Immunoassay reagent, immunoassay kit, and immunoassay method

Abstract

To provide an immunoassay reagent excellent in storage stability, an immunoassay kit containing the immunoassay reagent, and an immunoassay method using the kit.SOLUTION: An immunoassay reagent (X) contains a substance (FC) and at least one compound (D) selected from the group consisting of digitonin, cucurbitacin B, ouabain and saponin. The substance (FC) is a substance in which a substance (F) is labeled by a labeling substance (C) and the substance (F) is at least one substance selected from the group consisting of a substance (F1) to be measured, a substance (F2) analogous to the substance to be measured, and a substance (F3) uniquely binding the substance to be measured. In the immunoassay reagent (X), the content of the (D) in the (X) is 0.51 to 1.50 wt.% based on the weight of the (X).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an immunoassay reagent, an immunoassay kit, and an immunoassay method using the reagent. [Background technology]

[0002] In immunoassay reagents, a substance labeled with a labeling substance is a major component (Patent Document 1). In immunoassay reagents, it is desired that the substance labeled with a labeling substance maintains a certain level of activity. If the activity of the substance decreases over time or if the activity of the substance changes depending on the storage conditions, accurate measurement results cannot be expected, which is a major problem for immunoassay reagents. In the development of automated immunoassay devices aimed at simultaneous rapid testing of multiple samples, further improvement in storage stability is desired. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2010-127827 A Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present invention is to provide an immunoassay reagent having excellent storage stability, an immunoassay kit containing the immunoassay reagent, and an immunoassay method using the kit. [Means for solving the problem]

[0005] The present inventors conducted extensive research to achieve the above object and arrived at the present invention. That is, the present invention relates to a substance (FC) and at least one compound (D) selected from the group consisting of digitonin, cucurbitacin B, ouabain and saponin, wherein the substance (FC) is a substance obtained by labeling a substance (F) with a labeling substance (C), and the substance (F) is at least one substance selected from the group consisting of an analyte (F1), an analogue of the analyte (F2) and a substance (F3) that specifically binds to the analyte, The labeling substance (C) is peroxidase, An immunoassay reagent (X), in which the content of (D) in (X) is 0.51 to 1.50% by weight based on the weight of (X); an immunoassay kit comprising a labeled reagent (A) and a solid phase carrier reagent (E), wherein the labeled reagent (A) is the immunoassay reagent (X), and the solid phase carrier reagent (E) contains a solid phase carrier (BF) having a substance (F) immobilized on the surface of the solid phase carrier (B); and an immunoassay method using the immunoassay kit. Effect of the Invention

[0006] By using the immunoassay reagent and immunoassay kit of the present invention, the reagent can be stored for a long period of time, and loss of the reagent due to expiration of the expiration date can be reduced. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] <Immunoassay reagents> The immunoassay reagent (X) of the present invention is an immunoassay reagent (X) containing a substance (FC) and at least one compound (D) selected from the group consisting of digitonin, cucurbitacin B, ouabain and saponin, wherein the substance (FC) is a substance obtained by labeling a substance (F) with a labeling substance (C), and the substance (F) is at least one substance selected from the group consisting of a substance to be measured (F1), an analogue of the substance to be measured (F2) and a substance that specifically binds to the substance to be measured (F3), and the content of (D) in (X) is 0.51 to 1.50% by weight based on the weight of (X).

[0008] As described above, the substance (FC) in the present invention is a substance obtained by labeling the substance (F) with a labeling substance (C). The substance (F) includes at least one substance selected from the group consisting of the substance to be measured (F1), a substance similar to the substance to be measured (F2), and a substance that specifically binds to the substance to be measured (F3). The measurement target substance (F1) in the present invention includes substances contained in samples of biological origin [biological fluids (serum, blood, lymph, ascites, urine, etc.), various cells, culture fluids, etc.)]. Specifically, proteins (albumin, hemoglobin, myoglobin, transferrin, protein A, C-reactive protein (CRP), lipid proteins, enzymes, immunoglobulins, immunoglobulin fragments, blood coagulation-related factors, antibodies, antigens, hormones, etc.), Nucleic acids [deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), etc.] Drugs [antiepileptic drugs, antibiotics, theophylline, etc.] Viruses [Hepatitis C virus, Hepatitis B virus, Hepadnavirus, Adenovirus, Rhabdovirus, Flavivirus, Retrovirus, Herpesvirus, Osomyxovirus, etc.] Bacteria [O-157, Helicobacter pylori, Salmonella, etc.] Cells [adipocytes, ES cells, hepatocytes, stem cells, endothelial cells, epithelial cells, muscle cells, endocrine cells, exocrine cells, nerve cells, tumor cells, iPS cells, etc.]. Examples of the enzymes include alkaline phosphatase, amylase, acid phosphatase, γ-glutamyltransferase (γ-GTP), lipase, creatine kinase (CK), lactate dehydrogenase (LDH), glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), renin, protein kinase (PK), and tyrosine kinase. Examples of the lipid protein include high density lipoprotein (HDL), low density lipoprotein (LDL), and very low density lipoprotein. Examples of the immunoglobulins include IgG, IgM, IgA, IgD, and IgE. The immunoglobulin fragments include the Fc portion, the Fab portion, and the F(ab) 2 Department etc. Examples of the blood coagulation-related factors include fibrinogen, fibrin degradation products (FDP), prothrombin, thrombin, and the like. Examples of the antibody include anti-streptolysin O antibody, anti-human hepatitis B virus surface antigen antibody (HBs antigen), anti-human hepatitis C virus antibody, and anti-rheumatoid factor. The antigens include carcinoembryonic antigen (CEA) and the like. Examples of the hormones include thyroid stimulating hormone (TSH), thyroid hormones (FT3, FT4, T3, T4), parathyroid hormone (PTH), human chorionic gonadotropin (hCG), estradiol (E2), and the like. In addition, the above-mentioned antigens and antibodies also include substances known as cancer markers [PIVKA-II antigen, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), CA19-9, prostate-specific antigen (PSA), etc.] and heart disease markers [troponin T (TnT), N-terminal fragment of human brain natriuretic peptide precursor (NT-proBNP), etc.]. Among the above-listed examples, from the viewpoint of commercial needs, at least one selected from the group consisting of antibodies, hormones, cancer markers and heart disease markers is preferred.

[0009] In the present invention, the analogue (F2) of the measured substance may be any substance capable of binding to the "binding site for the measured substance (F1)" of the "substance (F3) which specifically binds to the measured substance", in other words, any substance having a binding site for the "substance (F3) which specifically binds to the measured substance" of the "measured substance (F1)", in other words, any substance capable of competing with the reaction between the "measured substance (F1)" and the "substance (F3) which specifically binds to the measured substance" when coexisting therewith.

[0010] In the present invention, the "substance (F3) that specifically binds to the measured substance" includes, for example, a substance that binds to the "measured substance or a substance similar thereto (F2)" through interactions such as an "antigen"-"antibody" reaction, a "sugar chain"-"protein" reaction, a "sugar chain"-"lectin" reaction, an "enzyme"-"inhibitor" reaction, a "protein"-"peptide chain" reaction, a "chromosome or nucleotide chain"-"nucleotide chain" reaction, and a "nucleotide chain"-"protein" reaction. In each of the above combinations, when one of them is a "substance to be measured (F1) or an analogue thereof (F2)", the other is a "substance that specifically binds to the substance to be measured (F3)". For example, when the "measurement target substance (F1) or its analogue (F2)" is an "antigen," the "substance (F3) that specifically binds to the measurement target substance" is an "antibody," and when the "measurement target substance (F1) or its analogue (F2)" is an "antibody," the "substance (F3) that specifically binds to the measurement target substance" is an "antigen" (the same applies to the other combinations mentioned below).

[0011] Examples of (F3) include the substances exemplified as the above-mentioned measurement target substance (F1) and antibodies against these substances. The antibodies used in the present invention include Fab and F(ab') fragments generated by proteolytic enzymes such as papain and pepsin, or by chemical degradation. 2 Decomposition products such as fragments are also included. When the substance to be measured (F1) is a PIVKA-II antigen, (F3) is preferably an anti-prothrombin monoclonal antibody (such as an anti-PIVKA-II monoclonal antibody).

[0012] As the substance (F3) that specifically binds to the above-mentioned measured substance, a substance that binds to the measured substance (F1) or its analogue (F2) through an "antigen"-"antibody" reaction or a "glycan-protein" reaction is preferred because it is easy to obtain. Specifically, antibodies against the analyte (F1) or its analogue (F2), antigens to which the analyte (F1) or its analogue (F2) bind, and proteins that bind to the analyte (F1) or its analogue (F2) are preferred, and antibodies against the analyte (F1) or its analogue (F2) and proteins that bind to the analyte (F1) or its analogue (F2) are even more preferred.

[0013] From the viewpoint of storage stability, the substance (F) is preferably an anti-prothrombin monoclonal antibody.

[0014] The labeling substance (C) used in the present invention is used to label the substance (F), and examples of such labeling substances include enzymes used in enzyme immunoassay (EIA), such as alkaline phosphatase, β-galactosidase, peroxidase (hereinafter sometimes abbreviated as POD), microperoxidase, glucose oxidase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, luciferase, tyrosinase, and acid phosphatase; Radioisotopes such as 99mTc, 131I, 125I, 14C, 3H, 32P, etc., used for example in radioimmunoassays (RIA); Fluorescent substances such as fluorescein, dansyl, fluorescamine, coumarin, naphthylamine or derivatives thereof used in fluorescence immunoassay (FIA), green fluorescent protein (GFP), etc.; Luminescent substances such as luciferin, isoluminol, luminol, bis(2,4,6-trifluorophenyl)oxalate, etc.; Substances that absorb in the ultraviolet region, such as phenol, naphthol, anthracene, or derivatives thereof; Examples of such substances include substances having 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. Among these, from the viewpoint of storage stability, enzymes and fluorescent substances are preferred, alkaline phosphatase, peroxidase and glucose oxidase are more preferred, and peroxidase is particularly preferred. (C) may be used alone or in combination of two or more kinds.

[0015] In the present invention, the substance (FC) is a substance obtained by labeling the above-mentioned substance (F) with a labeling substance (C). As a method for binding the labeling substance (C) to the substance (F) to obtain the substance (FC), a method generally used in this field (e.g., Medical Chemistry Experiment Lectures, Vol. 8, supervised by Yamamura Yuichi, 1st Edition, Nakayama Shoten, 1971; Illustrated Fluorescent Antibody, by Kawao Akira, 1st Edition, Soft Science Co., Ltd., 1983; Enzyme Immunoassay, edited by Ishikawa Eiji, Kawai Tadashi, and Muroi Kiyoshi, 2nd Edition, Igaku Shoin, 1982, etc.) may be used.

[0016] In the present invention, the content of the substance (FC) in the immunoassay reagent (X) is preferably 0.05 to 0.4 wt %, and more preferably 0.1 to 0.3 wt %, based on the weight of the immunoassay reagent (X) from the viewpoint of storage stability.

[0017] As described above, compound (D) in the present invention is at least one compound selected from the group consisting of digitonin, cucurbitacin B, ouabain, and saponin (steroid saponin, triterpenoid saponin, etc.).

[0018] In the present invention, the content of compound (D) in the immunoassay reagent (X) is 0.51 to 1.50% by weight, preferably 0.75 to 1.50% by weight, and more preferably 0.95 to 1.50% by weight, based on the weight of the immunoassay reagent (X) from the viewpoint of storage stability.

[0019] The immunoassay reagent (X) may further contain a protein, which is preferable because the inclusion of a protein can suppress the variation in the measured value from one measurement to the next (improvement of simultaneous reproducibility). From the viewpoint of simultaneous reproducibility, casein is preferred as the protein. The protein content in (X) is preferably 0.005 to 1.0% by weight, and more preferably 0.01 to 0.03% by weight, based on the weight of the immunoassay reagent (X) from the viewpoint of simultaneous reproducibility. From the viewpoint of further improving the simultaneous reproducibility, the ratio of the weight of casein to the weight of (D) [weight of casein / (total weight of compound (D))] is preferably 0.8 to 5.0.

[0020] The immunoassay reagent (X) of the present invention has high storage stability and can therefore be preferably used as the labeled reagent (A) in immunoassay.

[0021] The immunoassay kit of the present invention is an immunoassay kit comprising a labeled reagent (A) and a solid phase carrier reagent (E), wherein the labeled reagent (A) is the above-mentioned immunoassay reagent (X) of the present invention, and the solid phase carrier reagent (E) contains a solid phase carrier (BF) having a substance (F) immobilized on the surface of the solid phase carrier (B).

[0022] In the present invention, the solid phase carrier (B) is not particularly limited as long as it is one that is generally used in this field, and representative examples include glass beads, polystyrene beads, magnetic silica particles (B1), microplates, latex, etc. Examples of the magnetic silica particles (B1) include known magnetic silica particles described in JP-A-2014-210680 and JP-A-2013-019889. Of these, magnetic silica particles (B1) are preferred from the viewpoint of enabling measurement in a short period of time.

[0023] The magnetic silica particles (B1) are those in which a metal oxide having superparamagnetism and a volume average particle size of 1 to 15 nm is dispersed in a silica matrix. Superparamagnetism refers to a temporary magnetic field induced by the alignment of individual atomic magnetic moments of a substance in the presence of an external magnetic field, and when the external magnetic field is removed, the partial alignment is lost and the magnetic field is no longer exhibited. The volume average particle size of the metal oxide is the average value of particle sizes measured by observing 200 random particles with a scanning electron microscope (such as "JSM-7000F" manufactured by JEOL Ltd.).

[0024] Examples of superparamagnetic metal oxides having a volume average particle size of 1 to 15 nm and exhibiting superparamagnetism include oxides of iron, cobalt, nickel, and alloys thereof, among which iron oxide is particularly preferred due to its excellent sensitivity to magnetic fields. The superparamagnetic metal oxides may be used alone or in combination of two or more.

[0025] As the iron oxide, various known iron oxides can be used. Among the iron oxides, chemically Magnetite, γ-hematite, magnetite-α-hematite intermediate iron oxide, and γ-hematite-α-hematite intermediate iron oxide are preferred because they have excellent magnetic stability, and magnetite is more preferred because it has a large saturation magnetization and excellent sensitivity to an external magnetic field.

[0026] The lower limit of the content of the superparamagnetic metal oxide in the magnetic silica particles (B1) is preferably 60% by weight, more preferably 65% ​​by weight, and the upper limit is preferably 95% by weight, more preferably 80% by weight. If the content of the superparamagnetic metal oxide is 60% by weight or more, the magnetic properties of the magnetic silica particles (B1) obtained are sufficient, so that the separation operation in practical applications does not take much time, which is preferable, and if the content is 95% by weight or less, the synthesis is easy, which is preferable.

[0027] The method for producing the superparamagnetic metal oxide is not particularly limited, but the superparamagnetic metal oxide can be synthesized by a coprecipitation method using a water-soluble iron salt and ammonia based on the method reported by Massart (R. Massart, IEEE Trans. Magn. 1981, 17, 1247) or a method using an oxidation reaction in an aqueous solution of a water-soluble iron salt.

[0028] The volume average particle diameter of the magnetic silica particles (B1) is preferably 1 to 5 μm, more preferably 1 to 3 μm. If the volume average particle diameter is 1 μm or more, separation and recovery tend not to take much time. If the volume average particle diameter is 5 μm or less, the surface area becomes relatively large, and as a result, the amount of the immobilized substance (the substance to be measured, a substance similar to the substance to be measured, or a substance that specifically binds to the substance to be measured) bound also increases, and the binding efficiency tends to increase.

[0029] The volume average particle size of the magnetic silica particles (B1) is the average value of particle sizes measured by observing 200 random magnetic silica particles with a scanning electron microscope (JSM-7000F manufactured by JEOL Ltd.).

[0030] The volume average particle size of the magnetic silica particles (B1) can be controlled by adjusting the mixing conditions (shear force, etc.) when preparing the oil-in-water emulsion described below to adjust the particle size of the oil-in-water emulsion. The average particle size can also be adjusted to a desired value by changing the conditions of the water washing step when producing the magnetic silica particles or by a method such as general classification.

[0031] The magnetic silica particles (B1) can be obtained, for example, by mixing a dispersion containing 30 to 500% by weight of (alkyl)alkoxysilane and a dispersant based on the weight of superparamagnetic metal oxide particles having a volume average particle diameter of 1 to 15 nm with a solution containing water, a water-soluble organic solvent, a nonionic surfactant, and a catalyst for hydrolysis of (alkyl)alkoxysilane to form an oil-in-water emulsion, followed by hydrolysis reaction and condensation reaction of the (alkyl)alkoxysilane to obtain an aqueous dispersion of magnetic silica particles in which superparamagnetic metal oxide is encapsulated in silica, followed by solid-liquid separation of the aqueous dispersion of magnetic silica particles by centrifugation and / or magnetic collection, and washing with water, methanol, etc. Above and below, (alkyl)alkoxysilane means alkylalkoxysilane and / or alkoxysilane.

[0032] Since the superparamagnetic metal oxide of the magnetic silica particles (B1) is encapsulated in silica and not present on the particle surface, many target substances, substances similar to the target substances, or substances that specifically bind to the target substances can be immobilized on the surface.

[0033] In the present invention, at least one selected from the group consisting of a substance to be measured (F1), a substance similar to the substance to be measured (F2), and a substance that specifically binds to the substance to be measured (F3) is immobilized on a solid phase carrier (B) {preferably magnetic silica particles (B1)}, and the surface of the solid phase carrier (B) is Methods for producing the solid phase carrier (BF) onto which the substance (F) is immobilized include a method in which the target substance (F1) or the like is physically adsorbed onto the above-mentioned (B). From the viewpoint of more efficient immobilization of the target substance (F1) or the like, a preferred method is to bind at least one organic compound selected from the group consisting of glutaraldehyde, albumin, carbodiimide, streptavidin, biotin, and alkylalkoxysilanes having a functional group to the surface of (B) and immobilize the target substance (F1) or the like onto (B) via the organic compound. Of the organic compounds to be bonded to the surface of (B), from the viewpoint of bonding a specific target substance (F1) or the like, alkylalkoxysilanes having functional groups (ethylenically unsaturated groups, epoxy groups, amino groups, mercapto groups, isocyanate groups, and the like) are more preferred. Examples of 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 trimethoxysilane, N-2-(aminoethyl)-3-aminopropylmethyldimethoxysilane, N-2-(aminoethyl)-3-aminopropyltrimethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-triethoxysilyl-N-(1,3-dimethyl-butylidene)propylamine, N-phenyl-3-aminopropyltrimethoxysilane, tris-(trimethoxysilylpropyl)isocyanurate, 3-ureidopropyltrialkoxysilane, 3-mercaptopropylmethyldimethoxysilane, 3-mercaptopropyltrimethoxysilane, 3-isocyanatopropyltriethoxysilane, and the like.

[0034] The solid phase carrier reagent (E) contains a solid phase carrier (BF) having a substance (F) immobilized on the surface of the solid phase carrier (B). The substance (F) to be immobilized on the surface of the solid phase carrier (B) includes the substances (F) exemplified in the explanation of the substance (FC). The preferred substance (F) to be immobilized on the surface of the solid phase carrier (B) depends on the type of substance (FC) in the labeled reagent (A), but when the substance (FC) in the labeled reagent (A) is a POD-labeled anti-prothrombin monoclonal antibody, an anti-PIVKA-II monoclonal antibody is preferred.

[0035] The immunoassay kit of the present invention may further contain a buffer solution, a luminol luminescence reagent (K) and an aqueous peroxide solution (L) in addition to the above-mentioned solid phase carrier reagent (E) and labeling reagent (A). The luminol luminescence reagent (K) is selected based on the labeling substance (C) used to label the substance (FC) contained in the labeling reagent (A). For example, when the labeling substance (C) is peroxidase, the luminol luminescence reagent may contain a 2,3-dihydro-1,4-phthalazinedione compound and a chemiluminescence enhancer as essential components. The aqueous peroxide solution (L) is an aqueous peroxide solution containing a peroxide and water as essential components.

[0036] Buffer solutions used in the immunoassay kit of the present invention include Tris buffer, phosphate buffer, veronal buffer, borate buffer, Good's buffer, etc., which are commonly used in the field of immunoassays. The pH of the solution may be within a range that does not inhibit the effects of the present invention, and is preferably 5 to 9. In addition, such a buffer solution may contain salts (sodium chloride, etc.), albumin, globulin, water-soluble gelatin, polyethylene glycol, etc., as long as the effects of the present invention are not adversely affected. Stabilizers, surfactants, sugars, etc. may also be contained.

[0037] In the luminol luminescent reagent (K), the 2,3-dihydro-1,4-phthalazinedione compound may be, for example, the known 2,3-dihydro-1,4-phthalazinedione compounds described in JP-A-2-291299, JP-A-10-319015, JP-A-2000-279196, etc., or mixtures thereof. Of these, luminol, isoluminol, N-aminohexyl-N-ethylisoluminol (AHEI), N-aminobutyl-N-ethylisoluminol (ABEI) and metal salts thereof (such as alkali metal salts) are preferred, luminol and its metal salts are more preferred, and sodium salt of luminol is particularly preferred.

[0038] As the chemiluminescence enhancer, for example, known chemiluminescence enhancers described in JP-A-59-500252, JP-A-59-171839, JP-A-2-291299, etc., and mixtures thereof can be used. Of these, from the viewpoint of chemiluminescence enhancement effect, etc., phenol (including phenol derivatives) is preferred, more preferred are p-iodophenol, 4-(cyanomethylthio)phenol and 4-cyanomethylthio-2-chlorophenol, and particularly preferred is 4-(cyanomethylthio)phenol.

[0039] The luminol luminescent reagent (K) may contain a buffer solution and / or a chelating agent in addition to the 2,3-dihydro-1,4-phthalazinedione compound and the chemiluminescence enhancer.

[0040] As the buffer solution contained in the luminol luminescence reagent (K), for example, known buffer solutions as described in JP-A-10-319015 and JP-A-2003-279489, and mixtures thereof, etc. can be used. Among these, from the viewpoints of chemiluminescence enhancement effect and storage stability, 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid / sodium hydroxide buffer solution, 2-hydroxy-3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid monohydrate / sodium hydroxide buffer solution, and piperazinyl-1,4-bis(2-hydroxy-3-propanesulfonic acid) dihydrate / sodium hydroxide buffer solution are preferred, more preferred are 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid / sodium hydroxide buffer solution, and 2-hydroxy-3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid monohydrate / sodium hydroxide buffer solution, and particularly preferred is 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid / sodium hydroxide buffer solution.

[0041] As the chelating agent contained in the luminol luminescence reagent (K), for example, known chelating agents described in JP-A-9-75099 and JP-A-2003-279489 and mixtures thereof can be used. Among these, from the viewpoints of chemiluminescence enhancing effect and storage stability, etc., tetracoordinate chelating agents are preferred, more preferred are ethylenediaminetetraacetic acid (EDTA) and salts thereof (disodium ethylenediaminetetraacetate, trisodium ethylenediaminetetraacetate, tetrasodium ethylenediaminetetraacetate, dipotassium ethylenediaminetetraacetate, tripotassium ethylenediaminetetraacetate, etc.) and trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CyDTA), and particularly preferred is ethylenediaminetetraacetic acid (EDTA) and salts thereof.

[0042] The luminol luminescent reagent (K) is preferably a liquid, and is preferably alkaline from the viewpoint of the fluorescence intensity of the enzyme. The pH of the luminol luminescence reagent (K) is preferably 7 to 11, and more preferably 8 to 10. The pH is measured in accordance with JIS K0400-12-10:2000 (measurement temperature: 25° C.).

[0043] The luminol luminescent reagent (K) can be easily obtained by homogeneously mixing a 2,3-dihydro-1,4-phthalazinedione compound, a chemiluminescence enhancer, and, if necessary, a buffer solution and / or a chelating agent.

[0044] Examples of the peroxide contained in the aqueous peroxide solution (L) include aqueous solutions of known oxidizing agents such as inorganic peroxides (hydrogen peroxide, sodium perborate, potassium perborate, etc.), organic peroxides (dialkyl peroxides, acyl peroxides, etc.), and peroxoacid compounds (peroxosulfuric acid, peroxolinic acid, etc.), as described in JP-A-8-261943 and JP-A-2000-279196. Among these, from the viewpoint of storage stability, etc., an aqueous solution of hydrogen peroxide, an aqueous solution of sodium perborate and an aqueous solution of potassium perborate are preferred, and an aqueous solution of hydrogen peroxide is more preferred.

[0045] Examples of the water contained in the aqueous peroxide solution (L) include distilled water, reverse osmosis water, deionized water, etc. Among these, from the viewpoints of chemiluminescence enhancement effect and storage stability, etc., distilled water and deionized water are preferred, and deionized water is more preferred.

[0046] The aqueous peroxide solution (L) may contain a chelating agent and the like in addition to the peroxide and water. Examples of the chelating agent include the same ones as those exemplified as the chelating agents that can be contained in the above-mentioned luminol luminescence reagent (K), and the preferred ones are also the same. The aqueous peroxide solution (L) can be easily obtained by uniformly mixing a peroxide, water and, if necessary, a chelating agent.

[0047] The immunoassay kit of the present invention can be used in immunoassay methods for quantifying the analyte (F1) in a sample, which are generally performed in this field, and may be performed, for example, in accordance with the sandwich method and competitive method described in the literature [e.g., Enzyme Immunoassay Methods, 2nd Edition (edited by Ishikawa Eiji et al., Igaku-Shoin), 1982] and the measurement method described in JP-A-6-130063.

[0048] The sandwich method is carried out, for example, by contacting a sample containing a "substance to be measured (F1)", magnetic silica particles (B1F3) having a "substance (F3) that specifically binds to the substance to be measured" immobilized on the surface of the magnetic silica particles (B1), and a "substance (F3C) that specifically binds to the substance to be measured and is labeled with a labeling substance", thereby forming a labeled complex {(F3) / (F1) / (F3C)} of the "substance (F3) that specifically binds to the substance to be measured" supported on the surface of the magnetic silica particles (B1), the "substance to be measured (F1)", and the "substance (F3C) that specifically binds to the substance to be measured and is labeled with a labeling substance", subjecting the labeled complex to B / F separation, measuring the amount of the labeled substance in the complex, and quantifying the substance to be measured (F1) in the sample based on the results.

[0049] Specifically, for example, a "sample containing a target substance (F1)" is contacted with "magnetic silica particles (B1F3) having a substance (F3) that specifically binds to the target substance (F3) immobilized on the surface of the magnetic silica particles (B1)" to form a complex between the "substance (F3) that specifically binds to the target substance (F1)" carried on the surface of the magnetic silica particles (B1) and the "target substance (F1)". This complex is then contacted with a "substance (F3C) that specifically binds to the target substance (F3) labeled with a labeling substance" to form a labeled complex {(F3) / (F1) / (F3C)} between the "substance (F3) that specifically binds to the target substance (F1)" carried on the magnetic silica particles (B1), the "target substance (F1)", and the "substance (F3C) that specifically binds to the target substance (F3C) labeled with a labeling substance". The labeled complex is then B / F separated to measure the amount of the labeled substance in the labeled complex, and the target substance in the sample is quantified based on the results. In this method, the "measurement target substance (F1)" is reacted with magnetic silica particles (B1F3) on which a "substance (F3) that specifically binds to the measurement target substance" is immobilized, and then a "substance (F3C) that specifically binds to the measurement target substance labeled with a labeling substance" is reacted. However, it is also possible to react the "substance (F3C) that specifically binds to the labeled measurement target substance" with the "measurement target substance (F1)" and then react it with the magnetic silica particles (B1F3) on which a "substance (F3) that specifically binds to the measurement target substance" is immobilized, or to react these three simultaneously.

[0050] The B / F separation in the sandwich method means separation of the labeled complex {(F3) / (F1) / (F3C)} and the "substance (F3C) that specifically binds to the analyte labeled with a labeling substance" that was not involved in the formation of the labeled complex, specifically, separation of the "substance (F3) that specifically binds to the analyte supported on the magnetic silica particles (B1)," the "complex {(F3) / (F1)} of the substance (F3) that specifically binds to the analyte supported on the magnetic silica particles (B1) and the analyte (F1)," and the above "labeled complex {(F3) / (F1) / (F3C)}" from other components (components other than the analyte (F1) in the sample, substances (F3C) that specifically bind to the analyte labeled with a labeling substance that was not involved in the formation of the labeled complex, etc.). In addition, the B / F separation step is an essential step after the formation of the labeled complex {(F3) / (F1) / (F3C)}, but it can also be performed after the formation of a complex {(F3) / (F1)} between the "substance (F3) that specifically binds to the substance to be measured" supported on the surface of the magnetic silica particles (B1) and the "substance to be measured (F1)."

[0051] The competitive method involves contacting a sample containing a "substance to be measured (F1)," a "substance (F3C) that specifically binds to the substance to be measured labeled with a labeling substance," and magnetic silica particles (B1F1 / F2) having immobilized on their surfaces the "substance to be measured (F1) and / or an analogue of the substance to be measured (F2)," to form a labeled complex {(F1) / (F3C) and / or (F2) / (F3C)} between the "substance to be measured (F1) and / or its analogue (F2)" supported on the magnetic silica particles (B1) and the "substance (F3C) that specifically binds to the substance to be measured labeled with a labeling substance," and subjecting the magnetic silica particles supporting the labeled complex to B / F separation, measuring the amount of the labeled substance in the labeled complex, and quantifying the substance to be measured in the sample based on the results.

[0052] Specifically, for example, a sample containing a "measurement target substance (F1)" is contacted with a "substance (F3C) that specifically binds to the measurement target substance labeled with a labeling substance" and magnetic silica particles (B1F1 / F2) carrying the "measurement target substance (F1) and / or its analogue (F2)", so that the "substance (F3C) that specifically binds to the measurement target substance labeled with a labeling substance" reacts with the "measurement target substance (F1)" in the sample and the "measurement target substance (F1) and / or its analogue (F2)" carried on the surface of the magnetic silica particles (B1). The magnetic silica particles (B1) are subjected to a competitive reaction with the "substance (F1) and / or its analogue (F2)" carried on the surface of the magnetic silica particles (B1) to form a labeled complex {(F1) / (F3C) and / or (F2) / (F3C)} between the "substance (F1) to be measured and / or its analogue (F2)" carried on the surface of the magnetic silica particles (B1) and the "substance (F3C) which specifically binds to the substance to be measured and which is labeled with a labeling substance," the magnetic silica particles carrying the labeled complex are then subjected to B / F separation, the amount of the labeled substance in the labeled complex is measured, and the substance to be measured (F1) in the sample is quantified based on the result. In this method, the "measurement target substance (F1)", the "substance (F3C) that specifically binds to the measurement target substance labeled with a labeling substance", and the "measurement target substance (F1) and / or its analogue (F2)" immobilized on the surface of the magnetic silica particles (B1) are simultaneously subjected to a competitive reaction. Alternatively, the "measurement target substance (F1)" may be contacted with the "measurement target substance (F1) and / or its analogue (F2)" immobilized on the surface of the magnetic silica particles (B1) and then the "substance (F3C) that specifically binds to the measurement target substance labeled with a labeling target substance" may be added to perform the competitive reaction, or the "measurement target substance (F1)" may be contacted with the "substance (F3C) that specifically binds to the measurement target substance labeled with a labeling substance" and then the magnetic silica particles (B1F1 / F2) carrying the "measurement target substance (F1) and / or its analogue (F2)" may be added to perform the competitive reaction.

[0053] The B / F separation in the competitive method means separation of the labeled complex {(F1) / (F3C) and / or (F2) / (F3C)} from the "substance (F3C) that specifically binds to the analyte labeled with a labeling substance" that was not involved in the formation of the labeled complex, and the "complex {(F1) / (F3C)} of the substance (F3C) that specifically binds to the analyte labeled with a labeling substance and the analyte (F1)." Specifically, it means separation of the "analyte (F1) and / or its analogs" from the "substance (F3C) that specifically binds to the analyte labeled with a labeling substance" that was not involved in the formation of the labeled complex. This refers to separation of "magnetic silica particles carrying a substance (F2)" and "complexes {(B1F1 / F2) / (F3C)} of magnetic silica particles carrying a substance to be measured (F1) and / or its analogue (F2) and a substance (F3C) that specifically binds to the substance to be measured labeled with a labeling substance" from other components (components other than the substance to be measured (F1) in the sample, "substance (F3C) that specifically binds to the substance to be measured labeled with a labeling substance," complexes {(F1) / (F3C)}, etc.).

[0054] In another embodiment of the competitive method, a sample containing the "measurement target substance (F1)" is contacted with magnetic silica particles (B1F3) carrying the "measurement target substance and / or its analogue labeled with a labeling substance (F1 / F2C)" and the "substance that specifically binds to the measurement target substance (F3)" to form a labeled complex {(F3) / (F1 / F2C)} between the "substance that specifically binds to the measurement target substance (F3)" carried on the magnetic silica particles (B1) and the "measurement target substance and / or its analogue labeled with a labeling substance (F1 / F2C)", the magnetic silica particles carrying the labeled complex are subjected to B / F separation, the amount of the labeled substance in the labeled complex is measured, and the measurement target substance in the sample is measured based on the result.

[0055] Specifically, for example, a sample containing a "measurement target substance (F1)" is contacted with magnetic silica particles (B1F3) carrying a "measurement target substance and / or its analogues (F1 / F2C) labeled with a labeling substance" and a "substance (F3) that specifically binds to the measurement target substance," and the "measurement target substance (F1) in the sample" and the "measurement target substance and / or its analogues (F1 / F2C) labeled with a labeling substance" are competitively reacted with the "substance (F3) that specifically binds to the measurement target substance" carried by the magnetic silica particles (B1), A complex {(F3) / (F1)} is formed between the "substance (F3) that specifically binds to the measured substance" supported on the magnetic silica particles (B1) and the "measured substance (F1)" in the sample, as well as a labeled complex {(F3) / (F1 / F2C)} is formed between the "measured substance and / or its analogue labeled with a labeling substance (F1 / F2C)". The complex or the magnetic silica particles supporting the labeled complex are then subjected to B / F separation to measure the amount of labeled substance in the labeled complex, and the measured substance (F1) in the sample is measured based on the results. In this competitive method, the "measurement target substance (F1)," the "measurement target substance labeled with a labeling substance and / or its analogue (F1 / F2C)," and the magnetic silica particles (B1F3) carrying the "substance that specifically binds to the measurement target substance (F3)" are simultaneously subjected to a competitive reaction. After the "measurement target substance (F1)" is contacted with the magnetic silica particles carrying the "substance that specifically binds to the measurement target substance (F3)" {after the complex (F3) / (F1) is formed}, the "measurement target substance labeled with a labeling substance and / or its analogue (F1 / F2C)" is added to carry out the competitive reaction {magnetic silica particles}. In (B1F3), (F1 / F2C) may be reacted with (F3) to which the analyte (F1) in the sample has not reacted, or after contacting the "analyte and / or its analogue (F1 / F2C) labeled with a labeling substance" with magnetic silica particles (B1F3) carrying a "substance (F3) that specifically binds to the analyte" {after forming a complex (F3) / (F1 / F2C)}, the "analyte (F1)" may be added to cause a competitive reaction {in the magnetic silica particles (B1F3), the analyte (F1) in the sample may be reacted with (F3) to which (F1 / F2C) has not reacted}.

[0056] The B / F separation in the competitive method means separation of the labeled complex from the "measurement substance and / or its analogues labeled with a labeling substance (F1 / F2C)" that was not involved in the formation of the labeled complex, specifically, separation of the "magnetic silica particles (B1F3) carrying a substance (F3) that specifically binds to the measurement substance," the "complex {(F3) / (F1)} between the measurement substance (F3) on the magnetic silica particles (B1) and the measurement substance (F1), and the "complex {(F3) / (F1 / F2C)} between the substance (F3) that specifically binds to the measurement substance on the magnetic silica particles (B1) and the measurement substance labeled with a labeling substance and / or its analogues (F1 / F2C)" from other components (components other than the measurement substance (F1) in the sample, the measurement substance labeled with a labeling substance that was not involved in the formation of the labeled complex and / or its analogues (F1 / F2C), etc.). In addition, although the B / F separation process is an essential process after the formation of the labeled complex, it can also be performed after contacting the "substance to be measured (F1)" with magnetic silica particles carrying the "substance that specifically binds to the substance to be measured (F3)."

[0057] Furthermore, when the "substance to be measured (F1)" is an enzyme, the measurement may be performed using a method using an enzyme activity method other than the sandwich method or competitive method described above, for example, by contacting a sample containing the "substance to be measured (F1)" with magnetic silica particles (B1F3) having immobilized on their surface a "substance (F3) that specifically binds to the substance to be measured (e.g., a substance that can bind to an enzyme such as an antibody)" to form a complex {(F3) / (F1)} with the "substance (F3) that specifically binds to the substance to be measured" supported on the magnetic silica particles (B1), and after B / F separation of the magnetic silica particles supporting the complex, a substrate and a color developer according to the type of enzyme, and if necessary, a conjugated enzyme, are added, and the amount of the enzyme in the sample is measured based on the change in the substrate or the color development of the color developer. The substrate, coloring agent, and conjugated enzyme may be any known substance. For example, when the enzyme is peroxidase, hydrogen peroxide and luminol luminescence reagent may be used. The amounts of these used may be within the ranges generally used in this field. The B / F separation in the above method refers to the separation of a complex {(F3) / (F1)} of the "substance to be measured (F1)" and the "substance (F3) that specifically binds to the substance to be measured" immobilized on magnetic silica particles, from other components (components other than the substance to be measured (F1) in the sample, etc.).

[0058] In the immunoassay method of the present invention, the sample, the magnetic silica particles (B1F) to which the substance (F) is immobilized, the substance (F3C) that specifically binds to the labeled analyte, the labeled analyte (F1C) or its analogue (F2C), etc. may be contacted by generally performed treatments such as stirring, mixing, etc., so long as the magnetic silica particles are dispersed. The reaction time may be appropriately set depending on the analyte (F1), the substance (F3) that specifically binds to the analyte used, the sandwich method, the competitive method, etc., but is preferably 1 to 10 minutes, more preferably 1 to 5 minutes.

[0059] The B / F separation in the immunoassay method of the present invention is carried out, for example, by utilizing the magnetism of the magnetic silica particles, collecting the magnetic silica particles from the outside of the reaction vessel with a magnet or the like, discharging the reaction liquid, adding a washing liquid, removing the magnet, mixing and dispersing the magnetic silica particles, and washing them. The above operation may be repeated 1 to 3 times. As the washing liquid, a liquid generally used in this field (such as physiological saline) can be used. EXAMPLES

[0060] The present invention will be further described below with reference to examples, but the present invention is not limited thereto. Examples 2, 5, 8, and 11 are Reference Examples 1 to 4, respectively.

[0061] <Example 1> The solid phase carrier reagent (E) [anti-PIVKA-II monoclonal antibody] was prepared by the following method. The immunoassay kit (S1) of the present invention is composed of magnetic silica particles (BF) having immobilized thereon], a labeled reagent (A1) (POD-labeled anti-prothrombin monoclonal antibody reagent), an immune reaction buffer, a luminol luminescence reagent (K) and a hydrogen peroxide solution (L).

[0062] Preparation of solid support reagent (E): 40 mg of the magnetic silica particles (trade name: Magrapid, manufactured by Sanyo Chemical Industries, Ltd.) was added to a polystyrene bottle with a lid containing 40 mL of an aqueous solution containing 1 wt % γ-aminopropyltriethoxysilane, and the mixture was reacted at 25 ° C for 1 hour. After the magnetic silica particles were collected with a neodymium magnet, the liquid was removed by aspirating with an aspirator. Next, 40 mL of deionized water was added, the lid was placed, and the polystyrene bottle was slowly inverted and stirred twice, after which the magnetic silica particles were collected with a neodymium magnet, and the liquid was removed by aspirating with an aspirator to wash the magnetic silica particles. This washing operation was performed five times. Next, the washed magnetic silica particles were added to a polystyrene bottle with a lid 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 lid was placed, and the polystyrene bottle was slowly inverted and stirred twice, after which the magnetic silica particles were collected with a neodymium magnet, and the liquid was removed by aspirating with an aspirator to wash the magnetic silica particles. This washing operation was performed 10 times. Furthermore, the washed magnetic silica particles were added to a polystyrene bottle with a lid containing 120 mL of 0.02 M phosphate buffer (pH 8.7) containing anti-PIVKA-II monoclonal antibody (manufactured by Cosmo Bio Co., Ltd.) at a concentration of 10 μg / mL as substance (F), and reacted at 25 ° C for 1 hour. After the reaction, the magnetic silica particles (BF) with the substance (F) immobilized thereon were collected with a neodymium magnet, and the phosphate buffer containing the anti-PIVKA-II monoclonal antibody (F) was removed. Next, 40 mg of the obtained magnetic silica particles (BF) were added to a polyethylene bottle with a lid containing 40 mL of 0.02 M phosphate buffer (pH 7.0) containing 1 wt % Block Ace (manufactured by DS Pharma Biomedical Co., Ltd.), and immersed at 25 ° C for 12 hours. As a result, a solid phase carrier reagent (E) containing magnetic silica particles (BF) with anti-PIVKA-II monoclonal antibody (F) immobilized thereon was obtained, and stored in a refrigerator (2 to 10 ° C).

[0063] Preparation of labeling reagent (A1): Anti-prothrombin monoclonal antibody (Cosmo Bio Co., Ltd.) and horseradish-derived POD (Toyobo Co., Ltd.) were used to prepare POD-labeled anti-prothrombin monoclonal antibody (FC) according to the method described in the literature (S. Yoshitake, M. Imagawa, E. Ishikawa, Ethol; J. Biochem, Vol. 92, 1982, 1413-1424). This was diluted with 0.02 M phosphate buffer containing 0.95 wt% digitonin (Nacalai Tesque Co., Ltd.) as (D) to a POD-labeled anti-prothrombin monoclonal antibody (FC) concentration of 2.0 μg / mL to prepare labeled reagent (A1). The labeling reagent (A1) was prepared by storing it at 4°C for 7 days and at 35°C for 7 days. The content of digitonin in the labeling reagent (A1) was 0.95% by weight based on the weight of the labeling reagent (A1).

[0064] Preparation of immunoreaction buffer: A 0.02 M phosphate buffer solution (pH 7.0) containing 0.1 wt% bovine serum albumin (Boval Campany), 1 wt% Emulmin L-90-S (Sanyo Chemical Industries, Ltd.), and 0.85 wt% sodium chloride (Fujifilm Wako Pure Chemical Industries, Ltd.) was prepared and stored refrigerated (2-10°C).

[0065] Preparation of luminol luminescence reagent (K): 0.7 g of luminol sodium salt [Sigma-Aldrich Japan Co., Ltd.] and 0.1 g of 4-(cyanomethylthio)phenol were placed in a 1,000 mL measuring flask. 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid / sodium hydroxide buffer (10 mM, pH 8.6) was added to make the solution volume 1,000 mL, and the solution was mixed uniformly at 25°C to prepare luminol luminescence reagent (K). The solution was stored refrigerated (2-10°C) until it was used for measurement.

[0066] Preparation of hydrogen peroxide solution (L): 6.6 g of hydrogen peroxide [Fujifilm Wako Pure Chemical Industries, Ltd., special grade reagent, concentration 30% by weight] was placed in a 1,000 mL measuring flask. Deionized water was added to make the solution volume 1,000 mL, and the solution was mixed uniformly at 25°C to prepare hydrogen peroxide solution (L). The solution was stored refrigerated (2-10°C) until it was used for measurement.

[0067] <Examples 2 to 12> In "Preparation of labeled reagent (A1)" of Example 1, labeled reagents (A2) to (A12) were prepared in the same manner as in Example 1, except that the type and concentration of compound (D) in 0.02 M phosphate buffer were those shown in Table 1. These were combined with the same solid phase carrier reagent (E), immune reaction buffer, luminol luminescence reagent (K), and hydrogen peroxide solution (L) as in Example 1 to prepare immunoassay kits (S2) to (S12).

[0068] <Comparative Examples 1 to 3> In "Preparation of labeled reagent (A1)" of Example 1, labeled reagents (A'1) to (A'3) were prepared in the same manner as in Example 1, except that the type and concentration of compound (D) in 0.02 M phosphate buffer were those listed in Table 1. These were combined with the same solid phase carrier reagent (E), immune reaction buffer, luminol luminescence reagent (K) and hydrogen peroxide solution (L) as in Example 1 to prepare immunoassay kits (S'1) to (S'3).

[0069] In Table 1, the following components were used. Digitonin: Fujifilm Wako Pure Chemical Industries, Ltd. Cucurbitacin B: manufactured by Tokyo Chemical Industry Co., Ltd. Ouabain: Fujifilm Wako Pure Chemical Industries, Ltd. Saponin: Fujifilm Wako Pure Chemical Industries, Ltd.

[0070] Immunoassays were carried out using the immunoassay reagents (S1) to (S12) and (S'1) to (S'3) according to the following method, and the storage stability of the immunoassay kits was evaluated. The results are shown in Table 1.

[0071] <Examples 13 to 24 and Comparative Examples 4 to 6> <Immunoassay method> ○Process (1) 0.025 mL of each solid phase carrier reagent (E) of the immunoassay kit obtained in the examples and comparative examples was placed in a test tube, the magnetic silica particles were collected from the outside of the test tube with a neodymium magnet for 10 seconds, the liquid in the test tube was removed with an aspirator, and the neodymium magnet was sufficiently removed from the side. Then, 0.2 mL of immune reaction buffer and 0.025 mL of pooled serum prepared so that the concentration of the PIVKA-II antigen, which is the measurement target substance (F1), is 3000 mAU / mL, were placed in the test tube, mixed, and reacted in the test tube at 37°C for 3 minutes to form a complex of anti-PIVKA-II monoclonal antibody (F) / PIVKA-II antigen (F1) on the magnetic silica particles with the anti-PIVKA-II monoclonal antibody immobilized thereon. After the reaction, the magnetic silica particles were collected from the outside of the test tube with a neodymium magnet for 10 seconds, the liquid in the test tube was removed with an aspirator, and the neodymium magnet was sufficiently removed from the side. Thereafter, 0.5 mL of physiological saline was added to disperse the magnetic silica particles and collect the magnetic particles, after which a washing procedure was performed three times to remove the liquid using an aspirator.

[0072] ○Process (2) Next, 0.1 mL of the labeling reagent of each immunoassay kit [labeling reagent stored at 4°C for 7 days or labeling reagent stored at 35°C for 7 days] was injected into each test tube, and reacted in the test tube at 37°C for 3 minutes to form a complex of anti-PIVKA-II antibody (F) / PIVKA-II antigen (F1) / POD-labeled anti-prothrombin monoclonal antibody (FC) on the anti-PIVKA-II monoclonal antibody-immobilized magnetic silica particles. After the reaction, the magnetic silica particles were collected from the outside of the test tube with a neodymium magnet for 10 seconds, the liquid in the test tube was removed with an aspirator, and the neodymium magnet was sufficiently removed from the side. Then, 0.5 mL of physiological saline was added to disperse the magnetic silica particles and collect them, and a washing operation of removing the liquid with an aspirator was performed twice.

[0073] Chemiluminescence detection process Finally, 0.1 mL of luminol luminescence reagent (K) and 0.1 mL of hydrogen peroxide solution (L) were added at the same time, and the luminescence reaction was allowed to proceed at 37°C. The average amount of luminescence per second was measured for 40 to 45 seconds after the addition of the luminol luminescence reagent (K) and hydrogen peroxide solution (L) using a luminometer [Berthold Japan "Lumat LB9507"]. The results are shown in Table 1.

[0074] <Evaluation method for storage stability> The storage stability was evaluated according to the following criteria. ○: (average luminescence amount when using labeling reagent (A) stored at 35℃ for 7 days / average luminescence amount when using labeling reagent (A) stored at 4℃ for 7 days) × 100 = 90% to 110% ×: (average luminescence amount when using labeling reagent (A) stored at 35℃ for 7 days / average luminescence amount when using labeling reagent (A) stored at 4℃ for 7 days) × 100<90%, or >110% The closer the measured concentration / actual measured concentration is to 100%, the higher the storage stability is.

[0075] The results of the storage stability evaluated by the immunoassay method in Examples 13 to 24 and Comparative Examples 4 to 6 are shown in Table 1.

[0076] [Table 1] [Industrial Applicability]

[0077] The immunoassay kit and immunoassay method of the present invention have excellent storage stability and can be widely applied to clinical tests such as radioimmunoassay, enzyme immunoassay, fluorescent immunoassay and chemiluminescent immunoassay.

Claims

1. The immunoassay reagent (X) contains a substance (FC) and at least one compound (D) selected from the group consisting of digitonin, cucurbitacin B, ouabain and saponin, wherein the substance (FC) is a substance obtained by labeling a substance (F) with a labeling substance (C), the substance (F) is at least one substance selected from the group consisting of a substance to be measured (F1), an analogue of the substance to be measured (F2) and a substance that specifically binds to the substance to be measured (F3), the labeling substance (C) is peroxidase, and the content of (D) in (X) is 0.95 to 1.50% by weight based on the weight of (X).

2. 2. The reagent for an immunoassay according to claim 1, wherein the substance (F) is an anti-prothrombin monoclonal antibody.

3. An immunoassay kit comprising a labeled reagent (A) and a solid phase carrier reagent (E), wherein the labeled reagent (A) is the immunoassay reagent (X) described in claim 1 or 2, and the solid phase carrier reagent (E) contains a solid phase carrier (BF) having a substance (F) immobilized on the surface of the solid phase carrier (B).

4. The immunoassay kit according to claim 3, further comprising a luminol luminescence reagent (K) and an aqueous peroxide solution (L).

5. 5. The immunoassay kit according to claim 3, wherein the solid phase carrier (B) is a magnetic silica particle (B1).

6. An immunoassay method, which uses the immunoassay kit according to any one of claims 3 to 5.

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