JP7578741B2 - Immunoassay method, immunoassay kit and solid phase carrier reagent - Google Patents

Description

The present invention relates to an immunoassay method, an immunoassay kit, and a solid phase carrier reagent.

Conventionally, a method for detecting biological substances in a sample, such as proteins in a biological sample, has been known in which the biological substances in the sample are bound to the surface of magnetic particles to which the biological substances can bind, and the magnetic particles to which the biological substances are bound are washed to remove impurities other than the biological substances to be detected, and then recovered, and a signal (such as the amount of light emitted) derived from the magnetic particles is measured (Patent Document 1).
When detecting biological substances (such as α-fetoprotein) in blood, blood cells (red blood cells and white blood cells) in the blood are usually precipitated by centrifugation, and the supernatant, serum or plasma, is used as a sample. However, if blood cells cannot be completely separated and some of them are mixed into the sample, signals derived from peroxidase contained in the blood cells will also be detected. In this case, a phenomenon occurs in which a signal exceeding the signal intensity corresponding to the amount of the biological substance to be detected (false high value) is detected, or a signal is detected even when the biological substance is not present (false positive).

International Publication No. 2012/173002

The present invention aims to provide an immunoassay method, an immunoassay kit, and a solid phase carrier reagent that can suppress false positives and false high values caused by peroxidase derived from impurities (blood cells, etc.) in the sample.

As a result of intensive research to achieve the above object, the present inventors have arrived at the present invention.
Obtaining a first mixture containing a complex of an antigen and a solid phase carrier (α); and
An immunoassay method comprising a step of measuring the antigen using the first mixture and a labeled reagent (C),
The step of obtaining the first mixture comprises:
A step of mixing a sample containing the antigen, a peroxidase inhibitor, hydrogen peroxide and the solid phase carrier (α) to obtain a second mixture containing a complex of the antigen and the solid phase carrier (α); and
a step of removing the peroxidase inhibitor after the step of obtaining the second mixture;
An immunoassay kit for use in the immunoassay method of the present invention, comprising:
The method comprises the steps of: a solid phase carrier reagent (A); a reaction buffer solution (B); the labeling reagent (C); and a luminescent reagent (D);
The solid phase carrier reagent (A) contains the solid phase carrier (α),
At least one of the solid phase carrier reagent (A) and the reaction buffer solution (B) contains the peroxidase inhibitor;
At least one of the solid phase carrier reagent (A) and the reaction buffer solution (B) contains the hydrogen peroxide, or
a kit for immunoassay, wherein the solid phase carrier reagent (A) contains either one (β) of a catalyst or a substrate in a chemical reaction producing hydrogen peroxide, and the reaction buffer solution (B) contains the other one (γ) of the catalyst and the substrate, and hydrogen peroxide can be generated by mixing the (β) and the (γ);
An immunoassay kit for use in the immunoassay method of the present invention, comprising:
The method comprises the steps of: a solid phase carrier reagent (A); a reaction buffer solution (B); the peroxidase inhibitor; the labeling reagent (C); and a luminescent reagent (D);
The solid phase carrier reagent (A) contains the solid phase carrier (α),
At least one of the solid phase carrier reagent (A) and the reaction buffer solution (B) contains the hydrogen peroxide, or
a kit for immunological measurement, in which the solid phase carrier reagent (A) contains either (β) of a catalyst or a substrate in a chemical reaction to produce hydrogen peroxide, and the reaction buffer solution (B) contains the other (γ) of the catalyst and the substrate, and hydrogen peroxide can be generated by mixing (β) and (γ); and a solid phase carrier reagent (A) containing a solid phase carrier (α) and either (β) of a catalyst or a substrate in a chemical reaction to produce hydrogen peroxide.

The immunoassay method, immunoassay kit, and solid phase carrier reagent of the present invention can suppress false positives and false high values caused by peroxidase derived from impurities.

The immunological measurement method of the present invention, as described in detail below,
Obtaining a first mixture containing a complex of an antigen and a solid phase carrier (α); and
An immunoassay method comprising a step of measuring the antigen using the first mixture and a labeled reagent (C),
The step of obtaining the first mixture comprises:
A step of mixing a sample containing the antigen, a peroxidase inhibitor, hydrogen peroxide and the solid phase carrier (α) to obtain a second mixture containing a complex of the antigen and the solid phase carrier (α); and
After obtaining the second mixture, the method includes removing the peroxidase inhibitor.
The immunoassay method of the present invention can be carried out, for example, using the immunoassay kit of the present invention, which will be described in detail later.
The immunoassay kit of the present invention is used in the immunoassay method of the present invention, as described below,
The method comprises the steps of: a solid phase carrier reagent (A); a reaction buffer solution (B); the labeling reagent (C); and a luminescent reagent (D);
The solid phase carrier reagent (A) contains the solid phase carrier (α),
At least one of the solid phase carrier reagent (A) and the reaction buffer solution (B) contains the hydrogen peroxide, or
the solid phase carrier reagent (A) contains either one (β) of a catalyst or a substrate in a chemical reaction that produces hydrogen peroxide, and the reaction buffer solution (B) contains the other one (γ) of the catalyst and the substrate, and hydrogen peroxide can be generated by mixing the (β) and the (γ);
At least one of the solid phase carrier reagent (A) and the reaction buffer solution (B) further contains the peroxidase inhibitor (first embodiment); and
The method comprises the steps of: a solid phase carrier reagent (A); a reaction buffer solution (B); the peroxidase inhibitor; the labeling reagent (C); and a luminescent reagent (D);
The solid phase carrier reagent (A) contains the solid phase carrier (α),
At least one of the solid phase carrier reagent (A) and the reaction buffer solution (B) contains the hydrogen peroxide, or
The solid phase carrier reagent (A) may be any one of a catalyst and a substrate (β) in a chemical reaction that produces hydrogen peroxide, and the reaction buffer solution (B) may be the other of the catalyst and the substrate (γ), and hydrogen peroxide may be generated by mixing (β) and (γ) (second embodiment).
First, the solid phase carrier reagent (A), the reaction buffer solution (B), the labeling reagent (C), and the luminescent reagent (D) will be described.

<Solid Phase Carrier Reagent (A)>
The solid phase carrier reagent (A) in the present invention contains a solid phase carrier (α).
The material of the solid phase carrier (α) is not particularly limited as long as it is generally used in the field of immunoassay, and representative examples include glass beads, polystyrene beads, magnetic particles, microplates, latex, etc. Among these, it is preferable to use magnetic particles from the viewpoints of shortening the measurement time and improving the accuracy in immunoassay.

As the magnetic particles, silica particles containing a superparamagnetic metal oxide are preferred, and silica particles having superparamagnetic metal oxide particles dispersed in a silica matrix (hereinafter also simply referred to as "silica particles"), as described in Patent Document 1, JP-A-2014-210680, JP-A-2013-019889, and the like, are more preferred.
Superparamagnetism refers to the fact that in the presence of an external magnetic field, the individual atomic magnetic moments of a material align and exhibit an induced temporary magnetic field, but when the external field is removed, the partial alignment is lost and no magnetic field is exhibited.

The volume average particle diameter of the silica particles is preferably 1 to 5 μm, and more preferably 1 to 3 μm. If the volume average particle diameter is 1 μm or more, the solid phase carrier (α) tends to be separated and recovered in a short time, and if it is 5 μm or less, the surface area is appropriate, the amount of the substance to be immobilized [antigen, antibody, etc.] that can be bound can be appropriate, and binding efficiency is good.

The volume average particle size of the silica particles 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 volume average particle size can also be adjusted to a desired value by changing the conditions of the water washing step during the production of silica particles or by a method such as ordinary classification.
In this specification, the volume average particle size is the average value of particle sizes measured by observing 200 random particles with a scanning electron microscope (JSM-7000F manufactured by JEOL Ltd.).

Superparamagnetic metal oxides include oxides of iron, cobalt, nickel, and alloys thereof, among which iron oxide is particularly preferred due to its excellent sensitivity to magnetic fields. Superparamagnetic metal oxides may be used alone or in combination of two or more types.

As the iron oxide, various known iron oxides can be used.
Among iron oxides, at least one selected from the group consisting of magnetite, γ-hematite, magnetite-α-hematite intermediate iron oxide, and γ-hematite-α-hematite intermediate iron oxide is preferred because it has particularly excellent chemical stability, and magnetite is more preferred because it has large saturation magnetization and excellent sensitivity to an external magnetic field.

The method for producing superparamagnetic metal oxides is not particularly limited, but they can be synthesized by a coprecipitation method using 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 water-soluble iron salt.

The superparamagnetic metal oxide particles preferably have a volume average particle size of 1 to 20 nm. When the volume average particle size is 1 nm or more, synthesis is relatively easy, and when it is 20 nm or less, it is easy to uniformly disperse the particles in a silica matrix.
The volume average particle size of the superparamagnetic metal oxide particles can be controlled by adjusting the metal ion concentration during the preparation of the superparamagnetic metal oxide particles, and the volume average particle size can also be adjusted to a desired value by a conventional classification method or the like.

The lower limit of the content of superparamagnetic metal oxide in the silica particles is preferably 60% by weight, more preferably 65% by weight, based on the weight of the silica particles, and the upper limit is preferably 95% by weight, more preferably 85% by weight. If the content of superparamagnetic metal oxide is 60% by weight or more, the obtained silica particles have sufficient magnetism, and separation operations for practical applications can be carried out in a short time, which is preferable. In addition, if the content of superparamagnetic metal oxide is 95% by weight or less, the synthesis of silica particles is easy.

The silica particles can be obtained, for example, by mixing a dispersion containing superparamagnetic metal oxide particles having a volume average particle size of 1 to 20 nm, 30 to 500% by weight of an (alkyl)alkoxysilane based on the weight of the superparamagnetic metal oxide particles, and optionally a dispersant, with a solution containing water, a water-soluble organic solvent, a nonionic surfactant, and a catalyst for hydrolysis of the (alkyl)alkoxysilane to form an oil-in-water emulsion, followed by hydrolysis and condensation reactions of the (alkyl)alkoxysilane to obtain an aqueous dispersion of magnetic particles in which the superparamagnetic metal oxide particles are encapsulated in a silica matrix, followed by solid-liquid separation of the aqueous dispersion by centrifugation and/or magnetic force, and washing with water, methanol, or the like.
Furthermore, if necessary, the magnetic particles obtained by the above procedure, an (alkyl)alkoxysilane, water, a water-soluble organic solvent, a nonionic surfactant, and a catalyst for hydrolysis of the (alkyl)alkoxysilane may be mixed to carry out a hydrolysis reaction and a condensation reaction of the (alkyl)alkoxysilane, thereby producing silica particles having a core-shell structure.
In the above and following descriptions, the term "(alkyl)alkoxysilane" refers to an alkylalkoxysilane or an alkoxysilane. Examples of the (alkyl)alkoxysilane include tetraethoxysilane.

The above-mentioned silica particles have superparamagnetic metal oxide particles encapsulated in a silica matrix, and the amount of superparamagnetic metal oxide present on the silica particle surface is relatively small, so that a large amount of the substance to be immobilized can be immobilized on the particle surface.

The solid phase carrier (α) is preferably a solid phase carrier (α1) having a substance (antibody) that specifically binds to an antigen (a substance to be measured in a sample) immobilized on its surface.

<Antigen>
The antigen in the present invention is not particularly limited as long as it is generally measured in the field of immunological measurement, and examples thereof include nucleotide chains (oligonucleotide chains, polynucleotide chains) contained in samples derived from living organisms, such as biological fluids (e.g., serum, blood, plasma, urine, etc.), lymph, blood cells, and various types of cells; chromosomes; nucleic acids (e.g., deoxyribonucleic acid (DNA), ribonucleic acid (RNA), etc.); peptide chains (e.g., C-peptide, angiotensin I, etc.); proteins (e.g., procalcitonin, immunoglobulin A (IgA), immunoglobulin E (IgE), etc.); E), immunoglobulin G (IgG), immunoglobulin M (IgM), immunoglobulin D (IgD), β2-microglobulin, albumin, hemoglobin, myoglobin, transferrin, protein A, C-reactive protein (CRP), ferritin, troponin T (TnT), human brain natriuretic peptide precursor N-terminal fragment (NT-proBNP), and degradation products thereof]; blood coagulation-related factors (e.g., fibrinogen, fibrin degradation products, prothrombin, thrombin, etc.); enzymes (e.g., amylase (e.g., pancreatic type, salivary glandular type, X type, etc.), alkaline phosphatase (e.g. hepatic, osseous, placental, small intestinal, etc.), acid phosphatase (e.g. PAP, etc.), γ-glutamyltransferase (e.g. renal, pancreatic, hepatic, etc.), lipase (e.g. pancreatic type, gastric type, etc.), creatine kinase (e.g. CK-1, CK-2, mCK, etc.), lactate dehydrogenase (e.g. LDH1 to LDH5, etc.), glutamic oxaloacetic transaminase (e.g. ASTm, ASTs, etc.), glutamic pyruvic transaminase (e.g. ALTm, ALTs, etc.), cholinesterase (e.g., ChE1 to ChE5, etc.), leucine aminopeptidase (e.g., C-LAP, AA, CAP, etc.), renin, protein kinase, tyrosine kinase, etc.) and inhibitors of these enzymes; hormones (e.g., PTH, TSH, insulin, LH, FSH, estradiol, prolactin, etc.); receptors (e.g., receptors for estrogen, TSH, etc.); ligands (e.g., estrogen, TSH, etc.); bacteria (e.g., Mycobacterium tuberculosis, Streptococcus pneumoniae, Corynebacterium diphtheriae, Neisseria meningitidis, Neisseria gonorrhoeae, Staphylococcus aureus, Streptococcus enterica, Escherichia coli, Helicobacterium tuberculosis, and the like); pylori, etc.); viruses (e.g., rubella virus, herpes virus, hepatitis virus, ATL virus, AIDS virus, influenza virus, adenovirus, enterovirus, poliovirus, EB virus, HAV, HBV, HCV, HIV, HTLV, etc.); fungi (e.g., Candida, Cryptococcus, etc.); spirochetes (e.g., Leptospira, Treponema pallidum, etc.); microorganisms such as Chlamydia and Mycoplasma; protein, peptide, or sugar chain antigens derived from the above microorganisms; bronchial asthma, allergies Various allergens that cause allergies such as rhinitis and atopic dermatitis (e.g., house dust; mites such as house dust mite and house dust mite; pollen from cedar, cypress, paspalum, ragweed, timothy grass, Japanese silvergrass, rye, etc.; animals such as cats, dogs, crabs, etc.; foods such as rice and egg whites; allergens derived from fungi, insects, wood, drugs, chemicals, etc.); lipids (e.g., lipoproteins, etc.); proteases (e.g., trypsin, plasmin, serine proteases, etc.); tumor marker protein antigens (e.g., PSA, PGI, PGII, etc.); sugar chains Antigens (e.g., AFP (e.g., L1 to L3, etc.), hCG (hCG family), transferrin, IgG, thyroglobulin (Tg), decay-accelerating factor (DAF), carcinoembryonic antigen (e.g., CEA, NCA, NCA-2, NFA, etc.), CA19-9, PIVKA-II, CA125, prostate-specific antigen, tumor marker sugar chain antigens having special sugar chains produced by cancer cells, ABO sugar chain antigens, etc.); sugar chains (e.g., hyaluronic acid, β-glucan, sugar chains possessed by the above sugar chain antigens, etc.); proteins that bind to sugar chains (e.g. hyaluronic acid binding protein, β-glucan binding protein, etc.); phospholipids (e.g. cardiolipin, etc.); lipopolysaccharides (e.g. endotoxin, etc.); chemical substances (e.g. environmental hormones such as T3, T4, FT3, FT4, tributyltin, nonylphenol, 4-octylphenol, di-n-butyl phthalate, dicyclohexyl phthalate, benzophenone, octachlorostyrene, di-2-ethylhexyl phthalate, etc.); various drugs administered or inoculated to the human body and their metabolites; aptamers; nucleic acid binding substances; and antibodies thereto.

The antigen is preferably a substance contained in serum or plasma (thyroglobulin, thyroid peroxidase antibody, carcinoembryonic antigen, etc.) in order to fully exert the effects of the present invention.

In the present invention, an analogue of an antigen is any substance that can bind to the antigen-binding site of a substance (antibody) that specifically binds to the antigen, in other words, any substance that has the same binding site as the antigen, or in other words, any substance that can compete with the reaction between the antigen and the antibody when present during the reaction between the antigen and the antibody.

<Antibody>
The antibody in the present invention is a substance that specifically binds to the antigen in the present invention. Examples of the antibody include those that bind to an antigen (substance to be measured) or a similar substance in a sample through mutual reactions 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, or a "nucleotide chain"-"protein" reaction. In other words, in each of the above reactions, when one of the substances to be bound is an antigen (substance to be measured) or a similar substance, the other is an antibody (a substance that specifically binds to an antigen).
The antibodies used in the present invention also include decomposition products such as Fab and F(ab') ₂ fragments produced by proteolytic enzymes such as papain and pepsin, or by chemical decomposition.

In the present invention, examples of a method for immobilizing an antibody on the surface of a solid phase carrier to form the solid phase carrier (α1) include a method in which the antibody is physically adsorbed onto silica particles containing the above-mentioned superparamagnetic metal oxide. From the viewpoint of immobilizing the antibody more efficiently, however, it is preferable 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 the silica particles and immobilize the antibody to the silica particles via the organic compound, and more preferably, to immobilize the antibody via an alkylalkoxysilane having a functional group (such as an ethylenically unsaturated group, an epoxy group, an amino group, a mercapto group, or an isocyanate group).
The method for bonding the organic compound to the surface of the silica particles is not particularly limited, and examples thereof include the method described in Patent Document 1 and the like.
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 Examples of such silane include 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, etc. The alkylalkoxysilane may have two or more different functional groups in one molecule.

The method for immobilizing the antibody on the silica particles via the organic compound is not particularly limited, and examples include known methods (such as those described in JP 2014-210680 A and JP 2013-019889 A).

From the viewpoint of storage stability, the solid phase carrier reagent (A) preferably contains a solid phase carrier (α) and a liquid medium. From the viewpoint of washability of the solid phase carrier, the content of the solid phase carrier (α) in the solid phase carrier reagent (A) is preferably 0.001 to 10% by weight, and more preferably 0.01 to 1% by weight.

The liquid medium may include buffer solutions such as Tris buffer, phosphate buffer, veronal buffer, borate buffer, and Good's buffer, which are generally used in the field of immunoassays.

The liquid medium may also contain gelatin, proteins other than gelatin, serum (mouse serum, etc.), sugars, surfactants, inorganic salts, ethylenediaminetetraacetic acid, and the like.
Gelatin includes known gelatin, and is not limited in molecular weight and properties, and may be obtained from any animal (mammal, bird, fish, etc.). For example, acid-treated gelatin and alkali-treated gelatin produced by chemically treating collagen with acid or alkali and then heat-treating it can be mentioned. Furthermore, gelatin derivatives chemically modified by introducing functional groups such as amino groups, imino groups, carboxyl groups, mercapto groups, and hydroxyl groups into this gelatin using a known method can also be used. From the viewpoint of storage stability of the solid phase carrier reagent (A), the content of gelatin is preferably 1 to 8% by weight, more preferably 2 to 5% by weight, based on the weight of the solid phase carrier reagent (A).

The protein other than gelatin is not particularly limited as long as it is generally used in the field of immunological measurement, and examples thereof include bovine serum albumin (BSA), casein, and skim milk. One type of protein may be used alone, or two or more types may be used in combination. From the viewpoint of storage stability of the solid phase carrier reagent (A), the protein content is preferably 0 to 15% by weight, more preferably 0.1 to 15% by weight, based on the weight of the solid phase carrier reagent (A).
The content of serum is preferably 0 to 15% by weight, more preferably 0.1 to 10% by weight, based on the weight of the solid phase carrier reagent (A).

Sugars include monosaccharides, disaccharides and polysaccharides.
Examples of monosaccharides include trioses (ketotriose, etc.), tetroses (ketotetrose, etc.), pentoses (ketopentoses, aldopentoses, deoxysugars, etc.), hexoses [ketohexoses (psicose, fructose, sorbose, tagatose, etc.), aldohexoses (allose, altrose, glucose, mannose, gulose, idose, galactose, talose, etc.), and deoxysugars (fucose, fuculose, rhamnose, etc.)], and heptoses (sedoheptulose, etc.).
Disaccharides include those in which two molecules of the above-mentioned monosaccharides are dehydrated and condensed to form a glycosidic bond, and specific examples thereof include sucrose, lactose, maltose, and cellobiose.
Polysaccharides include those in which three or more molecules of the above-mentioned monosaccharides are dehydrated and condensed to form glycosidic bonds, and specific examples thereof include amylose, amylopectin, glycogen, cellulose, hyaluronic acid, chondroitin sulfate, and heparin.
The saccharides may be used alone or in combination of two or more kinds.
As the sugar, from the viewpoint of storage stability of the solid phase carrier reagent (A), disaccharides are preferred, and sucrose and lactose are more preferred.
From the viewpoint of storage stability of the solid phase carrier reagent (A), the content of the saccharide is preferably 5 to 40% by weight, more preferably 10 to 20% by weight, based on the weight of the solid phase carrier reagent (A).

The surfactant may be any of the surfactants exemplified in the description of the labeling reagent (C) described in detail below, and the preferred surfactants are the same. The content of the surfactant is preferably 0 to 2% by weight, and more preferably 0.01 to 1% by weight, based on the weight of the solid phase carrier reagent (A).

Examples of inorganic salts include alkali metal salts [halides (sodium chloride, potassium chloride, sodium bromide, sodium fluoride, etc.), sulfates (sodium sulfate, potassium sulfate, etc.), nitrates (sodium nitrate, potassium nitrate, etc.) and phosphates (sodium phosphate, potassium phosphate, etc.)], alkaline earth metal salts [halides (calcium chloride, magnesium chloride, etc.) and sulfates (magnesium sulfate, etc.)]. One type of inorganic salt may be used alone, or two or more types may be used in combination. Among these, from the viewpoint of storage stability of the solid phase carrier reagent (A), at least one type selected from the group consisting of sodium chloride, potassium chloride, magnesium chloride, sodium phosphate, sodium sulfate, potassium sulfate, sodium nitrate and potassium nitrate is preferred.
From the viewpoint of storage stability of the solid phase carrier reagent (A), the content of the inorganic salt is preferably 0.1 to 2% by weight, more preferably 0.5 to 1% by weight, based on the weight of the solid phase carrier reagent (A).

As ethylenediaminetetraacetic acid (EDTA), ethylenediaminetetraacetic acid, its salts (disodium ethylenediaminetetraacetate, trisodium ethylenediaminetetraacetate, tetrasodium ethylenediaminetetraacetate, dipotassium ethylenediaminetetraacetate, tripotassium ethylenediaminetetraacetate, etc.), and hydrates of the salts can be used. The content of ethylenediaminetetraacetic acid, its salts, and hydrates of the salts is preferably 0 to 2% by weight, and more preferably 0.01 to 1% by weight, based on the weight of the solid phase carrier reagent (A).

<Reaction Buffer (B)>
The reaction buffer solution (B) in the present invention contains a buffer solution generally used in the field of immunological measurement, such as Tris buffer solution, phosphate buffer solution, veronal buffer solution, borate buffer solution, Good's buffer solution, etc. The pH of the reaction buffer solution (B) may be in a range that does not inhibit the effects of the present invention, and is preferably 5 to 9.
In this specification, the pH is a value measured at a measurement temperature of 25° C. in accordance with JIS K0400-12-10:2000.
Furthermore, such a buffer solution may contain stabilizers such as salts (sodium chloride, ethylenediaminetetraacetic acid, and the like), albumin (bovine serum albumin, and the like), globulin, protein (casein hydrolysate), water-soluble gelatin, polyethylene glycol, and the like, surfactants (surfactants exemplified in the description of the labeling reagent (C) described below), and sugars (sugars exemplified in the description of the solid phase carrier reagent (A) above), as long as the effects of the present invention are not impaired.

At least one of the solid-phase carrier reagent (A) and the reaction buffer solution (B) in the immunoassay kit of the present invention contains hydrogen peroxide, or the solid-phase carrier reagent (A) contains either one (β) of a catalyst or a substrate in a chemical reaction that produces hydrogen peroxide, and the reaction buffer solution (B) contains the other one (γ) of the catalyst and the substrate, and hydrogen peroxide can be generated by mixing the above (β) and the above (γ). In this case, when the solid-phase carrier reagent (A) and the reaction buffer solution (B) are mixed, a mixture containing hydrogen peroxide is obtained.
Preferably, hydrogen peroxide enhances the inhibitory activity of the peroxidase inhibitor.
In addition, either one (β) of the catalyst and the substrate (γ) in the chemical reaction that produces hydrogen peroxide will hereinafter also be referred to as the “first hydrogen peroxide-generating component (β)” and the “second hydrogen peroxide-generating component (γ),” respectively.

An example of a chemical reaction that produces hydrogen peroxide is a reaction in which oxygen present in a reaction system acts as an electron acceptor to produce hydrogen peroxide. Examples of combinations of catalysts and substrates include those known for use in such reactions, such as combinations of oxidoreductases such as glucose oxidase, cholesterol oxidase, and alcohol oxidase with their substrates.
Among these, from the viewpoint of storage stability, it is preferable that the chemical reaction is an oxidation-reduction reaction, the catalyst is an oxidoreductase, and the substrate is a substrate that is oxidized in the oxidation-reduction reaction, and it is preferable that the oxidoreductase is glucose oxidase and the substrate is glucose (β-D-glucose, etc.).

When at least one of the solid phase carrier reagent (A) and the reaction buffer solution (B) contains hydrogen peroxide, the content of hydrogen peroxide (when both the solid phase carrier reagent (A) and the reaction buffer solution (B) contain hydrogen peroxide, the total content) is not particularly limited as long as it is within the range in which the effects of the present invention are exerted, but the total weight of hydrogen peroxide used in the reaction is preferably 0.05 to 60,000% by weight, and more preferably 0.9 to 20,000% by weight, based on the total weight of the peroxidase inhibitor used in the reaction.

When the solid phase carrier reagent (A) and the reaction buffer solution (B) each contain a first hydrogen peroxide generating component (β) and a second hydrogen peroxide generating component (γ), the contents of these components are not particularly limited as long as they are within a range in which a sufficient amount of hydrogen peroxide can be generated to exert the effects of the present invention. However, the amount of the catalyst used in the reaction is preferably 0.005 to 30,000% by weight, more preferably 0.005 to 100% by weight, and even more preferably 0.5 to 30% by weight, based on the weight of the substrate used in the reaction.
The amount of the substrate is preferably 0.001 to 5% by weight, more preferably 0.01 to 3% by weight, based on the weight of the component containing the substrate [solid phase carrier reagent (A) or reaction buffer solution (B)].

In addition, in the immunological measurement method of the present invention, a solid phase carrier reagent (A) and a reaction buffer solution (B) that do not contain hydrogen peroxide (or the first hydrogen peroxide generating component (β) and the second hydrogen peroxide generating component (γ)) can also be used. In this case, hydrogen peroxide (or the first hydrogen peroxide generating component (β) and the second hydrogen peroxide generating component (γ)) is added when the solid phase carrier reagent (A) and the reaction buffer solution (B) are mixed.

At least one of the solid phase carrier reagent (A) and the reaction buffer solution (B) in the immunoassay kit of the first embodiment further contains a peroxidase inhibitor, which inhibits (inactivates) the activity of peroxidase derived from impurities, thereby suppressing false positives and false high values caused by the peroxidase.
The peroxidase inhibitor is preferably one whose inhibitory activity is enhanced by hydrogen peroxide.
In the immunoassay kit of the second embodiment, the solid phase carrier reagent (A) and the reaction buffer solution (B) may not contain a peroxidase inhibitor. When the solid phase carrier reagent (A) and the reaction buffer solution (B) do not contain a peroxidase inhibitor, a peroxidase inhibitor is added when they are mixed.
In the immunological measurement method of the present invention, when a solid phase carrier reagent (A) and a reaction buffer solution (B) that do not contain a peroxidase inhibitor are used, a peroxidase inhibitor is added when the solid phase carrier reagent (A) and the reaction buffer solution (B) are mixed.

From the viewpoint of storage stability, it is preferable that either the solid phase carrier reagent (A) or the reaction buffer solution (B) in the immunological measurement kit of the present invention contains hydrogen peroxide, and the other contains a peroxidase inhibitor. However, as long as the effects of the present invention can be exhibited, either the solid phase carrier reagent (A) or the reaction buffer solution (B) may contain both hydrogen peroxide and a peroxidase inhibitor.

As the peroxidase inhibitor, known peroxidase inhibitors can be used, and examples thereof include hydrazine derivatives (including hydrazides, such as 4-aminobenzohydrazide, phenylhydrazine, hydrazine sulfate, propanoic acid hydrazide, adipic acid dihydrazide, indole-3-acetic acid hydrazide, 4-fluorobenzoic acid hydrazide, and 6-iodo-pyridine-2-carboxylic acid hydrazide), hydroxamic acid derivatives (salicylhydroxamic acid, 2-cyclohexylethanehydroxamic acid, acetohydroxamic acid, butyrylhydroxamic acid, 2-(3,5-bistrifluoromethylbenzylamino)-6-oxo-1H-pyrimidine-5-carbohydroxamic acid, 4-benzyl-2- hydroxybenzenecarbohydroxamic acid, 2-(benzyl amino)-6-oxo-3H-pyrimidine-5-carbohydroxamic acid, benzohydroxamic acid, 1-naphthohydroxamic acid, 2-naphthohydroxamic acid, etc.), azides (sodium azide, diphenylphosphoric acid azide, etc.), aromatic compounds capable of inhibiting the activity of peroxidase [indole derivatives (indole, tryptamine, serotonin, melatonin, etc.), flavonoids (quercetin, resveratrol, myricytosin, catechin, etc.), etc.], heterocyclic compounds capable of inhibiting the activity of peroxidase [isoniazid, thioxanthine derivatives (2-thioxanthine, 6-thioxanthine, 2-mercapto-8-phenyl-1,9-dihydro-6H-purin-6-one, etc.)], and the like.
Among these, from the viewpoint of the false positive suppression effect, those which can be classified into two or more of hydrazine derivatives, hydroxamic acid derivatives, and aromatic compounds are preferred, and aromatic compounds which are hydrazine derivatives (4-aminobenzohydrazide, phenylhydrazine, indole-3-acetic acid hydrazide, 4-fluorobenzoic acid hydrazide, 6-iodo-pyridine-2-carboxylic acid hydrazide, etc.) and aromatic compounds which are hydroxamic acid derivatives (salicylhydroxamic acid, 2-(3,5-bistrifluoromethylbenzylamino)-6-oxo-1H-pyrimidine-5-carbohydroxamic acid, 4-benzyl-2- hydroxybenzenecarbohydroxamic acid, 2-(benzylamino)-6-oxo-3H-pyrimidine-5-carbohydroxamic acid, benzohydroxamic acid, 1-naphthohydroxamic acid, 2-naphthohydroxamic acid, etc.) are more preferred.
Of these, 4-aminobenzohydrazide and salicylhydroxamic acid are particularly preferred.

The content of the peroxidase inhibitor to be used in the reaction (when both the solid phase carrier reagent (A) and the reaction buffer solution (B) contain a peroxidase inhibitor, the total content) is preferably 10 to 30,000 wt %, more preferably 50 to 15,000 wt %, further preferably 100 to 12,000 wt %, and particularly preferably 100 to 1,500 wt %, based on the total weight of the catalyst to be used in the reaction.
From the viewpoint of further improving the false positive suppression effect, when an aromatic compound which is a hydrazine derivative and an aromatic compound which is a hydroxamic acid derivative are used in combination, the weight ratio of the aromatic compound which is a hydrazine derivative and the aromatic compound which is a hydroxamic acid derivative to be subjected to the reaction (aromatic compound which is a hydrazine derivative/aromatic compound which is a hydroxamic acid derivative) is preferably 0.01 to 300, and more preferably 0.01 to 10.
From the viewpoint of further improving the effect of suppressing false positives, the content of the aromatic compound, which is a hydroxamic acid derivative, to be subjected to the reaction is preferably 100% by weight or less based on the total weight of the catalyst to be subjected to the reaction.

In addition, a solid phase carrier reagent (A) containing a solid phase carrier (α) and a first hydrogen peroxide generating component (β) is also one aspect of the present invention.
From the viewpoint of the false positive suppression effect, the first hydrogen peroxide generating component (β) is more preferably a catalyst, and the catalyst is further preferably glucose oxidase. From the viewpoint of the false positive suppression effect, it is particularly preferable that the solid phase carrier reagent (A) further contains a peroxidase inhibitor.
From the viewpoint of storage stability, the first hydrogen peroxide generating component (β) is more preferably a substrate, and the substrate is preferably glucose.
The solid phase carrier reagent (A) can also be used in the immunoassay kit or immunoassay method of the present invention.

<Labeling Reagent (C)>
The labeling reagent (C) in the present invention includes a substance labeled with a labeling substance. This labeled substance is a substance that can bind to at least one of the solid phase carrier (α) and the antigen to form a labeled complex. Therefore, the resulting labeled complex contains the labeling substance.
The substance labeled with the labeling substance includes an antibody labeled with a labeling substance (labeled antibody), or a labeled antigen or an analogue thereof (labeled antigen or an analogue thereof).
When the solid phase carrier (α) is a solid phase carrier (α1) having an antibody immobilized on its surface, the labeling reagent (C) preferably contains a labeled antibody or a labeled antigen or an analog thereof. When the labeling reagent (C) contains a labeled antibody, it can be used in an immunoassay method (hereinafter also referred to as a sandwich method) in which a complex formed between the solid phase carrier (α1) and an antigen derived from a sample is formed into a labeled complex that is a complex with the labeled antibody. When the labeling reagent (C) contains a labeled antigen or an analog thereof, it can be used in an immunoassay method (hereinafter also referred to as a first competitive method) in which a labeled complex is formed between the solid phase carrier (α1) and a labeled antigen or an analog thereof.

The antibodies, antigens, or similar substances to be labeled can be the same as those described above, and the preferred ones are also the same.

Examples of the labeling substance used for labeling include enzymes such as alkaline phosphatase, β-galactosidase, peroxidase, microperoxidase, glucose oxidase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, luciferase, tyrosinase, and acid phosphatase used in enzyme immunoassay (EIA); radioisotopes such as 99mTc, 131I, 125I, 14C, 3H, and 32P used in radioimmunoassay (RIA); and fluorescein, dansyl, fluorescamine, coumarin, naphthylamine, or derivatives thereof, and green fluorescent protein used in fluorescence immunoassay (FIA). Examples of such substances include fluorescent substances such as fluorescent protein (GFP); luminescent substances such as luciferin, isoluminol, luminol, and bis(2,4,6-trifluorophenyl)oxalate; substances that absorb in the ultraviolet region such as phenol, naphthol, anthracene, and derivatives thereof; and substances that have properties as spin labeling agents, represented by 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 sensitivity and the like, enzymes and fluorescent substances are preferred, alkaline phosphatase, peroxidase and glucose oxidase are more preferred, and peroxidase is particularly preferred.

To bind a labeling substance to an antibody, antigen, or a substance similar thereto, a method generally used in the field of immunological measurement, for example, a known labeling method generally used in known EIA, RIA, FIA, etc. [for example, the glutaraldehyde method, periodic acid method, maleimide method, or pyridyl disulfide method described in Medical Chemistry Experiment Lectures, Vol. 8, edited by Yamamura Yuichi, 1st Edition, Nakayama Shoten, 1971; Illustrated Fluorescent Antibodies, by Kawao Akira, 1st Edition, Soft Science Co., Ltd., 1983; Enzyme Immunoassay Method, edited by Ishikawa Eiji, Kawai Tadashi, and Muroi Kiyoshi, 2nd Edition, Igaku Shoin, 1982, etc.] may be used.

The amount of labeling substance used varies depending on the type of labeling substance, so it cannot be generalized. However, when peroxidase (hereinafter also referred to as POD) is used as the labeling substance, the antibody, antigen or similar substance and peroxidase are preferably used in a molar ratio of 1:1 to 1:20 (more preferably 1:1 to 1:10, particularly preferably 1:1 to 1:2), and these may be used by being contained in a buffer solution typically used in this field, such as Tris buffer, phosphate buffer, veronal buffer, borate buffer, Good's buffer, etc. The pH of the buffer may be in a range that does not inhibit the antigen-antibody reaction, and is preferably 5 to 9. In addition, such a buffer may contain stabilizers such as albumin, globulin, water-soluble gelatin, polyethylene glycol, surfactants, sugars, etc., as long as they do not inhibit the target antigen-antibody reaction.

From the viewpoint of sensitivity, the content of the labeled antibody or labeled antigen and its analogues in the labeled reagent (C) is preferably 0.01 to 40 μg/mL, and more preferably 0.1 to 20 μg/mL.

The labeling reagent (C) may contain, in addition to the above, a protein, a surfactant, and a polymer compound.
The protein is not particularly limited as long as it is one that is generally measured in the field of immunological measurement, and examples thereof include bovine serum albumin (BSA), casein, skim milk, etc. One type of protein may be used alone, or two or more types may be used in combination. From the viewpoints of sensitivity and storage stability of the reagent, the content of the protein is preferably 0.001 to 8% by weight based on the weight of the labeling reagent (C).

Examples of the surfactant include known nonionic surfactants, amphoteric surfactants, anionic surfactants, etc., but from the viewpoint of reducing nonspecific adsorption, water-soluble nonionic surfactants are preferred. Here, water-soluble means that 10 g of the surfactant dissolves in 100 g of water at 25° C.
Specific examples of the water-soluble nonionic surfactant include polyoxyethylene nonylphenyl ether, polyoxyethylene alkyl ether (polyoxyethylene octyl ether, etc.), and polyoxyethylene sorbitan aliphatic esters, each having an HLB of 12 or more.
In the present invention, HLB (hydrophile-lipophile balance) means an HLB that can be measured by the Griffin method [Griffin method described in "Introduction to Surfactants" (published by Sanyo Chemical Industries, Ltd. in 2007, written by Takehiko Fujimoto), p. 142].
From the viewpoint of washing properties of the particles, the content of the surfactant is preferably 0.001 to 4% by weight based on the weight of the labeling reagent (C).

The polymer compound is not particularly limited as long as it is generally used in the field of immunological measurements, and examples thereof include polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, Blockmaster (manufactured by JSR Corporation) and Lipidure (manufactured by NOF Corporation). The polymer compound may be used alone or in combination of two or more types. From the viewpoint of suppressing nonspecific adsorption, the content of the polymer compound is preferably 0.001 to 3 wt %, more preferably 0.5 to 1 wt %, based on the weight of the labeling reagent (C).

<Luminescent Reagent (D)>
In the immunoassay method of the present invention, a luminescent reagent (D) may be used to cause the labeled complex to emit light to detect or quantify the antigen. The immunoassay kit of the present invention contains the luminescent reagent (D).
The luminescent reagent (D) is selected based on the labeling substance. When the labeling substance is peroxidase, the luminescent reagent (D) may include a first chemiluminescent reagent solution containing a 2,3-dihydro-1,4-phthalazinedione derivative and a chemiluminescence enhancer as essential components, and a second chemiluminescent reagent solution containing an oxidizing agent and water as essential components.

As the 2,3-dihydro-1,4-phthalazinedione derivative, for example, known 2,3-dihydro-1,4-phthalazinedione derivatives described in JP-A-2-291299, JP-A-10-319015, JP-A-2000-279196, and mixtures thereof can be used.
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 metal salts thereof are more preferred, and sodium salt of luminol is particularly preferred.
The content of the 2,3-dihydro-1,4-phthalazinedione derivative in the luminescent reagent (D) is appropriately set depending on the type thereof and the measurement method and measurement conditions to be applied, but from the viewpoints of the chemiluminescence enhancing effect, storage stability, and the like, it is preferably 0.5 to 80 mM, more preferably 1.8 to 40 mM, and particularly preferably 3.5 to 21 mM.

Examples of the chemiluminescence enhancer that can be used include known chemiluminescence enhancers described in, for example, JP-A-59-500252, JP-A-59-171839, and JP-A-2-291299, and mixtures thereof. Among these, from the viewpoint of the chemiluminescence enhancing effect, etc., phenols are preferred, more preferred are p-iodophenol, 4-(cyanomethylthio)phenol, and 4-cyanomethylthio-2-chlorophenol, and particularly preferred is 4-(cyanomethylthio)phenol.
The content of the chemiluminescence enhancer in the luminescence reagent (D) is appropriately set depending on the type thereof and the measurement method and measurement conditions to be applied, but from the viewpoints of the chemiluminescence enhancing effect, storage stability, etc., it is preferably 0.1 to 15 mM, more preferably 0.3 to 7.0 mM, and particularly preferably 0.6 to 3.4 mM.

The first chemiluminescent reagent solution may contain a buffer solution and/or a chelating agent in addition to the 2,3-dihydro-1,4-phthalazinedione derivative and the chemiluminescent enhancer.

As the buffer solution, for example, known buffer solutions described in JP-A-10-319015 and JP-A-2003-279489 can be used. Among these, from the viewpoints of chemiluminescence enhancing effect and storage stability, etc., 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.
The content of the buffering agent in these buffer solutions is preferably 1 to 500 mM, more preferably 5 to 300 mM, and particularly preferably 10 to 200 mM, from the viewpoints of chemiluminescence enhancing effect and storage stability.

As the chelating agent, for example, known chelating agents described in JP-A-9-75099 and JP-A-2003-279489 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 are ethylenediaminetetraacetic acid (EDTA) and salts thereof.
When a chelating agent is contained, the content thereof is preferably 0.001 to 4% by weight, more preferably 0.01 to 2% by weight, and particularly preferably 0.05 to 1% by weight, based on the weight of the 2,3-dihydro-1,4-phthalazinedione derivative, from the viewpoints of the chemiluminescence enhancing effect and storage stability.

From the standpoint of fluorescence intensity, the first chemiluminescent reagent liquid is preferably alkaline, with a pH of preferably 7 to 11, and more preferably 8 to 10.

The first chemiluminescent reagent solution can be easily obtained by homogeneously mixing the 2,3-dihydro-1,4-phthalazinedione derivative, the chemiluminescence enhancer, and, if necessary, a buffer solution and/or a chelating agent.

Examples of the oxidizing agent contained in the second chemiluminescent reagent solution include known oxidizing agents described in JP-A-8-261943 and JP-A-2000-279196, etc. [inorganic peroxides (hydrogen peroxide, sodium perborate, potassium perborate, etc.), organic peroxides (dialkyl peroxides, acyl peroxides, etc.), peroxy acid compounds (peroxosulfuric acid, peroxolinic acid, etc.), etc.].
Among these, from the viewpoint of storage stability, hydrogen peroxide, sodium perborate and potassium perborate are preferred, and hydrogen peroxide is more preferred.

The concentration of the oxidizing agent in the second chemiluminescent reagent solution is set appropriately depending on the type of oxidizing agent and the measurement method and conditions to be applied, but from the viewpoint of the chemiluminescence enhancement effect, etc., it is preferably 0.5 to 40 mM, more preferably 1 to 20 mM, and particularly preferably 2.5 to 10 mM.

Examples of the water contained in the second chemiluminescent reagent liquid include distilled water, reverse osmosis water, and deionized water. Of these, from the viewpoints of chemiluminescence enhancement effect and storage stability, distilled water and deionized water are preferred, and deionized water is even more preferred.

The second chemiluminescent reagent liquid may contain a chelating agent and the like in addition to an oxidizing agent and water.
Examples of the chelating agent include the same ones as those exemplified as the chelating agents that can be contained in the first chemiluminescent reagent solution, and the preferred ones are also the same.
When a chelating agent is contained, the content thereof is preferably 0.2 to 100% by weight, more preferably 0.5 to 20% by weight, and particularly preferably 1 to 10% by weight, based on the weight of the oxidizing agent, from the viewpoints of the chemiluminescence enhancing effect and storage stability.
The second chemiluminescent reagent solution can be easily obtained by uniformly mixing an oxidizing agent, water and, if necessary, a chelating agent.

<Immunological measurement method>
The immunoassay method of the present invention can be used as a method for detecting or quantifying an antigen in a sample. Specifically, the method can be described in the literature [for example, Enzyme Immunoassay Method, 2nd Edition (edited by Eiji Ishikawa et al., Igaku Shoin Publishing Co., Ltd.]. ) 1982] and the immunoassay method described in JP-A-6-130063.

In the immunological measurement method of the present invention, first, a first mixture containing a complex of an antigen and a solid phase carrier (α) is obtained. The step of obtaining the first mixture includes a step of mixing a sample containing an antigen, a peroxidase inhibitor, hydrogen peroxide, and a solid phase carrier (α) to obtain a second mixture containing a complex of an antigen and a solid phase carrier (α), and a step of removing the peroxidase inhibitor after the step of obtaining the second mixture.

In the step of obtaining the second mixture, the mixing order of the antigen-containing sample, the peroxidase inhibitor, the hydrogen peroxide, and the solid phase carrier (α) is not particularly limited, and they may be mixed simultaneously or in two or more stages. In addition, hydrogen peroxide may be generated using the first hydrogen peroxide generating component (β) and the second hydrogen peroxide generating component (γ). In that case, the mixing order of the antigen-containing sample, the peroxidase inhibitor, the solid phase carrier (α), the first hydrogen peroxide generating component (β), and the second hydrogen peroxide generating component (γ) is also not particularly limited. For example, the first hydrogen peroxide generating component (β) and the second hydrogen peroxide generating component (γ) may each be mixed in advance with other components [such as the solid phase carrier (α), the peroxidase inhibitor, and the sample containing the antigen].
When these components are mixed in two or more stages, the following mixing order can be mentioned, for example.
(a) A sample containing an antigen is mixed with a solid phase carrier (α), and then a peroxidase inhibitor and hydrogen peroxide are simultaneously or sequentially mixed therewith.
(b) A sample containing an antigen is mixed with a peroxidase inhibitor, and then a solid phase carrier (α) and hydrogen peroxide are simultaneously or sequentially mixed therewith.
(c) A sample containing an antigen is mixed with hydrogen peroxide, and then a solid phase carrier (α) and a peroxidase inhibitor are simultaneously or sequentially mixed therewith.
(d) The solid phase carrier (α) is mixed with a peroxidase inhibitor, and then a sample containing an antigen and hydrogen peroxide are simultaneously or sequentially mixed therewith.
(e) The solid phase carrier (α) is mixed with hydrogen peroxide, and then a sample containing an antigen and a peroxidase inhibitor are simultaneously or sequentially mixed therewith.
(f) A peroxidase inhibitor and hydrogen peroxide are mixed, and then a sample containing an antigen and a solid phase carrier (α) are simultaneously or sequentially mixed therewith.
(g) The solid phase carrier (α), a peroxidase inhibitor and hydrogen peroxide are mixed, and then a sample containing an antigen is mixed therewith.
(h) A sample containing an antigen is mixed with a peroxidase inhibitor and hydrogen peroxide, and then mixed with a solid phase carrier (α).
(i) The solid phase carrier (α), a sample containing an antigen, and a peroxidase inhibitor are mixed, and then hydrogen peroxide is further mixed therewith.
(j) The solid phase carrier (α), a sample containing an antigen, and hydrogen peroxide are mixed, and further a peroxidase inhibitor is mixed therewith.
In the above (a) to (f), when the components are mixed simultaneously, the components to be mixed simultaneously may be premixed together (for example, in the case of (a), a mixture of a peroxidase inhibitor and hydrogen peroxide).
Moreover, instead of hydrogen peroxide, the first hydrogen peroxide generating component (β) and the second hydrogen peroxide generating component (γ) may be used as described above.
Also, each component may be mixed in two or more portions. For example, in the above (d), the peroxidase inhibitor may be mixed in two or more portions. That is, the solid phase carrier (α) and the peroxidase inhibitor may be mixed, and then, when the sample containing the antigen and hydrogen peroxide are mixed, the peroxidase inhibitor may be additionally mixed.

By mixing these components, the peroxidase inhibitor can inactivate the peroxidase derived from the impurities, thereby suppressing false positives and false high values caused by the peroxidase. In addition, the inhibitory activity of the peroxidase inhibitor can be improved by hydrogen peroxide. Furthermore, a complex consisting of the antigen in the sample and the solid phase carrier (α) can be formed. In this case, the solid phase carrier (α) is preferably a solid phase carrier (α1) having an antibody immobilized on its surface.

When using the immunoassay kit of the present invention, a mixture of the solid phase carrier (α), the peroxidase inhibitor, and hydrogen peroxide can be obtained by mixing the solid phase carrier reagent (A) with the reaction buffer (B) [when using a solid phase carrier reagent (A) and a reaction buffer (B) that do not contain a peroxidase inhibitor, by mixing the peroxidase inhibitor simultaneously with or after mixing the solid phase carrier reagent (A) with the reaction buffer (B)]. In this case, the inhibitory activity of the peroxidase inhibitor can be improved by the hydrogen peroxide.
When the solid phase carrier reagent (A) contains the solid phase carrier (α) and a liquid medium, the supernatant (part of the liquid medium) may be removed, and the precipitated solid phase carrier (α) and the remaining liquid medium may be mixed with the reaction buffer (B). In this case, from the viewpoint of quantitativeness, it is preferable that the entire amount of the solid phase carrier (α) contained in the solid phase carrier reagent (A) is mixed with the reaction buffer (B). In addition, when the solid phase carrier reagent (A) contains hydrogen peroxide or the first hydrogen peroxide generating component (β) and/or a peroxidase inhibitor, it is necessary to remove the supernatant so that at least a part of the peroxidase inhibitor and hydrogen peroxide remains in the mixture. The amount of the supernatant to be removed is preferably such that the amount of the remaining components to be subjected to the reaction [the first hydrogen peroxide generating component (β), the second hydrogen peroxide generating component (γ) and the peroxidase inhibitor, etc.] is in the above-mentioned preferred ratio. In addition, from the viewpoint of accuracy, it is preferable that the volume of the liquid remaining after removal is 10% or less with respect to the reaction buffer (B).
The mixture obtained above further contains a sample containing an antigen, which constitutes the second mixture of the present invention. In the second mixture, the peroxidase inhibitor inhibits the activity of peroxidase derived from impurities contained in the sample. Furthermore, a complex between the antigen in the sample and the solid phase carrier (α) is obtained by the procedure for obtaining the second mixture.
In the operation for obtaining the second mixture, the order of mixing the solid phase carrier reagent (A), the reaction buffer solution (B) and the sample containing the antigen is not limited, and they may be mixed simultaneously, or the sample may be mixed with either the solid phase carrier reagent (A) or the reaction buffer solution (B) and then mixed with the other. Of course, even when using a solid phase carrier reagent (A) and a reaction buffer solution (B) that do not contain a peroxidase inhibitor, the order of mixing the solid phase carrier reagent (A), the reaction buffer solution (B), the sample containing the peroxidase inhibitor and the antigen is not limited.
In the step of obtaining the first mixture, the temperature of the second mixture is preferably 20 to 50°C, and more preferably 25 to 45°C.
The lower limit of the time from obtaining the second mixture to performing the step of removing the peroxidase inhibitor is preferably 30 seconds or more, more preferably 1 minute or more, and the upper limit of the time from obtaining the second mixture to performing the step of removing the peroxidase inhibitor is not particularly limited, but may be 24 hours or less, 1 hour or less, 30 minutes or less, 10 minutes or less, 5 minutes or less, etc.

After obtaining the second mixture, the peroxidase inhibitor is removed.
When the labeling substance contained in the labeling reagent (C) or the labeled complex is peroxidase, it is necessary to remove the peroxidase inhibitor from the system before using the labeling reagent (C) in order to avoid inactivation of the labeling substance by the peroxidase inhibitor.
The B/F separation method can be used as a method for removing the peroxidase inhibitor. After removal, the residue may be washed. For example, when using a solid phase carrier (α1) that is a magnetic particle, after a complex is formed between the antigen in the sample and the solid phase carrier (α1), the magnetic particles are collected from the outside of the reaction vessel using a magnet or the like, the reaction liquid is discharged to remove the peroxidase inhibitor, and then a washing liquid such as physiological saline is added. Thereafter, the magnet is removed, and the magnetic particles are dispersed and washed. This operation may be repeated 1 to 3 times.
The first mixture is obtained by removing the peroxidase inhibitor from the second mixture. Note that, when removing the peroxidase inhibitor, hydrogen peroxide may be removed.

Next, the antigen is measured using the first mixture and the labeled reagent (C).
For example, a labeling reagent (C) is added to the obtained first mixture to form a labeled complex. Thereafter, the amount of the labeled substance in the labeled complex is measured, and the substance to be measured in the sample is detected or quantified based on the result.
Methods for measuring the amount of labeled substance in a labeled complex include radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA) and chemiluminescence immunoassay (CLIA and CLEIA). From the viewpoint of sensitivity in short-term immunological measurements, EIA, CLIA and CLEIA are preferred, and CLEIA is more preferred.

For example, in the sandwich method, in the step of obtaining the second mixture, the sample containing the antigen comes into contact with the solid phase carrier (α1), and the solid phase carrier (α1) binds to the antigen in the sample to form a complex. After this, the peroxidase inhibitor is removed and a labeling reagent (C) is added, whereby the labeled antibody comes into contact with the complex, forming a complex (labeled complex) of the solid phase carrier (α1), the antigen, and the labeled antibody.

In the first competitive method, in the step of obtaining the second mixture, the sample containing the antigen comes into contact with the solid phase carrier (α1), and a complex is formed between the antibody of a part of the solid phase carrier (α1) and the antigen in the sample. After this, when the peroxidase inhibitor is removed and the labeling reagent (C) is added, the antibody of the remaining solid phase carrier (α1) comes into contact with the labeled antigen or its analogue in the labeling reagent, and a complex (labeled complex) between the solid phase carrier (α1) and the labeled antigen or its analogue is formed.

Next, the antigen is detected or quantified by causing the labeled complex to emit light using, for example, a luminescent reagent (D).
In this step, first, the labeled antibody or labeled antigen or its analogue that was not involved in the formation of the labeled complex is separated, for example, by B/F separation. In the sandwich method, B/F separation means separation of the solid phase carrier (α1), the complex of the solid phase carrier (α1) and the antigen, and the labeled complex from other components (such as the labeled antibody that was not involved in the formation of the labeled complex). In the first competitive method, B/F separation means separation of the solid phase carrier (α1), the complex of the solid phase carrier (α1) and the antigen, and the labeled complex from other components (such as the labeled antigen or its analogue that was not involved in the formation of the labeled complex). B/F separation can be performed by the above-mentioned separation method using a magnet, etc.

Subsequently, for example, a luminescent reagent (D) is added, the accumulated amount of chemiluminescence over a certain period of time is measured with a chemiluminometer, and the amount of labeled substance is calculated based on the measured value. In this case, the amount of labeled substance can be easily calculated by using a calibration curve showing the relationship between the amount of labeled substance and the accumulated amount of chemiluminescence, which is prepared by a similar procedure using a solution containing a predetermined labeled substance as a sample. The amount of antigen in the sample can be quantified based on this amount of labeled substance.

The following items are disclosed in this specification:

The present disclosure (1) is an immunoassay method including a step of obtaining a first mixture containing a complex of an antigen and a solid phase carrier (α), and a step of measuring the antigen using the first mixture and a labeling reagent (C), in which the step of obtaining the first mixture includes a step of mixing a sample containing the antigen, a peroxidase inhibitor, hydrogen peroxide, and the solid phase carrier (α) to obtain a second mixture containing the complex of the antigen and the solid phase carrier (α), and a step of removing the peroxidase inhibitor after the step of obtaining the second mixture.

The present disclosure (2) is an immunoassay kit used in the immunoassay method described in the present disclosure (1), comprising a solid-phase carrier reagent (A), a reaction buffer solution (B), the labeling reagent (C), and a luminescent reagent (D), in which the solid-phase carrier reagent (A) contains the solid-phase carrier (α), at least one of the solid-phase carrier reagent (A) and the reaction buffer solution (B) contains the peroxidase inhibitor, and at least one of the solid-phase carrier reagent (A) and the reaction buffer solution (B) contains the hydrogen peroxide, or the solid-phase carrier reagent (A) contains either one of the catalyst and the substrate (β) in a chemical reaction that produces hydrogen peroxide, and the reaction buffer solution (B) contains the other one of the catalyst and the substrate (γ), and hydrogen peroxide can be generated by mixing the (β) and the (γ).

The present disclosure (3) is an immunoassay kit used in the immunoassay method described in the present disclosure (1), comprising a solid-phase carrier reagent (A), a reaction buffer solution (B), the peroxidase inhibitor, the labeling reagent (C), and a luminescent reagent (D), in which the solid-phase carrier reagent (A) contains the solid-phase carrier (α), and at least one of the solid-phase carrier reagent (A) and the reaction buffer solution (B) contains the hydrogen peroxide, or the solid-phase carrier reagent (A) contains either one (β) of the catalyst and the substrate in a chemical reaction that produces hydrogen peroxide, and the reaction buffer solution (B) contains the other (γ) of the catalyst and the substrate, and hydrogen peroxide can be generated by mixing the (β) and the (γ).

The present disclosure (4) is a kit for immunological measurement according to the present disclosure (2) or (3), in which the chemical reaction is an oxidation-reduction reaction, the catalyst is an oxidoreductase, and the substrate is a substrate that is oxidized in the oxidation-reduction reaction.

The present disclosure (5) is the immunoassay kit according to the present disclosure (4), in which the oxidoreductase is glucose oxidase and the substrate is glucose.

The present disclosure (6) is a kit for immunological measurement according to any one of the present disclosures (2) to (5), in which the solid phase carrier (α) is a solid phase carrier (α1) having a substance (antibody) that specifically binds to the antigen immobilized on its surface, and the labeling reagent (C) contains the antibody labeled with peroxidase (labeled antibody).

The present disclosure (7) is a kit for immunological measurement according to any one of the present disclosures (2) to (5), in which the solid phase carrier (α) is a solid phase carrier (α1) having a substance (antibody) that specifically binds to the antigen immobilized on its surface, and the labeling reagent (C) contains the antigen or an analog thereof labeled with peroxidase (labeled antigen or an analog thereof).

The present disclosure (8) is a solid-phase carrier reagent (A) containing a solid-phase carrier (α) and either a catalyst or a substrate (β) in a chemical reaction that produces hydrogen peroxide.

The present disclosure (9) is a solid phase carrier reagent (A) described in the present disclosure (8) that contains the above catalyst.

The present disclosure (10) is the solid phase carrier reagent (A) described in the present disclosure (9) in which the catalyst is glucose oxidase.

The present disclosure (11) is a solid phase carrier reagent (A) described in any one of the present disclosures (8) to (10), further containing a peroxidase inhibitor.

The present disclosure (12) is a solid phase carrier reagent (A) described in any one of the present disclosures (8) to (11) for obtaining an immunoassay kit described in any one of the present disclosures (2) to (7).

The present invention will be further explained below with reference to examples, but the present invention is not limited thereto. In the following, parts are parts by weight.

Example 1
By the method described below, an immunoassay kit (S-1) of the present invention was obtained, which is composed of a solid phase carrier reagent (A-1), a reaction buffer solution (B-1), a labeling reagent (C-1) and a luminescent reagent (D-1) [luminol luminescent reagent (D1) and hydrogen peroxide solution (D2)].

[Preparation of magnetic particles (PH-1)]
Preparation of superparamagnetic metal oxide particles In a reaction vessel, 186 parts of iron (III) chloride hexahydrate, 68 parts of iron (II) chloride tetrahydrate, and 1288 parts of water were charged and dissolved, and the temperature was raised to 50 ° C., and while maintaining the temperature under stirring at 50 to 55 ° C., 280 parts of 25 wt % ammonia water was dropped over 1 hour to obtain magnetite particles in water. 64 parts of oleic acid, a dispersant, was added to the obtained magnetite particles, and stirring was continued for 2 hours. After cooling to room temperature, the magnetite particles with oleic acid adsorbed thereon obtained by solid-liquid separation by decantation were washed with 1000 parts of water three times, and further washed with 1000 parts of acetone twice, and dried at 40 ° C. for 2 days to obtain superparamagnetic metal oxide particles with a volume average particle size of 15 nm.

Preparation of Core Layer: 80 parts of the obtained superparamagnetic metal oxide particles were added to 240 parts of tetraethoxysilane and dispersed to prepare dispersion (1).
Next, 5,050 parts of water, 3,500 parts of a 25% by weight aqueous ammonia solution, and 400 parts of a nonionic surfactant ("NSA-17", manufactured by Sanyo Chemical Industries, Ltd.) were added to a reaction vessel and mixed using Clearmix (manufactured by M Technique Co., Ltd.) to obtain solution (2). After heating to 50°C, the above dispersion liquid (1) was added dropwise to solution (2) over 1 hour while stirring at a rotation speed of 6,000 rpm of the Clearmix, and the reaction was allowed to proceed at 50°C for 1 hour. After the reaction, the mixture was centrifuged at 2,000 rpm for 20 minutes to remove the supernatant containing the fine particles, and a core layer was obtained.

Preparation of magnetic particles 80 parts of the core layer, 2500 parts of deionized water, 260 parts of 25 wt% aqueous ammonia, 2500 parts of ethanol, and 1200 parts of tetraethoxysilane were added to a reaction vessel and mixed using Clearmix (M Technique Co., Ltd.), and reacted for 2 hours while stirring at a rotation speed of 6,000 rpm. After the reaction, the mixture was centrifuged at 2,000 rpm for 20 minutes to remove the supernatant containing the fine particles. After centrifugation, 4000 parts of deionized water was added to the precipitated particles to redisperse the particles, and the dispersed particles were collected using a magnet, and the operation of removing the supernatant was repeated 10 times.
Next, 5,000 parts of water was added to the obtained solid phase to disperse the particles, and the mixture was centrifuged at 600 rpm for 10 minutes. The procedure of removing the supernatant containing the fine particles was repeated 20 times, and then 5,000 parts of water was added to the obtained solid phase to disperse the particles, and the mixture was centrifuged at 300 rpm for 10 minutes to allow particles with large particle sizes to settle and be removed, thereby classifying the particles.
Furthermore, the particles were collected using a magnet, and the supernatant was removed. Then, 5,000 parts of water was added to disperse the particles, and the particles were collected using a magnet, and the operation of removing the supernatant was repeated 10 times to obtain magnetic particles (PH-1) having a target volume average particle size of 2.0 μm. The content of superparamagnetic metal oxide particles in the obtained magnetic particles (PH-1) was measured, and the content was 81 wt %.

[Measurement of volume average particle size of particles (superparamagnetic metal oxide particles and magnetic particles)]
Using a scanning electron microscope (model number: JSM-7000F, manufacturer: JEOL Ltd.), 200 randomly selected particles were observed to measure their particle sizes, and the volume average particle size was calculated.

[Measurement of the content of superparamagnetic metal oxide particles in magnetic particles]
Any 20 magnetic particles were observed with a scanning electron microscope (model number JSM-7000F, manufacturer: JEOL Ltd.), and the content of superparamagnetic metal oxide particles was measured with an energy dispersive X-ray spectrometer (model number INCA Wave/Energy, manufacturer: Oxford University), and the average value was taken as the content S. The silica content was also measured by the same measurement, and the average value was taken as the content T. The content of superparamagnetic metal oxide particles was calculated using the following calculation formula (1).
Content of superparamagnetic metal oxide particles (weight %) = (S) / (S + T) × 100... (1)

[Preparation of anti-Tg monoclonal antibody F(ab')2]
10 mg of anti-Tg monoclonal antibody (mouse) (manufactured by HyTest) was dissolved in 2 ml of 0.1 M acetate buffer at pH 4.0, and 0.2 mg of pepsin was added and incubated for 3 hours at 37° C. An appropriate amount of 1 M carbonate buffer (pH 9.0) was added to neutralize (pH: 7.0) to terminate the reaction, and gel filtration was performed using an Ultragel AcA-44 (LKB) column (02, OX70 cm, manufactured by Sigma-Aldrich) equilibrated with 0.02 M phosphate buffer (pH 7.2) containing 0.2 M sodium chloride, followed by dialysis against 0.05 M phosphate buffer (pH 6.0) containing 5 mM EDTA.2Na to obtain anti-Tg monoclonal antibody F(ab')2.

[Preparation of solid phase carrier reagent (A-1) containing magnetic particles (H-1)]
40 mg of the magnetic particles (PH-1) produced was added to a polyethylene bottle with a lid containing 40 mL of 1 wt % γ-aminopropyltriethoxysilane-containing acetone solution, and the mixture was reacted at 25 ° C for 1 hour. The magnetic particles were collected with a neodymium magnet, and 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 particles were collected with a neodymium magnet, and the liquid was removed by aspirating with an aspirator to wash the magnetic particles. This washing operation was performed five times. Next, the washed magnetic particles were added to a polyethylene bottle with a lid containing 40 mL of a 2 wt % glutaraldehyde-containing aqueous solution, 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 particles were collected with a neodymium magnet, and the liquid was removed by aspirating with an aspirator to wash the magnetic particles. This washing operation was performed ten times.
Furthermore, these washed magnetic particles were added to a lidded polyethylene bottle containing 120 mL of 0.02 M phosphate buffer (pH 8.7) containing anti-Tg monoclonal antibody F(ab')2 at a concentration of 10 μg/mL and peroxidase at a concentration of 3.64 μg/mL, and reacted for 1 hour at 25° C. After the reaction, the magnetic particles (H-1) were collected with a neodymium magnet, and then the anti-Tg monoclonal antibody F(ab')2 and the peroxidase-containing phosphate buffer were removed.
Next, the solid phase carrier reagent (A-1) containing the magnetic particles (H-1) was diluted with a diluent for the solid phase carrier reagent (A-1) described below so that the concentration of the magnetic particles (H-1) became 0.01% by weight, thereby preparing a solid phase carrier reagent (A-1) containing the magnetic particles (H-1).
It was confirmed by the following method that peroxidase was bound to the magnetic particles (H-1). The magnetic particles (H-1) are a model of the state in which peroxidase derived from impurities is adsorbed to the solid phase carrier (α).

[Method for confirming that peroxidase is bound to magnetic particles (H-1)]
0.025 mL of solid phase carrier reagent (A-1) containing magnetic particles (H-1) was placed in a test tube, and the magnetic particles were collected from the outside of the test tube with a neodymium magnet for 10 seconds, after which the liquid in the test tube was removed with an aspirator. Then, 0.5 mL of physiological saline was added to disperse the magnetic particles, and the particles were collected again, and a washing operation of removing the liquid with an aspirator was performed twice.
Thereafter, 0.07 mL of luminol luminescence reagent (D1) and 0.07 mL of hydrogen peroxide solution (D2) were added simultaneously, and the luminescence behavior was confirmed at 37°C.
The above confirmation test was carried out using a "solid phase carrier reagent (AT-1) containing magnetic particles (HT-1)" described below instead of the "solid phase carrier reagent (A-1) containing magnetic particles (H-1)," and the luminescence behavior was confirmed at 37°C.
When the solid phase carrier reagent (AT-1) was used, almost no luminescence was observed, whereas when the solid phase carrier reagent (A-1) was used, luminescence was observed. Since peroxidase is bound to the magnetic particles (H-1), it is believed that luminescence occurs even without the addition of the labeling reagent (C).

[Preparation of diluent for solid phase carrier reagent (A-1)]
The components shown in "Dilution solution for solid phase carrier reagent (A)" in Table 1-1 were mixed with 10% by weight of BSA (bovine serum albumin), 0.1% by weight of Naroacty CL-100 [polyoxyalkylene alkyl ether, manufactured by Sanyo Chemical Industries, Ltd.], 0.1% by weight of EDTA salt (ethylenediamine-N,N,N',N'-tetraacetic acid disodium salt dihydrate, manufactured by Dojindo Laboratories, Ltd.), 5% by weight of mouse serum [manufactured by Cosmo Bio Co., Ltd.], and the remainder of 0.02 M sodium phosphate (pH 7.0) to achieve the weight ratios shown, and stored refrigerated (2 to 10°C).

[Preparation of reaction buffer solution (B-1)]
Each raw material shown in "Reaction buffer solution (B)" in Table 1-1 was mixed with 0.05% by weight of EDTA, 0.5% by weight of BSA (bovine serum albumin), and the remainder of 0.02 M sodium phosphate (pH 7.0) in the weight ratio shown, to give a reaction buffer solution (B-1).

[Preparation of labeling reagent (C-1)]
A POD-labeled anti-human IgG polyclonal antibody (rabbit) (F3-1) was prepared using an anti-human IgG polyclonal antibody (rabbit) (Agilent Technologies, Inc.) and horseradish-derived POD (Toyobo Co., Ltd.) by the method described in the literature (Yoshitake S. et al., J. Biochem., 92, 1413-1424 (1982)). This was diluted with 0.02 M phosphate buffer (pH 7.0) containing 0.5 wt % bovine serum albumin and 1 wt % Naroacty CL-100 as a surfactant so that the concentration of the POD-labeled anti-human IgG polyclonal antibody (rabbit) (F3-1) was 100 nM to prepare a labeled reagent (C-1), which was then stored in a refrigerator (2 to 10° C.).

[Preparation of luminol luminescence reagent (D1)]
0.7 g of luminol sodium salt [Sigma-Aldrich] and 0.1 g of 4-(cyanomethylthio)phenol were charged into a 1,000 mL measuring flask. Furthermore, 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid/sodium hydroxide buffer (10 mM, pH = 8.6) was charged so that the volume of the solution became 1,000 mL, and the mixture was mixed uniformly at 25°C to prepare luminol luminescence reagent (D1). The mixture was stored in a refrigerator (2 to 10°C) until it was used for the measurement.

[Preparation of hydrogen peroxide solution (D2)]
6.6 g of hydrogen peroxide [FUJIFILM Wako Pure Chemical Industries, Ltd., special grade reagent, concentration 30 wt%] was charged into a 1,000 mL measuring flask. Further, deionized water was charged so that the volume of the solution became 1,000 mL, and the solution was mixed uniformly at 25°C to prepare hydrogen peroxide solution (D2). The solution was stored in a refrigerator (2 to 10°C) until it was used for the measurement.

<Examples 2 to 27 and Reference Examples 1 and 2>
In the preparation of the immunoassay kit (S-1) in Example 1, the solid phase carrier reagent (A-1) and the reaction buffer solution (B-1) were changed as follows, but the procedure was the same as in Example 1 to obtain the immunoassay kits (S-2) to (S-27) and (S'-1) to (S'-2).
[Solid Phase Carrier Reagent (A)]
In preparing the dilution solution used in the production of the solid phase carrier reagent (A-1) of Example 1, the procedure of Example 1 was repeated except that the components and weight ratios were changed as shown in Table 1-1 or Table 1-2, to obtain solid phase carrier reagents (A-2) to (A-27) and (A'-1) to (A'-2).
[Reaction Buffer (B)]
The same procedure as in Example 1 was repeated to prepare reaction buffer solution (B-1) in Example 1, except that the components and weight ratios were changed as shown in Table 1-1 or Table 1-2, to obtain reaction buffer solutions (B-2) to (B-27) and (B'-1) to (B'-2).

<Example 28>
An immunoassay kit (S-28) was obtained in the same manner as in Example 1, except that in the production of the immunoassay kit (S-1) in Example 1, the solid phase carrier reagent (A-1), the reaction buffer solution (B-1) and the labeling reagent (C-1) were changed as follows.
[Solid Phase Carrier Reagent (A)]
The same procedure as in Example 1 was repeated except that, in the preparation of the solid phase carrier reagent (A-1) in Example 1, 120 mL of 0.02 M phosphate buffer (pH 8.7) containing 10 μg/mL of anti-CEA monoclonal antibody [manufactured by Agilent Technologies, Inc.] and 3.64 μg/mL of peroxidase was used in place of 120 mL of 0.02 M phosphate buffer (pH 8.7) containing 10 μg/mL of anti-Tg monoclonal antibody F(ab')2 and 3.64 μg/mL of peroxidase. As a result, a solid phase carrier reagent (A-28) containing magnetic particles (H-2) to which anti-CEA monoclonal antibody and peroxidase were immobilized was obtained.
[Reaction Buffer (B)]
The reaction buffer solution (B-1) in Example 1 was used as the reaction buffer solution (B-28).
[Labeling Reagent (C)]
The same procedure as in Example 1 was repeated to prepare the labeled reagent (C-1) in Example 1, except that an anti-CEA monoclonal antibody (manufactured by Agilent Technologies, Inc.) was used instead of the anti-human IgG polyclonal antibody. This resulted in a labeled reagent (C-2) in which the concentration of the POD-labeled anti-CEA monoclonal antibody (F3-2) was adjusted to 0.5 μg/mL.

<Evaluation of false positive suppression rate>
Immunological assays were performed using the immunological assay kits (S-1) to (S-28) obtained in Examples 1 to 28 or the immunological assay kits (S'-1) to (S'-2) obtained in Reference Examples 1 and 2 by the following method (sandwich method), and the false positive inhibition rate was evaluated.

0.025 g of solid phase carrier reagent (A) from the immunoassay kit was placed in a test tube, and the magnetic particles were collected from the outside of the test tube with a neodymium magnet for 10 seconds. 90% by volume of the liquid in the test tube was removed with an aspirator, and the neodymium magnet was moved sufficiently away from the side.
Next, 0.065 g of reaction buffer solution (B) was poured into the test tube, and the test tube was allowed to stand at 37° C. for 3 minutes.
After the reaction, the magnetic 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 moved far enough away from the side. Then, 0.5 mL of physiological saline was added to disperse the magnetic particles, and the particles were collected again. This washing procedure of removing the liquid with an aspirator was repeated twice.

Subsequently, 0.05 mL of the labeling reagent (C) corresponding to each immunoassay kit was poured into the test tube, and the tube was left to react at 37° C. for 3 minutes.
After the reaction, the magnetic 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 moved far enough away from the side. Then, 0.5 mL of physiological saline was added to disperse the magnetic particles, and the particles were collected again. This washing procedure of removing the liquid with an aspirator was repeated twice.
Finally, 0.07 mL of luminol luminescence reagent (D1) and 0.07 mL of hydrogen peroxide solution (D2) were added at the same time, and the luminescence reaction was allowed to proceed for 45 seconds at 37° C. The average luminescence intensity from 43 to 45 seconds after the addition of the luminol luminescence reagent (D1) and hydrogen peroxide solution (D2) was measured using a luminometer (Berthold Japan, “Lumat LB9507”). The average luminescence intensity at this time was designated as the average luminescence intensity (Z1).

In addition, in the above procedure, the average luminescence was measured with a luminometer in the same manner, except that instead of the solid phase carrier reagent (A) and reaction buffer (B), separately prepared solutions were used in which the catalyst, substrate (or hydrogen peroxide), and peroxidase inhibitor in the solid phase carrier reagent (A) and reaction buffer (B) were not added. The average luminescence at this time was designated as the average luminescence (Z2).
The value of 1-[mean luminescence amount (Z1)]/[mean luminescence amount (Z2)] was calculated and this value was taken as the false positive inhibition rate. The results are shown in Table 1-1 or Table 1-2.
A higher false positive suppression rate indicates that false positives due to peroxidase bound to magnetic particles (H) are more suppressed.

<Production of Solid Phase Carrier Reagents (AT-1) to (AT-27) and (AT'-1) to (AT'-2) for Preparation of Calibration Curve>
In the preparation of the solid phase carrier reagents (A-1) to (A-27) and (A'-1) to (A'-2) in Examples 1 to 27 and Reference Examples 1 and 2, solid phase carrier reagents (AT-1) to (AT-27) and (AT'-1) to (AT'-2) containing magnetic particles (HT-1) were prepared in the same manner, except that 120 mL of 0.02 M phosphate buffer (pH 8.7) containing anti-Tg monoclonal antibody F(ab')2 at a concentration of 10 μg/mL was used instead of 120 mL of 0.02 M phosphate buffer (pH 8.7) containing anti-Tg monoclonal antibody F(ab')2 at a concentration of 10 μg/mL and peroxidase at a concentration of 3.64 μg/mL.
These were used to prepare calibration curves in evaluating the accuracy of the immunoassay kits (S-1) to (S-27) and (S'-1) to (S'-2), respectively.

<Production of solid phase carrier reagent (AT-28) for preparing a calibration curve>
A solid phase carrier reagent (AT-28) containing magnetic particles (HT-2) was prepared in the same manner as in Example 28, except that 120 mL of 0.02 M phosphate buffer (pH = 8.7) containing anti-CEA monoclonal antibody [manufactured by Agilent Technologies, Inc.] at a concentration of 10 μg/mL was used in place of 120 mL of 0.02 M phosphate buffer (pH = 8.7) containing anti-CEA monoclonal antibody [manufactured by Agilent Technologies, Inc.] at a concentration of 10 μg/mL and peroxidase at a concentration of 3.64 μg/mL.
This was used to create a calibration curve in evaluating the accuracy of the immunoassay kit (S-28).

<Evaluation of accuracy of immunological assay kits (S-1) to (S-27) and (S'-1) to (S'-2)>
Immunoassay kits (S-1) to (S-27) obtained in Examples 1 to 27 and immunoassay kits (S'-1) to (S'-2) obtained in Reference Examples 1 and 2 were used to carry out immunoassays by the following method (sandwich method), and the accuracy was evaluated. The results are shown in Table 1-1 or Table 1-2.

<Immunological measurement method>
Step (1)
0.025 g of the solid phase carrier reagent (A) of each immunoassay kit was placed in a test tube, and magnetic particles were collected from the outside of the test tube with a neodymium magnet for 10 seconds. 90% by volume of the liquid in the test tube was collected. The air was removed using an aspirator, and the neodymium magnet was kept well away from the side.
Next, 0.065 g of reaction buffer solution (B) and 0.025 mL of pooled serum adjusted to a concentration of 15 ng/mL of the target substance, Tg (antigen), were mixed in a test tube. The mixture was reacted in a test tube at 37° C. for 3 minutes to bind the anti-Tg monoclonal antibody F(ab′) 2 on the magnetic particles to Tg, forming a complex between the magnetic particles and the antigen.
After the reaction, the magnetic 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 separated from the side. Then, 0.5 mL of physiological saline was added to collect the magnetic particles. After the particles were dispersed, they were collected again and a washing operation of removing the liquid with an aspirator was carried out twice.

Step (2)
Subsequently, 0.05 mL of the labeled reagent (C) of each immunoassay kit was poured into each test tube and reacted in the test tube at 37°C for 3 minutes to form a labeled complex consisting of the magnetic particles, Tg, and POD-labeled anti-human IgG polyclonal antibody (rabbit) (F3-1).
After the reaction, the magnetic 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 moved far enough away from the side. Then, 0.5 mL of physiological saline was added to disperse the magnetic particles, and the particles were collected again. This washing procedure of removing the liquid with an aspirator was repeated twice.

Finally, 0.07 mL of luminol luminescence reagent (D1) and 0.07 mL of hydrogen peroxide solution (D2) were added simultaneously, and the luminescence reaction was carried out at 37°C. The average amount of luminescence per second from 43 to 45 seconds after the addition of the luminol luminescence reagent (D1) and hydrogen peroxide solution (D2) was measured using a luminometer (Berthold Japan, "Lumat LB9507").

In addition, instead of the pooled serum adjusted to a Tg concentration of 15 ng/mL in the above step (1), the following standard solutions were used, and the corresponding solid phase carrier reagents (AT-1) to (AT-27) or (AT'-1) to (AT'-2) for preparing a calibration curve were used as the solid phase carrier reagent (A). The average luminescence amount was measured with a luminometer in the same manner, and a calibration curve showing the relationship between the luminescence amount and the Tg concentration was prepared. The measured concentration in the above immunological measurement method was determined from the obtained calibration curve. The results are shown in Table 1-1 or Table 1-2.
Standard solution: pooled serum with Tg concentration adjusted to 0, 1, 3, 10, 30, 100, 300, or 1000 ng/mL

<Accuracy assessment method>
Accuracy was judged based on the ratio between the measured concentration and the actual concentration (15 ng/mL) according to the following criteria.
◯: (measured concentration/actual concentration)×100(%) is within the range of 85 to 115. ×: (measured concentration/actual concentration)×100(%) is less than 85 or greater than 115.

<Evaluation of accuracy of immunoassay kit (S-28)>
Using the immunoassay kit (S-28) obtained in Example 28, immunoassay was carried out by the following method (sandwich method) and the accuracy was evaluated. The results are shown in Table 1-2.

<Immunological measurement method>
Step (1)
0.025 g of solid phase carrier reagent (A) from the immunoassay kit was placed in a test tube, and magnetic particles were collected from the outside of the test tube with a neodymium magnet for 10 seconds. 90% by volume of the liquid in the test tube was aspirated. The neodymium magnet was removed and placed well away from the side.
Next, 0.065 g of reaction buffer solution (B) and 0.025 mL of pooled serum adjusted to a concentration of 7 ng/mL of CEA (antigen), which is the substance to be measured, were mixed in a test tube. The mixture was reacted in a test tube at 37° C. for 3 minutes to bind the anti-CEA monoclonal antibody on the magnetic particles to CEA, forming a complex between the magnetic particles and the antigen.
After the reaction, the magnetic 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 separated from the side. Then, 0.5 mL of physiological saline was added to collect the magnetic particles. After the particles were dispersed, they were collected again and a washing operation of removing the liquid with an aspirator was carried out twice.

Step (2)
Next, 0.05 mL of the labeled reagent (C) of each immunoassay kit was poured into the test tube and reacted in the test tube at 37° C. for 3 minutes to form a labeled complex consisting of the magnetic particles, CEA, and the POD-labeled anti-CEA monoclonal antibody (F3-2).
After the reaction, the magnetic 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 moved far enough away from the side. Then, 0.5 mL of physiological saline was added to disperse the magnetic particles, and the particles were collected again. This washing procedure of removing the liquid with an aspirator was repeated twice.

Finally, 0.07 mL of luminol luminescence reagent (D1) and 0.07 mL of hydrogen peroxide solution (D2) were added simultaneously, and the luminescence reaction was carried out at 37°C. The average amount of luminescence per second from 43 to 45 seconds after the addition of the luminol luminescence reagent (D1) and hydrogen peroxide solution (D2) was measured using a luminometer (Berthold Japan, "Lumat LB9507").

In addition, in the above step (1), the following standard solution was used instead of the pooled serum adjusted to a CEA concentration of 7 ng/mL, and the corresponding solid phase carrier reagent (AT-28) for preparing a calibration curve was used as the solid phase carrier reagent (A) in the same manner, except that the average luminescence amount was measured with a luminometer and a calibration curve showing the relationship between the luminescence amount and the Tg concentration was prepared. The measured concentration in the above immunological measurement method was determined from the obtained calibration curve. The results are shown in Table 1-2.
Standard solution: Pooled serum with CEA concentration adjusted to 0, 5, 30, 50, 100, 500 or 1000 ng/mL

<Accuracy assessment method>
Accuracy was judged based on the ratio between the measured concentration and the actual concentration (7 ng/mL) according to the following criteria.
◯: (measured concentration/actual concentration)×100(%) is within the range of 85 to 115. ×: (measured concentration/actual concentration)×100(%) is less than 85 or greater than 115.

The immunoassay method and immunoassay kit of the present invention are highly accurate and sensitive, and therefore can be widely applied to clinical tests such as radioimmunoassay, enzyme immunoassay, fluorescent immunoassay, and chemiluminescent immunoassay.

Claims (7)

Obtaining a first mixture containing a complex of an antigen and a solid phase carrier (α); and
An immunoassay method comprising a step of measuring the antigen using the first mixture and a labeled reagent (C),
The step of obtaining the first mixture comprises:
A step of mixing a sample containing the antigen, a peroxidase inhibitor, hydrogen peroxide and the solid phase carrier (α) to obtain a second mixture containing a complex of the antigen and the solid phase carrier (α); and
removing the peroxidase inhibitor after obtaining the second mixture ;
an aromatic compound, wherein the peroxidase inhibitor is at least one hydrazine derivative selected from the group consisting of 4-aminobenzohydrazide, indole-3-acetic acid hydrazide, 4-fluorobenzoic acid hydrazide, and 6-iodo-pyridine-2-carboxylic acid hydrazide; and
An immunoassay method comprising the step of: preparing an immunoassay composition comprising at least one aromatic compound which is a hydroxamic acid derivative selected from the group consisting of salicylhydroxamic acid, 2-(3,5-bistrifluoromethylbenzylamino)-6-oxo-1H-pyrimidine-5-carbohydroxamic acid, 4-benzyl-2-hydroxybenzenecarbohydroxamic acid, 2-(benzylamino)-6-oxo-3H-pyrimidine-5-carbohydroxamic acid, benzohydroxamic acid, 1-naphthohydroxamic acid, and 2-naphthohydroxamic acid . An immunoassay kit for use in the immunoassay method according to claim 1, comprising:
The method comprises the steps of: a solid phase carrier reagent (A); a reaction buffer solution (B); the labeling reagent (C); and a luminescent reagent (D);
The solid phase carrier reagent (A) contains the solid phase carrier (α),
At least one of the solid phase carrier reagent (A) and the reaction buffer solution (B) contains the peroxidase inhibitor;
At least one of the solid phase carrier reagent (A) and the reaction buffer solution (B) contains the hydrogen peroxide, or
A kit for immunological measurement, wherein the solid phase carrier reagent (A) contains either (β) of a catalyst or a substrate in a chemical reaction that produces hydrogen peroxide, and the reaction buffer solution (B) contains the other (γ) of the catalyst and the substrate, and hydrogen peroxide can be generated by mixing (β) and (γ). An immunoassay kit for use in the immunoassay method according to claim 1, comprising:
The method comprises the steps of: (A) a solid phase carrier reagent; (B) a reaction buffer solution; (C) the peroxidase inhibitor; (D) the labeling reagent; and (D) a luminescent reagent.
The solid phase carrier reagent (A) contains the solid phase carrier (α),
At least one of the solid phase carrier reagent (A) and the reaction buffer solution (B) contains the hydrogen peroxide, or
A kit for immunological measurement, wherein the solid phase carrier reagent (A) contains either (β) of a catalyst or a substrate in a chemical reaction that produces hydrogen peroxide, and the reaction buffer solution (B) contains the other (γ) of the catalyst and the substrate, and hydrogen peroxide can be generated by mixing (β) and (γ). The immunoassay kit according to claim 2 or 3, wherein the chemical reaction is an oxidation-reduction reaction, the catalyst is an oxidoreductase, and the substrate is a substrate that is oxidized in the oxidation-reduction reaction. The immunoassay kit according to claim 4, wherein the oxidoreductase is glucose oxidase and the substrate is glucose. 4. The immunoassay kit according to claim 2 or 3, wherein the solid phase carrier (α) is a solid phase carrier (α1) having an antibody that specifically binds to the antigen immobilized on its surface, and the labeled reagent (C) contains a labeled antibody labeled with peroxidase. 4. The immunoassay kit according to claim 2 or 3, wherein the solid phase carrier (α) is a solid phase carrier (α1) having an antibody that specifically binds to the antigen immobilized on its surface, and the labeled reagent (C) contains a labeled antigen labeled with peroxidase or a substance analogous thereto.