonal antibody that binds to both IgA1 and IgA2, and the test animal to be measured preferably contains. When the antibody class is IgG, the detection antibody preferably comprises a polyclonal antibody or a monoclonal antibody that binds to all four subclasses of IgG1 to IgG4. It can be confirmed by using a known immune response measurement method that the polyclonal antibody or the monoclonal antibody cross-reacts with both IgA1 and IgA2, or cross-reacts with all four subclasses of IgG1 to IgG4.

Examples of the antibody that cross-reacts with all subclasses of immunoglobulin include the trade name Mouse Anti-Human IgG Fc-UNLB (Clone: JDC-10, manufactured by Southern Biotech). Recognition sites for antibodies that cross-react with all subclass immunoglobulins include the constant region and the Fc region.

Detection antibodies may include antibodies that cross-react with all subclass immunoglobulins of the particular class described above, as well as antibodies that specifically react with immunoglobulins of any one subclass of that particular class. preferable. When a particular class is IgA, detection antibodies, including antibodies that cross-react with both IgA1 and IgA2 and antibodies that specifically react with IgA1, and antibodies that cross-react with both IgA1 and IgA2 and are specific to IgA2. It is possible to select any of the detection antibodies including the antibody that reacts with the target.

If the particular class is IgG, you can choose from detection antibodies that include the combinations shown below.
(1) Detection antibody containing 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 Detection antibody (3) Detection antibody containing antibody that cross-reacts with all subclasses of IgG and antibody that specifically reacts with IgG3 (4) Antibodies that cross-react with all subclasses of IgG and IgG4 specifically Detection antibody containing reactive antibody (5) Detection antibody containing antibody that cross-reacts with all subclasses of IgG, antibody that specifically reacts with IgG1 and antibody that specifically reacts with IgG2 (6) All of IgG Detection antibody including antibody that cross-reacts with the subclass of IgG1 and antibody that specifically reacts with IgG3 (7) Antibodies that cross-react with all subclasses of IgG and react specifically with IgG1 Detection antibody containing antibody that specifically reacts with IgG4 (8) Detection containing antibody that cross-reacts with all subclasses of IgG, antibody that reacts specifically with IgG2, and antibody that reacts specifically with IgG3 Antibodies for detection (9) Antibodies for detection including antibodies that cross-react with all subclasses of IgG, antibodies that specifically react with IgG2, and antibodies that specifically react with IgG4 (10) Cross-react with all subclasses of IgG. Detection antibody including antibody that reacts specifically with IgG3 and antibody that reacts specifically with IgG4 (11) Antibody that cross-reacts with all subclasses of IgG and antibody that reacts specifically with IgG1 and IgG2 specifically Detection antibody containing antibody that reacts specifically with IgG3 (12) Antibodies that cross-react with all subclasses of IgG, antibody that reacts specifically with IgG1, and antibody that reacts specifically with IgG2 Detection antibody containing antibody that specifically reacts 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 IgG4. Detection antibody containing antibody that reacts with (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 react specifically with IgG4. Included detection antibodies (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 IgG4 specifically. Anti-antibody Detection antibody including corresponding antibody

Antibodies that specifically react with immunoglobulins of any one subclass of a specific class include, for example, IgG1 such as the trade name Mouse anti-Human IgG1 Fc Secondary Antibody (Clone: HP6070, manufactured by Thermo Fisher Scientific). Antibodies that specifically react with IgG2, trade name Mouse monoclonal [31-7-4] Anti-Human IgG2 Fc (Clone: 31-7-4, manufactured by Abcum), etc. Antibodies that specifically react with IgG2, trade name Antibodies that specifically react with IgG3 such as Mouse Anti-Human IgG3 Hinge-UNLB (Clone: HP6050, manufactured by Southern Biotech), trade name Mouse Anti-Human IgG4 Fc-UNLB (Clone: HP6025, manufactured by Southern Biotech), Product name: Mouse Anti-Human IgG4 pFc'-UNLB (Clone: HP6023, manufactured by Southern Biotech), Monoclonal Antibody to Human IgG4 (Clone: 5C3, manufactured by Yamasa Soy Sauce) and other antibodies that specifically react with IgG4. Be done. Examples of the recognition site of the antibody that specifically reacts with the immunoglobulin of any one subclass of the specific class include a constant region, an Fc region, a pFc'region, a hinge region, and the like.

The detection antibody may include a plurality of antibodies that specifically react with a specific subclass of immunoglobulin. When comprising a plurality of antibodies that specifically react with a particular subclass of immunoglobulin, each antibody is preferably an antibody that recognizes different epitopes of that subclass of immunoglobulin.

As the detection antibody, instead of using an antibody that cross-reacts with immunoglobulins of all the subclasses of the above specific class, an antibody that specifically reacts with the immunoglobulin of each subclass may be used in combination. That is, in the case of IgA, it may be a detection antibody containing an antibody that specifically reacts with IgA1 and an antibody that specifically reacts with IgA2, and in the case of IgG, an antibody that specifically reacts with IgG1 and IgG2 may be used. It may be a detection antibody containing an antibody that specifically reacts with IgG3, an antibody that specifically reacts with IgG3, and an antibody that specifically reacts with IgG4. An antibody that specifically reacts with immunoglobulin of any one or more subclasses may be added to the detection antibody having such a configuration to obtain a detection antibody.

Specific examples of the method for measuring the complex of the antigen, the test animal antibody, and the anti-test animal immunoglobulin antibody in step 2 include, for example, an enzyme-bound immunoassay measurement method (ELISA method) and an enzyme immunoassay. Method (EIA method), Radiation immunoassay (RIA method), Fluorescent enzyme immunoassay (FEIA method), Fluorescent immunoassay (FIA method), Chemiluminescent enzyme immunoassay (CLEIA method), Chemiluminescent immunoassay (CLEIA method) CLIA method), electrochemical luminescence immunoassay (ECLIA method), immunocomplex transfer method, immunochromatography method (ICA method), Luminescent Oxygen Channeling Immunoassay (LOCI method), Liquid-phase Binding Assay-ElectroKinetic Analyte (LBA-EATA) Method), capillary electrophoresis, western blot method, immunoassay method such as latex immunoassay (NIA method), immunoturbidimetric method such as latex immunoassay (TIA method), fine particle counting immunoaggregation measurement method Immunoaggregation method such as (PCIA method), surface plasmon resonance method (SPR method), AlphaLISA method, assay to detect the presence of target molecule using fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET), etc. It can be carried out by a known immunoassay method or the like.

By using the measurement method of the present invention, the correlation between the measured value of the test animal antibody to be measured and the neutralizing antibody is compared with the measurement method using a single detection antibody (anti-test animal immunoglobulin antibody). It can enhance sex. When the measurement method of the present invention is used, the correlation coefficient between the antibody measurement value and the neutralizing antibody is R = 0.7 or more, R = 0.75 or more, R = 0.8 or more, R = 0.85 or more, R = 0.9 or more. Is preferable. The evaluation method of the neutralizing antibody activity (neutralizing activity) differs depending on the antigen recognized by the test animal antibody to be measured, and a known neutralizing activity value measuring method is used depending on the antigen recognized by the test animal antibody. Can be measured.

〔kit〕
The present invention provides a kit for measuring a test animal antibody contained in a biological sample of a test animal (hereinafter referred to as "kit of the present invention"). By using the kit of the present invention, the measurement method of the present invention can be carried out easily and quickly. In the kit of the present invention, a plurality of reagents containing an anti-test animal immunoglobulin antibody for detecting an antibody of a test animal to be measured (hereinafter referred to as "antibody reagent for detection") are used for a specific class of immunoglobulin. It is characterized by being an antibody of.

The detection antibody reagent provides the detection antibody described above as a reagent. The detection antibody reagent is a combination of a plurality of antibodies as described above, and may be a reagent in which a mixture of a plurality of antibodies is provided in one container, or a form in which the plurality of antibodies are provided in separate containers. It may be used as a reagent of. The antibody 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 solution suitable for the antibody solution. When the reagent is in the form of a mixture, it can be prepared by mixing each antibody solution. As the buffer solution, for example, a Tris buffer solution having a pH of 6 to 9, preferably a pH of 7 to 8, a phosphate buffer solution, an acetate buffer solution, a borate buffer solution, a citrate buffer solution, a veronal buffer solution and the like can be used. .. The detection antibody reagent may be freeze-dried.

The kit of the present invention may include a reagent containing an antigen recognized by the test animal antibody to be measured (hereinafter referred to as "antigen reagent") in addition to the detection antibody reagent. Since the antigen varies depending on the test animal antibody to be measured, a kit containing the desired antigen reagent is individually provided. The antigen may be labeled. The antigen reagent can be prepared, for example, by dissolving the antigen in a buffer solution suitable for the antigen solution. Examples of the buffer solution suitable for the antigen solution include those similar to the buffer solution suitable for the antibody reagent for detection. The antigen reagent may be freeze-dried.

For example, when implementing a kit for measuring a test animal antibody that recognizes 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 gene recombination techniques.

In addition to the above, the kit of the present invention may contain a buffer solution for diluting a sample, reagents for measuring a labeling substance, a multi-well plate or tube, instructions for use, and the like.

Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited thereto.

[Example 1: Measurement of IgG antibody against SARS-CoV-2 S-RBD in COVID-19 patients]
Using 34 serums collected from COVID-19 patients who visited Fujita Medical University, the amount of IgG antibody against SARS-CoV-2 Spike protein receptor binding domain (S-RBD) in the serum was measured, and the amount of IgG antibody against the SARS-CoV-2 Spike protein receptor binding domain (S-RBD) was measured. The neutralization activity values for COVID-19 were compared.

1-1 Preparation of reagents The constituent reagents required for measurement were prepared using the following reagent raw materials. MES (2-morpholinoetan sulfonic acid, manufactured by Dojin Chemical Industries, Ltd.), sodium chloride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), BSA (manufactured by Sigma Aldrich Japan Co., Ltd.), boric acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) , Luminor sodium salt (manufactured by Sigma Aldrich Japan Co., Ltd.), Phosphoric acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), Hydrogen peroxide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)

(1) First reagent (antigen solid phase magnetic particle reagent)
Magnetic silica particles (trade name: Magrapid, manufactured by Sanyo Kasei Kogyo Co., Ltd.) are reacted with γ-aminopropyltriethoxysilane (manufactured by Shin-Etsu Chemical Co., Ltd.) and anhydrous apuccinic acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) one after another. Then, the particles were collected by a neodymium magnet and the supernatant was removed to obtain magnetic particles having a carboxyl group. Subsequently, using N-hydroxysuccinimide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and WSC (water-soluble carbodiimide, manufactured by Dojin Chemical Research Institute), SARS-CoV-2 S-RBD (manufactured by ACRO Biosystems). Was reacted at 26 ° C. for 12 to 16 hours, magnetized with a neodium magnet, and the supernatant was removed to prepare solid-phase magnetic particles of antigen, and the first reagent having the following composition was prepared.
・ S-RBD antigen solid phase magnetic particles 0.25mg / mL
・ 50mM MES (pH 5.5)
・ 500 mM sodium chloride

(2) Second reagent (reaction buffer)
A buffer solution having the following composition was prepared.
・ 50mM MES (pH 6.0)
・ 300 mM sodium chloride

(3) Third reagent (antibody reagent for detection)
As the third reagent constituting the reagent for measuring the amount of IgG antibody in the patient's serum, three kinds of reagents including the following peroxidase-labeled antibody were prepared.
Reagent A: Antibodies that recognize all subclasses of human IgG (hereinafter referred to as "anti-human IgG antibodies")
Reagent B: An antibody that specifically recognizes human IgG4 (hereinafter referred to as "anti-human IgG4 antibody")
Reagent C: Mixing of anti-human IgG antibody and anti-human IgG4 antibody

COVID-19 ELISA reagent (trade name: COVID-19 IgG test ELISA kit Wako (S-RBD), manufactured by Fujifilm Wako Junyaku Co., Ltd.) for the enzyme-labeled anti-human IgG antibody of the third reagent A and the third reagent C. The enzyme-labeled antibody included in the kit was diluted and used. The enzyme-labeled antibody included in this kit is an antibody that recognizes all subclasses of human IgG.

(3-1) Preparation of Third Reagent A The composition of Third Reagent A is shown below.
・ 53.3% enzyme-labeled anti-human IgG antibody solution ・ 50 mM MES (pH 6.5)
・ 150 mM sodium chloride ・ 2.0% BSA

(3-2) Preparation of Third Reagent B As an anti-human IgG4 antibody, a commercially available peroxidase-labeled anti-human IgG4 antibody (Clone: 5C3, manufactured by Yamasa Soy Sauce Co., Ltd.) was used. Using this antibody, a third reagent B having the following composition was prepared.
・ 1000ppm POD-labeled anti-human IgG4 antibody (diluted 1000 times)
・ 50mM MES (pH 6.5)
・ 150 mM sodium chloride ・ 2.0% BSA

(3-3) Preparation of Third Reagent C Reagent C having the following composition was prepared by mixing the third reagent A and the antibody used in the third reagent B.
・ 53.3% enzyme-labeled anti-human IgG antibody solution ・ 1000ppm POD-labeled anti-human IgG4 antibody (diluted 1000 times)
・ 50mM MES (pH 6.5)
・ 150 mM sodium chloride ・ 2.0% BSA

(4) Fourth reagent A fourth reagent having the following composition was prepared.
・ 200 mM boric acid (pH 8.55)
・ 150 mM sodium chloride ・ 1 g / L luminol sodium salt

(5) Fifth reagent A fifth reagent having the following composition was prepared.
・ 68 μL / L Phosphoric acid ・ 670 μL / L Hydrogen peroxide

(6) Other Reagents "Acura Seed B / F Separation Solution" (manufactured by Wako Pure Chemical Industries, Ltd.) was used as the cleaning solution. As a sample for calibration lines, 400 ng / mL of antibody against SARS-CoV-2 S-RBD recombinant to human IgG1 (manufactured by ACRO Biosystems) is defined as 1 U / mL, 1.4 U / mL, 14.3 U / mL, 28.6 U. Diluted samples of / mL, 52.4 U / mL, 76.1 U / mL and 104.7 U / mL were prepared.

1-2 Measurement of antibody concentration against SARS-CoV-2 S-RBD Using the first to fifth reagents and the washing solution, follow the procedure below to determine the IgG antibody concentration against SARS-CoV-2 S-RBD in human serum. Measurement was performed using an automatic chemiluminescent enzyme immunoassay device Accuraseed (registered trademark, manufactured by Fujifilm Techno-Products Co., Ltd.).

50 μL of the first reagent added to the reaction cuvette is magnetized using a neodymium magnet, the supernatant is removed, and then 140 μL of the second reagent and 10 μL of the measurement sample (COVID-19 patient serum or calibration curve sample) are added. The mixture was stirred and heated at 37 ° C. for 3 minutes. After the heating was completed, magnetism was collected using a neodymium magnet, reagents other than magnetic particles were removed, and the mixture was washed 3 times with a washing solution. Subsequently, 50 μL of the third reagent was added, and the mixture was heated at 37 ° C. for 3 minutes. After the heating was completed, magnetic collection was performed using a neodymium magnet, reagents other than magnetic particles were removed, and the mixture was washed 3 times with a washing solution. After the washing was completed, 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 amount of luminescence was measured. A calibration line showing the relationship between the IgG antibody content concentration for SARS-CoV-2 S-RBD and the amount of luminescence was created from the amount of luminescence when each sample for the calibration line was measured, and SARS-CoV in the serum of COVID-19 patients was created. -2 The IgG antibody content concentration for S-RBD was determined. As for the third reagent B, since it is not possible to prepare a calibration curve with a sample for a calibration curve, a study was conducted using the calibration curve prepared with the third reagent A.

1-3 Neutralization activity value for SARS-CoV-2 The neutralization activity value for SARS-CoV-2 is described in the "COVID-19 Serological Test Manual" published by the National Institute of Infectious Diseases. It was measured according to the Japanese test method.

1-4 Results Figure 1 shows the correlation between the antibody concentration and the neutralization activity value. (A) is the result when the third reagent A is used, (B) is the result when the third reagent B is used, and (C) is the result when the third reagent C is used. An improvement in correlation was observed when the third reagent C was used as compared with the case where the third reagent A or the third reagent B was used.

As the enzyme-labeled anti-human IgG antibody solution in the third reagent, Clone: JDC-10 (manufactured by Southern Biotech) was used instead of the enzyme-labeled antibody attached to the COVID-19 IgG test ELISA kit Wako (S-RBD). Similarly, it was confirmed that the correlation was improved when the third reagent C was used as compared with the case where the third reagent A or the third reagent B was used.

In addition, as the POD-labeled anti-human IgG4 antibody in the third reagent, Clone: HP6025 (manufactured by Southern Biotech) or Clone: HP6023 (manufactured by Southern Biotech) is used instead of the peroxidase-labeled anti-human IgG4 antibody (Clone: 5C3). Similarly, it was confirmed that the correlation was improved when the third reagent C was used as compared with the case where the third reagent A or the third reagent B was used.

In addition, as an enzyme-labeled anti-human IgG antibody solution, Clone: JDC-10 (manufactured by Southern Biotech) was used instead of the enzyme-labeled antibody attached to the COVID-19 IgG test ELISA kit Wako (S-RBD). Similarly, when Clone: HP6025 (manufactured by Southern Biotech) or Clone: HP6023 (manufactured by Southern Biotech) is 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 was improved when the third reagent C was used as compared with the case where the third reagent A or the third reagent B was used.

[Example 2: Measurement of IgG antibody against SARS-CoV-2 S-RBD in COVID-19 patients]
2-1 Reagents and Methods Commercially available as an antibody that specifically recognizes human IgG3 (hereinafter referred to as "anti-human IgG3 antibody") instead of the anti-human IgG4 antibody used in the third reagents B and C of Example 1. POD-labeled anti-human IgG3 antibody (Clone: HP6050, manufactured by Southern Biotech) was used to prepare a third reagent D containing only the anti-human IgG3 antibody and a third reagent E containing the anti-human IgG antibody and the anti-human IgG3 antibody. did.

The composition of the third reagent D is as follows.
・ 100ppm POD-labeled anti-human IgG3 antibody (10000-fold diluted)
・ 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 ・ 100ppm POD-labeled anti-human IgG3 antibody (10000-fold diluted)
・ 50mM MES (pH 6.5)
・ 150 mM sodium chloride ・ 2.0% BSA

SARS-CoV was used for 34 COVID-19 patient sera by the same method as in Example 1 except that the third reagent D was used instead of the third reagent B and the third reagent E was used instead of the third reagent C. -2 The antibody concentration against S-RBD and the neutralization activity value against SARS-CoV-2 were measured. As for the third reagent D, since it is not possible to prepare a calibration curve with a sample for a calibration curve, a study was conducted using the calibration curve prepared with the third reagent A.

2-2 Results Figure 2 shows the correlation between the antibody concentration and the neutralization activity value. (A) is the result when the third reagent A is used, (B) is the result when the third reagent D is used, and (C) is the result when the third reagent E is used. An improvement in correlation was observed when the third reagent E was used as compared with the case where the third reagent A or the third reagent D was used.

[Example 3: Measurement of IgG antibody against SARS-CoV-2 Spike protein in COVID-19 patients]
3-1 Reagents and Methods SARS-CoV-2 Spike protein Immobilization Plate (hereinafter referred to as "S-full Immobilization Plate") and COVID-19 ELISA Reagent (trade name: COVID-19 IgG Test ELISA Kit Wako (S) -RBD), manufactured by Fujifilm Wako Junyaku Co., Ltd.) to measure the amount of IgG antibody against SARS-CoV-2 Spike protein in the sera of 34 sera collected from COVID-19 patients who visited Fujita Medical University. The patient's serum was compared with the neutralizing activity value for COVID-19. The enzyme-labeled antibody included in this kit is an antibody that recognizes all subclasses of human IgG.

(1) Preparation of S-full immobilization plate SARS-CoV-2 Spike protein (manufactured by CerTest) dissolved in 50 mM carbonate buffer was reacted with a 96-well plate (manufactured by Thermo Fisher Scientific Co., Ltd.) to block 1.0%. An S-full immobilized plate was prepared by blocking with a phosphate buffer solution containing Ace (manufactured by KAC Co., Ltd.).

(2) Preparation of enzyme-labeled antibody The enzyme-labeled antibody included in the kit was used as it was for the evaluation of the third reagent A. For the evaluation of the third reagent E, the POD-labeled anti-human IgG3 antibody used in Example 2 was added to the enzyme-labeled antibody solution attached to the kit so as to be 1000 ppm.
(3) Preparation of cleaning solution The concentrated cleaning solution (10 ×) included in the kit was diluted 10-fold with purified water produced by a Milli-Q water production device (manufactured by Merck Millipore Co., Ltd.) to prepare a cleaning solution.

(4) Measurement method The same 34 COVID-19 patient sera as in Examples 1 and 2 were diluted 201-fold with the sample diluent included in the kit, or the standard product included in the kit was added to the S-full immobilized plate. , Reacted at room temperature for 1 hour. Next, wash 3 times with 300 μL of the washing solution, then add 50 μL each of the enzyme-labeled antibody solution for evaluating the third reagent A or the enzyme-labeled antibody solution for evaluating the third reagent E, and add 50 μL each to room temperature for 1 hour. It was reacted. After washing 3 times with 300 μL of the washing solution, 100 μL of the coloring solution was added, and after 10 minutes, 100 μL of the reaction stop solution was added, and the absorbance at the main wavelength of 450 nm and the sub-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 product.

3-2 Results Figure 3 shows the correlation between the amount of IgG antibody against S-full calculated above and the neutralizing activity value measured in Example 1. (A) is the result of evaluating the third reagent A, and (B) is the result of evaluating the third reagent E. Both the antibody that recognizes all subclasses of anti-human IgG and the anti-human IgG3 antibody (third reagent E) are compared with the case where the antibody that recognizes all subclasses of anti-human IgG alone (third reagent A) is used. When used, improvement in correlation was observed.

The present invention is not limited to the above-described embodiments and examples, and various modifications can be made within the scope of the claims, and the technical means disclosed in the different embodiments may be appropriately combined. The obtained embodiments are also included in the technical scope of the present invention. In addition, all of the academic and patent documents described in this specification are incorporated herein by reference.

Claims (11)

1. It is a method of measuring a test animal antibody contained in a biological sample of a test animal, in which the antigen and the biological sample of the test animal are brought into contact with an anti-test animal immunoglobulin antibody, and the antigen and the test animal antibody are used. The anti-test animal immunoglobulin antibody comprises measuring a complex with an anti-test animal immunoglobulin antibody, wherein the anti-test animal immunoglobulin antibody is a plurality of antibodies against a particular class of immunoglobulin and of all subclasses of said particular class. A method comprising an antibody that cross-reacts with an immunoglobulin.
2. The method according to claim 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 claim 1 or 2, wherein the particular class of immunoglobulin is IgG.
4. The method according to any one of claims 1 to 3, wherein the test animal antibody is an antibody against non-self or an autoantibody.
5. The method according to claim 4, wherein the non-self is a pathogenic microorganism, a virus or a parasite.
6. The method according to any one of claims 1 to 5, wherein the anti-test animal immunoglobulin antibody or antigen is labeled.
7. A kit for measuring a test animal antibody contained in a biological sample of a test animal, which comprises an antigen and an anti-test animal immunoglobulin antibody, wherein the anti-test animal immunoglobulin antibody is a specific class of immunoglobulin. A kit comprising a plurality of antibodies against the antibody that cross-reacts with immunoglobulins of all subclasses of said particular class.
8. The kit according to claim 7, 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.
9. The kit according to claim 7 or 8, wherein the particular class of immunoglobulin is IgG.
10. The kit according to any one of claims 7 to 9, wherein the antigen is a pathogenic microbial antigen, a viral antigen, a parasite, or an antigen involved in an autoimmune disease.
11. The kit according to any one of claims 7 to 10, wherein the anti-test animal immunoglobulin antibody or antigen is labeled.

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