Immunoassay methods and kits for immunoassays

Abstract

The present invention aims to provide an immunoassay method and an immunoassay kit with high measurement accuracy. [Solution] An immunoassay method that uses a solid-phase reactant having a substance (D) that reacts specifically with a substance (G) contained in a liquid sample, wherein the specific reaction between substance (G) and substance (D) is performed, and the immunoassay method is performed in the presence of (alkyl)benzenesulfonate and / or (polyoxyalkylene)alkyl ether sulfate, wherein the total weight ratio of (alkyl)benzenesulfonate and (polyoxyalkylene)alkyl ether sulfate is 0.005 to 5% by weight based on the total weight of the substances present in the reaction system where the specific reaction is performed.

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 analyte is bound to the surface of particles carrying a substance capable of binding the target analyte, the particles bound with the target analyte are recovered, and the binding amount is measured; and immunoassay methods in which the degree of aggregation of particles caused by binding of the target analyte to the particle surface is measured. It is known that biological samples contain substances (hereinafter referred to as interfering substances) that affect the reaction (also referred to as a specific reaction) between the target analyte and a substance capable of binding the target analyte present on the surface of particles or the like. As a method for improving the accuracy of immunoassay by suppressing the influence of interfering substances, a method of performing immunoassay in the presence of anethole sulfonate or a naphthalenesulfonic acid-formalin condensate salt is known (Patent Document 1). However, even in the immunoassay described in Patent Document 1, sufficiently accurate measurement cannot be performed due to the influence of non-specific reactions in which substances other than the measurement target react with the measurement reagent. [Prior Art Document] [Patent Document]

[0003] [Patent Document 1] Japanese Patent No. 4580180 [Summary of the Invention] [Problem 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 accuracy. [Means for Solving the Problem]

[0005] The inventors of this invention arrived at this present invention as a result of diligent research to achieve the above objective. 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, to perform a specific reaction between substance (G) and substance (D), an immunoassay method that performs the specific reaction in the presence of (alkyl)benzenesulfonate and / or (polyoxyalkylene)alkyl ether sulfate, an immunoassay method in which the total weight ratio of (alkyl)benzenesulfonate and (polyoxyalkylene)alkyl ether sulfate is 0.005 to 5% by weight based on the total weight of the substances present in the reaction system performing the specific reaction, and an immunoassay kit comprising a solid-phase support reagent (A) and an immunoassay buffer (W), an immunoassay kit in which the solid-phase support reagent (A) and / or the immunoassay buffer (W) contain (alkyl)benzenesulfonate and / or (polyoxyalkylene)alkyl ether sulfate. [Effects of the Invention]

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

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

[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.

[0010] Specific reactions include reactions that utilize interactions between specific chemical structures, such as reactions between "proteins (e.g., avidin)" and "ligands (e.g., biotin)."

[0011] A solid-phase reactant having a substance (D) that specifically reacts with a substance (G) contained in a liquid sample used in the reaction is a substance consisting of a solid support (a1) on which substance (D) is immobilized.

[0012] 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, etc., corresponding to substance (G). In other words, 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.

[0013] 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.

[0014] Substances similar to those contained in biological fluids (hereinafter also referred to as "similar substances") are substances that can react specifically with substances that biological fluids react specifically with, in the same way as those contained in biological fluids, and are substances that can bind to the binding sites of substances that biological fluids react specifically with; in other words, substances that have binding sites that can react specifically with substances that biological fluids react specifically with, in the same way as those contained in biological fluids; and in further terms, substances whose specific reaction with substances that biological fluids react specifically with competes with that of substances contained in biological fluids, but are different from those contained in biological fluids. For example, a substance that labels a substance contained in biological fluids is distinguished from the substance contained in biological fluids 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.

[0015] Substance (G2) may also be a labeled substance.

[0016] 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).

[0017] Examples of pretreatments performed on biological body fluids and the like include a treatment of mixing a biological body fluid or a biological-derived liquid sample with substances such as those of the following <Treatment 1> to <Treatment 2>, a treatment of adjusting the pH of a biological body fluid or a biological-derived liquid sample, and a treatment of adjusting the concentration of a biological body fluid or a biological-derived liquid sample. <処理1>A treatment of mixing a biological body fluid or the like with a substance (G2) that specifically reacts with a substance contained in the biological body fluid or the like, for the purpose of labeling, stabilizing, or increasing the sensitivity of a substance contained in the biological body fluid or the like <処理2>A treatment of mixing a biological body fluid or the like with a substance (G1+) similar to a substance contained in the biological body fluid or the like

[0018] The measurement method of the present invention, which performs a specific reaction using a solid-phase reaction substance in a liquid sample, includes the following reactions and the like.

[0019] <方法1>A measurement method in which a specific reaction between a substance (G1) contained in a biological body fluid or the like and a substance (D) is performed.

[0020] <方法2>A measurement method wherein, for a liquid sample containing an analogous substance (G1+), after obtaining the liquid sample containing the substance (G1+) by labeling a substance contained in the biological body fluid or the like by the method described below, or mixing a substance (G1+) similar to a substance contained in the biological body fluid or the like into the biological body fluid or the like, a specific reaction between the substance (G1+) contained in the liquid sample and the substance (D) is performed.

[0021] <方法3>A measurement method wherein the specific reaction in said <Method 1> and the specific reaction in said <Method 2> are performed simultaneously (that is, both the specific reaction between the substance (G1) contained in the biological body fluid or the like and the substance (D), and the specific reaction between the analogous substance (G1+) and the substance (D) are performed in the same sample).

[0022] <方法4>A measurement method wherein a substance (G2) that specifically reacts with a substance contained in a biological body fluid or the like is added to the biological body fluid or the like, and a specific reaction between the substance (G2) contained in the liquid sample after addition and the substance (D) is performed.

[0023] <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).

[0024] <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).

[0025] 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.

[0026] 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.

[0027] As magnetic particles, known particles described in Japanese Patent Publication No. 2023-105525, etc., can be used. From the viewpoint of shortening the measurement time and accuracy in immunoassays, 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 (Magrapid is a registered trademark of Sanyo Chemical Industries, Ltd.) can be used.

[0028] 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 or similar, which are available on the market.

[0029] 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.

[0030] 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).

[0031] 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.

[0032] 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.).

[0033] 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.

[0034] 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.

[0035] 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).

[0036] 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).

[0037] Labeling of substances contained in biological fluids, etc., with labeling substances (b), etc., can be carried out using methods commonly used in this field, such as known labeling methods that are commonly 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.].

[0038] 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.

[0039] Method 3 involves carrying out the specific reaction in Method 1 and the specific reaction in Method 2 simultaneously in the same reaction system.

[0040] 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.

[0041] Furthermore, in Method 4, the substance (G2) may also include a substance that undergoes a specific reaction, 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.

[0042] The solid support (a1) in Method 4 is the same as that exemplified in Method 1.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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).

[0047] The solid support (a1) in Method 5 is the same as that exemplified in Method 1.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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>.

[0052] 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).

[0053] 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 selected according to the type of substance that specifically reacts with the substance (G) bonded to the solid phase, which is then labeled with a labeling substance (b). Labeling with the labeling substance (b) can be carried out in the same manner as in Method 2.

[0054] The immunoassay methods of the present invention include known immunoassay methods such as direct methods, indirect methods, sandwich methods, competitive methods, and latex agglutination methods.

[0055] When the disease detection method is the sandwich method, for example, the method described as <Method 1> is included, and the following methods are also examples. This method involves measuring the concentration of a substance (G1) contained in a biological fluid, etc., by performing a specific reaction between a substance (G1) and a 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 the substance (G1) contained in the biological fluid, etc., thereby causing a bond between substance (D) and substance (G1) and labeling of the substance (G1) bonded to substance (D), and measuring the strength of the labeled substance. 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.

[0056] When the immunoassay method is a direct method, examples include the method described as <Method 6>, in which 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 specifically reacts with the substance contained in biological fluids, etc., are carried out in a liquid sample, and the concentration of the substance contained in the liquid sample derived from biological fluids, etc. is measured by measuring the intensity of the labeled substance.

[0057] When the immunoassay method is a competitive method, one example is the method described as <Method 3>, in which a specific reaction is carried out in the liquid sample between a substance (G1) contained in a biological fluid, a similar substance (G1+) added to the biological fluid or a liquid sample pretreated with the biological fluid, and a substance (D) immobilized on a solid support (a1), and the concentration of the substance (G1) contained in the biological liquid sample is measured by measuring the intensity of the labeled substance.

[0058] When the immunoassay is an indirect method, one example is a method in which a solid-phase 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., are carried out in a liquid sample, and then a labeled substance is reacted with the substance (G2) bound to the solid-phase reactant, and the concentration of the substance contained in the biological liquid sample is measured by measuring the intensity of the labeled substance.

[0059] When the immunoassay method is the latex agglutination method, one possible method is to perform a specific reaction between a substance (G1) contained in a biological liquid sample (Method 1) and particulate solid-phase reactive substances dispersed in an aqueous dispersion medium, and then quantify the amount of solid-phase reactive substances agglutinated by bond formation due to the specific reaction using turbidity or the like to measure the concentration of the substance contained in the biological liquid sample.

[0060] The immunoassay methods of the present invention also include immunoassay methods other than the direct method, indirect method, sandwich method, competitive method, and latex agglutination method described above.

[0061] The present invention relates to an immunoassay method that performs a specific reaction between substance (G) and substance (D) in the presence of (alkyl)benzene sulfonate and / or (polyoxyalkylene) alkyl ether sulfate. In this specification, (alkyl)benzenesulfonate means alkylbenzenesulfonate and / or benzenesulfonate, and (polyoxyalkylene)alkyl ether sulfate means polyoxyalkylene alkyl ether sulfate and / or alkyl ether sulfate.

[0062] In Method 1 described above, it is preferable to carry out the specific reaction between substance (G1) and substance (D) contained in biological fluids, etc., in a liquid sample containing (alkyl)benzenesulfonate and / or (polyoxyalkylene)alkyl ether sulfate.

[0063] In Method 2 described above, it is preferable to carry out 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) in a liquid sample containing (alkyl)benzenesulfonate and / or (polyoxyalkylene)alkyl ether sulfate.

[0064] In Method 3 described above, it is preferable to carry out both the specific reaction between a substance (G1) contained in a biological fluid, 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 a biological fluid, etc. and a substance (D) immobilized on a solid support (a1), simultaneously in a liquid sample containing (alkyl)benzenesulfonate and / or (polyoxyalkylene)alkyl ether sulfate.

[0065] In Method 4 described above, it is preferable to carry out the 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) in a liquid sample containing (alkyl)benzenesulfonate and / or (polyoxyalkylene)alkyl ether sulfate.

[0066] In Method 5 described above, it is preferable to carry out the specific reaction between the composite (G12) and the substance (D) immobilized on the solid support (a1) in a liquid sample containing (alkyl)benzenesulfonate and / or (polyoxyalkylene)alkyl ether sulfate.

[0067] Furthermore, in Method 5 described above, it is also preferable to carry out the specific reaction that forms a complex (G12) between a substance added to a biological fluid or a liquid sample pretreated with a biological fluid, and a substance contained in the biological fluid, in a liquid sample containing (alkyl)benzenesulfonate and / or (polyoxyalkylene)alkyl ether sulfate.

[0068] In Method 6 described above, it is preferable to carry out the specific reaction between a substance contained in a biological fluid, etc., immobilized as substance (D), and / or a substance similar to a substance contained in a biological fluid, etc., and a substance (G2) that specifically reacts with the substance contained in a biological fluid, etc., and / or a substance similar to a substance contained in a biological fluid, etc., in a liquid sample containing (alkyl)benzenesulfonate and / or (polyoxyalkylene)alkyl ether sulfate.

[0069] The immunoassay method of the present invention is thought to enable highly accurate immunoassays because, by carrying out a specific reaction in a liquid sample containing (alkyl)benzene sulfonate and / or (polyoxyalkylene) alkyl ether sulfate, the charge in the reaction system changes, making it less likely for substances that cause nonspecific reactions to be adsorbed onto the solid support.

[0070] In the immunoassay method of the present invention, if a substance that specifically reacts with substance (G) bound to a solid-phase reactant is to be further reacted with the substance (G) bound to the solid-phase reagent, the specific reaction with substance (G) bound to the solid-phase reagent may be carried out in a liquid sample containing (alkyl)benzenesulfonate and / or (polyoxyalkylene)alkyl ether sulfate.

[0071] Examples of alkylbenzene sulfonates used in the immunoassay method of the present invention include alkali metal salts of alkylbenzene sulfonic acid having an alkyl group with 1 to 14 carbon atoms, and amine salts of alkylbenzene sulfonic acid having an alkyl group with 1 to 14 carbon atoms.

[0072] Examples of alkylbenzenesulfonic acids having an alkyl group with 1 to 14 carbon atoms include toluenesulfonic acid, ethylbenzenesulfonic acid, propylbenzenesulfonic acid, butylbenzenesulfonic acid, hexylbenzenesulfonic acid, octylbenzenesulfonic acid, nonylbenzenesulfonic acid, decylbenzenesulfonic acid, dodecylbenzenesulfonic acid, tetradecylbenzenesulfonic acid, and octadecylbenzenesulfonic acid.

[0073] Examples of alkali metals that make up (alkyl)benzenesulfonates include lithium, sodium, and potassium.

[0074] Examples of amines constituting (alkyl)benzenesulfonates include ammonia and primary to tertiary aliphatic monoamines that may have hydroxyl groups (such as ethylamine, diethylamine, triethylamine, monoethanolamine, diethanolamine, triethanolamine, 2-amino-2-methylpropanol, dimethylethanolamine, and diethylethanolamine).

[0075] Examples of (alkyl)benzenesulfonates include sodium toluenesulfonate, sodium nonylbenzenesulfonate, nonylbenzenesulfonate triethanolamine, sodium dodecylbenzenesulfonate, and potassium dodecylbenzenesulfonate.

[0076] As the (alkyl)benzenesulfonate, alkali metal salts of alkylbenzenesulfonic acid having an alkyl group with 1 to 14 carbon atoms are preferred, and sodium dodecylbenzenesulfonate and potassium dodecylbenzenesulfonate are more preferred.

[0077] Among the (polyoxyalkylene) alkyl ether sulfates used in the immunoassay method of the present invention, examples of polyoxyalkylene alkyl ether sulfates include alkali metal salts of polyoxyalkylene alkyl ether sulfate and amine salts of polyoxyalkylene alkyl ether sulfate. Examples of polyoxyalkylene alkyl ether sulfates include polyoxyethylene decyl ether sulfate, polyoxyethylene lauryl ether sulfate, polyoxyethylene tridecyl ether sulfate, and polyoxyethylene oleyl cetyl ether sulfate.

[0078] The alkali metals and amines that constitute polyoxyalkylene alkyl ether sulfates are the same as those in (alkyl)benzene sulfonates.

[0079] Examples of alkyl ether sulfates include decyl ether sulfate, lauryl ether sulfate, tridecyl ether sulfate, and oleyl cetyl ether sulfate.

[0080] The alkali metals and amines that constitute alkyl ether sulfates are the same as those in (alkyl)benzene sulfonates.

[0081] As the (polyoxyalkylene)alkyl ether sulfate, polyoxyalkylene alkyl ether sulfate is preferred, and polyoxyethylene lauryl ether sodium sulfate is more preferred.

[0082] In the present invention, the specific reaction carried out in the presence of (alkyl)benzenesulfonate and / or (polyoxyalkylene)alkyl ether sulfate 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). • A 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 specific reaction between 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 substance (D) immobilized on a solid support (a1). • A 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.

[0083] Of the specific reactions carried out in a liquid sample containing (alkyl)benzenesulfonate and / or (polyoxyalkylene)alkyl ether sulfate, 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 (alkyl)benzenesulfonate and / or (polyoxyalkylene)alkyl ether sulfate.

[0084] From the viewpoint of suppressing non-specific reactions, the proportion of the total weight of the (alkyl)benzene sulfonate and (polyoxyalkylene) alkyl ether sulfate contained in the reaction system that performs the specific reaction is 0.005 to 5% by weight, based on the total weight of the substances present in the reaction system that performs the specific reaction. If the concentration is less than 0.005% by weight, the accuracy of the measurement will be poor when the substance (G) is at a low concentration, making accurate measurement impossible. If the concentration exceeds 5% by weight, the accuracy of the measurement will be poor when the substance (G) is at a high concentration, making accurate measurement impossible. From the viewpoint of measurement accuracy, the total weight percentage of (alkyl)benzene sulfonate and (polyoxyalkylene) alkyl ether sulfate is preferably 0.01 to 3% by weight. The total weight of substances present in the reaction system undergoing a specific reaction refers to the total weight of all components (including the solvent) that constitute the reaction solution undergoing the specific reaction. These components include the solvent of the liquid sample, the substance (G) in the liquid sample, the solid-phase reactant, (alkyl)benzene sulfonate and (polyoxyalkylene) alkyl ether sulfate, the following labeling reagent (F) used as needed, the immunoassay buffer (hereinafter also referred to as buffer) used as needed, and the following other components dissolved and / or dispersed in the liquid sample.

[0085] In the aforementioned specific reaction, the solid-phase reactant is preferably a solid-phase reactant contained in the solid-phase support reagent (A). 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 nonspecific reactions.

[0086] 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, and surfactants (excluding (alkyl)benzenesulfonates and (polyoxyalkylene)alkyl ether sulfates).

[0087] 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).

[0088] 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).

[0089] 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).

[0090] 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. Polymer compounds may be used individually or in combination of two or more types. From the viewpoint of measurement accuracy, the content of the polymer compound is preferably 0.001 to 5% by weight, and more preferably 0.01 to 5% by weight, based on the weight of the solid support reagent (A).

[0091] As surfactants other than (alkyl)benzene sulfonates and (polyoxyalkylene) alkyl ether sulfates, nonionic surfactants are preferred, 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.

[0092] When the immunoassay method of the present invention is performed using solid-phase carrier reagent (A), it is preferable that solid-phase carrier reagent (A) is an aqueous dispersion containing 0.001 to 10% by weight (preferably 0.01 to 1% by weight) of the solid-phase reactant.

[0093] When the reaction between substance (G) and solid-phase reactant is carried out using the solid-phase support reagent (A) described above, 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, such buffer solutions may contain stabilizers such as albumin, globulin, water-soluble gelatin, and polyethylene glycol, as well as surfactants and sugars, provided they do not inhibit the desired antigen-antibody reaction.

[0094] When the immunoassay method of the present invention is carried out using a solid-phase support reagent (A) and a buffer solution, it is preferable to carry out the reaction between the solid-phase reactant contained in the solid-phase support reagent (A) and substance (G) in the presence of (alkyl)benzene sulfonate and (polyoxyalkylene) alkyl ether sulfate. In this case, the (alkyl)benzene sulfonate and (polyoxyalkylene) alkyl ether sulfate 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 the buffer solution. To use the preferred amounts of (alkyl)benzene sulfonate and (polyoxyalkylene) alkyl ether sulfate as described above, the concentration in the reaction solution containing the solid support reagent (A) and buffer solution can be adjusted by diluting or adding the solution, and then the reaction can be carried out.

[0095] The immunoassay kit of the present invention is an immunoassay kit comprising a solid-phase support reagent (A) and an immunoassay buffer (W). 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.

[0096] The solid-phase support reagent (A) and / or immunoassay buffer (W), which constitute the immunoassay kit, contain (alkyl)benzenesulfonate and / or (polyoxyalkylene)alkyl ether sulfate. In other words, the solid-phase support reagent (A) may contain (alkyl)benzenesulfonate, (polyoxyalkylene)alkyl ether sulfate, or both; the immunoassay buffer (W) may contain (alkyl)benzenesulfonate, (polyoxyalkylene)alkyl ether sulfate, or both; and both the solid-phase support reagent (A) and the immunoassay buffer (W) may contain (alkyl)benzenesulfonate, (polyoxyalkylene)alkyl ether sulfate, or both.

[0097] The anionic surfactants contained in the solid-phase support reagent (A) and the immunoassay buffer (W) are the same as those described above for the immunoassay method, and the content of the anionic surfactants 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 the buffer described above for the immunoassay method.

[0098] In the immunoassay kit of the present invention, the solid-phase support reagent (A) described above in the immunoassay method can be used, and its composition and preferred ranges are the same.

[0099] As the immunoassay buffer (W) in the immunoassay kit of the present invention, the buffer described above in the immunoassay method can be used, and its composition and preferred ranges are the same.

[0100] The immunoassay kit of the present invention may further include a labeled reagent (F). Labeled reagent (F) is a reagent containing the labeling substance described above, and may also contain proteins, the surfactants and polymer compounds described above, etc. The proteins used are not particularly limited as long as they are commonly 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.

[0101] 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].

[0102] The present invention will be further described below with reference to examples, but the present invention is not limited thereto. Hereinafter, parts refer to parts by weight. The concentration (wt%) in this specification was calculated by converting the mass from the volume, assuming a solution density of 1.0 (g / mL).

[0103] <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.

[0104] <<Fabrication of magnetic particles>> 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% ammonia water (3.8 parts) and water (100 parts) was added dropwise over 1 hour. After addition, the mixture was stirred 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.

[0105] 39.0 parts of a 25% by weight aqueous ammonia solution, 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 alkyl ether ("Emarumin 200" manufactured by Sanyo Chemical Industries, Ltd.) were added to a reaction vessel 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. This procedure of removing the supernatant containing fine particles 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.

[0106] 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.).

[0107] 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 the silicon content obtained from the same measurement was defined as content T. The superparamagnetic metal oxide particle content was then calculated using the following formula. Content of superparamagnetic metal oxide particles (%)=(S) / (S+T)×100

[0108] <<Preparation of Solid-Phase Support Reagent (A-1)>> 40 mg of magnetic particles 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 polyethylene 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 polyethylene 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-AFP monoclonal antibody (manufactured by Agilent Technologies, Inc.), 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 the anti-AFP monoclonal antibody 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) with 1% by weight of bovine serum albumin (manufactured by Boval BioSolutions), and immersed at 25°C for 12 hours. This prepared the solid-phase support reagent (reagent containing anti-AFP antibody-conjugated magnetic particles) (A-1). The prepared solid-phase support reagent was stored refrigerated (2-10°C).

[0109] <<Preparation of Labeled Reagent (F-1)>> Using anti-AFP polyclonal antibody (Agilent Technologies, Inc.) and horseradish-derived POD (Toyobo Co., Ltd.), POD-labeled anti-AFP antibody was prepared using 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-AFP antibody in 0.02 M phosphate buffer (pH 7.0) containing 0.5 wt% bovine serum albumin (Boval BioSolutions), and the labeling reagent (F-1) was prepared and stored under refrigeration (2-10°C).

[0110] <<Preparation of immunoassay buffer (W-1)>> Bovine serum albumin (Boval BioSolutions) was mixed and dissolved in 0.02 M phosphate buffer (pH 7.0) to a concentration of 0.1 wt%, Neoperex G-25 (Kao Corporation) to a concentration of 2.24 wt%, and sodium chloride (Fujifilm Wako Pure Chemical Industries, Ltd.) to a concentration of 0.85 wt%. This prepared an immunoassay buffer (W-1) and was stored under refrigeration (2-10°C).

[0111] <<Preparation of Chemiluminescent Reagent Solution 1 (E-1)>> 0.7 g of luminol sodium salt [manufactured by Sigma-Aldrich Japan Co., Ltd.] and 0.1 g of 4-(cyanomethylthio)phenol were placed in a 1,000 mL volumetric flask. Furthermore, 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid / sodium hydroxide buffer (10 mM, pH 8.6) was added to a total 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).

[0112] <<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).

[0113] <Examples 2-3, Examples 5-7, Comparative Examples 1-7> Immunoassay buffers (W-2) to (W-3), (W-5) to (W-7), and (W'-1) to (W'-7) were prepared in the same manner as in "Preparation of immunoassay buffer (W-1)" of Example 1, with the types and concentrations of surfactants shown in Table 1. The immunoassay buffer (W-1) in Example 1 was changed to the prepared immunoassay buffers (W-2) to (W-3), (W-5) to (W-7), and (W'-1) to (W'-7), and these were 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-3), (S-5) to (S-7) of the present invention according to Examples 2 to 3 and Examples 5 to 7, and comparative immunoassay kits (H-1) to (H-7) according to Comparative Examples 1 to 7.

[0114] <Example 4> In the "Preparation of Solid-Phase Support Reagent (A-1)" of Example 1, after removing the phosphate buffer containing anti-AFP monoclonal antibody, 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 BioSolutions) and 3% by weight of polyethylene glycol 6000 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and the bottle was immersed at 25°C for 12 hours. The same procedure was used to prepare the solid-phase support reagent (A-2) in the same manner as in Example 1. The prepared solid-phase support reagent (A-2) was then replaced with the labeling reagent (F-1), immunoassay buffer (W-1), chemiluminescence reagent solution 1 (E-1), and chemiluminescence reagent solution 2 (E-2), and the immunoassay kit (S-4) according to Example 4 was prepared in the same manner as in Example 1.

[0115] [Table 1]

[0116] The surfactants used, as listed in Table 1, are as follows: • Neoperex G-25: 25% by weight aqueous solution of sodium dodecylbenzenesulfonate (manufactured by Kao Corporation) • Viewlight NA25S: 25% by weight aqueous solution of sodium polyoxyethylene lauryl ether sulfate (manufactured by Sanyo Chemical Industries, Ltd.) • Emal D-3-D: 26% by weight aqueous solution of sodium polyoxyethylene alkyl ether sulfate (manufactured by Kao Corporation) • Emulmin L-90S: Polyoxyethylene lauryl ether (manufactured by Sanyo Chemical Industries, Ltd.) • Naroacty CL100: Polyoxyalkylene alkyl ether with HLB=13 (manufactured by Sanyo Chemical Industries, Ltd.) • Mydol 12: 40% by weight aqueous solution of lauryl glucoside (manufactured by Kao Corporation) • Carribon L-400: Polycarboxylic acid-type surfactant (43% by weight aqueous solution of polycarboxylic acid salt) (manufactured by Sanyo Chemical Industries, Ltd.) • Perex NBL: 35% by weight aqueous solution of sodium alkylnaphthalene sulfonate (manufactured by Kao Corporation)

[0117] <<Immunoassay using an immunoassay kit>> Using the immunoassay kits (S-1) to (S-7) obtained in Examples 1 to 7 and the comparative immunoassay kits (H-1) to (H-7) obtained in Comparative Examples 1 to 7, the accuracy of the immunoassays was evaluated by performing immunoassays on three types of serum with AFP concentrations (AFP concentrations: 120 ng / mL, 450 ng / mL, and 1200 ng / mL) using the following method.

[0118] <<Evaluation of the accuracy of immunoassays using immunoassay kits>> 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. Next, 0.2 mL of immunoassay buffer (W) and 0.025 mL of serum with a known AFP concentration were injected into a test tube, and the mixture was allowed to react in the test tube at 37°C for 3 minutes to form a complex. 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.

[0119] 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], and the average luminescence amount was defined as the average luminescence amount (Z1).

[0120] <Calibration curve creation> Except for not using serum with a known AFP concentration and using the following seven standard samples instead of immunoassay buffer (W), the measurements were performed in the same manner, and a calibration curve showing the relationship between average luminescence and AFP concentration was created. Using the obtained calibration curve, the AFP concentration corresponding to the average luminescence (Z1) measured in the immunoassay for the accuracy evaluation above (hereinafter referred to as the measured concentration) was determined. Standard samples: Prepared by adding AFP antigen (G) to immunoassay buffer at concentrations of 0, 25, 50, 100, 500, 1000, and 2000 (ng / mL).

[0121] <Calculation of recall as an indicator of accuracy> For accuracy evaluation, the accuracy index was calculated as the ratio (percentage) of the measured concentration obtained from the calibration curve to the AFP concentration in serum with a known AFP concentration used in the immunoassay. This ratio, along with the concentration of the active chemical component of each surfactant in the solution used for the immunoassay, is listed as the recall rate in Table 2. A recall rate closer to 100% indicates superior accuracy and high measurement precision.

[0122] [Table 2]

[0123] Examples 1 to 7, which represent the immunoassay method of the present invention, show better reproducibility and higher measurement accuracy compared to the results in the comparative examples.

Claims

1. This is an immunoassay method that uses a solid-phase reactant having a substance (D) that reacts specifically with a substance (G) contained in a liquid sample, thereby performing a specific reaction between substance (G) and substance (D). This is an immunoassay method that carries out the above specific reaction in the presence of (alkyl)benzenesulfonate and / or (polyoxyalkylene)alkyl ether sulfate, An immunoassay method in which the total weight ratio of (alkyl)benzene sulfonate and (polyoxyalkylene) alkyl ether sulfate is 0.005 to 5% by weight, based on the total weight of the substances present in the reaction system undergoing a specific reaction.

2. 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 (alkyl)benzenesulfonate and / or (polyoxyalkylene) alkyl ether sulfate.

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