Chemiluminescent immunoassay method and kit for chemiluminescent immunoassay

Abstract

The present invention aims to provide a highly accurate chemiluminescence immunoassay method and a chemiluminescence immunoassay kit using magnetic particles. [Solution] A chemiluminescence immunoassay method performed using magnetic particles immobilized with a substance (D) that specifically reacts with a target substance (G) contained in a liquid sample, in the presence of a (co)polymer having a weight-average molecular weight of 10,000 to 1,000,000 and containing acrylic acid and / or acrylate as constituent monomers, wherein the proportion of the total weight of the (co)polymer 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.

Description

TECHNICAL FIELD

[0001] The present invention relates to a chemiluminescent immunoassay method and a kit for chemiluminescent immunoassay.

BACKGROUND ART

[0002] As methods for measuring substances such as proteins contained in biological samples such as body fluids, known are 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, as well as immunoassay methods for measuring the degree of particle aggregation caused by the binding of the target substance to the particle surface. It is known that biological samples contain substances (hereinafter referred to as interfering substances) that affect the reaction (also referred to as an immunoreaction) between the target substance and the substance capable of binding to the target substance present on the surface of particles or the like. Further, in the colloidal gold immunoassay method that uses colloidal gold particles as a carrier and utilizes the color tone change resulting from the antigen-antibody reaction occurring on the colloidal gold particles, a method of performing an immunoreaction in a solution containing 0.4 to 2.5 (W / W)% of a polyanion having an average molecular weight of about 20,000 or more or a salt thereof is known as a method for performing analysis while suppressing the influence of interfering substances (Patent Document 1). However, no method has been known for performing sufficiently highly accurate measurement while suppressing the influence of interfering substances in a chemiluminescent immunoassay method in which an antigen is reacted with an antibody supported on magnetic particles, a labeled antibody having a chemiluminescent substrate is further subjected to a secondary reaction with the antigen, and the luminescence intensity is measured.

PRIOR ART DOCUMENTS

PATENT DOCUMENTS

[0003]

PATENT DOCUMENT 1

SUMMARY OF THE INVENTION

PROBLEMS TO BE SOLVED BY THE INVENTION

[0004] The present invention aims to provide a highly accurate chemiluminescence immunoassay method and a chemiluminescence immunoassay kit that use magnetic particles. [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 a chemiluminescent immunoassay method performed using magnetic particles immobilized with a substance (D) that specifically reacts with a substance (G) contained in a liquid sample in the presence of a (co)polymer having a weight-average molecular weight of 10,000 to 1,000,000 and containing acrylic acid and / or an acrylate as constituent monomers, wherein the proportion of the total weight of the (co)polymer 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; and an immunoassay kit comprising a solid-phase support reagent (A), an immunoassay buffer (W), and a labeling reagent (F), wherein at least one selected from the group consisting of the solid-phase support reagent (A), the immunoassay buffer (W), and the labeling reagent (F) contains the (co)polymer. [Effects of the Invention]

[0006] The chemiluminescence immunoassay method and kit of the present invention enable a highly accurate chemiluminescence immunoassay method using magnetic particles. [Modes for carrying out the invention]

[0007] [Chemiluminescence Immunoassay Method] The present invention is a chemiluminescence immunoassay method performed using magnetic particles on which a substance (D) that specifically reacts with a substance (G) contained in a liquid sample is immobilized.

[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 antigen-antibody reactions. Chemiluminescent immunoassay is an immunoassay method that involves a secondary reaction between a labeled antibody containing a chemiluminescent substrate and an antigen, and then measuring the luminescence intensity.

[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, or 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 reactivity.

[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] The specific reaction in the measurement method of the present invention is, for example, a reaction between a substance (G) contained in a liquid sample, which is one of the following: an antigen, antibody, protein, or ligand, and magnetic particles on which a substance (D) that specifically reacts with the substance (G) is immobilized. The substance (D) immobilized on the magnetic particles 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.

[0012] [Substance (G)] Substances (G) that specifically react with the substance (D) possessed by magnetic particles 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 composites (G12) of the aforementioned substances contained in biological fluids, etc. and substances that specifically react with the aforementioned substances contained in biological fluids, etc.

[0013] 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 living body fluid reacts specifically with, in the same way as a living body fluid, and is capable of binding to the binding site of a substance that a living body fluid reacts specifically with. In other words, it is a substance that has a binding site that can react specifically with a substance that a living body fluid reacts specifically with, in the same way as a living body fluid, and further in other words, it is a substance whose specific reaction with a substance that a living body fluid reacts specifically with competes with that of a living body fluid, and is different from a living body fluid. 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.

[0014] Substances that react specifically with substances contained in biological fluids, etc. (G2) may also be labeled substances.

[0015] 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).

[0016] Pretreatments performed on biological fluids and the like include a treatment of mixing biological fluids and liquid samples derived from living organisms with substances such as <Treatment 1> to <Treatment 2> described below, a treatment of adjusting the pH of the biological fluids and the liquid samples derived from living organisms, a treatment of adjusting the concentration of the biological fluids and the liquid samples derived from living organisms, and the like. <Treatment 1> A treatment of mixing a substance (G2) or the like that specifically reacts with a substance contained in a biological fluid or the like with the biological fluid or the like, for the purpose of labeling, stabilizing, or enhancing the sensitivity of the substance contained in the biological fluid or the like. <Treatment 2> A treatment of mixing a substance (G1+) similar to a substance contained in a biological fluid or the like with the biological fluid or the like.

[0017] In the measurement method of the present invention, the specific reaction using magnetic particles in a liquid sample includes the reactions of <Method 1> to <Method 6> described below, and the like.

[0018] <Method 1> A chemiluminescent immunoassay method that performs a specific reaction between a substance (G1) contained in a biological fluid or the like and a substance (D).

[0019] <Method 2> A chemiluminescent immunoassay method that obtains 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 performs a specific reaction between the substance (G1+) contained in the liquid sample and a substance (D).

[0020] <Method 3> A chemiluminescent immunoassay method that simultaneously performs the specific reaction in <Method 1> described above and the specific reaction in <Method 2> described above (that is, performs 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).

[0021] <Method 4> A chemiluminescent immunoassay method that adds 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 performs a specific reaction between the substance (G2) contained in the liquid sample after the addition and a substance (D).

[0022] <Method 5> A chemiluminescent immunoassay method in which a substance that reacts specifically with a substance contained in a biological fluid is added to the biological fluid 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).

[0023] <Method 6> A chemiluminescent immunoassay method in which a substance (G2) that reacts specifically with a substance (D) and a substance (G2) that reacts specifically with

[0024] 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, glycogen 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, orchard 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-f Actor (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 of 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.

[0025] 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 (Magrapid is a registered trademark of Sanyo Chemical Industries, Ltd.) can be used.

[0026] 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).

[0027] In the above-described method 1, methods for immobilizing the substance (D) onto magnetic particles include physically adsorbing the substance (D) onto the magnetic particles, and reacting and bonding the substance (D) with functional groups derived from chemical substances bonded to the surface of the magnetic particles to immobilize it.

[0028] Examples of chemical substances bonded to the surface of magnetic particles having 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.).

[0029] 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, and 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. 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).

[0030] Examples of labeling substances (b) 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. Of these, from the viewpoint of sensitivity, etc., enzyme (b1) is preferred, more 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).

[0031] Labeling of substances contained in biological fluids, etc., with labeling substances (b), etc., can be carried out using known methods used in this field, for example, known labeling methods performed in EIA [e.g., 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.].

[0032] In Method 2 described above, the substance (D) and the magnetic particles on which the substance (D) is immobilized are the same as the substance (D) and magnetic particles exemplified in Method 1 described above.

[0033] Method 3 involves carrying out the specific reaction in Method 1 and the specific reaction in Method 2 simultaneously in the same reaction system.

[0034] 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.

[0035] 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.

[0036] The magnetic particles in Method 4 are the same as those exemplified in Method 1.

[0037] 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.

[0038] In Method 4, the method for immobilizing the substance (D) onto the magnetic particles can be the same as the method exemplified in Method 1.

[0039] 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.

[0040] 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).

[0041] The magnetic particles in Method 5 are the same as those exemplified in Method 1.

[0042] In Method 5, the method for immobilizing the substance (D) onto the magnetic particles can be the same as the method exemplified in Method 1. The magnetic particles in Method 6 are the same as those exemplified in Method 1.

[0043] 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 magnetic particles 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.

[0044] 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.

[0045] In the above-mentioned Method 1, the specific reaction may be carried out in a liquid sample containing a substance (G1) that is capable of an immunoreaction against a substance contained in biological fluids, etc., and is not immobilized on magnetic particles. By performing a specific reaction in a liquid sample containing a substance (G1) that is specifically reactive with a substance (D) that is not immobilized on magnetic particles, substance (G1) specifically bonds with substance (D) that is immobilized on magnetic particles, and then the substance (G1) that is bonded with substance (D) bonds with the substance (G1) that is specifically reactive with substance (G1) and not immobilized on magnetic particles. For a substance that is specifically reactive to substance (G1) and is not immobilized on magnetic particles, if substance (G1) is an antigen, then the corresponding antibody should be selected; 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 magnetic particles, 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>.

[0046] In the above-mentioned Methods 1 to 6, it is preferable from the viewpoint of reactivity to perform a specific reaction between substance (G) and substance (D), and then to react the substance (G) bonded to the substance (D) immobilized on magnetic particles with a substance that reacts specifically with it.

[0047] When performing a further specific reaction against a substance (G) bound to a magnetic particle, the substance that specifically reacts with the substance (G) bound to the magnetic particle is selected according to the type of substance that specifically reacts with the substance (G) bound to the magnetic particle. If the substance that specifically reacts with the substance (G) bound to the magnetic particle is an antigen, then the corresponding antibody should be selected. If the substance that specifically reacts with the substance (G) bound to the magnetic particle is an antibody, then the corresponding antigen should be selected. Furthermore, as the substance that specifically reacts with the substance (G) bonded to the magnetic particles, 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 magnetic particles, and labeling with the labeling substance (b) can be carried out in the same manner as in <Method 2>.

[0048] The chemiluminescence immunoassay method of the present invention includes known immunoassay methods such as the sandwich method, direct method, competitive method, and indirect method.

[0049] (1) The sandwich method includes, for example, the method described as <Method 1>, and the following methods, etc. A method for measuring the concentration of a substance (G1) contained in biological fluids, etc., by performing a specific reaction between a substance (G1) and a substance (D) immobilized on magnetic particles in the presence of a labeled substance (also called a labeled substance) capable of a specific reaction with the substance (G1) contained in biological fluids, etc., and measuring the intensity of the labeled substance based on the substance (G1) that has bonded to substance (D) and to 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 labeling substance, which binds to substance (D), and by measuring the intensity of the labeling substance based on the labeled substance (G1) that has bound to the labeling substance.

[0050] (2) As a direct method, there is a method described as <Method 6> in which magnetic particles immobilized with substances (D) contained in biological fluids, etc. as substance (D) and a labeled substance (G2) that reacts specifically with substances contained in biological fluids, etc., are carried out in a liquid sample, and the concentration of substances contained in a liquid sample is measured by measuring the intensity of the labeled substance.

[0051] (3) Examples of competing methods include the method described as <Method 3>, in which a specific reaction is carried out in the liquid sample between a substance (G1) contained in biological fluids, a similar substance (G1+) added to biological fluids or a liquid sample pretreated with biological fluids, and a substance (D) immobilized on magnetic particles, and the concentration of the substance (G1) contained in a biological liquid sample is measured by measuring the intensity of the labeled substance.

[0052] (4) As an indirect method, one example is a method in which a specific reaction is carried out in a liquid sample between magnetic particles immobilized with a substance contained in biological fluids, etc., as in Method 6, and a substance (G2) that specifically reacts with the substance contained in biological fluids, etc., and further a labeled substance is reacted with the substance (G2) bonded to the substance (D) immobilized on the magnetic particles, and the concentration of the substance contained in the biological liquid sample is measured by measuring the strength of the labeled substance.

[0053] The chemiluminescence immunoassay method of the present invention also includes immunoassay methods other than the sandwich method, direct method, competitive method, and indirect method described above.

[0054] The present invention provides a chemiluminescence immunoassay method that uses magnetic particles immobilized with a substance (D) that reacts specifically with a target substance (G) contained in a liquid sample, in the presence of a (co)polymer having a weight-average molecular weight of 10,000 to 1,000,000 and containing acrylic acid and / or acrylate as constituent monomers. In other words, this is a chemiluminescent immunoassay method that involves a specific reaction between a substance (G) and a substance (D) immobilized on magnetic particles in a liquid sample containing a (co)polymer that includes acrylic acid and / or an acrylate as constituent monomers. In this specification, (co)polymer means copolymer and / or polymer. Polymers containing acrylic acid and / or acrylates as constituent monomers include polyacrylic acid, polyacrylates, and copolymers of acrylic acid and / or acrylates. In the following, (co)polymers containing acrylic acid and / or acrylates as constituent monomers may be abbreviated as (co)polymers.

[0055] In Method 1 described above, it is preferable from the viewpoint of reactivity to carry out the specific reaction between the substance (G1) contained in biological fluids, etc. and the substance (D) immobilized on magnetic particles in a liquid sample containing a (co)polymer. It is also preferable to carry out the reaction with the labeling substance in a liquid sample containing a (co)polymer.

[0056] In Method 2 described above, it is preferable from the viewpoint of reactivity to carry out the specific reaction between a substance similar to a substance contained in biological fluids, etc. (G1+) and a substance immobilized on magnetic particles (D) in a liquid sample containing a (co)polymer.

[0057] In Method 3 described above, it is preferable from the viewpoint of reactivity to carry out both the specific reaction between a substance (G1) contained in biological fluids, etc. and a substance (D) immobilized on magnetic particles, and the specific reaction between a substance (G1+) similar to a substance contained in biological fluids, etc. and a substance (D) immobilized on magnetic particles, simultaneously in a liquid sample containing a (co)polymer.

[0058] In Method 4 described above, it is preferable from the viewpoint of reactivity 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 magnetic particles, in a liquid sample containing a (co)polymer.

[0059] In Method 5 described above, it is preferable from the viewpoint of reactivity to carry out the specific reaction between the composite (G12) and the substance (D) immobilized on the magnetic particles in a liquid sample containing the (co)polymer.

[0060] Furthermore, in Method 5 described above, it is also preferable from the viewpoint of reactivity to carry out the specific reaction in a liquid sample containing a (co)polymer 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.

[0061] In Method 6 described above, from the viewpoint of reactivity, it is preferable to carry out the specific reaction between a substance (D) contained in biological fluids, etc., immobilized on magnetic particles and / or a similar substance to a substance contained in biological fluids, etc., and a substance (G2) that specifically reacts with the substance contained in biological fluids, etc., and / or a similar substance to a substance contained in biological fluids, etc., in a liquid sample containing a (co)polymer.

[0062] By carrying out a specific reaction in a liquid sample containing a (co)polymer whose constituent monomers are acrylic acid and / or acrylate salts, the charge in the reaction system changes, making it less likely for substances causing nonspecific reactions to react with substance (D) immobilized on magnetic particles. This is thought to enable highly accurate immunoassays.

[0063] Furthermore, even if the (co)polymer is a copolymer that further contains constituent monomers other than acrylic acid and / or acrylate salts, the anionic properties derived from the structural units of acrylic acid and / or acrylate salts are imparted to the reaction system. This adjusts the charge state and interface state of the reaction system, making it less likely for components that cause nonspecific reactions (e.g., proteins contained in bodily fluids, etc.) to be nonspecifically adsorbed or bound to the magnetic particle surface or to the substance (D) immobilized on the magnetic particles. As a result, measurement errors such as false positives, falsely high values, or falsely low values ​​are reduced, and measurement accuracy is improved.

[0064] In this specification, "non-specific reaction" refers to a reaction in which binding, reaction, or signal generation occurs in any step of the measurement system without the original specific binding 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. 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 or rheumatoid factors) may nonspecifically adsorb or bind to substances (D) immobilized on magnetic particles, and / or substances that are specifically reactive to substance (G1) but are not immobilized on magnetic particles, 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.

[0065] In the measurement method of the present invention, when a substance that specifically reacts with a substance (G) bonded to a substance (D) immobilized on magnetic particles is further reacted, the specific reaction with the substance (G) bonded to the solid-phase reagent may be carried out in a liquid sample containing a (co)polymer.

[0066] [(Co)polymers containing acrylic acid and / or acrylates as constituent monomers] Examples of acrylates that constitute a (co)polymer containing acrylic acid and / or acrylate as constituent monomers in the chemiluminescence immunoassay method of the present invention include ammonium salts of acrylic acid and alkali metal salts of acrylic acid, among which alkali metal salts of acrylic acid are preferred from the viewpoint of reactivity, and sodium acrylate is even more preferred.

[0067] In the present invention, "(co)polymer containing acrylic acid and / or acrylate as constituent monomers" means a polymer containing acrylic acid and / or acrylate as constituent monomers, which may further contain other ethylenically unsaturated monomers other than acrylic acid and / or acrylate as constituent monomers (it may also be a copolymer).

[0068] Other constituent monomers besides acrylic acid and / or acrylate salts include, for example, (meth)acrylic acid and / or its salts, (meth)acrylamide, N-substituted (meth)acrylamide, hydroxyalkyl (meth)acrylate esters, vinyl carboxylic acids and / or their salts (maleic acid, itaconic acid, etc.), ethylenically unsaturated monomers having a sulfonic acid group and / or their salts (2-acrylamido-2-methylpropanesulfonic acid and / or its salt, styrenesulfonic acid and / or its salt, etc.). These may be used individually or in combination of two or more. Furthermore, the (co)polymer may optionally contain a small amount of a crosslinkable monomer (for example, N,N'-methylenebis(meth)acrylamide).

[0069] In the above (co)polymer, the content of structural units derived from acrylic acid and / or acrylate salts is preferably 10 mol% or more, more preferably 50 mol% or more, and even more preferably 80 mol% or more, relative to the total structural units, from the viewpoint of non-specific reaction suppression effect and reactivity. Note that the content ratio of the structural unit may be calculated based on the charging ratio of the raw material monomers, or may be determined by analysis such as nuclear magnetic resonance (NMR).

[0070] The weight average molecular weight of the (co)polymer containing acrylic acid and / or an acrylate as constituent monomers, which is used in the chemiluminescent immunoassay method of the present invention, is from 10,000 to 1,000,000, and preferably from the viewpoint of reactivity, is from 25,000 to 1,000,000.

[0071] The weight average molecular weight of the (co)polymer containing polyacrylic acid and / or a polyacrylate as constituent monomers is measured by gel permeation chromatography (GPC) under the following conditions. <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 {5 g / liter (relative to water / methanol)} Flow rate 1.0 (ml / min) Column; TSKgel G3000PWXL and TSKGel G50000PWXL (each having 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)}

[0072] The (co)polymer used in the chemiluminescent immunoassay method of the present invention can be obtained by an aqueous solution polymerization method, a reversed-phase suspension polymerization method, a bulk polymerization method, or the like. For example, it can be obtained by known methods described in JP-A-2017-25136, JP-A-56-26909, JP-A-2014-198793, and the like. Among the (co)polymers, polyacrylates and acrylic acid / acrylate copolymers may be produced by neutralizing polyacrylic acid or by using acrylates. The weight-average molecular weight of the (co)polymer can be adjusted by selecting the concentration of the polymerization initiator used in polymerization and the concentration of acrylic acid (salt) in the polymerization reaction.

[0073] The (co)polymer used in the measurement method of the present invention may be one that is available on the market, such as polyacrylic acid (Aquaric L:HL-415), sodium polyacrylate (Aquaric L:DL-453, DL-522) (both from Nippon Shokubai Co., Ltd.), sodium polyacrylate (Aron A-7100), polyacrylic acid (Aron A-10H), ammonium polyacrylate (Aron A-30) (both from Toagosei Co., Ltd.).

[0074] In the present invention, the specific reaction carried out in the presence of a (co)polymer containing acrylic acid and / or an acrylate as constituent monomers is preferably the following reaction. • Specific reaction between a substance (G1) contained in a biological liquid sample and a substance (D) immobilized on magnetic particles. • A specific reaction between a substance (G1+) similar to a substance contained in a biological liquid sample and a substance (D) immobilized on magnetic particles. • Specific reactions between a substance (G2) that specifically reacts with substances contained in a biological liquid sample and a substance (D) immobilized on magnetic particles. • 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 magnetic particles. • A specific reaction between a substance contained in a biologically derived liquid sample immobilized on magnetic particles and a substance (G2) that specifically reacts with the substance contained in the biologically derived liquid sample.

[0075] Of the specific reactions carried out in a liquid sample containing a (co)polymer, the preferred specific reaction described above can be carried out by mixing a liquid sample containing substance (G), magnetic particles immobilized with substance (D), and the (co)polymer.

[0076] From the viewpoint of suppressing non-specific reactions, the proportion of the total weight of (co)polymers containing acrylic acid and / or acrylates as constituent monomers in a system undergoing a specific reaction is 0.005 to 5% by weight, based on the total weight of the substances present in the reaction system undergoing 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 percentage of the total weight of the (co)polymer is preferably 0.01 to 1% by weight. The total weight of substances present in the reaction system that performs the specific reaction is the sum of the weight of the substance in the liquid sample (G), magnetic particles, (co)polymer, the solvent of the liquid sample if applicable, the labeling reagent (F) used if applicable, the immunoassay buffer (hereinafter also referred to as buffer) used if applicable, and the other components listed below that are dissolved and / or dispersed in the liquid sample.

[0077] [Magnetic particles] In the measurement method of the present invention, it is preferable from the viewpoint of reactivity to use magnetic particles contained in the solid-phase support reagent (A) as the magnetic particles, and the content of magnetic particles 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.

[0078] [Other ingredients] In addition to magnetic particles, the solid-phase support reagent (A) may also contain other components such as gelatin, proteins other than gelatin, serum (mouse serum, etc.), sugars, surfactants, inorganic salts, ethylenediaminetetraacetic acid, water, polymer compounds other than (co)polymers containing acrylic acid and / or acrylates as constituent monomers, and surfactants.

[0079] 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).

[0080] 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.

[0081] 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).

[0082] Other polymeric compounds (hereinafter abbreviated as polymeric compounds) that contain acrylic acid and / or acrylate as constituent monomers are not particularly limited as long as they are used in the field of immunoassays, and 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 reactivity. 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 (A).

[0083] As for the surfactant, nonionic surfactants are preferred from the viewpoint of reactivity, 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.

[0084] 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 magnetic particles in an amount of 0.001 to 10% by weight (preferably 0.01 to 1% by weight from the viewpoint of reactivity).

[0085] When the immunoassay method of the present invention is performed 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 its 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.

[0086] In the chemiluminescence immunoassay method of the present invention, it is preferable from the viewpoint of reactivity to use a labeling reagent (F) containing a labeling substance, and the labeling reagent (F) may contain proteins, and the above-mentioned surfactants and polymer compounds, 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.

[0087] When performing the chemiluminescence immunoassay method of the present invention using a solid-phase support reagent (A), a buffer solution, and a labeling reagent (F), it is preferable to carry out the reaction between the magnetic particles contained in the solid-phase support reagent (A) and substance (G), and the reaction between the magnetic particles contained in the solid-phase support reagent (A), substance (G), and the labeling substance, in the presence of a (co)polymer containing polyacrylic acid and / or polyacrylate as constituent monomers. In this case, the (co)polymer containing acrylic acid and / or acrylate as constituent monomers may be included in the solid support reagent (A), the buffer solution, the labeling reagent (F), or added separately from the solid support reagent (A), the buffer solution, and the labeling reagent (F). To obtain the desired amount of (co)polymer, the reaction can be carried out by adjusting the concentration of the reaction solution containing the solid support reagent (A), buffer solution, and labeling reagent (F), etc., by diluting or adding the reagents.

[0088] [Chemiluminescence Immunoassay Kit] The chemiluminescent immunoassay kit of the present invention is a chemiluminescent immunoassay kit comprising a solid-phase support reagent (A), an immunoassay buffer (W), and a labeling reagent (F). The solid-phase support reagent (A) contains magnetic particles, and a substance (D) that specifically binds to the target substance (G) is immobilized on the magnetic particles.

[0089] In a chemiluminescent immunoassay kit, at least one component selected from the group consisting of a solid-phase support reagent (A), an immunoassay buffer (W), and a labeling reagent (F) contains a (co)polymer. In other words, the solid support reagent (A) may contain a (co)polymer, the immunoassay buffer (W) may contain a (co)polymer, the labeling reagent (F) may contain a (co)polymer, two selected from the group consisting of the solid support reagent (A), the immunoassay buffer (W), and the labeling reagent (F) may contain a (co)polymer, or all of the solid support reagent (A), the immunoassay buffer (W), and the labeling reagent (F) may contain a (co)polymer.

[0090] The (co)polymer contained in the solid support reagent (A), the immunoassay buffer (W), and the labeling reagent (F) is the same as the (co)polymer containing acrylic acid and / or acrylate as constituent monomers as described above in the immunoassay method.

[0091] If the solid-phase support reagent (A) contains a (co)polymer, the weight percentage of the (co)polymer contained in the solid-phase support reagent (A) is preferably 0.01 to 2.5% by weight, based on the weight of the solid-phase support reagent (A), from the viewpoint of measurement accuracy.

[0092] If the immunoassay buffer (W) contains a (co)polymer, the weight percentage of the (co)polymer contained in the immunoassay buffer (W) is preferably 0.01 to 2.5% by weight, based on the weight of the immunoassay buffer (W), from the viewpoint of measurement accuracy.

[0093] In the chemiluminescent 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.

[0094] In the chemiluminescent immunoassay kit of the present invention, the immunoassay buffer (W) can be the buffer described above in the immunoassay method, and its composition and preferred ranges are the same.

[0095] In the chemiluminescent immunoassay kit of the present invention, the labeling reagent (F) can be the labeling substance (b) described above in the immunoassay method, and its composition and preferred ranges are the same.

[0096] The chemiluminescent 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]. [Examples]

[0097] 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.

[0098] <Manufacturing Example 1> A chemiluminescent immunoassay kit (S-1) 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.

[0099] <<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, 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 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.

[0100] 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 240" 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.

[0101] 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.).

[0102] <<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. After magnetizing the magnetic particles with a neodymium magnet, the liquid portion of the reaction mixture 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. After magnetizing the magnetic particles with a neodymium magnet, the liquid portion was removed by aspirator to wash the magnetic particles. This washing operation 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. After magnetizing the magnetic particles with a neodymium magnet, the liquid portion was removed by aspirator to wash the magnetic particles. This washing operation 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 Proliant Biologicals), 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).

[0103] <<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 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-AFP antibody in 0.02 M phosphate buffer (pH 7.0) containing 0.5 wt% bovine serum albumin (Proliant Biologicals), and the labeling reagent (F-1) was prepared and stored under refrigeration (2-10°C).

[0104] <<Preparation of Immunotherapy Buffer (W-1)>> To a 0.02 M phosphate buffer (pH 7.0), bovine serum albumin (Proliant Biologicals) was added to a concentration of 0.1% by weight. Then, a 45% aqueous solution of polyacrylic acid with a weight-average molecular weight of 10,000 (product name: Aquaric HL-415) was added to achieve a concentration of 0.01% by weight of the (co)polymer polyacrylic acid with a weight-average molecular weight of 10,000. Finally, sodium chloride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to a concentration of 0.85% by weight. The mixture was then mixed and dissolved to prepare immunoassay buffer (W-1), which was stored under refrigeration (2-10°C).

[0105] <<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. 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).

[0106] <<Preparation of Chemiluminescent Reagent Solution 2 (E-2)>> 6.6 g of hydrogen peroxide [manufactured by Fujifilm 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 solution volume 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).

[0107] <Manufacturing Examples 2-7, 9; Comparative Manufacturing Examples 1-2> In the "Preparation of Immunotherapy Buffer (W-1)" of Production Example 1, the polyacrylic acid used was changed to the polyacrylic acid with the weight-average molecular weight listed in Table 1, and its concentration was also changed to the concentration listed in Table 1. This resulted in the preparation of immunotherapy buffers (W-2) to (W-7), (W-9), and (W-11) to (W-12), where the molecular weight and concentration of the polyacrylic acid were as shown in Table 1. The solid support reagent (A-1), labeling reagent (F-1), chemiluminescent reagent solution 1 (E-1), and chemiluminescent reagent solution 2 (E-2), prepared in the same manner as in Production Example 1, were combined with immunoassay buffers (W-2) to (W-7), (W-9), and (W-11) to (W-12) in the same manner as in Production Example 1 to obtain immunoassay kits (S-2) to (S-7), and (S-9) for Production Examples 2 to 7 and Production Example 9, and chemiluminescent immunoassay kits (H-1) to (H-2) for Comparative Production Examples 1 to 2.

[0108] <Manufacturing Example 10> A chemiluminescent immunoassay kit (S-10) was obtained by combining a solid-phase support reagent (A-1), a labeling reagent (F-1), a chemiluminescent reagent solution 1 (E-1), and a chemiluminescent reagent solution 2 (E-2), prepared in the same manner as in Manufacturing Example 1, with an immunoassay buffer (W-10) prepared by the following method. The components of the chemiluminescent immunoassay kit (S-10) are shown in Table 1.

[0109] <<Preparation of acrylic acid-maleic acid copolymer used in immunoassay buffer (W-10)>> In a 0.50 L stainless steel separable flask equipped with a reflux condenser and stirrer, 36.57 g of deionized water and 11.78 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 48% NaOH (54.75 g) was gradually added dropwise to neutralize it, yielding an aqueous solution containing an acrylic acid (salt)-maleic acid (salt) copolymer with a degree of neutralization of 90% and a solid content of 55.0%.

[0110] <<Preparation of Immunotherapy Buffer (W-10)>> To 0.02 M phosphate buffer (pH 7.0), bovine serum albumin (Proliant Biologicals) was added to a concentration of 0.1% by weight. Then, a 55.0% aqueous solution of acrylic acid(salt)-maleic acid(salt) copolymer with a weight-average molecular weight of 11,900 was added to a concentration of 0.01% by weight. Finally, sodium chloride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to a concentration of 0.85% by weight. The mixture was then mixed and dissolved to prepare immunoassay buffer (W-10), which was stored under refrigeration (2-10°C).

[0111] <Manufacturing Example 8> A chemiluminescent immunoassay kit (S-8) was obtained by combining a solid-phase support reagent (A-1), chemiluminescent reagent solution 1 (E-1), and chemiluminescent reagent solution 2 (E-2), prepared in the same manner as in Manufacturing Example 1, with a labeling reagent (F-2) and immunoassay buffer (W-8) prepared by the following method. The components of the chemiluminescent immunoassay kit (S-8) are shown in Table 2.

[0112] <<Preparation of Labeling Reagent (F-2)>> A solution for labeling reagents was prepared by mixing 0.02 M phosphate buffer (pH 7.0) with an aqueous solution containing 26% by weight of polyacrylic acid (product name: Aron A-10H), which is a copolymer of bovine serum albumin (Proliant Biologicals) and has a weight-average molecular weight of 200,000. The same POD-labeled anti-AFP antibody used in labeling reagent (F-1) was diluted in the labeling reagent solution to a concentration of 1.0 μg / mL to prepare labeling reagent (F-2) containing polyacrylic acid, which was then stored in a refrigerator (2-10°C).

[0113] <<Preparation of Immunotherapy Buffer (W-8)>> Bovine serum albumin (Proliant Biologicals) was mixed and dissolved in 0.02 M phosphate buffer (pH 7.0) to a concentration of 0.1% by weight, and sodium chloride (Fujifilm Wako Pure Chemical Industries, Ltd.) was mixed and dissolved to a concentration of 0.85% by weight, to prepare immunoassay buffer (W-8), which was then stored under refrigeration (2-10°C).

[0114] <Comparative Manufacturing Example 3> A chemiluminescent immunoassay kit (H-3) according to Comparative Manufacturing Example 3 was obtained by combining a solid-phase support reagent (A-1), chemiluminescent reagent solution 1 (E-1), labeling reagent (F-1), and chemiluminescent reagent solution 2 (E-2), prepared in the same manner as in Manufacturing Example 1, with an immunoassay buffer (W-8), prepared in the same manner as in Manufacturing Example 8. The composition of the chemiluminescent immunoassay kit (H-3) is shown in Table 2.

[0115] [Table 1]

[0116] [Table 2]

[0117] The following polyacrylic acids were used, as listed in Tables 1 and 2. • Polyacrylic acid with a weight-average molecular weight of 10,000: Aqualic HL-415 (45% active ingredient), manufactured by Nippon Shokubai Co., Ltd. • Polyacrylic acid with a weight-average molecular weight of 25,000: Manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., reagent (100% active ingredient) • Polyacrylic acid with a weight-average molecular weight of 1,000,000: Manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., reagent (100% active ingredient) • Polyacrylic acid with a weight-average molecular weight of 200,000: Toagosei Co., Ltd., Aron A-10H (active ingredient 26%) • Polyacrylic acid with a weight-average molecular weight of 5,000: Manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., reagent (100% active ingredient) • Polyacrylic acid with a weight-average molecular weight of 1,600,000: Manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., reagent (100% active ingredient) Furthermore, the following (co)polymers containing acrylic acid and / or acrylates as constituent monomers, as listed in Tables 1 and 2, were used. • Acrylic acid-maleic acid copolymer with a weight-average molecular weight of 11,900 (55% active ingredient)

[0118] <Examples 1-9, Comparative Examples 1-4: Accuracy evaluation of chemiluminescence immunoassay using chemiluminescence immunoassay kits> Using the immunoassay kits (S-1) to (S-10) obtained in manufacturing examples 1 to 10, and the immunoassay kits (H-1) to (H-3) obtained in comparative manufacturing examples 1 to 3, chemiluminescence immunoassays were performed on three different serum samples with α-fetoprotein (AFP) concentrations (AFP concentrations: 80 ng / mL, 600 ng / mL, and 1500 ng / mL) using the following methods.

[0119] <<Chemiluminescent immunoassay using an immunoassay kit>> 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.

[0120] 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 43 to 45 seconds after the addition of the chemiluminescent reagents was measured using a luminometer [Lumat LB9507, manufactured by Berthold Japan], and this average luminescence amount was defined as the average luminescence amount (Z1). Using the calibration curve below, the AFP concentration corresponding to the average luminescence amount (Z1) measured in the immunoassay for the accuracy evaluation above (hereinafter referred to as the measured concentration) was determined.

[0121] <Calibration curve creation> Except for not using "serum with known AFP concentration" and replacing the immunoassay buffer (W) with the following seven standard samples, the measurements were performed in the same manner, and a calibration curve showing the relationship between average luminescence and AFP concentration was created. Standard samples: AFP antigen (G) was added to immunoassay buffer at concentrations of 0, 25, 50, 100, 500, 1000, and 2000 (ng / mL) to prepare samples, which were then used as standard samples.

[0122] <Calculation of recall as an indicator of accuracy> The ratio of the measured concentration to the serum AFP concentration (percentage) was calculated and is shown in Table 3 as the recall rate. A recall rate closer to 100% indicates better accuracy and higher measurement precision.

[0123] [Table 3]

[0124] The results of Examples 1-8, which use the chemiluminescence immunoassay method of the present invention, showed a near 100% recall rate at all AFP concentration levels. On the other hand, the results in the comparative examples showed poor accuracy at at least one of the three concentrations, and none of the three levels showed good accuracy. From these results, it can be seen that the chemiluminescence immunoassay method of the present invention has a wide measurement range and high measurement accuracy.

Claims

1. A chemiluminescence immunoassay method performed using magnetic particles immobilized with a substance (D) that specifically reacts with a target substance (G) contained in a liquid sample, in the presence of a (co)polymer having a weight-average molecular weight of 10,000 to 1,000,000 and containing acrylic acid and / or an acrylate as constituent monomers, wherein the proportion of the total weight of the (co)polymer 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.

2. An immunoassay kit comprising a solid-phase support reagent (A), an immunoassay buffer (W), and a labeling reagent (F), wherein at least one selected from the group consisting of the solid-phase support reagent (A), the immunoassay buffer (W), and the labeling reagent (F) contains a (co)polymer with a weight-average molecular weight of 10,000 to 1,000,000, which includes acrylic acid and / or an acrylate as constituent monomers.

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