Immunoassay methods and kits for immunoassays

Abstract

The present invention aims to provide an immunoassay method and an immunoassay kit with high measurement sensitivity. [Solution] An immunoassay method in which a solid-phase reactant having a substance (D) that specifically reacts with a substance (G) contained in a liquid sample is used to perform a specific reaction in the presence of an acrylic acid (salt) copolymer, wherein the acrylic acid (salt) copolymer is a copolymer containing constituent units derived from acrylic acid (salt) and constituent units derived from an unsaturated dibasic acid (salt), and / or a copolymer containing constituent units derived from acrylic acid (salt) and constituent units derived from a sulfonic acid (salt) group-containing monomer.

Description

Technical Field

[0001] The present invention relates to an immunoassay method and an immunoassay kit. Background Art

[0002] As methods for measuring substances such as proteins contained in biological samples such as body fluids, there are known immunoassay methods in which a target substance to be measured is bound to the surface of particles carrying a substance capable of binding to the target substance, the particles bound with the target substance are collected, and the binding amount is measured, immunoassay methods for measuring the degree of particle aggregation caused by the target substance binding to the particle surface, and the like. As an immunoassay method capable of measuring an antigen with high sensitivity and accuracy, a method of performing immunoassay in the presence of a polycarboxylic acid surfactant (a copolymer of maleic acid and / or maleic anhydride and diisobutylene, or a salt thereof) is known (Patent Document 1). However, even in the immunoassay described in Patent Document 1, sufficient sensitivity has not been obtained due to the influence of non-specific reactions in which substances other than the measurement target react with the measurement reagent. Prior Art Documents Patent Documents

[0003] Patent Document 1 Japanese Patent No. 5996234 Summary of Invention Problems to be Solved by the Invention

[0004] An object of the present invention is to provide an immunoassay method and an immunoassay kit with high measurement sensitivity. Means for Solving the Problems

[0005] As a result of intensive studies to achieve the above object, the present inventors have arrived at the present invention. In other words, the present invention relates to an immunoassay method that uses a solid-phase reactant having a substance (D) that specifically reacts with a substance (G) contained in a liquid sample, and performs a specific reaction in the presence of an acrylic acid (salt) copolymer, wherein the acrylic acid (salt) copolymer is a copolymer containing constituent units derived from acrylic acid (salt) and constituent units derived from an unsaturated dibasic acid (salt), and / or a copolymer containing constituent units derived from acrylic acid (salt) and constituent units derived from a sulfonic acid (salt) group-containing monomer; and an immunoassay kit comprising a solid-phase support reagent (A) and an immunoassay buffer (W), wherein the solid-phase support reagent (A) and / or the immunoassay buffer (W) contain a copolymer containing constituent units derived from acrylic acid (salt) and constituent units derived from an unsaturated dibasic acid (salt), and / or a copolymer containing constituent units derived from acrylic acid (salt) and constituent units derived from a sulfonic acid (salt) group-containing monomer. [Effects of the Invention]

[0006] The immunoassay method and immunoassay kit of the present invention enable highly sensitive immunoassays. [Modes for carrying out the invention]

[0007] The present invention is an immunoassay method that performs a specific reaction using a solid-phase reactant having a substance (D) that specifically reacts with a substance (G) contained in a liquid sample.

[0008] In the present invention, the immunoassay method is a measurement method that measures the concentration of a target substance in a liquid sample by performing measurements using specific reactions such as immune reactions represented by the antigen-antibody reaction.

[0009] The specific reactions of the present invention include antigen-antibody reactions, glycan-protein reactions, glycan-lectin reactions, enzyme-inhibitor reactions, protein-peptide chain reactions, chromosome or nucleotide chain-nucleotide chain reactions, and nucleotide chain-protein reactions. Among these, antigen-antibody reactions and glycan-protein reactions are preferred from the viewpoint of specificity.

[0010] In this specification, "non-specific reaction" refers to a reaction in which binding, reaction, or signal generation occurs in any step of the immunoassay system without being due to the intended specific reaction to the target being measured (antigen and / or antibody, etc.), resulting in measurement errors such as false positives, false highs, false negatives, or false lows.

[0011] The forms of nonspecific reactions are not limited to these, but as an example, nonspecific components contained in biological fluids, etc. (e.g., heterophilic antibodies, rheumatoid factors, etc.) nonspecifically adsorb or bind to substances that are not immobilized on the solid support (a1) and are specifically reactive to substances (D) and / or substances (G1) immobilized on the solid support (a1) (magnetic particles, etc.), resulting in a signal that suggests the presence of substance (G1) even though substance (G1), the substance to be measured, is not actually present in the biological fluids, etc.

[0012] Specific reactions include reactions that utilize interactions between specific chemical structures, such as reactions between "proteins (e.g., avidin)" and "ligands (e.g., biotin)."

[0013] A solid-phase reactant having a substance (D) that specifically reacts with a substance (G) contained in a liquid sample is a substance consisting of a solid support (a1) on which substance (D) is immobilized.

[0014] The specific reaction using a solid-phase reactant in the immunoassay method of the present invention is, for example, a reaction between a substance (G) which is one of the following contained in a liquid sample: an antigen, antibody, protein, or ligand, and a solid-phase reactant which is a solid-phase support (a1) on which a substance (D) that specifically reacts with the substance (G) is immobilized. The substance (D) immobilized on the solid support (a1) is an antigen, antibody, protein, or ligand corresponding to substance (G). That is, if substance (G) is an antigen, then substance (D) is the corresponding antibody; if substance (G) is an antibody, then substance (D) is the corresponding antigen; and if substance (G) is a ligand, then substance (D) is the corresponding protein.

[0015] Substances (G) that specifically react with substances (D) contained in solid-phase reactants include substances (G1) contained in biological fluids such as serum, blood, plasma, and urine, liquid samples derived from living organisms such as lymph, blood cells, and various types of cells, and liquid samples obtained by processing non-liquid biological samples such as feces (hereinafter, biological fluids, liquid samples derived from living organisms, and liquid samples obtained by processing biological samples may be collectively referred to as biological fluids, etc.), substances similar to substances contained in biological fluids, etc. (G1+) [hereinafter also referred to as similar substances (G1+)], substances that specifically react with the aforementioned substances contained in biological fluids, etc. (G2), and complexes (G12) of the aforementioned substances contained in biological fluids, etc. and substances that specifically react with the aforementioned substances contained in biological fluids, etc.

[0016] A substance similar to a substance contained in a living body fluid (G1+) is a substance that can react specifically with a substance that a substance contained in a living body fluid specifically reacts with, and is capable of binding to the binding site of the substance that a substance contained in a living body fluid specifically reacts with; in other words, it is a substance that has a binding site that can react specifically with a substance that a substance contained in a living body fluid specifically reacts with, in the same way as a substance contained in a living body fluid; or, to put it another way, it is any substance that competes with the substance contained in a living body fluid for its specific reaction with the substance that a substance contained in a living body fluid specifically reacts with, but is different from the substance contained in a living body fluid. For example, a substance that labels a substance contained in a living body fluid is distinguished from the substance contained in a living body fluid as a similar substance. In other words, the analogous substance (G1+) may be a substance that has been labeled with a substance contained in biological fluids, etc.

[0017] Substances that react specifically with substances contained in biological fluids, etc. (G2) may also be labeled substances.

[0018] In the measurement method of the present invention, the liquid sample containing substance (G) includes a liquid sample that has been pretreated from a biological fluid or the like (including a liquid sample obtained by treating a non-liquid biological sample).

[0019] Pretreatments performed on biological fluids and the like include a process of mixing a biological fluid or a liquid sample derived from a living body with a substance such as those described in <Process 1> to <Process 2> below, a process of adjusting the pH of the biological fluid or the liquid sample derived from a living body, a process of adjusting the concentration of the biological fluid or the liquid sample derived from a living body, and the like. <Process 1>A process of mixing a biological fluid or the like with a substance (G2) or the like that specifically reacts with a substance contained in the biological fluid or the like, for the purpose of labeling, stabilizing, or enhancing the detection sensitivity of the substance contained in the biological fluid or the like. <Process 2>A process of mixing a biological fluid or the like with a substance (G1+) similar to the substance contained in the biological fluid or the like.

[0020] The measurement method of the present invention includes the reactions and the like of <Method 1> to <Method 6> below.

[0021] <Method 1>A measurement method of performing a specific reaction between a substance (G1) contained in a biological fluid or the like and a substance (D).

[0022] <Method 2>A measurement method comprising obtaining a liquid sample containing a substance (G1+) by labeling a substance contained in a biological fluid or the like by a method described later, or mixing a substance (G1+) similar to the substance contained in the biological fluid or the like into the biological fluid or the like, and then performing a specific reaction between the substance (G1+) contained in the liquid sample and the substance (D).

[0023] <Method 3>A measurement method of simultaneously performing the specific reaction in the above <Method 1> and the specific reaction in the above <Method 2> (that is, performing both the specific reaction between the substance (G1) contained in the biological fluid or the like and the substance (D), and the specific reaction between the similar substance (G1+) and the substance (D) in the same sample).

[0024] <Method 4>A measurement method comprising adding a substance (G2) that specifically reacts with a substance contained in a biological fluid or the like to the biological fluid or the like, and performing a specific reaction between the substance (G2) contained in the liquid sample after the addition and the substance (D).

[0025] <Method 5> A measurement method in which a substance that reacts specifically with substances contained in biological fluids is added to biological fluids to prepare a liquid sample containing a complex (G12), and a specific reaction is performed between the substance (G12) contained in the liquid sample and substance (D).

[0026] <Method 6> A measurement method that uses a solid-phase reactive material in which a substance contained in biological fluids, etc. and / or a similar substance to a substance contained in biological fluids, etc. is immobilized as substance (D), and performs a specific reaction between substance (G2), which is a substance that specifically reacts with a substance contained in biological fluids, etc. and / or a similar substance to a substance contained in biological fluids, etc., and substance (D).

[0027] In the above-mentioned Method 1, the substance (G1) contained in biological fluids, etc., that specifically reacts with substance (D) may include nucleotide chains (oligonucleotide chains, polynucleotide chains), chromosomes, nucleic acids [deoxyribonucleic acid (DNA), ribonucleic acid (RNA), etc.], peptide chains (e.g., C-peptide, angiotensin I, etc.), proteins [procalcitonin, immunoglobulin A (IgA), immunoglobulin E (IgE), immunoglobulin G (IgG), immunoglobulin M (IgM), immunoglobulin D (IgD), β2-microglobulin, albumin, hemoglobin, etc.]. Oglobin, transferrin, protein A, C-reactive protein (CRP), ferritin, troponin T (TnT), human brain natriuretic peptide precursor N-terminal fragment (NT-proBNP), and their degradation products, etc., blood coagulation-related factors (fibrinogen, fibrin degradation products, prothrombin, thrombin, etc.), enzymes [amylase (pancreatic type, salivary gland type, X type, etc.), alkaline phosphatase (hepatic, bone, placental, small intestinal, etc.), acid phosphatase (PAP, etc.), γ-glutamyl transferase (renal, pancreatic, hepatic, etc.), lipase (pancreatic type, gastric type) [etc.], creatine kinase (CK-1, CK-2, mCK, etc.), lactate dehydrogenase (LDH1-LDH5, etc.), glutamate oxaloacetate transaminase (ASTm, ASTs, etc.), glutamate pyruvate transaminase (ALTm, ALTs, etc.), cholinesterase (ChE1-ChE5, etc.), leucine aminopeptidase (C-LAP, AA, CAP, etc.), renin, protein kinase, tyrosine kinase, etc.] and inhibitors of these enzymes, hormones (PTH, TSH, insulin, LH, FSH, estradiol, prolactin, etc.) Receptors (receptors for estrogen, TSH, etc.), ligands (estrogen, TSH, etc.), bacteria [Mycobacterium tuberculosis, Streptococcus pneumoniae, Neisseria diphtheriae, Neisseria meningitidis, Neisseria gonorrhoeae, Staphylococcus, Streptococcus, Enterobacteriaceae, Escherichia coli, Helicobacter pylori, spirochetes (Leptospirosis, Treponema pallidum, etc.), Chlamydia, Mycoplasma, etc.], viruses (Rubella virus, Herpes virus, Hepatitis virus, ATL virus, AIDS virus, Influenza virus, Adenovirus, Enterovirus, Poliovirus, EB virus, HAV, HBV, HCV,HIV, HTLV, etc.), fungi (Candida, Cryptococcus, etc.), chemical substances derived from the aforementioned microorganisms (bacteria, viruses, fungi, etc.) (proteins, peptides, glycosylation antigens, etc.), various allergens that cause allergic symptoms (bronchial asthma, allergic rhinitis, atopic dermatitis, etc.) [house dust, mites (house dust mites, dust mites, etc.), pollen (cedar, cypress, barnyard grass, ragweed, timothy grass, sweet vernal grass, rye, etc.)] Other allergens (substances derived from animals, food, fungi, insects, wood, drugs, and chemicals, etc.), lipids (lipoproteins, etc.), proteases (trypsin, plasmin, serine protease, etc.), tumor marker protein antigens (PSA, PGI, PGII, etc.), glycosylation antigens [AFP (L1-L3, etc.), hCG (hCG family), transferrin, IgG, thyroglobulin, Decay-accelerating-fa [Cyctor (DAF), carcinoembryonic antigens (CEA, NCA, NCA-2, NFA, etc.), CA19-9, PIVKA-II, CA125, prostate-specific antigen, tumor marker glycan antigens with special glycans produced by cancer cells, ABO glycan antigens, etc.], glycans (hyaluronic acid, β-glucan, glycans possessed by the aforementioned glycan antigens, etc.), proteins that bind to glycans (hyaluronic acid-binding protein, β-glucan-binding protein, etc.), phospholipids (cardiolipin, etc.), Examples include lipopolysaccharides (endotoxins, etc.), various drugs administered or inoculated into the human body and their metabolites, aptamers, nucleic acid-binding substances, and other chemical substances [environmental hormones (T3, T4, FT3, FT4, tributyltin, nonylphenol, 4-octylphenol, di-n-butyl phthalate, dicyclohexyl phthalate, benzophenone, octachlorostyrene, di-2-ethylhexyl phthalate, etc.)], and antibodies against these substances.

[0028] In Method 1 described above, the solid support (a1) used to fix the substance (D) can be any known support used in the field of immunoassays, without any particular limitations. Typical examples include glass beads, polystyrene beads, magnetic particles, microplates, and latex.

[0029] As magnetic particles, known particles described in Japanese Patent Publication No. 2023-126848 and others can be used, and from the viewpoint of shortening the measurement time and accuracy in immunoassay, it is preferable to use silica particles containing metal oxides described in Japanese Patent Publication No. 2014-210680 and Japanese Patent Publication No. 2013-019889. As magnetic particles, commercially available analytical magnetic particles [such as Magrapid (Magrapit is a registered trademark of Sanyo Chemical Industries, Ltd.)] can be used.

[0030] As the microplate, a known microplate described in Japanese Patent Publication No. 3584616, etc., can be used. Alternatively, you may use microplates manufactured by Sumitomo Bakelite Corporation, which are available on the market.

[0031] As the latex, known latex described in Japanese Patent Publication No. 2024-52454 and Japanese Patent Publication No. 2022-158904, etc., can be used. As the latex, commercially available latex particles such as the IMMUTEX series (IMMUTEX is a registered trademark of JSR Corporation) and micromer (manufactured by Corefront Corporation) can be used.

[0032] In the above-described Method 1, substance (D) is selected according to the type of substance (G1) contained in the body fluid, etc. If substance (G1) is an antigen, an antibody corresponding to substance (G1) should be selected; if substance (G1) is an antibody, an antigen corresponding to substance (G1) should be selected. Monoclonal antibodies, polyclonal antibodies, bispecific antibodies, mini-bodies, domain antibodies (dAb), single-domain antibodies (sdAb), synthetic antibodies, chimeric antibodies, humanized antibodies, proteolytic enzymes (papain and pepsin, etc.), and degradation products such as Fab and F(ab')2 fragments produced by chemical degradation can also be used as antibodies corresponding to substance (G1).

[0033] In the above-described method 1, methods for immobilizing the substance (D) on the solid support (a1) include physically adsorbing the substance (D) onto the solid support (a1), and reacting and bonding the substance (D) with functional groups derived from chemical substances bonded to the surface of the solid support (a1) to immobilize it.

[0034] Examples of chemical substances bonded to the surface of a solid support (a1) that have functional groups that react with and bond with substance (D) include glutaraldehyde, albumin, carbodiimide, streptavidin, biotin, and alkylalkoxysilanes having functional groups, and preferably alkylalkoxysilanes having functional groups (ethylenically unsaturated groups, epoxy groups, amino groups, mercapto groups, and isocyanate groups, etc.).

[0035] Alkylalkoxysilanes having such functional groups include vinyltrimethoxysilane, vinyltriethoxysilane, 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane, 3-glycidoxypropylmethyldimethoxysilane, 3-glycidoxypropyltrimethoxysilane, 3-glycidoxypropylmethyldiethoxysilane, 3-glycidoxypropyltriethoxysilane, p-styryltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-acryloxypropyl Examples include trimethoxysilane, N-2-(aminoethyl)-3-aminopropylmethyldimethoxysilane, N-2-(aminoethyl)-3-aminopropyltrimethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-triethoxysilyl-N-(1,3-dimethylbutylidene)propylamine, N-phenyl-3-aminopropyltrimethoxysilane, tris-(trimethoxysilylpropyl)isocyanurate, 3-ureidopropyltrialkoxysilane, 3-mercaptopropylmethyldimethoxysilane, 3-mercaptopropyltrimethoxysilane, and 3-isocyanatetopropyltriethoxysilane.

[0036] The solid support (a1) may have polymer chains made of a water-soluble polymer (such as polyethylene glycol) immobilized on it in addition to substance (D). The immobilization method can be any known method, including chemical bonding to the surface of the solid support, immobilization by electrical interactions such as ionic bonding to the surface of the solid support, and using a compound having polymer chains as a raw material for the solid support. Examples of solid support (a1) with immobilized polymer chains made of a water-soluble polymer (such as polyethylene glycol) include magnetic particles described in Japanese Patent Application Publication No. 2022-159923 and latex described in Japanese Patent Application Publication No. 2017-122684.

[0037] The analogous substance (G1+) in Method 2 above includes the substance (G1) contained in biological fluids, etc., as exemplified in Method 1, which is further labeled with a labeling substance (b).

[0038] The labeling substance (b) may include enzymes (b1) used in enzyme immunoassay (EIA), such as alkaline phosphatase, β-galactosidase, peroxidase, microperoxidase, glucose oxidase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, luciferase, tyrosinase, and acid phosphatase; radioisotopes such as 99mTc, 131I, 125I, 14C, 3H, and 32P used in radioimmunoassay (RIA); and fluorescein, dansyl, fluorescein, coumarin, naphthylamine, or derivatives thereof used in fluorescence immunoassay (FIA), such as green fluorescent protein (GF). Examples include fluorescent substances such as P), luminescent substances such as luciferin, isoluminol, luminol, and bis(2,4,6-trifluorophenyl)oxalate, substances that absorb in the ultraviolet region such as phenol, naphthol, anthracene, or derivatives thereof, and substances that have properties as spin labeling agents, such as compounds having an oxyl group, such as 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl, 3-amino-2,2,5,5-tetramethylpyrrolidine-1-oxyl, and 2,6-di-t-butyl-α-(3,5-di-t-butyl-4-oxo-2,5-cyclohexadiene-1-ylidene)-p-trioxyl. Of these, from the viewpoint of sensitivity, etc., enzyme (b1) and fluorescent substances are preferred, more preferably enzyme (b1), and particularly preferably at least one selected from the group consisting of alkaline phosphatase, peroxidase and glucose oxidase, and most preferably peroxidase (hereinafter sometimes abbreviated as POD).

[0039] Labeling of substances contained in biological fluids, etc., with labeling substances (b), etc., can be carried out using known methods used in this field, such as known labeling methods used in EIA, RIA, or FIA [for example, Medical Chemistry Experiment Course, Vol. 8, supervised by Yuichi Yamamura, 1st edition, Nakayama Shoten, 1971; Illustrated Fluorescent Antibodies, by Akira Kawai, 1st edition, Soft Science Co., Ltd., 1983; Enzyme Immunoassay, edited by Eiji Ishikawa, Tadashi Kawai, and Kiyoshi Muroi, 2nd edition, Igaku Shoin, 1982, etc.].

[0040] In Method 2 described above, the substance (D) and the solid support (a1) on which the substance (D) is immobilized are the same as the substance (D) and solid support (a1) exemplified in Method 1 described above.

[0041] Method 3 involves carrying out the specific reaction in Method 1 and the specific reaction in Method 2 simultaneously in the same reaction system.

[0042] In Method 4, the substance (G2) that specifically reacts with a substance contained in the body fluid is selected according to the type of substance contained in the body fluid. If the substance contained in the body fluid is an antigen, the corresponding antibody should be selected; if the substance contained in the body fluid is an antibody, the corresponding antigen should be selected.

[0043] Furthermore, in Method 4, the substance (G2) may also include a substance that reacts specifically, selected according to the type of substance contained in biological fluids, etc., and is further labeled with a labeling substance (b). Labeling with labeling substance (b) can be carried out in the same manner as in Method 2.

[0044] The solid support (a1) in Method 4 is the same as that exemplified in Method 1.

[0045] In Method 4, substance (D) should be selected according to the type of substance (G2), such that if substance (G2) is an antigen, the corresponding antibody should be selected, and if substance (G2) is an antibody, the corresponding antigen should be selected.

[0046] In Method 4, the method for immobilizing the substance (D) onto the solid support (a1) can be the same as the method exemplified in Method 1.

[0047] In Method 5, the substance added to the body fluid, etc., which specifically reacts with the substance contained in the body fluid, etc., to form a complex (G12), is selected according to the type of substance contained in the body fluid, etc. If the substance contained in the body fluid, etc., is an antigen, the corresponding antibody should be selected, and if the substance contained in the body fluid, etc., is an antibody, the corresponding antigen should be selected. The substance added to the body fluids, etc., to form a complex (G12) may be one type, two or more types, or it may be a substance labeled with a labeled substance (b) similar to the substance (G2) in Method 4.

[0048] In Method 5, substance (D) can be selected according to the type of complex (G12) from substances that can react specifically with the complex (G12). This selection is made from substances that react specifically with substances contained in biological fluids, substances that react specifically with substances added to biological fluids, and substances that react specifically with any of the substances that react specifically with the complex (G12). If the binding site between the complex (G12) and substance (D) is located in a structure derived from a substance contained in the body fluids, etc., then if the substance contained in the body fluids, etc. is an antigen, then the corresponding antibody should be selected; if the substance contained in the body fluids, etc. is an antibody, then the corresponding antigen should be selected. If the binding site between the complex (G12) and substance (D) is located in a structure derived from a substance added to a biological fluid, then if the added substance is an antigen, the corresponding antibody should be selected; if the added substance is an antibody, the corresponding antigen should be selected. Furthermore, if the substance added to bodily fluids or other biological fluids contains biotin in its structure, then a protein capable of binding to biotin (such as avidin) should be selected as substance (D).

[0049] The solid support (a1) in Method 5 is the same as that exemplified in Method 1.

[0050] In Method 5, the method for immobilizing the substance (D) onto the solid support (a1) can be the same as the method exemplified in Method 1. The solid support (a1) in Method 6 is the same as that exemplified in Method 1.

[0051] In Method 6, the method for immobilizing the substance (D) contained in biological fluids, etc., and similar substances to the substance contained in biological fluids, etc., onto the solid support (a1) can be the same as the method exemplified in Method 1. The similar substances to the substance contained in biological fluids, etc., are the same as substance (G1+) in Method 2.

[0052] In Method 6, the substance (G2) is the same as the substance (G2) in Method 4, and is selected according to the type of substance contained in the body fluid, etc. If the substance contained in the body fluid, etc. is an antigen, the corresponding antibody should be selected, and if the substance contained in the body fluid, etc. is an antibody, the corresponding antigen should be selected. Furthermore, in Method 6, the substance (G2) may be a substance selected according to the type of substance contained in biological fluids, etc., and labeled with labeling substance (b). Labeling with labeling substance (b) can be carried out in the same manner as in Method 2.

[0053] In Method 1 described above, a specific reaction using a solid-phase reagent may be carried out in a liquid sample containing a substance (G1) that is capable of specific reaction with a substance contained in a biological fluid or the like and is not immobilized on a solid-phase support (a1). In a liquid sample containing a substance (G1) present in biological fluids, etc., and a substance not immobilized on a solid support (a1), a specific reaction is carried out using a solid-phase reagent. This causes substance (G1) to specifically bond with substance (D) immobilized on a solid support (a1), and then the substance (G1) bonded to substance (D) is further bonded to the substance (G1) that is specifically reacted with substance (G1) and not immobilized on a solid support (a1). For a substance that is specifically reactive to substance (G1) and is not immobilized on a solid support (a1), if substance (G1) is an antigen, then the corresponding antibody should be selected, and if substance (G1) is an antibody, then the corresponding antigen should be selected. Furthermore, as a substance that can react specifically with substance (G1) and is not immobilized on a solid support (a1), it is preferable to use a substance labeled with labeling substance (b), and labeling with labeling substance (b) can be carried out in the same manner as in <Method 2>.

[0054] In the above-described methods 1 to 6, after carrying out a specific reaction between substance (G) and substance (D), a substance that reacts specifically with the substance (G) bonded to the substance (D) immobilized on the solid support (a1) [hereinafter, this may be referred to as the substance (G) bonded to the solid-phase reactant, or the substance (G) bonded to the solid phase] may be further reacted with another substance that reacts specifically with the substance (G).

[0055] When performing a further specific reaction with a substance (G) bound to a solid phase, the substance that specifically reacts with the substance (G) bound to the solid phase is selected according to the type of substance that specifically reacts with the substance (G). If the substance that specifically reacts with the substance (G) bound to the solid phase is an antigen, then the corresponding antibody should be selected. If the substance that specifically reacts with the substance (G) bound to the solid phase is an antibody, then the corresponding antigen should be selected. Furthermore, as the substance that specifically reacts with the substance (G) bonded to the solid phase, it is preferable to use a substance that has been labeled with a labeling substance (b) according to the type of substance that specifically reacts with the substance (G) bonded to the solid phase, and labeling with the labeling substance (b) can be carried out in the same manner as in <Method 2>.

[0056] The immunoassay methods of the present invention include known immunoassay methods such as the sandwich method, direct method, competitive method, indirect method, and latex agglutination method.

[0057] (1) The sandwich method includes the method described as <Method 1>, and the following methods are also examples. <1> A method for measuring the concentration of substance (G1) contained in biological fluids, etc., by performing a specific reaction between substance (G1) and substance (D) immobilized on a solid support (a1) in the presence of a labeled substance (also called a labeled substance) capable of a specific reaction with substance (G1) contained in biological fluids, etc., thereby causing bonding between substance (D) and substance (G1) and labeling of the substance (G1) bonded to substance (D), and measuring the strength of the labeled substance. <2> A method for measuring the concentration of substance (G1) contained in biological fluids, etc., by performing a specific reaction between substance (G1) and substance (D), then reacting with a labeled substance, and measuring the intensity of the labeled substance.

[0058] (2) Examples of direct methods include the method described in <Method 6>, which involves a solid support (a1) on which a substance (D) contained in biological fluids, etc. is immobilized as substance (D), and a labeled substance (G2) that reacts specifically with the substance contained in biological fluids, etc., in a liquid sample, and measuring the intensity of the labeled substance to measure the concentration of the substance contained in the liquid sample derived from biological fluids, etc.

[0059] (3) As a competitive method, the concentration of the substance (G1) contained in the biological fluid, etc., in the biological fluid, etc., is measured by carrying out a specific reaction in the liquid sample between the substance (G1) contained in the biological fluid, etc., a similar substance (G1+) added to the biological fluid, etc., or a liquid sample that has been pretreated with the biological fluid, etc., and a substance (D) immobilized on a solid support (a1), as described in <Method 3>, and measuring the intensity of the labeled substance.

[0060] (4) As an indirect method, a specific reaction is carried out in the liquid sample between a solid support (a1) on which a substance contained in biological fluids, etc. is immobilized, and a substance (G2) that specifically reacts with the substance contained in biological fluids, etc., as described in <Method 6>. Furthermore, a labeled substance is reacted with the substance (G2) bonded to the solid reactant, and the concentration of the substance contained in the biological liquid sample is measured by measuring the intensity of the labeled substance.

[0061] (5) As a latex agglutination method, a specific reaction is carried out between the substance (G1) contained in the biological liquid sample, which is <Method 1>, and particulate solid-phase reactive material dispersed in an aqueous dispersion medium. The concentration of the substance contained in the biological liquid sample is measured by quantifying the amount of solid-phase reactive material agglutinated by bond formation due to the specific reaction using turbidity or the like.

[0062] The immunoassay methods of the present invention also include immunoassay methods other than the sandwich method, direct method, competitive method, indirect method, and latex agglutination method described above.

[0063] The immunoassay method of the present invention is an immunoassay method that performs a specific reaction in the presence of an acrylic acid (salt) copolymer, wherein the acrylic acid (salt) copolymer is a copolymer containing constituent units derived from acrylic acid (salt) and constituent units derived from an unsaturated dibasic acid (salt), and / or a copolymer containing constituent units derived from acrylic acid (salt) and constituent units derived from a sulfonic acid (salt) group-containing monomer.

[0064] Furthermore, copolymers containing constituent units derived from acrylic acid (salt) and unsaturated dibasic acid (salt) include copolymers (i.e., ternary or more copolymers) that contain constituent units derived from other monomers in addition to those derived from acrylic acid (salt) and unsaturated dibasic acid (salt).

[0065] The aforementioned other monomers (third monomers) are not particularly limited as long as they are copolymerizable ethylenically unsaturated monomers, and examples include aromatic vinyl monomers (styrene, substituted styrene, etc.), (meth)acrylamide monomers (acrylamide, N-substituted (meth)acrylamide, etc.), (meth)acrylate monomers, (meth)allyl monomers, etc. These may be used individually or in combination of two or more types. Specifically, examples include copolymers containing acrylic acid (salt), unsaturated dibasic acid (salt), and constituent units derived from aromatic vinyl monomers.

[0066] In a copolymer containing constituent units derived from acrylic acid (salt) and constituent units derived from unsaturated dibasic acid (salt), acrylic acid (salt) represents acrylic acid and / or acrylic acid salts, and unsaturated dibasic acid (salt) represents unsaturated dibasic acid and / or unsaturated dibasic acid salts.

[0067] Examples of acrylate salts include alkali metal (lithium, sodium, and potassium, etc.) salts of acrylic acid, ammonium salts of acrylic acid, and organic amine (triethylamine, etc.) salts of acrylic acid.

[0068] Examples of unsaturated dibasic acids include maleic acid, fumaric acid, and itaconic acid.

[0069] Examples of unsaturated dibasic salts include alkali metal salts, ammonium salts, and organic amine salts of the aforementioned unsaturated dibasic acids.

[0070] As the acrylic acid (salt) that constitutes the copolymer containing constituent units derived from acrylic acid (salt) and constituent units derived from an unsaturated dibasic acid (salt), sodium acrylic acid is preferred from the viewpoint of solubility, and as the unsaturated dibasic acid (salt), sodium maleic acid is preferred.

[0071] Copolymers containing constituent units derived from acrylic acid (salt) and constituent units derived from sulfonic acid (salt) group-containing monomers include copolymers that contain constituent units derived from other monomers in addition to the constituent units derived from acrylic acid (salt) and sulfonic acid (salt) group-containing monomers. The constituent units derived from other monomers are the same as those described above. Specifically, examples include copolymers containing constituent units derived from acrylic acid (salt) / (meth)acrylamide monomers / sulfonic acid (salt) group-containing monomers.

[0072] In a copolymer containing constituent units derived from acrylic acid (salt) and constituent units derived from a sulfonic acid (salt) group-containing monomer, acrylic acid (salt) represents acrylic acid and / or acrylic acid salt, and sulfonic acid (salt) group-containing monomer represents a sulfonic acid group-containing monomer and / or a sulfonate salt-containing monomer.

[0073] Examples of monomers containing sulfonic acid groups include vinyl sulfonic acid, styrene sulfonic acid, (meth)allyl sulfonic acid, 3-(meth)allyloxy-2-hydroxypropanesulfonic acid, 3-(meth)allyloxy-1-hydroxypropanesulfonic acid, 2-(meth)allyloxyethylene sulfonic acid, and 2-acrylamido-2-methylpropanesulfonic acid.

[0074] Examples of monomers containing sulfonates include alkali metal (lithium, sodium, and potassium, etc.) salts, ammonium salts, and organic amine salts of monomers having the aforementioned sulfonic acid group.

[0075] As a monomer containing a sulfonic acid (salt) group, sodium 3-(meth)allyloxy-2-hydroxypropanesulfonate is preferred from the viewpoint of copolymerizability.

[0076] In a copolymer comprising a constituent unit derived from acrylic acid (salt) and a constituent unit derived from a monomer containing a sulfonic acid (salt) group, sodium acrylate is preferred as the acrylic acid (salt) from the viewpoint of solubility, and sodium 3-(meth)allyloxy-2-hydroxypropanesulfonate is preferred as the monomer containing a sulfonic acid (salt) group.

[0077] In a copolymer containing constituent units derived from acrylic acid (salt) and constituent units derived from unsaturated dibasic acid (salt), the ratio of the total number of moles of acrylic acid to the total number of moles of unsaturated dibasic acid to the total number of moles of unsaturated dibasic acid to the total number of moles of unsaturated dibasic acid is preferably 10 / 90 to 95 / 5, and more preferably 50 / 50 to 95 / 5, from the viewpoint of measurement sensitivity.

[0078] The aforementioned ratio [acrylic acid (salt) / unsaturated dibasic acid (salt)] is defined as the ratio of the total number of moles of constituent units derived from acrylic acid (salt) to the total number of moles of constituent units derived from the corresponding unsaturated dibasic acid (salt), even if the copolymer further contains constituent units derived from a third monomer.

[0079] The weight average molecular weight of the copolymer containing a constituent unit derived from acrylic acid (salt) and a constituent unit derived from an unsaturated dibasic acid (salt) is preferably from 1,000 to 100,000, more preferably from 15,000 to 90,000, from the viewpoint of measurement sensitivity.

[0080] In the copolymer containing a constituent unit derived from acrylic acid (salt) and a constituent unit derived from a sulfonic acid (salt) group-containing monomer, the ratio of the total number of moles of acrylic acid and acrylate to the total number of moles of the sulfonic acid group-containing monomer and the sulfonate group-containing monomer is preferably from 1 / 99 to 95 / 5, more preferably from 10 / 90 to 95 / 5, and particularly preferably from 50 / 50 to 95 / 5, from the viewpoint of measurement sensitivity.

[0081] The aforementioned ratio [acrylic acid (salt) / sulfonic acid (salt) group-containing monomer] is defined as the ratio of the total number of moles of the constituent unit derived from acrylic acid (salt) to the total number of moles of the corresponding constituent unit derived from the sulfonic acid (salt) group-containing monomer, even when the copolymer further contains a constituent unit derived from a third monomer.

[0082] The weight average molecular weight of the copolymer containing a constituent unit derived from acrylic acid (salt) and a constituent unit derived from a sulfonic acid (salt) group-containing monomer is preferably from 2,000 to 200,000, more preferably from 20,000 to 180,000, from the viewpoint of measurement sensitivity.

[0083] The weight average molecular weight (Mw) of the copolymer containing a constituent unit derived from acrylic acid (salt) and a constituent unit derived from an unsaturated dibasic acid (salt), and the copolymer containing a constituent unit derived from acrylic acid (salt) and a constituent unit derived from a sulfonic acid (salt) group-containing monomer is measured by a gel permeation chromatography (GPC) method under the following conditions.

[0084] <GPC Measurement Conditions> Measuring instrument: GPC system manufactured by Waters [Pump: Model 510, Detector: waters410] Eluent; Type: Water / Methanol (70 / 30 (volume ratio)) + Sodium Acetate {5g / liter (relative to water / methanol)} Flow rate 1.0 (ml / min) Columns: TSKgel G3000PWXL and TSKGel G50000PWXL (each with an inner diameter of 7.8 mm and a length of 30 cm) Column temperature: 40°C Standard substance: Polyethylene glycol {Mw: 2.4×10⁴, 5.0×10⁴, 1.07×10⁵, 2.5×10⁵, 5.4×10⁵, 9.0×10⁵ (TSK standard polyethylene oxide SE-2, SE-5, SE-8, SE-30, SE-70, SE-150 manufactured by Tosoh Corporation)}

[0085] Among acrylic acid (salt) copolymers, those containing constituent units derived from acrylic acid (salt) and constituent units derived from unsaturated dibasic acid (salt) are the same as copolymers produced using acrylic acid (salt) and unsaturated dibasic acid (salt).

[0086] Copolymers containing constituent units derived from acrylic acid (salt) and constituent units derived from unsaturated dibasic acid (salt) can be produced by methods described in Japanese Patent No. 6814616, Japanese Patent No. 4485237, Japanese Patent No. 5722460, etc. Copolymers containing constituent units derived from acrylic acid (salt) and constituent units derived from sulfonic acid (salt) group-containing monomers can be produced by the method described in Japanese Patent No. 7507701, etc.

[0087] Copolymers containing acrylic acid salts and unsaturated dibasic acid salts as constituent monomers can also be obtained by neutralizing a copolymer of acrylic acid and an unsaturated dibasic acid with a base (such as alkali metal hydroxides, alkaline earth metal hydroxides, ammonia, and organic amines). Copolymers containing acrylate and sulfonate-containing monomers as constituent monomers can also be obtained by neutralizing a copolymer of acrylic acid and a sulfonic acid group-containing monomer with a base (alkali metal hydroxide, alkaline earth metal hydroxide, ammonia, and organic amines, etc.).

[0088] When a copolymer of acrylic acid and an unsaturated dibasic acid is neutralized with a base, the degree of neutralization (the ratio of salt formation to the total amount of carboxyl groups in the copolymer) is preferably 50% to 100% from the viewpoint of solubility. When a copolymer of an acrylate salt and a sulfonate-containing monomer is neutralized with a base, a degree of neutralization of 10% to 100% is preferred from the viewpoint of solubility.

[0089] Copolymers containing constituent units derived from acrylic acid (salt) and constituent units derived from unsaturated dibasic acid (salt), and copolymers containing constituent units derived from acrylic acid (salt) and constituent units derived from sulfonic acid (salt) group-containing monomers, can be copolymers available on the market. Examples of copolymers of acrylic acid (salt) and unsaturated dibasic acid (salt) available on the market include Aquaric L-TL-200 and Aquaric L-TL-213 (Nippon Shokubai Co., Ltd.). Examples of copolymers of acrylic acid (salt) and sulfonic acid (salt) group-containing monomers available on the market include Aquaric L-GH234 and Aquaric L-GH366 (Nippon Shokubai Co., Ltd.).

[0090] The types of monomers constituting copolymers available on the market, their molar ratios, and weight-average molecular weights can be determined by known compositional analysis methods such as GC-MS and NMR, as well as by performing the aforementioned GPC measurements.

[0091] In Method 1 described above, a specific reaction between a substance (G1) contained in a biological fluid, etc., and substance (D) is carried out in the presence of the acrylic acid (salt) copolymer.

[0092] In Method 2 described above, a specific reaction is carried out in the presence of the acrylic acid (salt) copolymer between a substance (G1+) similar to a substance contained in biological fluids, etc., and a substance (D) immobilized on a solid support (a1).

[0093] In Method 3 described above, both the specific reaction between a substance (G1) contained in biological fluids, etc. and a substance (D) immobilized on a solid support (a1), and the specific reaction between a substance (G1+) similar to a substance contained in biological fluids, etc. and a substance (D) immobilized on a solid support (a1) are carried out simultaneously in the presence of the acrylic acid (salt) copolymer.

[0094] In Method 4 described above, a specific reaction between a substance (G2) that specifically reacts with substances contained in biological fluids, etc., and a substance (D) immobilized on a solid support (a1) is carried out in the presence of the acrylic acid (salt) copolymer.

[0095] In Method 5 described above, it is preferable from the viewpoint of measurement sensitivity to carry out the specific reaction between the composite (G12) and the substance (D) immobilized on the solid support (a1) in the presence of the acrylic acid (salt) copolymer.

[0096] Furthermore, in Method 5 described above, it is also preferable from the viewpoint of measurement sensitivity to carry out the specific reaction in the presence of the acrylic acid (salt) copolymer in which a substance added to a biological fluid or a liquid sample pretreated with a biological fluid forms a complex (G12) with a substance contained in the biological fluid.

[0097] In Method 6 described above, a specific reaction between substance (D) and substance (G2), which is a substance contained in an analytical reagent and specifically reacts with a substance contained in a liquid sample of biological origin, is carried out in the presence of the acrylic acid (salt) copolymer.

[0098] By carrying out a specific reaction in a liquid sample containing the aforementioned acrylic acid (salt) copolymer, which is a copolymer containing constituent units derived from acrylic acid (salt) and constituent units derived from an unsaturated dibasic acid (salt), and / or a copolymer containing constituent units derived from acrylic acid (salt) and constituent units derived from a sulfonic acid (salt) group-containing monomer, the charge in the reaction field changes, making it less likely for substances that cause nonspecific reactions to be adsorbed onto the solid support. This makes it easier for specific reactions between antigens and antibodies to occur, and it is thought that highly sensitive immunoassays can be performed.

[0099] Even if the copolymer further contains constituent units derived from a third monomer, nonspecific reactions can be suppressed by changes in the charge interactions between constituent units derived from acrylic acid (salt) and constituent units derived from unsaturated dibasic acid (salt), or constituent units derived from acrylic acid (salt) and constituent units derived from sulfonic acid (salt) group-containing monomers, as well as by changes in the interaction between the solid support surface and nonspecific components. The constituent units derived from the third monomer may affect the hydrophilicity / hydrophobicity, steric hindrance, solution viscosity, etc., of the copolymer, but the effects of the present invention can be obtained by including the constituent units derived from acrylic acid (salt) as described above.

[0100] In the measurement method of the present invention, if a substance that specifically reacts with substance (G) bound to a solid-phase reactant having substance (D) is to be reacted further, the specific reaction with substance (G) bound to the solid-phase reagent may be carried out in the presence of the acrylic acid (salt) copolymer.

[0101] In the present invention, the specific reaction carried out in the presence of the acrylic acid (salt) copolymer is preferably the following reaction. • Specific reaction between a substance (G1) contained in a biological liquid sample and a substance (D) immobilized on a solid support (a1) • Specific reaction between a substance (G1+) similar to a substance contained in a biological liquid sample and a substance (D) immobilized on a solid support (a1). • Specific reaction between a substance (G2) that specifically reacts with substances contained in a biological liquid sample and a substance (D) immobilized on a solid support (a1). • A composite (G12) of a substance that specifically reacts with a substance contained in a biologically derived liquid sample and a substance contained in the biologically derived liquid sample, and a specific reaction between the substance (D) immobilized on a solid support (a1) and the composite (G12) of the substance contained in the biologically derived liquid sample. • Specific reaction between a substance contained in a biological liquid sample immobilized on a solid support (a1) and a substance (G2) that specifically reacts with the substance contained in the biological liquid sample.

[0102] Among the specific reactions carried out in the presence of an acrylic acid (salt) copolymer [i.e., a copolymer containing constituent units derived from acrylic acid (salt) and constituent units derived from an unsaturated dibasic acid (salt), and / or a copolymer containing constituent units derived from acrylic acid (salt) and constituent units derived from a sulfonic acid (salt) group-containing monomer], the preferred specific reaction described above can be carried out by mixing a liquid sample containing substance (G), a solid support (a1) on which substance (D) is immobilized, and the acrylic acid (salt) copolymer.

[0103] From the viewpoint of suppressing non-specific reactions, the proportion of the total weight of the acrylic acid (salt) copolymer contained in the liquid sample when performing a specific reaction is preferably 0.005 to 5% by weight, based on the total weight of the substances present in the system in which the specific reaction is performed. From the viewpoint of measurement sensitivity, the total weight percentage of the acrylic acid (salt) copolymer is more preferably 0.01 to 4% by weight, and particularly preferably 0.015 to 3% by weight. The total weight of substances present in the system undergoing the specific reaction can also be expressed as the total weight of the solution used for the immunoassay, and is the total weight of the solvent in the liquid sample, the substance in the liquid sample (G), the solid-phase reactant, the acrylic acid (salt) copolymer, the labeling reagent (F) used as needed, the immunoassay buffer (hereinafter also referred to as buffer) used as needed, and the other components dissolved and / or dispersed in the liquid sample.

[0104] In the aforementioned specific reaction, the solid-phase reactant is preferably a solid-phase reactant contained in the solid-phase support reagent (A) from the viewpoint of operability, and the content of the solid-phase reactant in the solid-phase support reagent (A) is preferably 0.001 to 10% by weight, and more preferably 0.01 to 1% by weight, from the viewpoint of suppressing non-specific reactions.

[0105] In addition to the solid-phase reactant, the solid-phase support reagent (A) may contain other components such as gelatin, proteins other than gelatin, serum (mouse serum, etc.), sugars, surfactants, inorganic salts, ethylenediaminetetraacetic acid, water, polymer compounds (excluding the aforementioned acrylic acid (salt) copolymers), and surfactants.

[0106] The gelatin may include any known gelatin, and is not limited in terms of molecular weight or properties; it may be obtained from any animal (mammals, birds, fish, etc.). Examples of gelatin include acid-treated gelatin and alkali-treated gelatin, which are produced by chemically treating collagen with an acid or alkali followed by heat treatment. Furthermore, gelatin derivatives can also be used, which are chemically modified by introducing functional groups such as amino groups, imino groups, carboxyl groups, mercapto groups, and hydroxyl groups using well-known methods. From the viewpoint of the storage stability of the solid-phase support reagent (A), the gelatin content is preferably 0.0001 to 8% by weight, and more preferably 0.01 to 5% by weight, based on the weight of the solid-phase support reagent (A).

[0107] Other proteins besides gelatin are not particularly limited as long as they are commonly used in the field of immunoassays, and examples include bovine serum albumin (BSA), casein, and skim milk. One protein may be used alone, or two or more may be used in combination. From the viewpoint of the storage stability of the solid support reagent (A), the protein content is preferably 0 to 15% by weight, and more preferably 0.1 to 15% by weight, based on the weight of the solid support reagent (A). Furthermore, if serum is included, its content is preferably 0 to 15% by weight, and more preferably 0.01 to 10% by weight, based on the weight of the solid-phase carrier reagent (A), from the viewpoint of storage stability.

[0108] Sugars include monosaccharides, disaccharides, and polysaccharides. Examples of monosaccharides include trioses (ketotriose, etc.), tetroses (ketotetrose, etc.), pentoses (ketopentoses, aldopentoses, and deoxy sugars, etc.), hexoses [ketohexoses (psicose, fructose, sorbose, and tagatose, etc.), aldohexoses (allose, altrose, glucose, mannose, gross, idose, galactose, and talose, etc.), and deoxy sugars (fucose, fuculose, and rhamnose, etc.)] and heptoses (sedoheptulose, etc.). Disaccharides include those formed by the dehydration condensation of two molecules of the above-mentioned monosaccharides to form a glycosidic bond. Specifically, examples include sucrose, lactose, maltose, and cellobiose. Polysaccharides include those formed by the dehydration condensation of three or more molecules of the above-mentioned monosaccharides to form glycosidic bonds. Specifically, examples include amylose, amylopectin, glycogen, cellulose, hyaluronic acid, chondroitin sulfate, and heparin. Sugars may be used individually or in combination of two or more types. As for the sugars, disaccharides are preferred from the viewpoint of storage stability of the solid-phase support reagent (A), and sucrose and lactose are more preferred. From the viewpoint of the storage stability of the solid-phase support reagent (A), the sugar content is preferably 0.1 to 40% by weight, and more preferably 0.5 to 20% by weight, based on the weight of the solid-phase support reagent (A).

[0109] The polymer compound is not particularly limited as long as it is used in the field of immunoassays, and examples include polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, Blockmaster (manufactured by JSR Corporation), and Lipidure (manufactured by NOF Corporation), with polyethylene glycol being preferred from the viewpoint of measurement sensitivity. Polymer compounds may be used individually or in combination of two or more types. From the viewpoint of measurement sensitivity, the content of the polymer compound is preferably 0.001 to 3% by weight, and more preferably 0.01 to 2% by weight, based on the weight of the solid support (A).

[0110] As for the surfactant, nonionic surfactants are preferred from the viewpoint of suppressing nonspecific reactions, and polyoxyethylene nonylphenyl ether, polyoxyethylene alkyl ether (such as polyoxyethylene octyl ether), and polyoxyethylene sorbitan aliphatic esters with an HLB of 12 or higher can be preferably used.

[0111] When the immunoassay method of the present invention is performed using solid-phase support reagent (A), it is preferable that solid-phase support reagent (A) is an aqueous dispersion containing 0.001 to 10% by weight (preferably 0.01 to 1% by weight from the viewpoint of suppressing non-specific reactions) of solid-phase reactant.

[0112] When the immunoassay method of the present invention is performed using a solid-phase support reagent (A), a buffer solution may also be used. As the buffer solution, those used in the field of immunoassays (such as Tris buffer, phosphate buffer, Veronal buffer, borate buffer, and Good's buffer) can be used, and the pH should be within a range that does not suppress the antigen-antibody reaction, preferably between 5 and 9. Furthermore, the buffer may contain stabilizers such as albumin, globulin, water-soluble gelatin, and polyethylene glycol, as well as surfactants and sugars, provided that they do not inhibit the target antigen-antibody reaction.

[0113] When the immunoassay method of the present invention is performed using the solid-phase support reagent (A) and buffer solution, the acrylic acid (salt) copolymer [i.e., a copolymer containing constituent units derived from acrylic acid (salt) and constituent units derived from an unsaturated dibasic acid (salt), and / or a copolymer containing constituent units derived from acrylic acid (salt) and constituent units derived from a sulfonic acid (salt) group-containing monomer] may be included in the solid-phase support reagent (A), included in the buffer solution, or added separately from the solid-phase support reagent (A) and buffer solution. When performing the immunoassay method of the present invention using the solid-phase support reagent (A) and buffer solution, if the amount of acrylic acid (salt) copolymer is to be the preferred amount described above, the concentration in the reaction solution containing the solid-phase support reagent (A) and buffer solution can be adjusted by diluting or adding the copolymer, etc., before proceeding with the reaction.

[0114] The immunoassay kit of the present invention is an immunoassay kit comprising a solid-phase support reagent (A) and an immunoassay buffer (W), wherein the solid-phase support reagent (A) and / or the immunoassay buffer (W), which constitute the immunoassay reagent of the immunoassay kit, include a copolymer comprising a constituent unit derived from acrylic acid (salt) and a constituent unit derived from an unsaturated dibasic acid (salt), and / or a copolymer comprising a constituent unit derived from acrylic acid (salt) and a constituent unit derived from a sulfonic acid (salt) group-containing monomer. The solid-phase support reagent (A) contains a solid-phase support (a1) on which the solid-phase reactant, i.e., a substance (D) that specifically binds to the substance to be measured (G), is immobilized.

[0115] The immunoassay kit of the present invention may contain, in addition to the above, a solid-phase support reagent (A) which may contain a copolymer comprising constituent units derived from acrylic acid (salt) and constituent units derived from an unsaturated dibasic acid (salt), or a copolymer comprising constituent units derived from acrylic acid (salt) and constituent units derived from a sulfonic acid (salt) group-containing monomer, or both thereof; and in addition to the solid-phase support reagent (A) and the immunoassay buffer (W), each of the solid-phase support reagent (A) and the immunoassay buffer (W) may contain a copolymer comprising constituent units derived from acrylic acid (salt) and constituent units derived from an unsaturated dibasic acid (salt), or a copolymer comprising constituent units derived from acrylic acid (salt) and constituent units derived from a sulfonic acid (salt) group-containing monomer, or both thereof.

[0116] The copolymers containing acrylic acid (salt)-derived structural units and unsaturated dibasic acid (salt)-derived structural units, and copolymers containing acrylic acid (salt)-derived structural units and sulfonic acid (salt)-containing monomers, as described above in the immunoassay method, are the same as the copolymers containing acrylic acid (salt)-derived structural units and unsaturated dibasic acid (salt)-derived structural units, and copolymers containing acrylic acid (salt)-derived structural units and sulfonic acid (salt)-containing monomers, and the preferred ones are also the same. The content of copolymers containing constituent units derived from acrylic acid (salt) and unsaturated dibasic acid (salt), and copolymers containing constituent units derived from acrylic acid (salt) and sulfonic acid (salt) group-containing monomers, contained in the solid-phase support reagent (A) and the immunoassay buffer (W) is the same as the content in the solid-phase support reagent (A) and buffer described above in the immunoassay method.

[0117] The composition and preferred range of the solid-phase support reagent (A) in the immunoassay kit of the present invention are the same as those of the solid-phase support reagent (A) described in the immunoassay method.

[0118] The composition and preferred range of the immunoassay buffer (W) in the immunoassay kit of the present invention are the same as those of the buffer described in the immunoassay method.

[0119] The immunoassay kit of the present invention may further include a labeled reagent (F). Labeling reagent (F) is a reagent containing the labeling substance described in the immunoassay method described above. Labeling reagent (F) may contain the acrylic acid (salt) copolymer described above, or it may contain proteins, and the surfactants and polymer compounds described above. The proteins used are not particularly limited as long as they are measured in the field of immunoassays. Examples include bovine serum albumin (BSA), casein, and skim milk, and one type may be used alone or two or more types may be used in combination. From the viewpoint of sensitivity and storage stability of the reagent, the protein content is preferably 0.001 to 8% by weight, based on the weight of the labeled reagent.

[0120] The immunoassay kit of the present invention may further include a chemiluminescent reagent (E). Examples of chemiluminescent reagents (E) include chemiluminescent reagents composed of a luminol luminescent reagent [a chemiluminescent reagent (solution 1) in which the 2,3-dihydro-1,4-phthalazinedione compound is luminol and / or its metal salt] and a hydrogen peroxide solution [a chemiluminescent reagent (solution 2) in which the oxidizing agent is hydrogen peroxide].

[0121] The present invention will be further described below with reference to examples, but the present invention is not limited thereto. In the following, "parts" refers to parts by weight.

[0122] <Manufacturing Example 1> In a 0.50 L stainless steel separable flask equipped with a reflux condenser and stirrer, 36.57 g of deionized water and 11.80 g of maleic acid (hereinafter also referred to as "MA") were charged, and then 10.0 g of 48% sodium hydroxide aqueous solution (hereinafter also referred to as "48% NaOH") was added. The mixture was then heated to 85°C under stirring. After reaching 85°C, the temperature was maintained at 85°C, and 80% acrylic acid aqueous solution (hereinafter also referred to as "80%AA") (82.45g), 48% NaOH (19.17g), 35% sodium pyrosulfite aqueous solution (hereinafter also referred to as "35%Na2S2O5") (0.17g), and 15% sodium persulfite aqueous solution (hereinafter also referred to as "15%Na2S2O8") (0.12g) were added dropwise from separate dropping nozzles, with 80%AA added over 73 minutes, 48% NaOH over 79 minutes, 35% Na2S2O5 over 9 minutes, and 15% Na2S2O8 over 11 minutes. After the addition was complete, the temperature was maintained at 85°C for another 30 minutes to allow maturation and polymerization to occur. After polymerization was complete, the reaction solution was cooled and neutralized by gradually adding 48% NaOH (54.75 g) dropwise to obtain an aqueous solution containing an acrylic acid (salt)-maleic acid (salt) copolymer (copolymer 1) with a degree of neutralization of 90% and a solid content of 55%. The physical properties of the obtained copolymer 1 were measured, and the results are summarized in Table 1.

[0123] <Manufacturing Examples 2-3> Copolymer 2 and copolymer 3 were obtained using the same method as in Production Example 1, except that the monomer composition ratio was changed as shown in Table 1, and the polymerization conditions, such as the dropping time, were also changed as shown in Table 1. The physical properties of the obtained copolymer 2 and copolymer 3 were measured, and the results are summarized in Table 1.

[0124] [Table 1]

[0125] <Manufacturing Example 4> In a 0.5 L stainless steel separable flask equipped with a reflux condenser and stirrer, 140 g of deionized water and 53.29 g of Mohr's salt (hereinafter also referred to as "(NH4)2Fe(SO4)2·6H2O") were charged, and the temperature was raised to 85°C while stirring. Next, while maintaining the temperature at 85°C under stirring, 80% AA (81.63 g), an aqueous solution containing 40% sodium 3-allyloxy-2-hydroxypropanesulfonate (hereinafter also referred to as "40% HAPS") (75.41 g), 35% Na2S2O5 (0.21 g), and 15% Na2S2O8 (0.14 g) were added dropwise from separate dropping nozzles, with the 80% AA added over 61 minutes, the 40% HAPS over 60 minutes, the 35% Na2S2O5 over 22 minutes, and the 15% Na2S2O8 over 10 minutes. After the addition of 15% Na2S2O8 dropwise was completed, the reaction solution was held at 85°C for a further 30 minutes (aged) to complete the polymerization, and an aqueous solution containing a copolymer (copolymer 4) of acrylic acid (salt) with a degree of neutralization of 40% and a monomer containing sulfonic acid (salt) group was obtained with a solid content concentration of 42%. The physical properties of the obtained copolymer 4 were measured, and the results are summarized in Table 2.

[0126] <Manufacturing Example 5> An aqueous solution containing a copolymer of acrylic acid (salt) and a sulfonic acid (salt) group-containing monomer (copolymer 5) was obtained in the same manner as in Example 4, except that the monomer composition ratio was changed as shown in Table 2, and the polymerization conditions such as the dropping time were also changed as shown in Table 2. The physical properties of the obtained copolymer 5 were measured, and the results are summarized in Table 2.

[0127] [Table 2]

[0128] <Example 1> The immunoassay kit (S-1) of the present invention was obtained by combining the solid-phase support reagent (A-1), labeling reagent (F-1), immunoassay buffer (W-1), chemiluminescent reagent solution 1 (E-1), and chemiluminescent reagent solution 2 (E-2), which were prepared by the method described below.

[0129] <<Fabrication of magnetic particles (PH-1)>> In a reaction vessel, 2.7 parts iron(III) chloride hexahydrate, 1.0 part iron(II) chloride tetrahydrate, and 375 parts water were charged, dissolved, and heated. While stirring, the temperature was maintained at 50-55°C, and a solution of 25% aqueous ammonia (3.8 parts) and water (100 parts) was added dropwise over 1 hour. After addition, stirring was continued for 1 hour to obtain an aqueous dispersion of magnetite particles. Oleic acid (10.5 parts) was added to the obtained aqueous dispersion of magnetite particles as a dispersant, and stirring was continued for 2 hours. After cooling to room temperature, solid-liquid separation was performed by decantation, and the solid was recovered to obtain magnetite particles with adsorbed oleic acid. The obtained magnetite particles were washed with 50 parts water three times. The resulting magnetite particles with adsorbed oleic acid were then charged into a container, and 5.7 parts decane and 2.2 parts tetraethoxysilane were added and mixed to prepare a dispersion.

[0130] In a reaction vessel, 39.0 parts of 25% aqueous ammonia, 55.4 parts of isopropanol, 2.9 parts of sorbitan monooleate ("Ionet S-80" manufactured by Sanyo Chemical Industries, Ltd.), and 2.0 parts of polyoxyethylene (20 moles added) alkyl ether ("Emarumin 200" manufactured by Sanyo Chemical Industries, Ltd.) were added and mixed using Clear Mix (manufactured by M-Technique Co., Ltd.). After raising the temperature to 50°C, the dispersion was added dropwise over 1 hour while stirring with the Clear Mix at a rotation speed of 6,000 rpm, and the mixture was reacted at 50°C for 1 hour. After the reaction, the mixture was centrifuged at 2,000 rpm for 5 minutes to remove the supernatant containing fine particles. Water (50 parts) was added to the obtained solid phase to disperse the particles, and the mixture was centrifuged at 1,000 rpm for 10 minutes, after which the supernatant containing fine particles was removed. This procedure was repeated 10 times. Next, water (50 parts) was added to the obtained solid phase to disperse the particles, and the mixture was centrifuged at 500 rpm for 5 minutes to settle the larger particle sizes, and the supernatant (1) containing particles of the desired particle size was collected. The remaining solid phase was then centrifuged at 500 rpm for 5 minutes, and the supernatant (2) was collected. This process was repeated twice to collect particles of the desired particle size present in the solid phase. Next, the particles in supernatants (1) and (2) were magnetized using a magnet, and the magnetized particles were dried at 80°C for 8 hours to obtain magnetic particles.

[0131] The particle size of the magnetic particles was determined by averaging the particle sizes of 200 arbitrary particles observed using a scanning electron microscope (model number: JSM-7000F, manufacturer: JEOL Ltd.).

[0132] The magnetic particle content was determined by observing 20 arbitrary magnetic particles with a scanning electron microscope (model JSM-7000F, manufacturer JEOL Ltd.) and measuring the superparamagnetic metal oxide content using an energy-dispersive X-ray spectrometer (model INCAWave / Energy, manufacturer Oxford). The average value of these 20 particles was defined as content S, and the average value of silicon content obtained from the same measurement was defined as content T. The magnetic particle content was then calculated using the following formula. Content of magnetic particles (%)=(S) / (S+T)×100

[0133] <<Preparation of Solid-Phase Support Reagent (A-1)>> 40 mg of magnetic particles (PH-1) were added to a lidded polyethylene bottle containing 40 mL of an acetone solution containing 1 wt% γ-aminopropyltriethoxysilane. The mixture was reacted at 25°C for 1 hour. The magnetic particles were then magnetized with a neodymium magnet, and the liquid was removed by aspirator. Next, 40 mL of deionized water was added, the bottle was sealed, and the polystyrene bottle was slowly inverted and stirred twice. The magnetic particles were then magnetized with a neodymium magnet, and the liquid was removed by aspirator to wash them. This washing procedure was repeated 5 times. Next, the washed magnetic particles were added to a lidded polyethylene bottle containing 40 mL of an aqueous solution containing 2 wt% glutaraldehyde, and the mixture was reacted at 25°C for 1 hour. Then, 40 mL of deionized water was added, the bottle was sealed, and the polystyrene bottle was slowly inverted and stirred twice. The magnetic particles were then magnetized with a neodymium magnet, and the liquid was removed by aspirator to wash them. This washing procedure was repeated 10 times. Furthermore, the washed magnetic particles were added to a lidded polyethylene bottle containing 120 mL of 0.02 M phosphate buffer (pH 8.7) with a concentration of 10 μg / mL of anti-CEA monoclonal antibody (manufactured by Hytest), which is substance (D) in the present invention, and reacted at 25°C for 1 hour. After the reaction, the magnetic particles were magnetized with a neodymium magnet, and the phosphate buffer containing anti-CEA monoclonal antibody (manufactured by Hytest) was removed. Next, the magnetic particles were added to a lidded polyethylene bottle containing 40 mL of 0.02 M phosphate buffer (pH 7.0) containing 1% by weight of bovine serum albumin (manufactured by Boval Company), and immersed at 25°C for 12 hours. This prepared the solid-phase support reagent (reagent containing anti-CEA antibody-bound magnetic particles) (A-1). The prepared solid-phase support reagent was stored refrigerated (2-10°C).

[0134] <<Preparation of Labeled Reagent (F-1)>> Using anti-CEA monoclonal antibody (Hytest) and horseradish-derived POD (Toyobo Co., Ltd.), POD-labeled anti-CEA antibody was prepared according to the method described in the literature (S. Yoshitake, M. Imagawa, E. Ishikawa, Etol; J. Biochem, Vol. 92, 1982, 1413-1424). This was diluted to a concentration of 1.0 μg / mL as POD-labeled anti-CEA antibody in 0.02 M phosphate buffer (pH 7.0) containing 0.5 wt% bovine serum albumin (Boval Company), and the labeling reagent (F-1) was prepared and stored under refrigeration (2-10°C).

[0135] <<Preparation of Immunotherapy Buffer (W-1)>> Bovine serum albumin (manufactured by Boval Company) was mixed and dissolved in 0.02 M phosphate buffer (pH 7.0) to prepare an immunoassay buffer (W-1) with a concentration of 0.1% by weight, a concentration of copolymer 1 of 2.2% by weight, and a concentration of sodium chloride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) of 0.85% by weight. This buffer was then stored under refrigeration (2-10°C).

[0136] <<Preparation of Chemiluminescent Reagent Solution 1 (E-1)>> 0.7 g of sodium luminol [manufactured by Sigma-Aldrich Japan Co., Ltd.] and 0.1 g of 4-(cyanomethylthio)phenol were placed in a 1,000 mL volumetric flask. 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid / sodium hydroxide buffer (10 mM, pH 8.6) was added to a volume of 1,000 mL, and the mixture was homogenized at 25°C to prepare chemiluminescent reagent solution 1 (E-1), which was then stored in a refrigerator (2-10°C).

[0137] <<Preparation of Chemiluminescent Reagent Solution 2 (E-2)>> 6.6 g of hydrogen peroxide [manufactured by Wako Pure Chemical Industries, Ltd., reagent grade, concentration 30% by weight] was placed in a 1,000 mL volumetric flask. Deionized water was added to bring the total volume of the solution to 1,000 mL, and the mixture was homogenized at 25°C to prepare chemiluminescent reagent solution 2 (E-2), which was then stored in a refrigerator (2-10°C).

[0138] <Examples 2-12> In the preparation of immunoreaction buffer (W-1) in Example 1, immunoreaction buffers (W-2) to (W-12) were obtained in the same manner as in Example 1, except that the type and concentration of the acrylic acid (salt) copolymer in the immunoreaction buffer were changed to those shown in Table 3. Each of the obtained immunoassay buffers (W-2) to (W-12) was combined with the solid support reagent (A-1), labeling reagent (F-1), chemiluminescent reagent solution 1 (E-1), and chemiluminescent reagent solution 2 (E-2) in the same manner as in Example 1 to prepare the immunoassay kits (S-2) to (S-12) according to Examples 2 to 12.

[0139] <Comparative Example 1> In the preparation of immunoreaction buffer (W-1) in Example 1, immunoreaction buffer (W'-1) was obtained in the same manner as in Example 1, except that acrylic acid (salt) copolymer was not used in the immunoreaction buffer. The obtained immunoassay buffer (W'-1) was combined with the solid support reagent (A-1), labeling reagent (F-1), chemiluminescent reagent solution 1 (E-1), and chemiluminescent reagent solution 2 (E-2) in the same manner as in Example 1 to prepare a comparative immunoassay kit (H-1) that does not contain the acrylic acid (salt) copolymer, as described in Comparative Example 1.

[0140] <Comparative Example 2> In the preparation of immunoreaction buffer (W-1) in Example 1, immunoreaction buffer (W'-2) was obtained in the same manner as in Example 1, except that the type and concentration of the acrylic acid (salt) copolymer in the immunoreaction buffer were changed to the type and concentration of the comparative polymer shown in Table 4. The obtained immunoassay buffer (W'-2) was combined with the solid support reagent (A-1), labeling reagent (F-1), chemiluminescent reagent solution 1 (E-1), and chemiluminescent reagent solution 2 (E-2) in the same manner as in Example 1 to prepare an immunoassay kit (H-2) that does not contain the acrylic acid (salt) copolymer of Comparative Example 2 but contains a comparative polymer.

[0141] [Table 3]

[0142] [Table 4]

[0143] Of the acrylic acid (salt) copolymers listed in Tables 3 and 4, the following acrylic acid (salt) copolymers other than copolymers 1 to 5 produced in Production Examples 1 to 5 were used. • Aqualic L-TL-200 (40% aqueous solution of sodium acrylic acid / maleic acid copolymer, weight-average molecular weight: 60,000, manufactured by Nippon Shokubai Co., Ltd.) • Aqualic L-TL-213 (36% aqueous solution of sodium acrylic acid / maleic acid copolymer, weight-average molecular weight: 10,000, manufactured by Nippon Shokubai Co., Ltd.) • Aqualic L-GH234 (40% aqueous solution of sodium salt of acrylic acid / sulfonic acid monomer copolymer, weight-average molecular weight: 140,000, manufactured by Nippon Shokubai Co., Ltd.) • Aqualic L-GH366 (50% aqueous solution of sodium salt of acrylic acid / sulfonic acid monomer copolymer, weight-average molecular weight: 5,000, manufactured by Nippon Shokubai Co., Ltd.) • Polystar OMR (25% aqueous solution of sodium salt of maleic acid / diisobutylene copolymer, manufactured by NOF Corporation)

[0144] <<Immunoassay using an immunoassay kit>> Using the immunoassay kits (S-1) to (S-12) obtained in Examples 1 to 12 and the comparative immunoassay kits (H-1) to (H-2) obtained in Comparative Examples 1 to 2, immunoassays were performed on serum containing carcinoembryonic antigen (hereinafter referred to as CEA) at a concentration of 1.0 ng / mL (hereinafter referred to as serum sample 1) and serum without CEA (CEA concentration: 0 ng / mL) (hereinafter referred to as serum sample 2) using the following methods.

[0145] 0.025 mL of the solid support reagent (A) from the immunoassay kit was placed in a test tube, and magnetic particles were collected from the outside of the test tube using a neodymium magnet for 10 seconds. The liquid in the test tube was removed with an aspirator, and the neodymium magnet was moved sufficiently away from the side. Furthermore, 0.2 mL of immunoassay buffer (W) and 0.025 mL of serum sample 1 were placed in a test tube, and the immunoassay was performed in the test tube at 37°C for 3 minutes. After the reaction, the magnetic particles were collected from the outside of the test tube using a neodymium magnet for 10 seconds, the liquid inside the test tube was removed with an aspirator, and the neodymium magnet was moved sufficiently away from the side. Subsequently, 0.5 mL of physiological saline was added to disperse the magnetic particles and collect the magnetization, followed by two washes using an aspirator to remove the liquid. Next, 0.05 mL of the labeled reagent (F) was added to the test tube and reacted in the test tube at 37°C for 3 minutes. After the reaction, magnetic particles were collected from the outside of the test tube using a neodymium magnet for 10 seconds, the liquid in the test tube was removed with an aspirator, and the neodymium magnet was moved sufficiently away from the side. Then, 0.5 mL of physiological saline was added to disperse the magnetic particles, and the washing procedure of removing the liquid with an aspirator was repeated twice after magnetization. Next, 0.1 mL of chemiluminescent reagent solution 1 (E-1) and 0.1 mL of chemiluminescent reagent solution 2 (E-2) were added simultaneously, and the luminescence reaction was allowed to proceed at 37°C for 45 seconds. The average luminescence amount from 43 to 45 seconds after the addition of the chemiluminescent reagents was measured using a luminometer [Lumat LB9507, manufactured by Berthold Japan].

[0146] The same procedure was performed on serum sample 2.

[0147] The sensitivity was calculated as the ratio of the luminescence obtained from measuring serum sample 1 to the luminescence obtained from measuring serum sample 2 [(luminescence obtained from measuring serum sample 1) / (luminescence obtained from measuring serum sample 2)] and is listed in Table 5. A higher ratio value indicates better sensitivity. In Table 5, the "Concentration of acrylic acid (salt) copolymer or comparative polymer in the immunoassay system" refers to the concentration in the entire reaction system used for the immunoassay, and is a value converted according to the mixing ratio of the reagents.

[0148] [Table 5]

[0149] Examples 1 to 12, which represent the immunoassay method of the present invention, show higher measurement sensitivity compared to the results in the comparative examples.

Claims

1. An immunoassay method comprising using a solid-phase reactant having a substance (D) that specifically reacts with a substance (G) contained in a liquid sample, wherein the specific reaction is carried out in the presence of an acrylic acid (salt) copolymer, wherein the acrylic acid (salt) copolymer is a copolymer containing constituent units derived from acrylic acid (salt) and constituent units derived from an unsaturated dibasic acid (salt), and / or a copolymer containing constituent units derived from acrylic acid (salt) and constituent units derived from a sulfonic acid (salt) group-containing monomer.

2. The immunoassay method according to claim 1, wherein the copolymer contains constituent units derived from acrylic acid (salt) and constituent units derived from an unsaturated dibasic acid (salt), and the ratio of the total number of moles of constituent units derived from acrylic acid (salt) to the total number of moles of constituent units derived from an unsaturated dibasic acid (salt) [acrylic acid (salt) / unsaturated dibasic acid (salt)] is 10 / 90 to 95 / 5.

3. The immunoassay method according to claim 1, wherein the acrylic acid (salt) copolymer is a copolymer of acrylic acid (salt) and an unsaturated dibasic acid (salt), and the weight-average molecular weight (Mw) of the copolymer of acrylic acid (salt) and an unsaturated dibasic acid (salt) is 1,000 to 100,000.

4. The immunoassay method according to claim 1, wherein the copolymer comprises constituent units derived from acrylic acid (salt) and constituent units derived from a sulfonic acid (salt) group-containing monomer, and the ratio of the total number of moles of constituent units derived from acrylic acid (salt) to the total number of moles of constituent units derived from the sulfonic acid (salt) group-containing monomer [acrylic acid (salt) / sulfonic acid (salt) group-containing monomer] is 1 / 99 to 95 / 5.

5. The immunoassay method according to claim 1, wherein the acrylic acid (salt) copolymer is a copolymer of acrylic acid (salt) and a sulfonic acid (salt) group-containing monomer, and the weight-average molecular weight (Mw) of the copolymer of acrylic acid (salt) and the sulfonic acid (salt) group-containing monomer is 2,000 to 200,000.

6. The immunoassay method according to claim 1, wherein the acrylic acid (salt) copolymer is 0.005 to 5% by weight based on the total weight of the substances present in the reaction system that undergoes a specific reaction.

7. An immunoassay kit comprising a solid-phase support reagent (A) and an immunoassay buffer (W), An immunoassay kit comprising a solid-phase support reagent (A) and / or an immunoassay buffer (W) comprising a copolymer containing constituent units derived from acrylic acid (salt) and constituent units derived from an unsaturated dibasic acid (salt), and / or a copolymer containing constituent units derived from acrylic acid (salt) and a monomer containing a sulfonic acid (salt) group.

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