JP2021071476A - Immunoassay reagent, immunoassay kit, and immunoassay method - Google Patents

Abstract

To provide an immunoassay reagent excellent in storage stability, an immunoassay kit using the immunoassay reagent, and an immunoassay method.SOLUTION: The immunoassay reagent contains a solid-phase carrier (E) for immunoassay, sugar (A) and, water. In the immunoassay reagent (M), the weight ratio of the water is 20-60% by weight with respect to the weight of the immunoassay reagent. The immunoassay reagent (M) preferably further contains protein (B). Further, in the immunoassay reagent (M), the weight ratio of the sugar (A) is preferably 20 to 60% by weight with respect to the weight of the immunoassay reagent.SELECTED DRAWING: None

Description

The present invention relates to immunoassay reagents, immunoassay kits and immunoassay methods.

In immunoassay reagents, a substance to be measured, a substance similar to the substance to be measured, or a substance that specifically binds to the substance to be measured, for example, an antigen, is used as a main component on the surface of magnetic particles or glass beads that serve as a solid phase carrier. And some have immobilized antibodies. In the immunoassay reagent, it is desired that the substance immobilized on the solid phase carrier maintains a certain activity. If the activity of the substance decreases with time or the activity of the substance changes depending on the storage state, accurate measurement results cannot be expected, which is a big problem as an immunoassay reagent.

Various stabilization methods have been proposed for the antigen or antibody substance itself immobilized on the solid phase carrier in each form, such as a liquid reagent dissolved in a buffer solution or the like, a freeze-dried reagent, or the like. In the case of a liquid reagent, a method of adding bovine serum albumin (BSA) to an IgM reagent solution to stabilize it (Patent Document 1), a method of stabilizing a labeled antibody by adding an amino acid and a derivative thereof (Patent Document 2), etc. Has been proposed. Further, in the case of a freeze-drying reagent, a method of adding sucrose, trehalose or dextran to an enzyme-labeled antibody solution and freeze-drying to stabilize it (Patent Document 3) has been proposed. However, it is not satisfactory as a stabilization method in the case of immobilizing these substances on a solid phase carrier and storing them in a liquid state for a long period of time. Further improvement in stability is desired in the development of an automated immunoassay device for the purpose of simultaneous rapid testing of a large number of samples.

Japanese Unexamined Patent Publication No. 9-127114 Japanese Unexamined Patent Publication No. 2011-241206 Japanese Unexamined Patent Publication No. 60-149972

An object of the present invention is to provide an immunoassay reagent having excellent storage stability, an immunoassay kit and an immunoassay method using the immunoassay reagent.

The present inventors have arrived at the present invention as a result of diligent studies to achieve the above object.
That is, the present invention is an immunoassay reagent containing an immunoassay solid-phase carrier (E), a sugar (A), and water.
The weight ratio of the water is 20 to 60% by weight based on the weight of the immunoassay reagent (M); an immunoassay kit containing the immunoassay reagent (M); the immunity. This is an immunoassay method using a measurement reagent (M).

Since the immunoassay reagent contained in the immunoassay kit of the present invention is excellent in storage stability, it is possible to reduce reagent loss due to expiration of the effective period.

<Reagent for immunoassay>
The immunoassay reagent (M) of the present invention contains an immunoassay solid-phase carrier (E), a sugar (A), and water.
In the present invention, the immunoassay reagent (M) is a reagent constituting an immunoassay kit described in detail later, and an immunoassay method (immunosassay method for measuring the concentration of a substance to be measured in a sample) described in detail later. ) Can be used.
The substance to be measured is not particularly limited as long as it is generally measured in the field of immunoassay, for example, biological fluids such as serum, blood, plasma and urine, lymph fluid, blood cells, various cells and the like. Nucleotide chains (oligonucleotide chains, polynucleotide chains); chromosomes; nucleic acids (deoxyribonucleic acid (DNA), ribonucleic acid (RNA), etc.); peptide chains (eg, C-peptide, angiotensin I, etc.) ); Proteins [eg, procalcitonin, immunoglobulin A (IgA), immunoglobulin E (IgE), immunoglobulin G (IgG), immunoglobulin M (IgM), immunoglobulin D (IgD), β2-microglobulin, albumin, Hemoglobin, myoglobin, transferase, protein A, C reactive protein (CRP), ferritin, troponin T (TnT), human brain sodium diuretic peptide precursor N-terminal fragment (NT-proBNP), degradation products thereof]; Factors (eg fibrinogen, fibrin degradation products, prothrombin, thrombin, etc.); Enzymes (eg, amylase (eg, pancreatic, plasma gland, X, etc.), alkaline phosphatase (eg, hepatic, osseous, placenta, small bowel, etc.), acidic Phosphatase (eg, PAP, etc.), γ-glutamyl transferase (eg, renal, pancreatic, hepatic, etc.), lipase (eg, pancreatic, gastric, etc.), creatine kinase (eg, CK-1, CK-2, mCK, etc.), Lactated dehydrogenase (eg LDH1-LDH5, etc.), glutamate oxaloacetate transaminase (eg, ASTm, ASTs, etc.), glutamate pyruvate transaminase (eg, ALTm, ALTs, etc.), choline esterase (eg, ChE1-ChE5, etc.), leucine aminopeptidase (eg, ChE1-ChE5, etc.) C-LAP, AA, CAP, etc.), renin, protein kinase, tyrosine kinase, etc.] and inhibitors of these enzymes; hormones (eg, PTH, TSH, insulin, LH, FSH, estradiol, prolactin, etc.); receptors (eg, estrogen, TSH, etc.) Receptors for etc.); Ligands (eg estrogen, TSH, etc.); Bacteria (eg, tuberculosis, pneumonia, diphtheria, meningitis, gonococcus, staphylococcus, lensasphere, enterobacteria, Escherichia coli, helicobacter pyrori, etc.) Viruses (eg, rubella virus, herpes virus, hepatitis virus, ATL virus, AI DS virus, influenza virus, adenovirus, enterovirus, poliovirus, EB virus, HAV, HBV, HCV, HIV, HTLV, etc.; fungi (eg, candida, cryptococcus, etc.); spirochete (eg, leptspira, syphilis treponema, etc.); chlamydia, Microorganisms such as mycoplasma; proteins or peptides or sugar chain antigens derived from the microorganisms; various allergens causing allergies such as bronchial asthma, allergic rhinitis, and atopic dermatitis (for example, house dust, for example, ticks such as Kona leopard mite and Yake leopard mite) For pollen such as cedar, hinoki, suzumenohie, butakusa, sardine, hargaya, limegi, for example, for animals such as cats, dogs, crabs, for example, foods such as rice, egg white, fungi, insects, wood, chemicals, chemical substances, etc. Derived allergens, etc.); Lipids (eg, lipoproteins, etc.); Proteas (eg, trypsin, plasmin, serine proteases, etc.); Tumor marker protein antigens (eg, PSA, PGI, PGII, etc.); Sugar chain antigens [eg, from AFP (eg, L1) L3 etc.), hCG (hCG family), transferase, IgG, thyroglobulin (Tg), Decay-accelerating-factor (DAF), cancer fetal antigen (eg CEA, NCA, NCA-2, NFA etc.), CA19-9, PIVKA-II, CA125, prostate-specific antigen, tumor marker sugar chain antigen having a special sugar chain produced by cancer cells, ABO sugar chain antigen, etc.]; Sugar chain (for example, hyaluronic acid, β-glucan, the above sugar chain antigen, etc.) Sugar chains, etc.); Proteins that bind to sugar chains (eg, hyaluronic acid-binding protein, β-glucan-binding protein, etc.); Phosphorlipids (eg, cardiolipin, etc.); Lipopolysaccharides (eg, endotoxin, etc.); Chemical substances (eg, T3, T4) , FT3, FT4, tributyltin, nonylphenol, 4-octylphenol, di-n-butyl phthalate, dicyclohexyl phthalate, benzophenone, octachlorostyrene, environmental hormones such as di-2-ethylhexyl phthalate); Various drugs and their metabolites; antigeners; nucleic acid-binding substances; these antibodies and the like. Among the above, antibodies, hormones, cancer markers, heart disease markers and the like are preferable.

Examples of the sugar (A) contained in the immunoassay reagent (M) of the present invention include monosaccharides, disaccharides, polysaccharides and sugar derivatives (sugar alcohols and the like).
Examples of monosaccharides include triose (ketotriose, etc.), tetrose (ketotetrose, etc.), pentose (ketopentose, aldopentose, deoxy sugar, etc.), hexose [ketohexose (psicose, fructose, sorbose, tagatous, etc.), aldhexose (allose, alto, etc.). Loin, glucose, mannose, growth, idose, galactose, tetrose, etc.), deoxy sugar (fucose, fukurosu, ramnorse, etc.), etc.], hexose (sedhepturose, etc.) and the like can be mentioned.
Examples of the disaccharide include those in which two molecules of the above monosaccharides are dehydrated and condensed to form a glycosidic bond, and specific examples thereof include trehalose, sucrose, lactose, maltose and cellobiose.
Examples of the polysaccharide include those in which three or more molecules of the above monosaccharides are dehydrated and condensed to form a glycosidic bond. Specifically, oligosaccharides (fructooligosaccharides, etc.), amylose, amylopectin, glycogen, cellulose, etc. Examples thereof include hyaluronic acid, chondroitin sulfate and heparin.
Examples of sugar alcohols include glycerol, erythritol and mannitol.

The chemical formula amount or weight average molecular weight of the sugar (A) is preferably 1000 or less.
The weight average molecular weight of the sugar (A) can be measured by gel permeation chromatography (GPC) under the following conditions.
Measuring device: "HLC-8120" manufactured by Tosoh Corporation
Columns: "TSKgel GMHXL" manufactured by Tosoh Corporation (2 pcs) and "T SKgel Multipore HXL-M" manufactured by Tosoh Corporation (1 pcs)
Sample solution: 0.25% by mass THF solution Injection amount of sample solution into the column: 100 μl
Flow velocity: 1 ml / min Measurement temperature: 40 ° C
Detector: Refractive index detector Reference material: Standard polystyrene (TSK standard POLYSTYRENE) manufactured by Tosoh Corporation 12 points (number average molecular weight: 500, 1050, 2800, 5970, 9100, 18100, 37900, 96400, 190000, 355000, 1090000 , 2890000).

The immunoassay solid-phase carrier (E) contained in the immunoassay reagent (M) of the present invention is a solid-phase carrier, and is not particularly limited as long as it is generally used in this field. For example, glass. Typical examples include beads, polystyrene beads, magnetic particles, microplates, and latex. Among these, from the viewpoint of measurement sensitivity and shortening of measurement time, magnetic particles (H) having an antigen (C) or antibody (D) described later [magnetic particles on which an antigen (C) or antibody (D) is immobilized, etc. ], And from the viewpoint of shortening the measurement time in immunoassay, for example, the antigen (C) or antibody (D) described in JP-A-2014-210680 and JP-A-2013-019889 can be used. Silica particles having [silica particles containing a metal oxide] are more preferable.

As the silica particles containing the metal oxide, a metal oxide having a volume average particle diameter of 1 to 20 nm and having superparamagnetism is dispersed in the silica matrix from the viewpoint of shortening the measurement time in immunoassay. Those are preferable. Hypernormal magnetism refers to a temporary magnetic field in which individual atomic magnetic moments of a substance are aligned and induced in the presence of an external magnetic field, and when the external magnetic field is removed, partial alignment is impaired and the magnetic field is no longer shown. Say.
The volume average particle size of the metal oxide is an average value of the particle size measured by observing any 200 particles with a scanning electron microscope (“JSM-7000F” manufactured by JEOL Ltd.).

Examples of superparamagnetic metal oxides having an average particle size of 1 to 20 nm and exhibiting superparamagnetism include oxides such as iron, cobalt, nickel and alloys thereof, but they are oxidized because of their excellent sensitivity to magnetic fields. Iron is particularly preferred. As the superparamagnetic metal oxide, one type may be used alone or two or more types may be used in combination.
As the iron oxide, various known iron oxides can be used.
Of 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 because of its excellent chemical stability. Is preferable, and magnetite is more preferable because it has a large saturation magnetization and is excellent in sensitivity to an external magnetic field.

The lower limit of the content of the supernormal magnetic metal oxide in the silica particles containing the metal oxide is preferably 60% by weight, more preferably 65% by weight, based on the weight of the silica particles containing the metal oxide. The upper limit is preferably 95% by weight, more preferably 80% by weight.
When the content of the superparamagnetic metal oxide is 60% by weight or more, the magnetism of the obtained silica particles containing the metal oxide is sufficient, and the separation operation in the actual application can be performed in a short time, which is preferable. .. Those having a weight of 95% by weight or less are easy to synthesize.

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

The volume average particle size of the silica particles containing the metal oxide is preferably 1 to 5 μm, more preferably 1 to 3 μm. When the volume average particle size is 1 μm or more, the time for separation and recovery tends to be relatively short. Further, when it is 5 μm or less, as a result of the relatively large surface area, the amount of the substance to be immobilized (the target substance, the substance similar to the substance to be measured or the substance specifically bound to the substance to be measured) is also increased, and the binding amount is increased. Efficiency tends to increase.

The volume average particle size of the silica particles containing the metal oxide is controlled by adjusting the particle size of the oil-in-water emulsion by adjusting the mixing conditions (shearing force, etc.) when producing the oil-in-water emulsion described later. can do. Further, the volume average particle diameter can be set to a desired value by changing the conditions of the water washing process at the time of producing silica particles containing a metal oxide or by a method such as ordinary classification.

The volume average particle diameter of the silica particles containing a metal oxide in the present invention is a particle measured by observing any 200 magnetic particles with a scanning electron microscope (“JSM-7000F” manufactured by JEOL Ltd.). It is the average value of the diameter.

The silica particles containing the metal oxide in the present invention are, for example, supernormal magnetic metal oxide particles having a volume average particle diameter of 1 to 20 nm, and 30 to 500% by weight (alkyl) based on the weight of the supernormal magnetic metal oxide particles. ) A dispersion containing an alkoxysilane and, if necessary, a dispersant, and a solution containing water, a water-soluble organic solvent, a nonionic surfactant and a catalyst for hydrolysis of (alkyl) alkoxysilane are mixed. After forming an oil-in-water emulsion, a hydrolysis reaction and a condensation reaction of (alkyl) alkoxysilane are carried out to obtain an aqueous dispersion of magnetic particles in which a supernormal magnetic metal oxide is contained in silica, and then an aqueous dispersion of magnetic particles is obtained. It is obtained by solid-liquid separation of the body by centrifugation and / or collection of magnetism, and washing with water, methanol or the like.
Further, if necessary, the magnetic particles obtained by the above operation, (alkyl) alkoxysilane, water, a water-soluble organic solvent, a nonionic surfactant, and a catalyst for hydrolysis of (alkyl) alkoxysilane are further mixed. Then, the hydrolysis reaction and the condensation reaction of the (alkyl) alkoxysilane may be carried out to obtain silica particles having a core-shell structure.
In the above and below, (alkyl) alkoxysilane means alkylalkoxysilane or alkoxysilane.

In the present invention, the antigen (C) is not particularly limited as long as it is generally measured in this field, and specifically includes the antigen used for the following immunoassay.
Nucleotide chains (oligonucleotide chains, polynucleotide chains) contained in biological samples such as serum, blood, plasma, urine, lymph fluid, blood cells, and various cells; chromosomes; nucleic acids (deoxyribonucleic acid (DNA)) , Ribonucleic acid (RNA), etc.); Peptide chains (eg, C-peptide, angiotensin I, etc.); Proteins [eg, procalcitonin, immunoglobulin A (IgA), immunoglobulin E (IgE), immunoglobulin G (IgG), immunity Globulin M (IgM), Immunoglobulin D (IgD), β2-microglobulin, albumin, hemoglobin, myoglobin, transferase, protein A, C reactive protein (CRP), ferritin, troponin T (TnT), human brain sodium diuretic peptide Precursor N-terminal fragments (NT-proBNP), degradation products thereof]; Blood coagulation-related factors (eg, fibrinogen, fibrin degradation products, prothrombin, thrombin, etc.); Enzymes [eg, amylase (eg, pancreatic, salivary gland, X, etc.) , Alkaline phosphatase (eg, hepatic, osseous, placenta, small intestine, etc.), Acid phosphatase (eg, PAP, etc.), γ-glutamyl transferase (eg, renal, pancreatic, hepatic, etc.), Lipase (eg, pancreatic, gastric) Types, etc.), creatine kinase (eg, CK-1, CK-2, mCK, etc.), lactate dehydrogenase (eg, LDH1-LDH5, etc.), glutamate oxaloacetate transaminase (eg, ASTm, ASTs, etc.), glutamate pyruvate transaminase (eg, ASTm, etc.) ALTm, ALTs, etc.), choline esterase (eg, ChE1-ChE5, etc.), leucine aminopeptidase (eg, C-LAP, AA, CAP, etc.), renin, protein kinase, tyrosine kinase, etc.] and inhibitors of these enzymes; hormones (eg, PTH, TSH, insulin, LH, FSH, estradiol, prolactin, etc.); Receptors (eg, receptors for estrogen, TSH, etc.); Ligands (eg, estrogen, TSH, etc.); Bacteria (eg, tuberculosis, pneumonia, diphtheria, meningitis) , Goco, staphylococcus, lensasphere, enterobacteria, Escherichia coli, Helicobacter pylori, etc.); Viruses (eg, rubella virus, herpes virus, hepatitis virus, ATL virus, AIDS virus, influenza virus, adenovirus, enterovirus, poliovirus) , EB Will Su, HAV, HBV, HCV, HIV, HTLV, etc.); Fungi (eg, candida, cryptococcus, etc.); Spiroheta (eg, leptspira, syphilis treponema, etc.); Microorganisms such as chlamydia, mycoplasma; Proteins or peptides or sugars derived from the microorganisms Chain antigens; various allergens that cause allergies such as bronchial asthma, allergic rhinitis, and atopic dermatitis (for example, house dust, for example, ticks such as Kona leopard mite, Yake leopard mite, etc. Pollen such as limes, animals such as cats, dogs, crabs, etc., foods such as rice, egg white, etc., allergens derived from fungi, insects, wood, drugs, chemical substances, etc.); lipids (eg, lipoproteins, etc.); proteases (For example, trypsin, plasmin, serine protease, etc.); Tumor marker protein antigen (for example, PSA, PGI, PGII, etc.); Glycan antigen [for example, AFP (for example, L1 to L3, etc.), hCG (hCG family), transferase, IgG, thyroglobulin, etc.) (Tg), Decay-accelerating-factor (DAF), cancer fetal antigens (eg NCA, NCA-2, NFA, etc.), PIVKA-II, CA125, prostate-specific antigens, having special sugar chains produced by cancer cells Tumor marker sugar chain antigen, ABO sugar chain antigen, etc.]; Sugar chain (for example, hyaluronic acid, β-glucan, sugar chain of the above sugar chain antigen, etc.); Protein that binds to the sugar chain (for example, hyaluronic acid-binding protein, β Glucan-binding protein, etc.); Phosphorlipids (eg, cardiolipin, etc.); Lipopolysaccharides (eg, endotoxin, etc.); Chemical substances (eg, T3, T4, FT3, FT4, tributyltin, nonylphenol, 4-octylphenol, di-n-butyl phthalate, etc.) Environmental hormones such as dicyclohexyl phthalate, benzophenone, octachlorostyrene, di-2-ethylhexyl phthalate); various drugs administered / inoculated to the human body and their metabolites; aptamers; nucleic acid-binding substances and the like.

In the present invention, the antibody (D) is not particularly limited as long as it is generally measured in this field, and specific examples thereof include antibodies against those exemplified as the antigen (C). The antibody used in the present invention also includes proteolytic enzymes such as papain and pepsin, and degradation products such as _{Fab and F (ab') 2 fragments generated by chemical degradation.}

In the present invention, as a method of immobilizing an antigen (C) or an antibody (D) on a magnetic particle to form a magnetic particle (H), the antigen (C) or the antibody (D) is physically adsorbed on the above-mentioned magnetic particle. Although a method can be mentioned, glutalaldehyde, albumin, carbodiimide, streptavidin, biotin and functional groups can be used from the viewpoint of immobilizing the antigen (C) or antibody (D) more efficiently, such as the substance to be measured. It is preferable that at least one organic compound selected from the group consisting of alkylalkoxysilanes having is bonded to the surface of magnetic particles, and the antigen (C) or antibody (D) is immobilized on the magnetic particles via them. More preferably, it is immobilized via an alkylalkoxysilane having a functional group (ethylene unsaturated group, epoxy group, amino group, mercapto group, isocyanate group, etc.).
Examples of the alkylalkoxysilane having such a functional group include vinyltrimethoxysilane, vinyltriethoxysilane, 2- (3,4-epoxycyclohexyl) ethyltrimethoxysilane, 3-glycidoxypropylmethyldimethoxysilane, and 3-. Glycydoxypropyltrimethoxysilane, 3-glycidoxypropylmethyldiethoxysilane, 3-glycidoxypropyltriethoxysilane, p-styryltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyl Trimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-acryloxypropyltrimethoxysilane, N-2- (aminoethyl) -3-aminopropylmethyldimethoxysilane, N -2- (Aminoethyl) -3-aminopropyltrimethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-triethoxysilyl-N- (1,3-dimethyl-butylidene) propyl Amin, N-phenyl-3-aminopropyltrimethoxysilane, tris- (trimethoxysilylpropyl) isocyanurate, 3-ureidopropyltrialkoxysilane, 3-mercaptopropylmethyldimethoxysilane, 3-mercaptopropyltrimethoxysilane, 3 -Isocyanate propyltriethoxysilane and the like can be mentioned.

Further, as the magnetic particles (H) having the antigen (C) or the antibody (D), the magnetic particles (H1) formed by binding to the magnetic particles via the antibody (CA) against the antigen (C) and / Alternatively, it is preferably a magnetic particle (H2) formed by binding to the magnetic particle via an antigen (DA) against the antibody (D).
Examples of the antigen (DA) include those similar to the above-mentioned antigen (C), and preferred ones are also the same.
Moreover, as the antibody (CA), the same thing as the said antibody (D) can be mentioned, and the preferable one is also the same.
_{Further, F (ab') 2} and Fab are preferable as the antibody (CA) in the magnetic particles (H1).

As a method for producing the magnetic particles (H1), first, an antibody (CA) is provided by a known method (methods described in JP-A-2014-210680 and JP-A-2013-019889, etc.). A method of producing magnetic particles (H) [magnetic particles on which an antibody (CA) is immobilized], and then binding an antigen (C) to an antibody (CA) on the magnetic particles (H) by a known method or the like. And so on.
Further, as a method for producing the magnetic particles (H2), first, an antigen (DA) is produced by a known method (methods described in JP-A-2014-210680 and JP-A-2013-019889, etc.). The magnetic particles (H) [magnetic particles on which the antigen (DA) is immobilized] are produced, and then the antibody (D) is bound to the antigen (DA) on the magnetic particles (H) by a known method or the like. There is a method of making it.

The weight ratio of water in the immunoassay reagent (M) is 20 to 60% by weight based on the weight of the immunoassay reagent (M) from the viewpoint of storage stability. If it is less than 20% by weight or more than 60% by weight, the storage stability deteriorates. The weight ratio of water in the immunoassay reagent (M) is preferably 20 to 50% by weight based on the weight of the immunoassay reagent (M) from the viewpoint of further enhancing storage stability.
The weight ratio of the sugar (A) in the immunoassay reagent (M) is preferably 20 to 60% by weight, more preferably 25, based on the weight of the immunoassay reagent (M) from the viewpoint of storage stability. ~ 55% by weight.
The weight ratio of the immunoassay solid phase carrier (E) in the immunoassay reagent (M) is redispersibility in the immunoassay step based on the weight of the immunoassay reagent (M). When (E) is a magnetic particle, it is preferably 0.001 to 10% by weight, more preferably 0.01 to 1% by weight, from the viewpoint of magnetic collection and redispersibility].

The weight ratio of sugar (A) to water [sugar (A) / water] is preferably 0.33 to 3.0, preferably 0.80 to 3.0, from the viewpoint of storage stability. Is more preferable.

The pH of the immunoassay reagent (M) is preferably 5 to 10, more preferably 5 to 7, from the viewpoint of storage stability.
The immunoassay reagent (M) preferably contains a buffer (2-morpholinoetan sulfonic acid or the like) so as to have the above pH.

The immunoassay reagent (M) contains other components {protein (B) [bovine serum albumin, casein (or its hydrolyzate) and collagen peptide (or its enzymatic degradation product) as long as it does not interfere with storage stability. ), Etc.], inorganic salts (sodium chloride, etc.), surfactants (polyoxyethylene sorbitan monostearate, etc.), preservatives (sodium azide, etc.) and non-specific reaction inhibitors (IgG antibody, etc. derived from normal animals) At least one selected from the group consisting of} may be contained.
When the immunoassay reagent (M) contains a protein (B), an inorganic salt, a surfactant, a preservative and a non-specific reaction inhibitor, the content of each is based on the weight of the protein (M). The content of B) is 0.001 to 30% by weight, the content of inorganic salt is 0.001 to 5% by weight, the content of surfactant is 0.001 to 10% by weight, and the content of preservative is The content of the non-specific reaction inhibitor is preferably 0.001 to 0.1% by weight, preferably 0.0001 to 5% by weight.
When the immunoassay reagent (M) contains a protein (B), the weight ratio of the protein (B) to water [protein (B) / water] is 0.1 to 0 from the viewpoint of storage stability. It is preferably 1.0.

Examples of the surfactant contained in the immunoassay reagent (M) include known nonionic surfactants, amphoteric surfactants and anionic surfactants, and examples of the surfactant include non-specific adsorption. From the viewpoint of reduction, a water-soluble nonionic surfactant is preferable.
In addition, water-soluble means that 10 g is dissolved in 100 g of water at 25 ° C.
Specific examples of the water-soluble nonionic surfactant include polyoxyethylene nonylphenyl ether having an HLB of 12 or more, polyoxyethylene alkyl ether (polyoxyethylene octyl ether, etc.), polyoxyethylene sorbitan aliphatic ester, and the like. Can be mentioned.

<Immunoassay kit>
The immunoassay kit of the present invention is an immunoassay kit containing the immunoassay reagent (M) of the present invention.

In addition to the immunoassay reagent (M), the immunoassay kit of the present invention includes an immunoassay labeling reagent (F), a chemical luminescent reagent (I), and an immune reaction buffer reagent (Y) [immunity measurement described later]. In the step, a buffer solution for mixing with the immunoassay solid phase carrier (E) extracted from the immunoassay reagent (M)], a standard reagent (solution of a measurement object having a known concentration, etc.), a reagent for dilution (high concentration). A reagent for diluting a sample, etc.), a cleaning reagent (a reagent for cleaning a reaction vessel, etc.) and the like may be included, and an instruction manual, a jig necessary for measurement (for example, a sample container, etc.) may be included.

As the immunoassay labeling reagent (F), as described in the description of specific immunoassay methods (sandwich method, competitive method, etc.) described later, the measurement target substance labeled with the labeling substance and the labeling substance labeled with the labeling substance. It is preferable to use a labeling reagent containing a substance similar to the substance to be measured or a substance labeled with the labeling substance and specifically binding to the substance to be measured.

In the immunoassay labeling reagent (F), the labeling substance used for labeling is
For example, alkaline phosphatase, β-galactosidase, peroxidase (hereinafter, may be abbreviated as POD), microperoxidase, glucose oxidase, glucose-6-phosphate dehydrogenase, and malic acid dehydration used in enzyme immunoassay (EIA). Enzymes such as elementary enzymes, luciferase, tyrosinase, acid phosphatase;
For example, radioisotopes such as 99mTc, 131I, 125I, 14C, 3H, 32P used in Radioimmunoassay (RIA);
Fluorescent substances such as fluorescein, dancil, fluorescamine, coumarin, naphthylamine or derivatives thereof, green fluorescent protein (GFP) used in fluorescence immunoassay (FIA);
Luminescent substances such as luciferin, isolminol, luminol, bis (2,4,6-trifluorophenyl) oxalate;
Substances with ultraviolet absorption, such as phenol, naphthol, anthracene or derivatives thereof;
For example, 4-amino-2,2,6,6-tetramethylpiperidin-1-oxyl, 3-amino-2,2,5,5-tetramethylpyrrolidin-1-oxyl, 2,6-di-t-butyl. As a spin labelant typified by a compound having an oxyl group such as −α- (3,5-di-t-butyl-4-oxo-2,5-cyclohexadiene-1-ylidene) -p-trioxyl. Examples include substances having properties.
Of these, enzymes and fluorescent substances are preferable from the viewpoint of sensitivity and the like, alkaline phosphatase, POD and glucose oxidase are more preferable, and POD is particularly preferable.

In order to bind a labeling substance to a substance to be measured, a substance similar to the substance to be measured, and a substance that specifically binds to the substance to be measured, a method generally used in this field, for example, EIA, RIA and FIA known per se, etc. A self-known labeling method commonly used in [For example, Medical Chemistry Experiment Course, Volume 8, Supervision by Yuichi Yamamura, 1st Edition, Nakayama Shoten, 1971; Illustrated Fluorescent Antibody, by Akira Kawao, 1st Edition, ( Soft Science Co., Ltd., 1983; Enzyme Immunoassay, Eiji Ishikawa, Tadashi Kawai, Kiyoshi Muroi, 2nd Edition, Medical School, 1982, etc.] and the like may be used.

The amount of labeling substance used varies depending on the type of labeling substance used, so it cannot be said unconditionally. For example, when POD is used as a labeling substance, it is specific to the substance to be measured, a substance similar to the substance to be measured, or the substance to be measured. The substance to be specifically bound and the labeling substance are, for example, preferably in a molar ratio of 1: 1 to 20, more preferably in a molar ratio of 1: 1 to 10, and particularly preferably in a molar ratio of 1: 1 to 2. , A buffer solution (a buffer solution generally used in this field such as a Tris buffer solution, a phosphate buffer solution, a veronal buffer solution, a boric acid buffer solution, and a Good buffer solution) may be used.
The pH of the buffer solution may be in a range that does not suppress the antigen-antibody reaction, and is preferably 5 to 9.
Further, such a buffer solution contains a stabilizer (albumin, globulin, water-soluble gelatin, polyethylene glycol, etc.), a surfactant, a saccharide, or the like as long as it does not inhibit the target antigen-antibody reaction. May be left

The chemiluminescent reagent (I) is selected based on the above-mentioned labeling substance. For example, when the labeling substance is peroxidase, 2,3-dihydro-1,4-phthalazinedione compound and chemiluminescence enhancer are essential constituents. It contains the first chemiluminescent reagent solution and the second chemiluminescent reagent solution containing an oxidizing agent and water as essential constituents.

Examples of the 2,3-dihydro-1,4-phthalazinedione compound include known 2,391299, JP-A-10-31901, and JP-A-2000-279196, which are described in JP-A-2-291299 and JP-A-2000-279196. 3-Dihydro-1,4-phthalazinedione compounds and mixtures thereof can be used.
Of these, luminol, isolminol, N-aminohexyl-N-ethylisoluminol (AHEI), N-aminobutyl-N-ethylisoluminol (ABEI) and metal salts thereof (alkali metal salts, etc.) are preferable. More preferred is luminol and a metal salt thereof, and particularly preferred is a sodium salt of luminol.

Examples of the chemiluminescence enhancer include known chemiluminescence enhancers described in JP-A-59-500252, JP-A-59-171839, JP-A-2-291299, and the like, and mixtures thereof. Can be used. Of these, phenol is preferable from the viewpoint of chemiluminescence enhancing effect and the like, more preferably p-iodophenol, 4- (cyanomethylthio) phenol and 4-cyanomethylthio-2-chlorophenol, and particularly preferably 4-. (Cyanomethylthio) Phenol.

The first liquid of the chemiluminescent reagent is preferably a liquid, and is preferably alkaline from the viewpoint of the fluorescence intensity of the enzyme. The pH of the first liquid is preferably 7 to 11, and more preferably 8 to 10.
The pH is measured in accordance with JIS K040-12-1-10: 2000 (measurement temperature 25 ° C.).

Examples of the oxidizing agent contained in the second liquid of the chemical luminescent reagent include known oxidizing agents described in JP-A-8-261943 and JP-A-2000-279196 [Inorganic peroxide (hydrogen peroxide). , Sodium perborate, potassium perborate, etc.), organic peroxides (dialkyl peroxide, acyl peroxide, etc.), peroxic acid compounds (peroxosulfate, peroxolic acid, etc.), etc.].
Of these, from the viewpoint of storage stability and the like, an aqueous solution of hydrogen peroxide, an aqueous solution of sodium perborate and an aqueous solution of potassium perborate are preferable, and an aqueous solution of hydrogen peroxide is more preferable.

As the chemiluminescent reagent (I), from the viewpoint of sensitivity, the chemiluminescent reagent 1st solution in which the luminol luminescent reagent (I1) [2,3-dihydro-1,4-phthalazinedione compound is luminol and / or a metal salt thereof. ] And the hydrogen peroxide solution (I2) [the second solution of the chemiluminescent reagent in which the oxidizing agent is hydrogen peroxide] are preferably contained.

<Immunoassay method>
The immunoassay method of the present invention is an immunoassay measurement method using the immunoassay reagent (M) of the present invention, and immediately before performing immunoassay, the immunoassay reagent (M) is used as an immunoassay solid-state carrier ( It is preferable to carry out the step of extracting E).
Specifically, the immunoassay method is performed by extracting the immunoassay solid phase carrier (E) in the immunoassay reagent (M) by magnetic force or the like and then mixing it with the immunoassay buffer reagent (Y). The solid phase carrier reagent (Z) for immunoassay used in the above can be obtained.
Using the solid-phase carrier reagent (Z) for immunoassay thus obtained, a known immunoassay method (specifically, the sandwich method and the competitive method described later) and the like can be carried out.
In addition to the buffer solution, the buffer solution reagent (Y) for an immune reaction includes salts (sodium chloride, etc.), ethylenediamine tetraacetic acid, serum (mouse serum, etc.), and albumin (bovine) as long as the effects of the present invention are not impaired. Serum albumin, etc.), globulin, other proteins (casein hydrolyzate), water-soluble gelatin, stabilizers such as polyethylene glycol, surfactants [surfactants exemplified in the above description of the immunoassay reagent (M)). Etc.] and sugar [the above-mentioned sugar (A) etc.] and the like may be contained.
The weight ratio of the immunoassay solid support carrier (E) in the immunoassay solid support reagent (Z) is 0.001 to 10% by weight based on the weight of the immunoassay solid support reagent (Z). It is preferable, more preferably 0.01 to 1% by weight.

The method of immunoreaction using the solid-phase carrier reagent (Z) for immunoassay in the present invention is described in the literature generally used in the field of immunoassay [for example, Enzyme Immunoassay 2nd Edition (edited by Eiji Ishikawa et al., Igaku-Shoin) 1982] The sandwich method, the competitive method and the method described in JP-A-6-130063, specifically, the following three methods are included.

Among the immunoassay methods in the present invention, the following three types are specifically used as a method using magnetic particles (H) having an antigen (C) or an antibody (D) as a solid-phase carrier (E) for immunoassay. Method is included.
<First method: Sandwich method>
As magnetic particles (H) (preferably magnetic particles containing a metal oxide), a substance that specifically binds to a substance to be measured {a substance to be measured-binding substance; specifically, an antigen (C) or an antibody (D). } Is immobilized on the surface of the particles.
Then, "a sample containing a substance to be measured (for example, a biological sample)", "magnetic particles (H)", and "a substance labeled by a labeling substance that specifically binds to the substance to be measured (labeled measurement)". Contact with the target substance binding substance).
As a result, a complex (labeled complex) of the "measurement target substance binding substance immobilized on the magnetic particles (H)", the "measurement target substance", and the "labeled measurement target substance binding substance" is formed.
Then, the magnetic particles (H) carrying the labeled complex are separated by B / F, the amount of the labeled substance in the labeled complex is measured, and the substance to be measured in the sample is measured based on the result.

<Second method: Competitive method 1>
As the magnetic particles (H) (preferably magnetic particles containing a metal oxide), a substance to be measured and / or a substance similar to the substance to be measured {specifically, an antigen (C) or an antibody (D)} is used as particles. Use the one that is fixed on the surface of.
Then, "a sample containing a substance to be measured (for example, a biological sample)", "a substance labeled with a labeling substance that specifically binds to the substance to be measured (labeled substance binding substance to be measured)", and " It is brought into contact with "magnetic particles (H)".
As a result, the "labeled substance-binding substance to be measured" is made to compete with the "substance to be measured and / or a substance similar to the substance to be measured" immobilized on the magnetic particles (H) and the "substance to be measured", and " A complex (labeled complex) of the "substance to be measured and / or a substance similar to the substance to be measured" immobilized on the magnetic particles (H) and the "substance to be labeled and the substance to be measured" is formed.
Then, the magnetic particles (H) carrying the labeled complex are separated by B / F, the amount of the labeled substance in the labeled complex is measured, and the substance to be measured in the sample is measured based on the result.

<Third method: Competitive method 2>
As magnetic particles (H) (preferably magnetic particles containing a metal oxide), a substance that specifically binds to a substance to be measured {a substance to be measured-binding substance; specifically, an antigen (C) or an antibody (D). } Is immobilized on the surface of the particles.
Then, "a sample containing a substance to be measured (for example, a biological sample)" and "a substance to be measured labeled with a labeled substance and / or a substance similar to a substance to be measured labeled with a labeled substance (a substance to be labeled or similar thereof). The substance) ”and the“ magnetic particle (H) ”are brought into contact with each other.
As a result, the "measurement target substance" and the "labeled measurement target substance or a similar substance" are competitively reacted with the "measurement target substance binding substance immobilized on the magnetic particles (H)", and the "magnetic particles (H)) A complex (labeled complex) of the "measurement target binding substance" immobilized on the "labeled measurement target substance or a similar substance thereof" is formed.
Then, the magnetic particles (H) carrying the labeled complex are separated by B / F, the amount of the labeled substance in the labeled complex is measured, and the substance to be measured in the sample is measured based on the result.

As the similar substance (analog) of the substance to be measured in the present invention, the substance that can be bonded to the "bonding site with the substance to be measured" possessed by the "substance that specifically binds to the substance to be measured", in other words, the "measurement target". A substance that has a binding site with a "substance that specifically binds to the substance to be measured" that the "substance" has, in other words, at the time of reaction between the "substance to be measured" and the "substance that specifically binds to the substance to be measured" When coexisting, there are substances that can compete with the reaction between the "substance to be measured" and the "substance that specifically binds to the substance to be measured".

The "substance that specifically binds to the substance to be measured" in the present invention is preferably an antibody (D) when the substance to be measured is an antigen (C), and is preferably an antibody (D) when the substance to be measured is an antibody (D). Is preferably the antigen (C).

The immunoassay method of the present invention includes chemiluminescent enzyme immunoassay (CLEIA), enzyme immunoassay (EIA), radioimmunosassay (RIA), chemiluminescent immunoassay (ECLIA), and fluorescent enzyme immunoassay. (FEIA), chemiluminescent immunoassay (CLIA), latex optical immunoassay (LPIA), latex particle counting immunoassay (CIA) and the like.

Hereinafter, the present invention will be further described with reference to Examples, but the present invention is not limited thereto. Hereinafter, unless otherwise specified, parts indicate parts by weight.

<Example 1>
The immunoassay reagent (M-1) was obtained as follows.

Preparation of magnetic particles (PH-1):
<Preparation of magnetic metal oxide particles>
186 parts of iron (III) chloride hexahydrate, 68 parts of iron (II) chloride tetrahydrate and 1288 parts of water are charged in a reaction vessel and dissolved to raise the temperature to 50 ° C., and the temperature under stirring is 50 to 55. While maintaining the temperature at ° C., 280 parts of 25 wt% aqueous ammonia was added dropwise over 1 hour to obtain magnetite particles in the water. 64 parts of oleic acid as a dispersant was added to the obtained magnetite particles, and stirring was continued for 2 hours. After cooling to room temperature, the operation of washing the magnetite particles adsorbed with oleic acid obtained by solid-liquid separation by decantation with 1000 parts of water was performed three times, and further the operation of washing with 1000 parts of acetone was performed twice. By drying at ° C. for 2 days, superparamagnetic metal oxide particles having a volume average particle diameter of 15 nm were obtained.

<Preparation of core layer>
80 parts of superparamagnetic metal oxide particles were added to 240 parts of tetraethoxysilane and dispersed to prepare a dispersion liquid (a1).
Next, 5050 parts of water, 3500 parts of a 25 wt% ammonia aqueous solution, and 400 parts of a nonionic surfactant (“NSA-17”, manufactured by Sanyo Chemical Industries, Ltd.) were added to the reaction vessel to clear mix (M-Technique Co., Ltd.). ) Was mixed to obtain a solution (a2). After raising the temperature to 50 ° C., the dispersion liquid (a1) was added dropwise to the solution (a2) over 1 hour while stirring at a rotation speed of 6,000 rpm of the clear mix, and then reacted at 50 ° C. for 1 hour. After the reaction, the mixture was centrifuged at 2,000 rpm for 20 minutes to remove the supernatant in which fine particles were present, and a core layer was obtained.

<Making magnetic particles>
80 parts of the core layer, 2500 parts of deionized water, 260 parts of a 25 wt% ammonia aqueous solution, 2500 parts of ethanol, and 1200 parts of tetraethoxysilane were added to the reaction vessel and mixed using Clearmix (manufactured by M-Technique Co., Ltd.). The reaction was carried out for 2 hours with stirring at a clear mix rotation speed of 6,000 rpm. After the reaction, the supernatant containing fine particles was removed by centrifugation at 2,000 rpm for 20 minutes. After centrifugation, 4000 parts of deionized water was added to the precipitated particles to redisperse the particles, and the dispersed particles were collected 10 times by using a magnet to collect the particles and remove the supernatant.
Next, 5000 parts of water was added to the obtained solid phase to disperse the particles, and after centrifugation at 600 rpm for 10 minutes, the operation of removing the supernatant in which the fine particles were present was performed 20 times, and then the obtained solid phase was obtained. Classification was performed by precipitating and removing particles having a large particle size by adding 5000 parts of water to disperse the particles and centrifuging at 300 rpm for 10 minutes.
Further, the particles were collected using a magnet to remove the supernatant liquid. Then, after adding 5000 parts of water to disperse the core-shell particles, the operation of collecting the particles using a magnet and removing the supernatant was performed 10 times, and the target magnetic particles (PH) having a volume average particle diameter of 2.0 μm were performed. -1) was obtained. As a result of measuring the content of the superparamagnetic metal oxide particles in the obtained magnetic particles (PH-1), the content was 81% by weight.

<Measurement method of volume average particle size of particles (superparamagnetic metal oxide particles and magnetic particles)>
Using a scanning electron microscope (model number: JSM-7000F, manufacturer name: JEOL Ltd.), observe any 200 superparamagnetic metal oxide particles and measure the particle size to obtain the volume average particle size. It was.
For magnetic particles, the volume average particle diameter was determined by the same method.

<Method of measuring the content of superparamagnetic metal oxide particles>
Observe any 20 magnetic particles with a scanning electron microscope (model number JSM-7000F, manufacturer name JEOL Ltd.) and use an energy dispersive X-ray spectrometer (model number INCA Wave / Energy, manufacturer name Oxford). The content of the ultranormal magnetic metal oxide particles was measured, and the average value thereof was taken as the content S. Further, the silica content was measured by the same measurement, and the average value thereof was taken as the content T. The content of the superparamagnetic metal oxide particles was determined by the following calculation formula (1).
Content of superparamagnetic metal oxide particles (% by weight) = (S) / (S + T) × 100 ... (1)

<Preparation of anti-Tg monoclonal antibody F (ab') _2>
10 mg of an anti-Tg monoclonal antibody (mouse) (manufactured by Hytest) was dissolved in 2 ml of 0.1 M acetate buffer having a pH of 4.0, 0.2 mg of pepsin was added, and the mixture was inked at 37 ° C. for 3 hours. After stopping the reaction by adding an appropriate amount of 1M carbonate buffer (pH 9.0) to neutralize (pH: 7.0), use 0.02M phosphate buffer (pH 7.2) containing 0.2M sodium chloride. Gel filtration was performed on an equilibrated Ultragel AcA-44 (LKB) column (02, OX70 cm, manufactured by Sigma Aldrich), followed by 0.05 M phosphate buffer (pH 6.0) containing 5 mM EDTA.2Na. Dialysis was performed to obtain anti-Tg monoclonal antibody F (ab') _2.

Preparation of magnetic particles (H1-1) and immunoassay reagent (M-1):
Add 40 mg of the produced magnetic particles (PH-1) to a polyethylene bottle with a lid containing 40 mL of an acetone solution containing 1 wt% γ-aminopropyltriethoxysilane, react at 25 ° C. for 1 hour, and collect the magnetic particles with a neodymium magnet. After magnetism, the liquid was removed by suction with an aspirator. Next, 40 mL of deionized water was added to cover the bottle, and the polystyrene bottle was slowly inverted and stirred twice. After collecting the magnetic particles with a neodymium magnet, the liquid was attracted and removed with an aspirator to wash the magnetic particles. This cleaning operation was performed 5 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 to cover the bottle, and the polystyrene bottle was slowly inverted and stirred twice. After collecting the magnetic particles with a neodymium magnet, the liquid was attracted and removed with an aspirator to wash the magnetic particles. This cleaning operation was performed 10 times.
Further, the washed magnetic particles were added to a polyethylene bottle with a lid 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.} The reaction was carried out at 25 ° C. for 1 hour. After the reaction, the magnetic particles were collected with a neodymium magnet, and then the phosphate buffer solution containing _{anti-Tg monoclonal antibody F (ab') 2 was removed.}
The magnetic particles were then placed in a covered polyethylene bottle containing 40 mL of 0.02 M phosphate buffer (pH 7.0) containing 0.001 wt% Tg (thyroglobulin, Hytest) and 1 wt% bovine serum albumin. In addition, the magnetic particles (H1-1) to which the Tg-anti-Tg monoclonal antibody F (ab') ₂ complex is bound by standing at 25 ° C. for 12 hours (Tg is the anti-Tg monoclonal antibody F (ab'). ) Those bound to the magnetic particles via _{2) were adjusted.} After collecting magnetic particles (H1-1) with a neodymium magnet, the supernatant was removed.
Next, each component shown in Table 1 is diluted with a magnetic particle storage solution (X1) containing parts by weight shown in Table 1 so that the concentration of the magnetic particles (H1-1) becomes 0.01% by weight. , A reagent for immunoassay (M-1) containing magnetic particles (H1-1) was prepared.

The following components were used as the components in Table 1.
Sugar (A-1): Gglycerol Sugar (A-2): Trehalose Sugar (A-3): Lactose Sugar (A-4): Fructo-oligosaccharide [manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., Wako first-class, molecular weight (chemical formula) A mixture containing a compound having a molecular weight (amount) of 504, a compound having a molecular weight (chemical formula) of 666, and a compound having a molecular weight (chemical formula) of 828]
MES: 2-morpholinoetan sulfonic acid NaCl: salt protein (B-1): BSA (bovine serum albumin)
Protein (B-2): Casein [Made of milk, made by Nacalai Tesque Co., Ltd.]
Protein (B-3): Casein hydrolyzate [Merck & Co., Ltd.]
Protein (B-4): Collagen peptide enzymatically decomposed product [manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.]

<Examples 2-9, 16, 19-21 and Comparative Examples 1-2>
In Example 1, instead of the magnetic particle preservative solution (X1), the magnetic particle preservative solutions (X2) to (X9), (X16), (X16), which contain each component shown in Table 1 by weight as shown in Table 1. Using X19) to (X21) or (X'1) to (X'2), the concentration of the magnetic particles (H) in the immunoassay reagent (M) was changed from 0.01% by weight to the concentration shown in Table 1. Except for the changes, the same procedure as in Example 1 was carried out, and immunoassay reagents (M-2) to (M-9), (M-16), (M-19) to (M-21) and comparisons were carried out. Reagents for immunoassay (M'-1) to (M'-2) were obtained.

<Example 10 and Comparative Example 3>
40 mg of the magnetic particles (PH-1) produced in Example 1 were added to a polyethylene bottle with a lid containing 40 mL of an acetone solution containing 1 wt% γ-aminopropyltriethoxysilane, and the mixture was reacted at 25 ° C. for 1 hour. After collecting the magnetic particles with a neodymium magnet, the liquid was attracted and removed with an aspirator. Next, 40 mL of deionized water was added to cover the bottle, and the polystyrene bottle was slowly inverted and stirred twice. After collecting the magnetic particles with a neodymium magnet, the liquid was attracted and removed with an aspirator to wash the magnetic particles. This cleaning operation was performed 5 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 to cover the bottle, and the polystyrene bottle was slowly inverted and stirred twice. After collecting the magnetic particles with a neodymium magnet, the liquid was attracted and removed with an aspirator to wash the magnetic particles. This cleaning operation was performed 10 times.
Further, the washed magnetic particles were added to a polyethylene bottle with a lid containing 120 mL of 0.02 M phosphate buffer (pH 8.7) containing Tg (thyroglobulin, manufactured by Hytest) at a concentration of 10 μg / mL, and the temperature was 25 ° C. Was reacted for 1 hour. After the reaction, the magnetic particles were collected with a neodymium magnet, and then the Tg-containing phosphate buffer solution was removed to obtain magnetic particles (H-1) in which Tg was bonded.
Next, the magnetic particle preservative solution (X10) or (X) containing each component shown in Table 1 by weight according to Table 1 so that the concentration of the magnetic particles (H-1) becomes 0.1% by weight. Diluted with '3) to prepare an immunoassay reagent (M-10) containing magnetic particles (H-1) and an immunoassay reagent (M'-3) for comparison.

<Example 11 or Comparative Example 4>
In the preparation of the magnetic particles (H1-1) and the immunoassay reagent (M-1) in Example 1, Tg (thyroglobulin, manufactured by Hytest) was used instead _{of the anti-Tg monoclonal antibody F (ab') 2.} By using an anti-Tg monoclonal antibody (mouse) (manufactured by Hytest) instead of (thyroglobulin, manufactured by Hytest), the anti-Tg monoclonal antibody-Tg complex is bound instead of the magnetic particles (H1-1). (H2-1) (anti-Tg monoclonal antibody bound to magnetic particles via Tg) is obtained, and each component shown in Table 1 is shown in place of the magnetic particle storage solution (X1). Table 1 shows the concentration of the magnetic particles (H) in the immunoassay reagent (M) from 0.01% by weight using the magnetic particle preservative solution (X11) or (X'4) contained in 1 part by weight. An immunoassay reagent (M-11) and a comparative immunoassay reagent (M'-4) were obtained in the same manner as in Example 1 except that the concentration was changed to.

<Example 12>
The magnetic particles in Example 2 except that the input amount of the superparamagnetic metal oxide particles was changed from 80 parts to 63 parts in <Preparation of the core layer> during the production of the magnetic particles (PH-1) in Example 2. The same procedure as in the preparation of (PH-1) was carried out to obtain magnetic particles (PH-2) having a volume average particle diameter of 2.0 μm. As a result of measuring the content of the superparamagnetic metal oxide particles in the obtained magnetic particles (PH-2), the content was 61% by weight.
In the subsequent operations of Example 2, the same procedure was carried out except that the magnetic particles (PH-2) were used instead of the magnetic particles (PH-1), and Tg was used instead of the magnetic particles (H1-1). - containing the anti-Tg monoclonal antibody F (ab _{') 2} complex formed by binding magnetic particles (H1-2) (Tg anti Tg monoclonal antibody F (ab' that bound to the magnetic particles via _{a) 2)} Then, an immunoassay reagent (M-12) diluted with a magnetic particle storage solution (X12) was obtained.

<Example 13>
The magnetic particles in Example 2 except that the input amount of the superparamagnetic metal oxide particles was changed from 80 parts to 92 parts in <Preparation of the core layer> during the production of the magnetic particles (PH-1) in Example 2. The same procedure as in the production of (PH-1) was carried out to obtain magnetic particles (PH-3) having a volume average particle diameter of 2.0 μm. As a result of measuring the content of the superparamagnetic metal oxide particles in the obtained magnetic particles (PH-3), the content was 90% by weight.
In the subsequent operations of Example 2, the same procedure was carried out except that the magnetic particles (PH-3) were used instead of the magnetic particles (PH-1), and Tg was used instead of the magnetic particles (H1-1). - containing the anti-Tg monoclonal antibody F (ab _{') 2} complex formed by binding magnetic particles (H1-3) (Tg anti Tg monoclonal antibody F (ab' that bound to the magnetic particles via _{a) 2)} Then, an immunoassay reagent (M-13) diluted with a magnetic particle storage solution (X13) was obtained.

<Example 14>
In <Preparation of magnetic particles> during production of magnetic particles (PH-1) in Example 2, a step of "centrifuging at 1200 rpm for 10 minutes" was carried out instead of the step of "centrifuging at 600 rpm for 10 minutes". In addition, the same procedure as in the production of the magnetic particles (PH-1) in Example 2 was carried out except that the step of "centrifuging at 600 rpm for 10 minutes" was carried out instead of the step of "centrifuging at 300 rpm for 10 minutes". Then, magnetic particles (PH-4) having a volume average particle diameter of 1.0 μm were obtained. As a result of measuring the content of the superparamagnetic metal oxide particles in the obtained magnetic particles (PH-4), the content was 81% by weight.
In the subsequent operations of Example 2, the same procedure was carried out except that the magnetic particles (PH-4) were used instead of the magnetic particles (PH-1), and Tg was used instead of the magnetic particles (H1-1). - containing the anti-Tg monoclonal antibody F (ab _{') 2} complex formed by binding magnetic particles (H1-4) (Tg anti Tg monoclonal antibody F (ab' that bound to the magnetic particles via _{a) 2)} Then, an immunoassay reagent (M-14) diluted with a magnetic particle storage solution (X14) was obtained.

<Example 15>
In <Preparation of magnetic particles> during production of magnetic particles (PH-1) in Example 2, a step of "centrifuging at 200 rpm for 10 minutes" was carried out instead of the step of "centrifuging at 600 rpm for 10 minutes". In addition, the same procedure as in the production of the magnetic particles (PH-1) in Example 2 was carried out except that the step of "centrifuging at 100 rpm for 10 minutes" was carried out instead of the step of "centrifuging at 300 rpm for 10 minutes". Then, magnetic particles (PH-5) having a volume average particle diameter of 5.0 μm were obtained. As a result of measuring the content of the superparamagnetic metal oxide particles in the obtained magnetic particles (PH-5), the content was 81% by weight.
In the subsequent operations of Example 2, the same procedure was carried out except that the magnetic particles (PH-5) were used instead of the magnetic particles (PH-1), and Tg was used instead of the magnetic particles (H1-1). - containing the anti-Tg monoclonal antibody F (ab _{') 2} complex formed by binding magnetic particles (H1-5) (Tg anti Tg monoclonal antibody F (ab' that bound to the magnetic particles via _{a) 2)} Then, an immunoassay reagent (M-15) diluted with a magnetic particle storage solution (X15) was obtained.

<Example 17>
In the preparation of the magnetic particles (H1-1) and the immunoassay reagent (M-1) in Example 2, the anti-Tg monoclonal antibody Fab prepared by the following method was used instead _{of the anti-Tg monoclonal antibody F (ab') 2.} A magnetic particle (H1-6) (Tg) formed by binding a Tg-anti-Tg monoclonal antibody Fab complex instead of the magnetic particle (H1-1) was carried out in the same manner as in Example 2 except that it was used. An immunoassay reagent (M-17) containing the anti-Tg monoclonal antibody Fab bound to the magnetic particles and diluted with the magnetic particle storage solution (X17) was prepared.
<Preparation of anti-Tg monoclonal antibody Fab>
10 mg of an anti-Tg monoclonal antibody (mouse) (manufactured by Hytest) was dissolved in 2 ml of 0.02 M phosphate buffer (pH 7.2) containing 0.2 M sodium chloride, 0.2 mg of papain was added, and the temperature was 37 ° C. for 3 hours. Ink buffered. Iodoacetamide was added to the reaction solution so that the final concentration was 0.03 M, completely dissolved to stop the reaction, and then equilibrated with 0.02 M phosphate buffer (pH 7.2) containing 0.2 M sodium chloride. Gel filtration was performed on an Ultragel AcA-44 (LKB) column (02, OX70 cm, manufactured by Sigma Aldrich), followed by application to NAb ^TM Protein A Plus Spin Volume (5 mL, manufactured by Thermo Fisher), and the column was passed through. By recovering the solution, an anti-Tg monoclonal antibody Fab was obtained.

<Example 18>
To a polyethylene bottle with a lid containing 40 mL of an acetone solution containing 1 wt% γ-aminopropyltriethoxysilane, 40 mg of the magnetic particles (PH-1) obtained in Example 1 was added and reacted at 25 ° C. for 1 hour to generate a neodymium magnet. After collecting the magnetic particles with, the liquid was sucked and removed with an aspirator. Next, 40 mL of deionized water was added to cover the bottle, and the polystyrene bottle was slowly inverted and stirred twice. After collecting the magnetic particles with a neodymium magnet, the liquid was attracted and removed with an aspirator to wash the magnetic particles. This cleaning operation was performed 5 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 to cover the bottle, and the polystyrene bottle was slowly inverted and stirred twice. After collecting the magnetic particles with a neodymium magnet, the liquid was attracted and removed with an aspirator to wash the magnetic particles. This cleaning operation was performed 10 times.
Further, the washed magnetic particles were placed in a polyethylene bottle with a lid containing 120 mL of 0.02 M phosphate buffer (pH 8.7) containing an anti-Tg monoclonal antibody (mouse) (manufactured by Hytest) at a concentration of 10 μg / mL. In addition, the reaction was carried out at 25 ° C. for 1 hour. After the reaction, the magnetic particles were collected with a neodymium magnet, and then the anti-Tg monoclonal antibody-containing phosphate buffer was removed to obtain magnetic particles (H-2) to which the anti-Tg monoclonal antibody was bound.
Next, each component shown in Table 1 is diluted with a magnetic particle preservative solution (X18) containing parts by weight shown in Table 1 so that the concentration of the magnetic particles (H-2) becomes 0.1% by weight. , A reagent for immunoassay (M-18) containing magnetic particles (H-2) was prepared.

Table 1 shows the pHs of the immunoassay reagents (M-1) to (M-21) and the comparative immunoassay reagents (M'-1) to (M'-4).

<Example 22>
The immunoassay reagent (M-1) obtained in Example 1, and the following immune reaction buffer reagent (Y-1) and labeling reagent (F-1) (POD-labeled anti-human IgG polyclonal antibody-containing reagent). , The chemiluminescent reagent first solution (I-1) and the chemiluminescent reagent second solution (I-2) were combined to obtain the immunoassay kit (S-1) of the present invention.

Preparation of buffer reagent (Y-1) for immune reaction:
10% by weight BSA, 0.1% by weight Naroacty CL-100 [polyoxyalkylene alkyl ether, manufactured by Sanyo Kasei Kogyo Co., Ltd.], 0.1% by weight EDTA (ethylenediamine-N, N, N', N'-Disodium tetraacetate dihydrate, manufactured by Dojin Chemical Laboratory Co., Ltd., 5% by weight mouse serum [manufactured by Cosmo Bio Co., Ltd.] and 0.02M sodium phosphate (pH 7.0) The buffer solution for immune reaction (Y-1) containing the above was prepared and stored in a refrigerator (2 to 10 ° C.).

Preparation of labeling reagent (F-1):
Using anti-human IgG polyclonal antibody (rabbit) (manufactured by Dako Japan Co., Ltd.) and horseradish-derived POD (manufactured by Toyobo Co., Ltd.), literature (S Yoshitake, M Imagawa, E Ishikawa, Etol; Jay Biochem , Vol. 92, 1982, 1413-1424) to prepare a POD-labeled anti-human IgG polyclonal antibody (rabbit). A 0.02M phosphate buffer (pH 7.0) containing 0.5% by weight of bovine serum albumin and 1% by weight of Naroacty CL-100 as a surfactant was used as a POD-labeled anti-human IgG polyclonal antibody (rabbit). ) Concentration was diluted to a concentration of 100 nM to prepare a labeling reagent (F-1), which was stored in a refrigerator (2 to 10 ° C.).

Preparation of chemiluminescent reagent first solution (I-1):
0.7 g of luminol sodium salt [manufactured by Sigma-Aldrich Japan Co., Ltd.] and 0.1 g of 4- (cyanomethylthio) phenol were placed in a 1,000 mL volumetric flask. Add 3- [4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid / sodium hydroxide buffer (10 mM, pH 8.6) to a solution volume of 1,000 mL and homogenize at 25 ° C. The chemiluminescent reagent first solution (I-1) was prepared by mixing. It was stored refrigerated (2-10 ° C) until used for measurement.

Preparation of chemiluminescent reagent second liquid (I-2):
1,000 mL and 6.6 g of hydrogen peroxide [manufactured by Wako Pure Chemical Industries, Ltd., special grade reagent, concentration 30% by weight] were charged into a 1,000 mL volumetric flask. Deionized water was charged so that the volume of the solution was 1,000 mL, and the mixture was uniformly mixed at 25 ° C. to prepare a chemiluminescent reagent second solution (I-2). It was stored refrigerated (2-10 ° C) until used for measurement.

<Examples 23 to 31, 33 to 38 and 40 to 42, and Comparative Examples 5 to 7>
In Example 22, instead of the immunoassay reagent (M-1), the immunoassay reagents (M-2) to (M-10), (M-12) to (M-17) or (M-19) )-(M-21) or the comparative immunoassay reagents (M'-1)-(M'-3) were carried out in the same manner as in Example 22, and the immunoassay kit of the present invention was used. (S-2) to (S-10), (S-12) to (S-17) and (S-19) to (S-21), and a comparative immunoassay kit (S'-1) to (S'-3) was obtained.

<Examples 32 and 39 and Comparative Example 8>
In Example 22, instead of the immunoassay reagent (M-1), an immunoassay reagent (M-11) or (M-18) or a comparative immunoassay reagent (M'-4) was used. The immunoassay kit (S-11) and (S) of the present invention were carried out in the same manner as in Example 22 except that the following labeling reagent (F-2) was used instead of the labeling reagent (F-1). -18) and a comparative immunoassay kit (S'-4) were obtained.

Preparation of labeling reagent (F-2):
In the preparation of the labeling reagent (F-1) in Example 1, an anti-Tg mouse monoclonal antibody (manufactured by Hytest) was used instead of the anti-human IgG polyclonal antibody (rabbit) (manufactured by Dako Japan Co., Ltd.). The same procedure was carried out to prepare a labeling reagent (F-2) [reagent containing 100 nM POD-labeled anti-Tg mouse monoclonal antibody] and stored in a refrigerator (2 to 10 ° C.).

Immunoassay kits (S-1) to (S-21) obtained in Examples 22 to 42 or comparative immunoassay kits (S'-1) to (S'-) obtained in Comparative Examples 5 to 8. Using 4), immunoassay was carried out by the following method, and the storage stability of the immunoassay reagent (M) was evaluated.
<Evaluation method of storage stability of immunoassay reagent (M)>
Immunoassay was performed by the following sandwich method, and the storage stability of the immunoassay reagent (M) was evaluated.
After production, 0.05 mL of the immunoassay reagent (M) stored in a refrigerator (4 to 7 ° C.) for 5 days was dispensed into a test tube, magnetic particles were collected with a magnet, and the supernatant was removed with an aspirator. The buffer reagent for immune reaction (Y-1) was added to disperse the magnetic particles to obtain a solid phase carrier reagent (Z) for immunomeasurement [the concentration of the magnetic particles is the solid phase carrier reagent (Z) for immunomeasurement). The concentration was adjusted to the value shown in Table 2 based on the weight of.]
Next, pooled sera [Examples 22-31, 33-38 and 40-42 and Comparative Examples 5-7 used pooled sera containing 300 IU / mL of anti-thyroglobulin antibody (TgAb), and used Examples 32 and 39. Further, in Comparative Example 8, 0.05 mL [using pooled serum containing 10 ng / mL Tg] was injected into a test tube and reacted at 37 ° C. for 3 minutes in the test tube, and the complex [Examples 22 to 31, In 33-38 and 40-42 and Comparative Examples 5 to 7, the Tg-immobilized magnetic particles / TgAb complex, and in Examples 32 and 39 and Comparative Example 8, the anti-Tg monoclonal antibody-immobilized magnetic particles / Tg. Complex] was formed.
After the reaction, 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 surface. Then, 0.5 mL of physiological saline was added to disperse the magnetic particles, and after collecting the magnetism, the washing operation of removing the liquid with an aspirator was performed twice.

Subsequently, the labeling reagent (F) contained in the immunoassay kit [Examples 22 to 31, 33 to 38 and 40 to 42, and Comparative Examples 5 to 7 use the labeling reagent (F-1), and Examples 32 and 32 and In 39 and Comparative Example 8, 0.05 mL using the labeling reagent (F-2) was injected into a test tube and reacted at 37 ° C. for 3 minutes in the test tube, and the complex [Examples 22 to 31, 33 to In 38 and 40-42 and Comparative Examples 5 to 7, Tg-immobilized magnetic particles / TgAb / POD-labeled anti-human IgG polyclonal antibody complex, and in Examples 32 and 39 and Comparative Example 8, anti-Tg monoclonal antibody was immobilized. Transformed magnetic particles / Tg / POD-labeled anti-Tg mouse monoclonal antibody complex] were formed.
After the reaction, 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 surface. Then, 0.5 mL of physiological saline was added to disperse the magnetic particles, and after collecting the magnetism, the washing operation of removing the liquid with an aspirator was performed twice.
Finally, 0.07 mL of the chemiluminescent reagent first solution (I-1) and 0.07 mL of the chemiluminescent reagent second solution (I-2) are added at the same time, and the chemiluminescent reagent is luminescence-reacted at 37 ° C. for 45 seconds to prepare the chemiluminescent reagent. The average chemiluminescence amount 43 to 45 seconds after the addition was measured with a luminometer [“Lumat LB9507” manufactured by Berthold Japan Co., Ltd.]. The average amount of light emitted at this time was defined as the average amount of light emitted (Y1).

Instead of the immunoassay reagent (M) stored in a refrigerator (4 to 7 ° C.) for 5 days after production, the immunoassay reagent (M) stored at 35 ° C. for 5 days (accelerated test) after production was used. The average light emission amount was measured in the same manner as in the above, and this value was taken as the average light emission amount (Y2).

The storage stability of the immunoassay reagent (M) was calculated from the luminescence amount ratio obtained by the following formula. The results are shown in Table 2.
Light emission ratio (%) = (Y2 / Y1) x 100

Regarding the immunoassay reagent (M) containing magnetic particles in which an antigen (C) is immobilized as a substance that specifically binds to the substance to be measured, the substance to be measured is water in the reagent (M) for immunoassay. Comparing Examples 22 to 31, 33 to 38 and 40 to 42 in which the weight ratio of water is 20 to 60% by weight with Comparative Examples 5 to 7 in which the weight ratio of water is outside the range of 20 to 60% by weight. It can be seen that Examples 22 to 31, 33 to 38 and 40 to 42 are superior in storage stability of the immunoassay reagent (M).
Further, with respect to the immunoassay reagent (M) containing magnetic particles in which an antibody (D) is immobilized as a substance that specifically binds to the measurement target substance and the measurement target substance is an antigen, the immunoassay reagent (M) 32 and 39 in which the weight ratio of water in the above is 20 to 60% by weight and Comparative Example 8 in which the weight ratio of water is outside the range of 20 to 60% by weight are compared with Examples 32 and 39. However, it can be seen that the immunoassay reagent (M) is excellent in storage stability.

Since the immunoassay reagent, immunoassay kit, and immunoassay method of the present invention are excellent in storage stability, radioimmunoassay, enzyme immunoassay, fluorescence immunoassay, chemoluminescence immunoassay, etc. Widely applicable to clinical tests.

Claims (9)

An immunoassay reagent containing a solid-phase carrier (E) for immunoassay, sugar (A), and water.
The immunoassay reagent (M) in which the weight ratio of the water is 20 to 60% by weight based on the weight of the immunoassay reagent. The immunoassay reagent according to claim 1, further comprising a protein (B). The immunoassay reagent according to claim 1 or 2, wherein the ratio of the weight of the sugar (A) is 20 to 60% by weight based on the weight of the immunoassay reagent. The reagent for immunoassay according to any one of claims 1 to 3, wherein the solid-phase carrier (E) for immunoassay is a magnetic particle (H) having an antigen (C) or an antibody (D). The magnetic particles (H) are magnetic via the magnetic particles (H1) formed by binding to the magnetic particles via an antibody (CA) against the antigen (C) and / or via an antigen (DA) against the antibody (D). The immunoassay reagent according to claim 4, which is a magnetic particle (H2) formed by binding to a particle. The reagent for immunoassay according to claim 5, wherein the antibody (CA) in the magnetic particles (H1) is F (ab') _{2 and / or Fab.} The immunoassay according to any one of claims 4 to 6, wherein the magnetic particles (H) are magnetic particles containing 60 to 95% by weight of a superparamagnetic metal oxide having a volume average particle diameter of 1 to 20 nm. reagent. An immunoassay kit comprising the reagent for immunoassay according to any one of claims 1 to 7. An immunoassay method using the immunoassay reagent according to any one of claims 1 to 7.