JP2018021903A - Reagent for immunoassay, kit for immunoassay and immunoassay method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide regent for immunoassay excellent in storage stability for a substance F labeled with enzyme, a kit for immunoassay containing the reagent for immunoassay, and an immunoassay method using the kit.SOLUTION: Regent X for immunoassay contains substance FC and hydrophilic protein D. The substance FC is substance labeled with substance F and enzyme C. The substance F is at least one kind of substance selected from a group consisting of measurement object substance F1, analogue F2 of the measurement object substance and substance F3 specifically bonding to the measurement object substance. The hydrophilic protein D is protein dissolved by 2.0 g or more in 100 g of water with pH of 7.0 at 25°C. The content of the hydrophilic protein D in the regent X for immunoassay is within a range of 2-6 wt.% with a weight of X as reference.SELECTED DRAWING: None

Description

  The present invention relates to an immunoassay reagent, an immunoassay kit, and an immunoassay method.

In recent years, measurement methods using an immune reaction have been frequently used in fields such as clinical examinations and immunology because of their simple operation. Among them, enzyme immunoassay and chemiluminescence immunoassay are often used from the viewpoint of high sensitivity. In enzyme immunoassay, an enzyme-labeled antibody is generally used (Non-patent Document 1).
Enzyme-labeled antibodies are often unstable, especially at low concentrations, and are known to degrade during storage. Several techniques are known to solve this problem. For example, there have been reports on labeling by the maleimide hinge method, use of a citrate buffer, lyophilization of labeled antibodies, and the like. In addition, a method of allowing a liberated enzyme to coexist in a solution containing an enzyme-labeled antibody has also been proposed (Patent Document 1 and Patent Document 2).
Various attempts have been made to stabilize the enzyme-labeled antibody as described above, but few have sufficient storage stability to withstand use as a commercially available reagent requiring stability of at least one year. Further, although stability is maintained by freeze-drying, another problem arises that the operation becomes complicated when used, and further improvement is desired.

Immunochemical identification method 3rd edition, J. MoI. Published by Clausen, 1993

Japanese Patent Laid-Open No. 9-80051 JP 2003-057238 A

  The object of the present invention is the group consisting of an enzyme-labeled substance (F) {measurement target substance (F1), a similar substance (F2) of the measurement target substance, and a substance (F3) that specifically binds to the measurement target substance. Another object of the present invention is to provide an immunoassay reagent excellent in storage stability of at least one kind of substance selected from}, an immunoassay kit containing the immunoassay reagent, and an immunoassay method using the kit.

The inventor of the present invention has arrived at the present invention as a result of intensive studies to achieve the above object.
That is, the present invention is an immunoassay reagent (X) containing a substance (FC) and a hydrophilic protein (D), wherein the substance (FC) is labeled with the substance (F) with the enzyme (C). The substance (F) is at least one substance selected from the group consisting of the measurement target substance (F1), a similar substance (F2) of the measurement target substance, and a substance (F3) that specifically binds to the measurement target substance. Yes, the hydrophilic protein (D) is a protein in which 2.0 g or more is dissolved in 100 g of water at 25 ° C. and pH 7.0, and the content of the hydrophilic protein (D) in the immunoassay reagent (X) is (X 2) to 6% by weight based on the weight of the immunoassay reagent (X); an immunoassay kit comprising a labeling reagent (A) and a solid phase carrier reagent (E), wherein the labeling reagent (A) comprises The reagent for immunoassay of any one of Claims 1-3 X), and a solid phase carrier reagent (E) containing a solid phase carrier (BF) having a substance (F) immobilized on the surface of the solid phase carrier (B); This is an immunoassay method using a measurement kit.

  The immunoassay reagent of the present invention is extremely excellent in storage stability of a substance labeled with an enzyme.

  The immunoassay reagent (X) of the present invention is an immunoassay reagent (X) containing a substance (FC) and a hydrophilic protein (D), wherein the substance (FC) is a substance (F) is an enzyme (C). ), And the substance (F) is selected from the group consisting of the measurement target substance (F1), the similar substance (F2) of the measurement target substance, and the substance (F3) that specifically binds to the measurement target substance. The hydrophilic protein (D) is a protein in which 2.0 g or more is dissolved in 100 g of water at 25 ° C. and pH 7.0, and the hydrophilic protein (D) in the reagent for immunoassay (D) ) Content is 2 to 6% by weight based on the weight of (X).

The substance (FC) in the present invention is a substance formed by labeling the substance (F) with the enzyme (C). The substance (F) includes at least one substance selected from the group consisting of a measurement target substance (F1), a similar substance (F2) of the measurement target substance, and a substance (F3) that specifically binds to the measurement target substance It is.
The measurement target substance (F1) in the present invention includes proteins, lipid proteins, nucleic acids, immunoglobulins contained in biological samples such as serum, blood, plasma, urine and the like, biological samples such as lymph, blood cells, and various cells. Representative examples include blood coagulation-related factors, antibodies, enzymes, hormones, cancer markers, heart disease markers, and various drugs. More specifically, for example, proteins such as albumin, hemoglobin, myoglobin, transferrin, protein A, C-reactive protein (CRP), such as high density lipoprotein (HDL), low density lipoprotein (LDL), ultra low density lipo Lipid proteins such as proteins, such as nucleic acids such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), such as alkaline phosphatase, amylase, acid phosphatase, γ-glutamyltransferase (γ-GTP), lipase, creatine kinase (CK), lactic acid Enzymes such as dehydrogenase (LDH), glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), renin, protein kinase (PK), tyrosine kinase, such as IgG, IgM, IgA, I Blood coagulation-related factors such as immunoglobulins such as gD and IgE (or fragments thereof such as Fc part, Fab part and F (ab) 2 part) such as fibrinogen, fibrin degradation product (FDP), prothrombin and thrombin For example, anti-streptridine O antibody, anti-human hepatitis B virus surface antigen antibody (HBs antigen), anti-human hepatitis C virus antibody, anti-rheumatic factor and other antibodies such as thyroid stimulating hormone (TSH), thyroid hormone [free bird Hormones such as iodothyronine (FT3), free thyroxine (FT4), triiodothyronine (T3), thyroxine (T4)], parathyroid hormone (PTH), human chorionic gonadotropin (hCG) estradiol (E2), for example α-fetoprotein (AFP), carcinoembryonic antigen (CEA), CA19 9. Cancer markers such as prostate specific antigen (PSA) such as troponin T (TnT), heart disease markers such as human brain natriuretic peptide precursor N-terminal fragment (NT-proBNP) such as antiepileptic drugs, antibiotics, theophylline And the like.
Among the above, at least one selected from the group consisting of antibodies, hormones, cancer markers, and heart disease markers is preferable from the viewpoint of commercial needs.

  The similar substance (analog) (F2) of the measurement target substance in the present invention is capable of binding to the binding site with the measurement target substance (F1) possessed by the substance (F3) specifically binding to the measurement target substance, in other words, A substance having a binding site for the substance (F3) that specifically binds to the substance to be measured included in the substance to be measured (F1), in other words, specifically binds to the substance to be measured (F1) and the substance to be measured. Any substance may be used as long as it can compete with the reaction when it coexists with the substance (F3).

Examples of the substance (F3) that specifically binds to the measurement target substance in the present invention include an “antigen”-“antibody” reaction, “sugar chain”-“protein” reaction, and “sugar chain”-“lectin”. Reaction, “enzyme”-“inhibitor” reaction, “protein”-“peptide chain” reaction or “chromosome or nucleotide chain”-“nucleotide chain” reaction, “nucleotide chain”-“protein” reaction, etc. Examples include those that bind to the substance to be measured or its similar substance (F2) by reaction, and when any one of the above combinations is the substance to be measured (F1) or its similar substance (F2) One is a substance (F3) that specifically binds to the substance to be measured.
For example, when the measurement target substance (F1) or its similar substance (F2) is “antigen”, the substance (F3) that specifically binds to the measurement target substance is “antibody”, and the measurement target substance (F1) or When the similar substance (F2) is an “antibody”, the substance (F3) that specifically binds to the substance to be measured is an “antigen” (the same applies to the other combinations described above).

  Specifically as (F3), for example, nucleotide chain (oligonucleotide chain, polynucleotide chain); chromosome; peptide chain (for example, C-peptide, angiotensin I, etc.), protein [for example, procalcitonin, immunoglobulin A (IgA), Immunoglobulin E (IgE), immunoglobulin G (IgG), immunoglobulin M (IgM), immunoglobulin D (IgD), β2-microglobulin, albumin, degradation products thereof, serum proteins such as ferritin]; enzyme [for example Amylase (eg pancreatic type, salivary gland type, type X), alkaline phosphatase (eg hepatic, osseous, placental, small intestine etc.), acid phosphatase (eg PAP etc.), γ-glutamyltransferase (eg renal, pancreatic) , Hepatic, etc.), lipase (eg pancreatic, gastric), Creatine kinase (eg, CK-1, CK-2, mCK, etc.), lactate dehydrogenase (eg, LDH1-LDH5, etc.), glutamic acid oxaloacetate transaminase (eg, ASTm, ASTs, etc.), glutamic acid pyruvate transaminase (eg, ALTm, ALTs, etc.) ), Cholinesterase (eg, ChE1-ChE5), leucine aminopeptidase (eg, C-LAP, AA, CAP, etc.), renin, protein kinase, tyrosine kinase, etc.) and inhibitors of these enzymes, hormones (eg, PTH, TSH, insulin, etc.) LH, FSH, prolactin, etc.), receptors (for example, receptors for estrogen, TSH, etc.); ligands (for example, estrogen, TSH, etc.); for example, bacteria (for example, tuberculosis, pneumococci, diphte) A, Neisseria meningitidis, Neisseria gonorrhoeae, Staphylococcus, Streptococcus, Enterobacteriaceae, Escherichia coli, Helicobacter pylori, etc.), viruses (eg, rubella virus, herpes virus, hepatitis virus, ATL virus, AIDS virus, influenza virus, Adenovirus, enterovirus, poliovirus, EB virus, HAV, HBV, HCV, HIV, HTLV, etc.), fungi (eg, Candida, cryptococcus, etc.), spirochete (eg, leptospira, syphilis treponema, etc.), microorganisms such as chlamydia, mycoplasma; Proteins or peptides derived from microorganisms or sugar chain antigens; allergens that cause allergies such as bronchial asthma, allergic rhinitis, atopic dermatitis (for example, house dust such as mite mite, mushroom mite) Ticks such as Japanese cedar, Japanese cypress, Japanese spruce, ragweed, green frog, hargaya, rye, etc., such as cats, dogs, crabs, etc., such as rice, egg white, food, fungi, insects, wood, drugs, chemicals Lipids (eg, lipoproteins, etc.); proteases (eg, trypsin, plasmin, serine proteases, etc.); tumor marker protein antigens (eg, PSA, PGI, PGII, etc.); sugar chain antigens (eg, AFP (eg, AFP, etc.) L1 to L3, etc.), hCG (hCG family), transferrin, IgG, thyroglobulin, decay-accelerating-factor (DAF), carcinoembryonic antigen (eg CEA, NCA, NCA-2, NFA, etc.), CA19-9, PIVKA -II, CA125, upright Gland specific antigen, tumor marker sugar chain antigen having a special sugar chain produced by cancer cells, ABO sugar chain antigen, etc.]; sugar chain (eg, hyaluronic acid, β-glucan, sugar chain possessed by the above sugar chain antigen, etc.) A protein that binds to a sugar chain (eg, hyaluronic acid-binding protein, β-glucan-binding protein); phospholipid (eg, cardiolipin); lipopolysaccharide (eg, endotoxin); chemical (eg, T3, T4, eg, tributyltin, nonylphenol, Environmental hormones such as 4-octylphenol, di-n-butyl phthalate, dicyclohexyl phthalate, benzophenone, octachlorostyrene, di-2-ethylhexyl phthalate); various drugs administered and inoculated to the human body and their metabolites; Examples include aptamers; nucleic acid-binding substances; and antibodies to these. The antibodies used in the present invention include proteolytic enzymes such as papain and pepsin, and degradation products such as Fab and F (ab ') 2 fragments generated by chemical degradation.

As the substance (F3) that specifically binds to the substance to be measured as described above, the substance to be measured (F1) or a similar substance (F2) by the “antigen”-“antibody” reaction or “sugar chain-protein” reaction. ) Is preferable because it is easily available.
Specifically, an antibody against the measurement target substance (F1) or a similar substance (F2), an antigen to which the measurement target substance (F1) or a similar substance (F2) binds, and a measurement target substance (F1) or a similar substance ( A protein that binds to F2) is preferred, and an antibody against the substance to be measured (F1) or a similar substance (F2) and a protein that binds to the substance to be measured (F1) or a similar substance (F2) are more preferred.

Examples of the enzyme (C) used in the present invention include alkaline phosphatase, β-galactosidase, peroxidase (hereinafter sometimes abbreviated as POD), microperoxidase, glucose oxidase, used in enzyme immunoassay (EIA), Examples of the enzyme include glucose-6-phosphate dehydrogenase, malate dehydrogenase, luciferase, tyrosinase, and acid phosphatase.
Of these, alkaline phosphatase, peroxidase and glucose oxidase are preferable from the viewpoint of sensitivity and the like, and peroxidase is particularly preferable.

  In the present invention, the substance (FC) is a substance formed by labeling the substance (F) with the enzyme (C). A method of binding an enzyme (C) to a substance (F) to form a substance (FC), a method generally used in this field [for example, medical chemistry experiment course, Vol. 8, supervised by Yuichi Yamamura, first edition, Nakayama Shoten, 1971; Illustrated Fluorescent Antibody, Akira Kawaio, 1st Edition, Soft Science, Inc., 1983; Enzyme Immunoassay, Eiji Ishikawa, Tadashi Kawai, Kiyoshi Muroi, 2nd Edition, Medical School, 1982 Etc.] may be used.

  In the present invention, the content of the substance (FC) in the immunoassay reagent (X) is 0.005 to 0.005 from the viewpoint of measurement sensitivity in the enzyme immunoassay based on the weight of the immunoassay reagent (X). 0.8 weight% is preferable, More preferably, it is 0.01 to 0.3 weight%.

The hydrophilic protein (D) in the present invention is a protein that is dissolved in an amount of 2.0 g or more in 100 g of water at 25 ° C. and pH 7.0.
(D) may be used alone or in combination of two or more.
Examples of the hydrophilic protein (D) include hydrophilic casein protein (D1), peptide (D2), albumin (D3) and the like.
Examples of (D1) include casein hydrolyzate, κ-casein, and block ace (manufactured by DS Pharma Biomedical Co., Ltd.).
Examples of the peptide (D2) include collagen peptides, milk peptides, and soybean peptides.
Examples of albumin (D3) include serum albumin such as egg albumin and bovine serum albumin (BSA), and milk albumin.
Among the above (D), the hydrophilic casein protein (D1) is preferable from the viewpoint of storage stability, and a casein hydrolyzate is more preferable.

In the present invention, the content of the hydrophilic protein (D) in the immunoassay reagent (X) is 2 to 6% by weight based on the weight of the immunoassay reagent (X), and is labeled with an enzyme. From the viewpoint of storage stability of the substance, it is preferably 2 to 6% by weight, more preferably 3 to 5% by weight.
When the content of (D) is 2% by weight or more, the hydrophilic protein (D) can improve the hydrophilicity of the substance (FC) in the immunoassay reagent (X), and the substance (FC) Therefore, the storage stability as the immunoassay reagent (X) is improved. Moreover, when the content of (D) is 6% by weight or less, dissolution in the epidemic measurement reagent (X) is easy. Moreover, the dispersibility of the magnetic silica particle (B1) in the enzyme immunoassay method using the magnetic silica particle (B1) can be improved by being in the above range.

  The immunoassay reagent (X) of the present invention is preferable as a labeled reagent (A) in immunoassay because the storage stability of the substance (FC) which is a substance labeled with the enzyme in (X) is extremely excellent. Can be used.

The immunoassay kit of the present invention is an immunoassay kit containing a labeling reagent (A) and a solid phase carrier reagent (E), wherein the labeling reagent (A) is the immunoassay reagent (X) of the present invention. A solid phase carrier reagent (E) is an immunoassay kit containing a solid phase carrier (BF) in which a substance (F) is immobilized on the surface of a solid phase carrier (B).

In the present invention, the solid phase carrier (B) is not particularly limited as long as it is generally used in this field. For example, glass beads, polystyrene beads, magnetic silica particles (B1), microplates, latex, etc. A typical example.
Examples of the magnetic silica particles (B1) include known magnetic silica particles described in JP 2014-210680 A and JP 2013-019889 A.
Among these, the magnetic silica particles (B1) are preferable from the viewpoint of being hardly affected by the hydrophilic protein (D) during immunoassay.

  The magnetic silica particles (B1) are those in which a metal oxide having a superparamagnetism having a number average particle diameter of 1 to 15 nm is dispersed in a silica matrix. Superparamagnetism refers to a temporary magnetic field that is induced by the individual atomic magnetic moments of a material being aligned in the presence of an external magnetic field, and that when the external magnetic field is removed, partial alignment is impaired and no magnetic field is exhibited. Say.

  Examples of superparamagnetic metal oxides having a number average particle diameter of 1 to 15 nm and exhibiting superparamagnetism include oxides such as iron, cobalt, nickel, and alloys thereof. Iron oxide is particularly preferred. A superparamagnetic metal oxide may be used individually by 1 type, or may use 2 or more types together.

  As iron oxide, various known iron oxides can be used. Among iron oxides, magnetite, γ-hematite, magnetite-α-hematite intermediate iron oxide and γ-hematite-α-hematite intermediate iron oxide are preferred because of their excellent chemical stability, and have a large saturation magnetization. Magnetite is more preferable because of its excellent sensitivity to an external magnetic field.

The lower limit of the content of the superparamagnetic metal oxide in the magnetic silica particles (B1) is preferably 60% by weight, more preferably 65% by weight, and the upper limit is preferably 95% by weight, more preferably 80% by weight. It is.
When the content of the superparamagnetic metal oxide is 60% by weight or more, the magnetic silica particles (B1) obtained are sufficiently magnetized, so that it does not take time for the separation operation in actual use, which is preferable. Moreover, since it is easy to synthesize | combine as it is 95 weight% or less, it is preferable.

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

  The number average particle diameter of the magnetic silica particles (B1) is preferably 1 to 5 μm, more preferably 1 to 3 μm. If the average particle size is 1 μm or more, it tends to take less time for separation and recovery, and if it is 5 μm or less, the surface area becomes large and the substance to be immobilized (target substance, similar substance to the measurement target substance or measurement target) The binding amount of the substance that specifically binds to the substance tends to increase and the binding efficiency tends to increase.

  The number average particle size of the magnetic silica particles (B1) is an average of the particle sizes measured by observing an arbitrary 200 magnetic silica particles with a scanning electron microscope (“JSM-7000F” manufactured by JEOL Ltd.). Value.

  The number average particle size of the magnetic silica particles (B1) is controlled by adjusting the particle size of the oil-in-water emulsion by adjusting the mixing conditions (shearing force, etc.) when preparing the oil-in-water emulsion described later. be able to. Further, the average particle diameter can be set to a desired value by a method such as a change in conditions of the water washing step at the time of producing the magnetic silica particles or a general classification method.

The magnetic silica particles (B1) are, for example, superparamagnetic metal oxide particles having a number average particle diameter of 1 to 15 nm, 30 to 500% by weight of (alkyl) alkoxysilane based on the weight of the superparamagnetic metal oxide particles, and After forming a oil-in-water emulsion by mixing a dispersion containing a dispersant and a solution containing water, a water-soluble organic solvent, a nonionic surfactant and a catalyst for hydrolysis of (alkyl) alkoxysilane, After hydrolyzing and condensing the (alkyl) alkoxysilane to obtain an aqueous dispersion of magnetic silica particles in which the superparamagnetic metal oxide is included in silica, the aqueous dispersion of magnetic silica particles is centrifuged and / or Alternatively, it is obtained by solid-liquid separation by magnetism collection and washing with water or methanol.
Above and below, (alkyl) alkoxysilane means alkylalkoxysilane or alkoxysilane.

  Since the magnetic silica particles (B1) contain superparamagnetic metal oxides in silica and do not exist on the surface of the particles, they specifically bind to many substances to be measured, similar substances to the substances to be measured, or substances to be measured. The substance to be immobilized can be immobilized on the surface.

  In the present invention, the solid phase carrier (B) {preferably magnetic silica particles (B1)} is bound to the measurement target substance (F1), a similar substance (F2) of the measurement target substance, and a substance that specifically binds to the measurement target substance ( F3) At least one selected from the group consisting of (measurement substance (F1), etc.) is immobilized to produce a solid phase carrier (BF) in which the substance (F) is immobilized on the surface of the solid phase carrier (B) Examples of the method for performing the above-mentioned (B) include a method of physically adsorbing the measurement target substance (F1) and the like. From the viewpoint of immobilizing the measurement target substance (F1) and the like more efficiently, glutaraldehyde, albumin, At least one organic compound selected from the group consisting of carbodiimide, streptavidin, biotin, and an alkylalkoxysilane having a functional group is bound to the surface (B) and measured through them. Preferable to immobilize the elephant substance (F1) or the like (B). Of these organic compounds, alkylalkoxysilane having a functional group is more preferable from the viewpoint of bonding a specific measurement target substance (F1) and the like.

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

In the luminol luminescent reagent (K), examples of 2,3-dihydro-1,4-phthalazinedione compounds include JP-A-2-291299, JP-A-10-319015, and JP-A-2000-279196. The known 2,3-dihydro-1,4-phthalazinedione compounds described in the above, and mixtures thereof can be used.
Of these, luminol, isoluminol, N-aminohexyl-N-ethylisoluminol (AHEI), N-aminobutyl-N-ethylisoluminol (ABEI), and metal salts thereof (alkali metal salts, etc.) At least one selected from the above is preferred, luminol and / or metal salts thereof are more preferred, and sodium salt of luminol is particularly preferred.

Examples of the chemiluminescence enhancer include known chemiluminescence enhancers described in JP-A-59-500262, JP-A-59-171839 and JP-A-2-291299, and mixtures thereof. Can be used.
Among these, from the viewpoint of chemiluminescence enhancing effect, etc., phenol is preferable, and more preferable is at least selected from the group consisting of P-iodophenol, 4- (cyanomethylthio) phenol and 4-cyanomethylthio-2-chlorophenol. One type, particularly preferred is 4- (cyanomethylthio) phenol.

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

As the buffer solution, for example, known buffer solutions described in JP-A-10-31915 and JP-A-2003-279489 can be used.
Among these, from the viewpoint of chemiluminescence enhancing effect and storage stability, 3- [4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid / sodium hydroxide buffer, 2-hydroxy-3- [ 4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid monohydrate / sodium hydroxide buffer and piperazinyl-1,4-bis (2-hydroxy-3-propanesulfonic acid) dihydrate At least one selected from the group consisting of a product / sodium hydroxide buffer, more preferably 3- [4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid / sodium hydroxide buffer and 2 -Hydroxy-3- [4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid monohydrate / sodium hydroxide buffer Is at least one selected from the group consisting of, particularly preferred is 3- [4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid / sodium hydroxide buffer.

As the chelating agent, for example, known chelating agents described in JP-A-9-75099 and JP-A-2003-279489 can be used.
Of these, 4-coordinate chelating agents are preferred from the viewpoint of chemiluminescence enhancing effect and storage stability, and more preferred are ethylenediaminetetraacetic acid (EDTA) and salts thereof (ethylenediaminetetraacetic acid disodium, ethylenediaminetetraacetic acid trisodium. , Ethylenediaminetetraacetic acid tetrasodium, ethylenediaminetetraacetic acid dipotassium and ethylenediaminetetraacetic acid tripotassium, etc.) and trans-1,2 diaminocyclohexane-N, N, N ′, N′-tetraacetic acid (CyDTA). At least one is preferable, and ethylenediaminetetraacetic acid (EDTA) and / or a salt thereof is particularly preferable.

The luminol luminescence reagent (K) 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, more preferably 8 to 10. The pH is measured in accordance with JIS K0400-12-10: 2000 (measurement temperature 25 ° C.).

  Luminol luminescence reagent (K) can be easily obtained by uniformly mixing a 2,3-dihydro-1,4-phthalazinedione compound, a chemiluminescence enhancer, and if necessary, a buffer and / or a chelating agent.

Examples of the peroxide contained in the aqueous peroxide solution (L) include known oxidizers described in JP-A-8-261944 and JP-A-2000-279196, etc. [Inorganic peroxide (peroxide Hydrogen peroxide, sodium perborate, potassium perborate, etc.), organic peroxides (dialkyl peroxide, acyl peroxide, etc.), peroxo acid compounds (peroxosulfuric acid, peroxophosphoric acid, etc.), etc.].
Among these, from the viewpoint of storage stability and the like, a hydrogen peroxide aqueous solution, a sodium perborate aqueous solution and a potassium perborate aqueous solution are preferable, and a hydrogen peroxide aqueous solution is more preferable.

  Examples of water contained in the peroxide aqueous solution (L) include distilled water, reverse osmosis water, and deionized water. Of these, distilled water and deionized water are preferable from the viewpoint of chemiluminescence enhancing effect and storage stability, and more preferable is deionized water.

The aqueous peroxide solution (L) can contain a chelating agent and the like in addition to the peroxide and water.
As a chelating agent, the thing similar to what was illustrated as a chelating agent which can be contained in the above-mentioned luminol luminescent reagent is mentioned, A preferable thing is also the same.
The aqueous peroxide solution (L) can be easily obtained by uniformly mixing the peroxide, water and, if necessary, the chelating agent.

  The immunoassay kit of the present invention can be used in a method generally performed in this field as an immunoassay method for quantifying a measurement target substance (F1) in a sample. For example, literature [for example, enzyme immunoassay method The second method (edited by Eiji Ishikawa et al., Medical School) 1982] can be performed according to the sandwich method, competition method and measurement method described in JP-A-6-130063.

  The sandwich method includes, for example, a sample containing a measurement target substance (F1), and magnetic silica particles (B1F3) in which a substance (F3) that specifically binds to the measurement target substance is immobilized on the surface of the magnetic silica particles (B1). The substance (F3C) that specifically binds to the measurement target substance on the surface of the magnetic silica particles (B1) by contacting the substance (F3C) that specifically binds to the measurement target substance labeled with the enzyme and the measurement target substance A complex {(F3) / (F1) / (F3C)} of (F1) and a substance (F3C) that specifically binds to the substance to be measured labeled with an enzyme is formed, and the labeled complex is converted to B / F is separated, the amount of the labeling substance in the complex is measured, and the substance to be measured (F1) in the sample is quantified based on the result.

  Specifically, for example, a sample containing the measurement target substance (F1) and a magnetic silica particle (B1F3) in which a substance (F3) that specifically binds to the measurement target substance is immobilized on the surface of the magnetic silica particle (B1). By contacting, a complex of the substance (F3) that specifically binds to the substance to be measured and the substance to be measured (F1) is formed on the surface of the magnetic silica particles (B1), and the complex is further labeled with an enzyme. A substance (F3), a measurement target substance (F1), and an enzyme that specifically bind to the measurement target substance supported on the magnetic silica particles (B1) by contacting a substance (F3C) that specifically binds to the measurement target substance. To form a labeled complex {(F3) / (F1) / (F3C)} with a substance (F3C) that specifically binds to the substance to be measured labeled by B, and B / F separates the labeled complex. Measure the amount of labeling substance in the labeling complex It may be quantified analyte in the sample based on the result. In this method, the measurement target substance (F1) is reacted with magnetic silica particles (B1F3) on which a substance (F3) that specifically binds to the measurement target substance is reacted, and then labeled with an enzyme. The substance (F3C) that specifically binds to the measurement target substance is reacted, but the measurement is performed after the substance (F3C) that specifically binds to the labeled measurement target substance and the measurement target substance (F1) are reacted. The magnetic silica particles (B1F3) on which the substance (F3) that specifically binds to the target substance is immobilized may be reacted, or these three may be reacted simultaneously.

The B / F separation in the sandwich method refers to the above-mentioned labeled complex {(F3) / (F1) / (F3C)} and a substance to be measured labeled with an enzyme that was not involved in the formation of the labeled complex. Is specifically separated from the substance (F3C) that specifically binds, specifically, the substance (F3) that specifically binds to the substance to be measured supported on the magnetic silica particles (B1), and the magnetic silica particles (B1). A complex {(F3) / (F1)} of the substance (F3) and the substance to be measured (F1) that specifically binds to the substance to be measured carried on the label {F3) / (F1) ) / (F3C)} and other components (components other than the measurement target substance (F1) in the sample, substances that specifically bind to the measurement target substance labeled with an enzyme that was not involved in the formation of the labeled complex) (F3C) etc.).
In addition, the B / F separation step is an essential step after the formation of the labeled complex {(F3) / (F1) / (F3C)}, but it is specific to the measurement target substance on the surface of the magnetic silica particles (B1). It can be carried out even after the complex {(F3) / (F1)} of the substance (F3) to be bound and the substance to be measured (F1) is formed.

  The competitive method includes a sample containing the measurement target substance (F1), a substance that specifically binds to the measurement target substance labeled with an enzyme (F3C), and a measurement target substance (F1) or a similar substance to the measurement target substance ( F2) is brought into contact with the magnetic silica particles (B1F1) or (B1F2) immobilized on the surface thereof, and the substance to be measured (F1) or a similar substance (F2) and an enzyme on the magnetic silica particles (B1) A labeled complex {(F1) / (F1) / (F3C) or (F2) / (F1) / (F3C)} with a substance (F3C) that specifically binds to the labeled substance to be measured; The magnetic silica particles carrying the labeled complex are subjected to B / F separation, the amount of the labeled substance in the labeled complex is measured, and the measurement target substance in the sample is quantified based on the result.

  Specifically, for example, a sample containing a measurement target substance (F1), a substance (F3C) that specifically binds to a measurement target substance labeled with an enzyme, a measurement target substance (F1), or a similar substance (F2) The substance to be measured (F1) and the magnetic substance in the sample are bonded to the substance (F3C) that specifically binds to the substance to be measured labeled with the enzyme by contacting the magnetic silica particles (B1F1) or (B1F2) carrying The measurement target substance (F1) or its similar substance (F2) on the surface of the silica particle (B1) is subjected to a competitive reaction, and the measurement target substance (F1) or its similar substance (F2) and the enzyme are reacted on the surface of the magnetic silica particle (B1) A labeled complex {(F1) / (F1) / (F3C) or (F2) / (F1) / (F3C)} with a substance (F3C) that specifically binds to the labeled substance to be measured; The labeling complex The lifting the magnetic silica particles in B / F separation, the amount of the labeling substance labeled complex was measured, the measurement target substance in the sample (F1) may be quantified based on the result. In this method, a measurement target substance (F1), a substance that specifically binds to a measurement target substance labeled with an enzyme (F3C), and a measurement target substance immobilized on the surface of magnetic silica particles (B1) ( F1) or a similar substance (F2) is simultaneously competitively reacted, but the measurement target substance (F1) immobilized on the surface of the measurement target substance (F1) and the magnetic silica particles (B1) or a similar substance (F2) Even if a substance (F3C) that specifically binds to the measurement target substance labeled with the enzyme is added and subjected to a competitive reaction, the measurement target substance (F1) and the measurement target substance labeled with the enzyme After bringing the substance (F3C) that specifically binds into contact, magnetic silica particles carrying the substance to be measured (F1) or a similar substance (F2) may be added to cause a competitive reaction.

  The B / F separation in the competition method involves the formation of a labeled complex with the labeled complex {(F1) / (F1) / (F3C) or (F2) / (F1) / (F3C)}. A substance that specifically binds to a measurement target substance labeled with an enzyme (F3C), and a substance that specifically binds to a measurement target substance labeled with an enzyme (F3C) and a measurement target substance (F1) This means separation from the complex {(F1) / (F3C)}, specifically, magnetic silica particles carrying the measurement target substance (F1) or a similar substance (F2), and the measurement target substance (F1) Alternatively, a complex {(B1F1) / (F3C) or (B1F2) / (B1F1) / (F3C) or (F3C), which is a substance (F3C) that specifically binds to a measurement target substance labeled with an enzyme. F3C)} and other components (in the sample Components other than the analyte (F1), an enzyme with a labeled analyte and the substance specifically binding to (F3C), means the separation of the composite) of analyte (F1).

  Further, as another aspect of the competitive method, a sample containing a measurement target substance (F1), a measurement target substance (F1C) labeled with an enzyme, or a similar substance (F2C), and a measurement target substance are specifically bound. The substance to be measured (F3) that specifically binds to the substance to be measured on the magnetic silica particle (B1) by contacting with the magnetic silica particle (B1F3) carrying the substance (F3) to be measured and the enzyme to be measured A labeled complex {(F3) / (F1C) or (F3) / (F2C)} with (F1C) or a similar substance (F2C) is formed, and magnetic silica particles supporting the labeled complex are converted to B / F is separated, the amount of labeling substance in the labeling complex is measured, and based on the result, the substance to be measured in the sample is measured.

  Specifically, for example, a sample containing a measurement target substance (F1), a measurement target substance (F1C) labeled with an enzyme or a similar substance (F2C), and a substance (F3) that specifically binds to the measurement target substance The substance to be measured (F1) and the enzyme in the sample are brought into contact with the substance (F3) that specifically binds to the substance to be measured on the magnetic silica particle (B1) by contacting with the magnetic silica particles (B1F3) supporting Measurement in a sample with a substance (F3) that specifically binds to the substance to be measured on the magnetic silica particle (B1) by causing a competitive reaction with the substance to be measured (F1C) or its similar substance (F2C) labeled with Complex {(F3) / (F1)} with target substance (F1) and labeled complex {(F3) / (F1C) with target substance (F1C) or its similar substance (F2C) labeled with an enzyme Or (F3) / (F C)} and B / F separation of the magnetic silica particles carrying the complex or the labeled complex, and the amount of the labeled substance in the labeled complex is measured, and the measurement in the sample based on the result What is necessary is just to measure a target substance (F1). In the competition method, a measurement target substance (F1), an enzyme-labeled measurement target substance (F1C) or a similar substance (F2C), and a substance that specifically binds to the measurement target substance (F3) are supported. The magnetic silica particles (B1F3) that have been subjected to the competitive reaction simultaneously are brought into contact with the measurement target substance (F1) and the magnetic silica particles carrying the substance (F3) that specifically binds to the measurement target substance. {After the complex (F3) / (F1) is formed}, the target substance (F1C) or its similar substance (F2C) labeled with an enzyme is added to the competitive reaction {in the magnetic silica particles (B1F3), Even if (F1C) or (F2C) is reacted with (F3) which does not react with the measurement target substance (F1) in the sample, the measurement target substance (F1C) labeled with the enzyme or a similar substance (F2C) ) After contacting the magnetic silica particles (B1F3) carrying the substance (F3) that specifically binds to the substance to be measured, {complex (F3) / (F1C) or (F3) / (F2C) is formed The measurement target substance (F1) was added to the competitive reaction {the magnetic silica particles (B1F3) (F1C) and (F2C) did not react (F3), and the measurement target substance (F1 ) May be reacted}.

The B / F separation in the competition method means separation of the labeled complex from a measurement target substance (F1C) or an analogous substance (F2C) labeled with an enzyme that was not involved in the formation of the labeled complex. Specifically, the magnetic silica particles (B1F3) carrying the substance (F3) that specifically binds to the substance to be measured, and the substances that specifically bind to the substance to be measured on the magnetic silica particles (B1) ( F3) and a complex {(F3) / (F1)} of the measurement target substance (F1), a substance (F3) that specifically binds to the measurement target substance on the magnetic silica particles (B1), and an enzyme A complex {(F3) / (F1C) or (F3) / (F2C)} with a labeled measurement target substance (F1C) or a similar substance (F2C), and other components (measurement target substance ( Concerning the formation of components other than F1) and labeled complexes It means the separation of the labeled analyte (F1C) or analogs thereof (F2C), etc.) by the enzyme did not.
Further, the B / F separation step is an essential step after the formation of the labeled complex, but the magnetic silica particles carrying the measurement target substance (F1) and the substance (F3) that specifically binds to the measurement target substance It can also be carried out after the contact.

  When the measurement target substance (F1) is an enzyme, a method using an enzyme activity method other than the sandwich method or the competition method, for example, a sample containing the measurement target substance (F1) and a measurement target substance The substance to be measured (F3) (for example, a substance capable of binding to an enzyme such as an antibody) is brought into contact with the magnetic silica particle (B1F3) immobilized on the surface, and the substance to be measured is specific on the magnetic silica particle (B1). After forming a complex {(F3) / (F1)} with a substance (F3) that binds electrically and separating the magnetic silica particles carrying the complex by B / F, or a substrate corresponding to the type of enzyme, or Substrates and color formers depending on the type of enzyme, and if necessary, a conjugated enzyme may be added, and the amount of enzyme in the sample may be measured based on the change in the substrate or the color development result of the color former. . In addition, what is necessary is just to use a well-known thing as a substrate, a coloring agent, and a conjugate enzyme, for example, when a enzyme is a peroxidase, what is necessary is just to use hydrogen peroxide, a luminol luminescent reagent, etc. These usage amounts may be in a range generally used in this field. B / F separation in the above method refers to a complex {(F3) / () of a measurement target substance (F1) and a substance (F3) that specifically binds to the measurement target substance immobilized on magnetic silica particles. F1)} and other components (components other than the measurement target substance (F1) in the sample).

  In the immunoassay method of the present invention, a sample, a magnetic silica particle (B1F) on which a substance (F) is immobilized, a substance that specifically binds to a labeled measurement target substance (F3C), and a measurement target labeled with an enzyme As a method of bringing the substance (F1C) or its similar substance (F2C) into contact, the magnetic silica particles may be dispersed by a treatment such as stirring and mixing generally performed. The reaction time may be appropriately set according to the difference between the measurement target substance (F1), the substance that specifically binds to the measurement target substance used (F3), the sandwich method, the competition method, etc. Is preferable, and more preferably 1 to 5 minutes.

  The B / F separation in the immunoassay method of the present invention uses, for example, the magnetic properties of magnetic silica particles, collects the magnetic silica particles with a magnet or the like from the outside of the reaction tank, discharges the reaction solution, and adds the washing solution. Thereafter, the magnet is removed, and the magnetic silica particles are mixed and dispersed and washed. The above operation may be repeated 1 to 3 times. The cleaning liquid is not particularly limited as long as it is usually used in this field.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited to this. Below, a part shows a weight part.

<Example 1>
Solid phase carrier reagent (E-1) (reagent containing magnetic silica particles (B1F3) on which anti-CEA monoclonal antibody (F3) is immobilized), labeling reagent (A-1) which is reagent for immunoassay (X) The immunoassay kit (S-1) of the present invention comprising (POD-labeled anti-CEA antibody (F3C) reagent), luminol luminescence reagent (K-1) and hydrogen peroxide solution (L-1) was obtained.

Production of magnetic silica particles (B1-1):
In a reaction vessel, 186 parts of iron (III) chloride hexahydrate, 68 parts of iron (II) chloride tetrahydrate and 1288 parts of water are charged and dissolved, and the temperature is raised to 50 ° C. While maintaining the temperature, 280 parts of 25 wt% aqueous ammonia was added dropwise over 1 hour to obtain magnetite particles in 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, magnetite particles adsorbed with oleic acid obtained by solid-liquid separation by decantation were washed 3 times with 1000 parts of water, and further washed twice with 1000 parts of acetone. Superparamagnetic metal oxide particles were obtained by drying at 2 ° C. for 2 days.

  80 parts of superparamagnetic metal oxide particles were added to and dispersed in 240 parts of tetraethoxysilane to prepare dispersion (a1). Next, 5050 parts of water, 3500 parts of 25% by weight ammonia aqueous solution and 400 parts of NSA-17 (manufactured by Sanyo Chemical Industries) are added to the reaction vessel and mixed using a clear mix (manufactured by MTechnic Co., Ltd.). ) After raising the temperature to 50 ° C., the dispersion (a1) was added dropwise to the solution (a2) over 1 hour while stirring the clear mix at a rotation speed of 6,000 rpm, 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 containing fine particles, thereby obtaining a core layer.

  Add 80 parts of the core layer, 2500 parts of deionized water, 260 parts of 25% by weight aqueous ammonia, 2500 parts of ethanol, and 1200 parts of tetraethoxysilane to the reaction vessel and mix using Clearmix (Mtechnic Co., Ltd.). The reaction was carried out for 2 hours while stirring at 6,000 rpm. After the reaction, centrifugation was performed at 2,000 rpm for 20 minutes to remove the supernatant containing fine particles, and the operation of collecting the particles using a magnet and removing the supernatant was performed 10 times. 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 containing fine particles was performed 20 times. Classification was performed by adding 5000 parts of water to disperse the particles and centrifuging at 300 rpm for 10 minutes to settle and remove particles having a large particle size. Furthermore, the operation of collecting particles using a magnet and removing the supernatant was performed 10 times to obtain magnetic silica particles (B1-1) having a target volume average particle diameter.

Production of solid phase carrier reagent (E-1) containing magnetic silica particles (B1F3) on which anti-CEA monoclonal antibody (F3) is immobilized:
40 mg of magnetic silica particles (B1-1) produced in a polyethylene bottle with a lid containing 40 mL of an acetone solution containing 1% by weight γ-aminopropyltriethoxysilane was added, reacted at 25 ° C. for 1 hour, and magnetic silica particles with a neodymium magnet. After collecting the liquid, the liquid was sucked and removed by an aspirator. Next, 40 mL of deionized water is added, the cap is closed, the polystyrene bottle is slowly inverted and stirred twice, and after collecting magnetic silica particles (B1-1) with a neodymium magnet, the liquid is sucked and removed with an aspirator. The particles were washed. This washing operation was performed 5 times. Next, the washed magnetic silica particles were added to a polyethylene bottle with a lid containing 40 mL of an aqueous solution containing 2% by weight glutaraldehyde, and reacted at 25 ° C. for 1 hour. Then, after adding 40 mL of deionized water and capping, the polystyrene bottle was slowly inverted and stirred twice, and after collecting the magnetic silica particles (B1-1) with a neodymium magnet, the liquid was attracted and removed with an aspirator. Silica particles (B1-1) were washed. This washing operation was performed 10 times. Further, the magnetic silica particles (B1-1) after washing have different recognition sites from the anti-CEA monoclonal antibody (manufactured by Dako Japan Co., Ltd., used in the labeling reagent (A-1) described later. ) (F3-1 ′) 0.02M phosphate buffer solution (pH 8.7) contained at a concentration of 10 μg / mL was added to a polyethylene bottle with a lid and allowed to react at 25 ° C. for 1 hour. After the reaction, magnetic silica particles (B1F3) on which the anti-CEA monoclonal antibody was immobilized with a neodymium magnet were collected, and then the residual anti-CEA monoclonal antibody (F3-1 ′)-containing phosphate buffer was removed. Next, the magnetic silica particles (B1F3) were added to a polyethylene bottle with a lid containing 40 mL of 0.02M phosphate buffer (pH 7.0) containing 1% by weight of bovine serum albumin, and immersed for 12 hours at 25 ° C. After collecting silica particles (B1F3) with a neodymium magnet, a phosphate buffer solution containing 1% by weight of bovine serum albumin was removed. Dilute to 1.0 mg / mL concentration of magnetic silica particles (B1F3) immobilized with anti-CEA monoclonal antibody, prepare solid phase carrier reagent (E-1), and store in refrigerator (2-10 ° C) did.

Preparation of labeling reagent (A-1):
Using anti-CEA monoclonal antibody (Dako Japan Co., Ltd.) (F3-1) and horseradish-derived POD (Toyobo Co., Ltd.) (C-1), literature (S Yoshitake, M Imagawa, Ei Ishikawa, Etol; J. Biochem, Vol. 92, 1982, 1413-1424) was used to prepare a POD-labeled anti-CEA antibody (F3C). This was diluted with 0.02M phosphate buffer (pH 7.0) containing 3.0% by weight of casein hydrolyzate (D-1) to a concentration of 20 nM as the concentration of the POD-labeled anti-CEA antibody (F3C). Then, a labeling reagent (A-1) corresponding to the immunoassay reagent (X) was prepared and stored in a refrigerator (2 to 10 ° C.).

Preparation of luminol reagent (K):
A 1,000 mL volumetric flask was charged with 0.7 g of a sodium salt of luminol [manufactured by Sigma Aldrich Japan Co., Ltd.] and 0.1 g of 4- (cyanomethylthio) phenol. 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 was 1,000 mL, and even at 25 ° C. By mixing, a luminol luminescent reagent (K-1) was prepared. It was stored refrigerated (2-10 ° C.) until used for measurement.

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

<Example 2>
Example 1 except that the “casein hydrolyzate (D-1)” in the production of the labeling reagent (A-1) was changed to “Block Ace (DS Pharma Biomedical Co., Ltd.) (D-2)”. In the same manner as described above, the solid phase carrier reagent (E-1), the labeling reagent (A-2) corresponding to the immunoassay reagent (X), the luminol luminescence reagent (K-1), and the hydrogen peroxide solution (L-1) An immunoassay kit (S-2) comprising:

<Example 3>
Except for changing “3.0 wt% casein hydrolyzate (D-1)” to “6.0 wt% casein hydrolyzate (D-1)” in the production of the labeling reagent (A-1). In the same manner as in Example 1, the solid phase carrier reagent (E-1), the labeling reagent (A-3) corresponding to the immunoassay reagent (X), the luminol luminescence reagent (K-1), and the hydrogen peroxide solution ( L-1) and an immunoassay kit (S-3) was obtained.

<Example 4>
“3.0 wt% casein hydrolyzate (D-1)” in the production of the labeling reagent (A-1) is referred to as “3.0 wt% bovine serum albumin (hereinafter abbreviated as BSA) (D-3). Except that the solid phase carrier reagent (E-1), the labeling reagent (A-4) corresponding to the immunoassay reagent (X), and the luminol luminescence reagent (K-1) are the same as in Example 1. ) And hydrogen peroxide solution (L-1), an immunoassay kit (S-4) was obtained.

<Example 5>
“3.0 wt% casein hydrolyzate (D-1)” in the production of the labeling reagent (A-1) was changed to “4.0 wt% bovine serum albumin (BSA) (D-3)”. Except for the above, in the same manner as in Example 1, the solid phase carrier reagent (E-1), the labeling reagent (A-5) corresponding to the immunoassay reagent (X), the luminol luminescence reagent (K-1), and hydrogen peroxide An immunoassay kit (S-5) composed of water (L-1) was obtained.

<Example 6>
Except for changing “3.0 wt% casein hydrolyzate (D-1)” to “2.0 wt% casein hydrolyzate (D-1)” in the production of the labeling reagent (A-1). In the same manner as in Example 1, the solid phase carrier reagent (E-1), the labeling reagent (A-6) corresponding to the immunoassay reagent (X), the luminol luminescence reagent (K-1), and the hydrogen peroxide solution ( An immunoassay kit (S-6) comprising L-1) was obtained.

<Example 7>
In the production of the solid phase carrier reagent (E-1), the “anti-CEA monoclonal antibody (Dako Japan Co., Ltd.) (F3-1 ′)” was changed to “anti-PSA monoclonal antibody (Dako Japan Co., Ltd., labeling reagent ( A recognition site is different from that of the anti-PSA monoclonal antibody used in A-7).) (F3-2 ′) ”to obtain a solid phase carrier reagent (E-2), and a labeling reagent (A-1 )), Except that “anti-CEA monoclonal antibody (Dako Japan Co., Ltd.) (F3-1)” is changed to “anti-PSA monoclonal antibody (Dako Japan Co., Ltd.) (F3-2)”, In the same manner as in Example 1, the solid phase carrier reagent (E-2), the labeling reagent (A-7) corresponding to the immunoassay reagent (X), the luminol luminescence reagent (K-1), and the hydrogen peroxide solution (L -1) immunoassay kit (S- ) Was obtained.

<Example 8>
In the preparation of the solid phase carrier reagent (E-1), “anti-CEA monoclonal antibody (Dako Japan Co., Ltd.) (F3-1 ′) 10 μg / mL” was changed to “anti-T4 monoclonal antibody (Dako Japan Co., Ltd.) ( F3-3) 2 μg / mL ”to obtain a solid phase carrier reagent (E-3), and in the preparation of the labeling reagent (A-1),“ anti-CEA monoclonal antibody (manufactured by Dako Japan Co., Ltd.) ( F3-1) "was changed to" L-thyroxine sodium pentahydrate (manufactured by Wako Pure Chemical Industries, Ltd.) (hereinafter referred to as T4) (F1-1) ", and" POD-labeled anti-CEA antibody (F3C The solid phase carrier reagent (E-3) was changed in the same manner as in Example 1 except that “diluted to a concentration of 20 nM” was changed to “diluted to a concentration of 2 nM as the concentration of POD-labeled T4 (F1C)”. , Labeling test corresponding to immunoassay reagent (X) (A-8), was obtained luminol luminescent reagent (K-1) and hydrogen peroxide solution (L-1) immunoassay kit composed of (S-8).

<Example 9>
Changed “anti-CEA monoclonal antibody (Dako Japan Co., Ltd.) (F3-1 ′) 10 μg / mL” to “T4 (F1-1) 2 μg / mL” in preparation of solid phase carrier reagent (E-1) Thus, a solid phase carrier reagent (E-4) was obtained, and in the preparation of the labeling reagent (A-1), “anti-CEA monoclonal antibody (Dako Japan Co., Ltd.) (F3-1)” was designated as “anti-T4 monoclonal antibody”. Changed to “Antibody (Dako Japan Co., Ltd.) (F3-3)”, “POD-labeled anti-CEA antibody (F3C) was diluted to a concentration of 20 nM” and “POD-labeled anti-T4 antibody (F3C) concentration” The labeling reagent (A-9) corresponding to the solid phase carrier reagent (E-4), the immunoassay reagent (X), and luminol luminescence, except for changing to “diluted to a concentration of 2 nM” as in Example 1. Reagent (K-1) and hydrogen peroxide solution (L- ) Was obtained composed immunoassay kit (S-9) from.

<Example 10>
In the preparation of the solid phase carrier reagent (E-1), “anti-CEA monoclonal antibody (Dako Japan Co., Ltd.) (F3-1 ′) 10 μg / mL” was changed to “anti-T4 monoclonal antibody (Dako Japan Co., Ltd.) ( F3-3) 2 μg / mL ”to obtain a solid phase carrier reagent (E-3), and in the preparation of the labeling reagent (A-1),“ anti-CEA monoclonal antibody (manufactured by Dako Japan Co., Ltd.) ( F3-1 ') "was changed to"3,3', 5-triiodo-L-thyronine (manufactured by Wako Pure Chemical Industries, Ltd.) (hereinafter referred to as T3) (F2-1) " The solid phase carrier reagent (E) was changed in the same manner as in Example 1 except that “diluted to a concentration of 20 nM as an anti-CEA antibody (F3C) concentration” was changed to “diluted to a concentration of 2 nM as a POD-labeled T3 (F2C) concentration”. -3) Labeling test corresponding to immunoassay reagent (X) An immunoassay kit (S-10) comprising a drug (A-10), a luminol luminescence reagent (K-1) and a hydrogen peroxide solution (L-1) was obtained.

<Comparative Example 1>
“3.0 wt% casein hydrolyzate (D)” in the preparation of the labeling reagent (A-1) was changed to “1.0 wt% casein sodium salt”, and the casein sodium salt was dissolved at pH 10.0. The solid phase carrier reagent (E-1), the labeling reagent (A-11), which is a comparative immunoassay reagent, and the luminol luminescence reagent (K-), except that the pH was adjusted to 7.0 later. A comparative immunoassay kit (H-1) composed of 1) and hydrogen peroxide solution (L-1) was obtained. Casein sodium salt has a solubility in 100 g of water at pH 7.0 of 1.0 g or less, and is a hydrophobic protein.

<Comparative example 2>
Except for changing “3.0 wt% casein hydrolyzate (D)” to “not adding casein hydrolyzate (D)” in the production of the labeling reagent (A-1), the same as in Example 1 A solid phase carrier reagent (E-1), a labeling reagent (A-12) which is a comparative immunoassay reagent, a luminol luminescence reagent (K-1) and a hydrogen peroxide solution (L-1). A comparative immunoassay kit (H-2) was obtained.

<Comparative Example 3>
Except for changing “3.0 wt% casein hydrolyzate (D)” to “0.5 wt% casein hydrolyzate (D-1)” in the production of the labeling reagent (A-1). In the same manner as in Example 1, the solid phase carrier reagent (E-1), the comparative immunoassay reagent, the labeling reagent (A-13), the luminol luminescence reagent (K-1), and the hydrogen peroxide solution (L-1 A comparative immunoassay kit (H-3) was obtained.

  Using the obtained immunoassay kits (S-1) to (S-6) and (H-1) to (H-3), storage stability in immunoassay and dispersion of magnetic silica particles by the following methods Sex was evaluated. The results are shown in Table 1.

<Stability Evaluation Method 1 of Immunoassay Kit of the Present Invention (Measuring Substance 1 (F1): CEA)>
Standard CEA (F1) having a CEA (F1) concentration of 1.0 ng / mL prepared with 0.025 mL of solid phase carrier reagent (E-1) containing magnetic silica particles and 0.02 M phosphate buffer (pH 7.0) ) 0.025 mL of the solution was placed in a test tube, mixed, and reacted in the test tube at 37 ° C. for 3 minutes to form an anti-CEA antibody (F3) -bound magnetic silica particle / CEA (F1) complex. After the reaction, collect the magnetic silica particles from the outside of the test tube with a neodymium magnet for 10 seconds, remove the liquid in the test tube with an aspirator, sufficiently separate the neodymium magnet from the side, add 0.5 mL of physiological saline, and add the magnetic silica particles. After collecting and magnetizing, the washing operation of removing the liquid with an aspirator was performed three times.

Subsequently, labeling reagent (An) (n = 1 in S-1, n = 2 in S-2, n = 3 in S-3, n = 4 in S-4, S-5 N = 5 in S-6, n = 6 in S-6, n = 11 in H-1, n = 12 in H-2 and n = 13 in H-3) 0.025 mL is injected into the test tube The mixture was reacted in a test tube at 37 ° C. for 3 minutes to form a complex of anti-CEA antibody (F3) / CEA (F1) / POD-labeled anti-CEA antibody (F3C) on magnetic silica particles (B1). After the reaction, magnetic silica particles are collected from the outside of the test tube with a neodymium magnet for 10 seconds, the liquid in the test tube is removed with an aspirator, the neodymium magnet is sufficiently separated from the side surface, and 0.5 mL of physiological saline is added to the magnetic silica particles. After the magnetic flux was dispersed and the magnetic flux was collected, the washing operation for removing the liquid with an aspirator was performed twice.
Finally, 0.07 mL of luminol luminescence reagent (K-1) and 0.07 mL of hydrogen peroxide solution (L-1) were added at the same time, and the luminol luminescence reagent (K-1) and luminescence reaction were performed at 37 ° C. for 43 seconds. After adding hydrogen peroxide solution (L-1), the average amount of luminescence for 43 to 45 seconds was measured with a luminometer [“Lumat LB9507” manufactured by Bertoled Japan Co., Ltd.].

The value measured using an immunoassay kit within 5.0 hours after the preparation of the immunoassay kit is the initial luminescence amount, the immunoassay kit is 4 ° C for 1 year (365 days), 40 ° C for 5 days, or 40 ° C. The stability rate was calculated by the following formula, using the value measured using the product after storage for 10 days as the amount of luminescence after storage for 1 year, 5 days or 10 days.
Stability rate (%) = B / A × 100
(A: initial light emission amount, B: light emission amount after storage for 5 days or 10 days for one year)

  Using the obtained immunoassay kit (S-7), the storage stability in the immunoassay was evaluated by the following method. The results are shown in Table 1.

<Stability Evaluation Method for Immunoassay Kit of the Present Invention (Measuring Substance 2 (F1): PSA)>
Standard PSA (F1) having a PSA (F1) concentration of 0.1 ng / mL prepared with 0.025 mL of solid phase carrier reagent (E-2) containing magnetic silica particles and 0.02 M phosphate buffer (pH 7.0) ) 0.025 mL of the solution was put in a test tube, mixed, and reacted in the test tube at 37 ° C. for 3 minutes to form an anti-PSA antibody (F3) -bound magnetic silica particle / PSA (F1) complex. After the reaction, collect the magnetic silica particles from the outside of the test tube with a neodymium magnet for 10 seconds, remove the liquid in the test tube with an aspirator, sufficiently separate the neodymium magnet from the side, add 0.5 mL of physiological saline, and add the magnetic silica particles. After collecting and magnetizing, the washing operation of removing the liquid with an aspirator was performed three times.

Subsequently, 0.025 mL of the labeling reagent (A-7) was injected into the test tube, reacted at 37 ° C. for 3 minutes in the test tube, and the anti-PSA antibody (F3) / PSA (on the magnetic silica particle (B-1). F1) / POD-labeled anti-PSA antibody (F3C) complex was formed. After the reaction, magnetic silica particles are collected from the outside of the test tube with a neodymium magnet for 10 seconds, the liquid in the test tube is removed with an aspirator, the neodymium magnet is sufficiently separated from the side surface, and 0.5 mL of physiological saline is added to the magnetic silica particles. After the magnetic flux was dispersed and the magnetic flux was collected, the washing operation for removing the liquid with an aspirator was performed twice.
Finally, 0.07 mL of luminol luminescence reagent (K-1) and 0.07 mL of hydrogen peroxide solution (L-1) were added at the same time, and the luminol luminescence reagent (K-1) and luminescence reaction were performed at 37 ° C. for 43 seconds. After adding hydrogen peroxide solution (L-1), the average amount of luminescence for 43 to 45 seconds was measured with a luminometer [“Lumat LB9507” manufactured by Bertoled Japan Co., Ltd.].

The value measured using an immunoassay kit within 5.0 hours after the preparation of the immunoassay kit is the initial luminescence amount, the immunoassay kit is 4 ° C for 1 year (365 days), 40 ° C for 5 days, or 40 ° C. The stability rate was calculated by the following formula, using the value measured using the product after storage for 10 days as the amount of luminescence after storage for 1 year, 5 days or 10 days.
Stability rate (%) = B / A × 100
(A: initial light emission amount, B: light emission amount after storage for 5 days or 10 days for one year)

  Using the obtained immunoassay kit (S-8), the storage stability in immunoassay was evaluated by the following method. The results are shown in Table 1.

<Stability Evaluation Method for Immunoassay Kit of the Present Invention (Measurement Substance 3 (F1): FT4)>
Standard FT4 (F1) solution having an FT4 (F1) concentration of 1 ng / dL prepared with 0.025 mL of solid phase carrier reagent (E-3) containing magnetic silica particles and 0.02 M phosphate buffer (pH 7.0) 0.025 mL and 0.025 mL of the labeling reagent (A-8) are mixed in a test tube, reacted in the test tube at 37 ° C. for 6 minutes, and anti-T4 antibody (F3) is applied onto the magnetic silica particles (B-1). ) / T4 (F1) complex or anti-T4 antibody (F3) / POD-labeled T4 (F1C) complex. After the reaction, collect the magnetic silica particles from the outside of the test tube with a neodymium magnet for 10 seconds, remove the liquid in the test tube with an aspirator, sufficiently separate the neodymium magnet from the side, add 0.5 mL of physiological saline, and add the magnetic silica particles. After collecting and magnetizing, the washing operation of removing the liquid with an aspirator was performed three times. Finally, 0.07 mL of luminol luminescence reagent (K-1) and 0.07 mL of hydrogen peroxide solution (L-1) were added at the same time, and the luminol luminescence reagent (K-1) and luminescence reaction were performed at 37 ° C. for 43 seconds. After adding hydrogen peroxide solution (L-1), the average amount of luminescence for 43 to 45 seconds was measured with a luminometer [“Lumat LB9507” manufactured by Bertoled Japan Co., Ltd.].

The value measured using an immunoassay kit within 5.0 hours after the preparation of the immunoassay kit is the initial luminescence amount, the immunoassay kit is 4 ° C for 1 year (365 days), 40 ° C for 5 days, or 40 ° C. The stability rate was calculated by the following formula, using the value measured using the product after storage for 10 days as the amount of luminescence after storage for 1 year, 5 days or 10 days.
Stability rate (%) = B / A × 100
(A: initial light emission amount, B: light emission amount after storage for 5 days or 10 days for one year)

  Using the obtained immunoassay kit (S-9), the storage stability in the immunoassay was evaluated by the following method. The results are shown in Table 1.

<Stability Evaluation Method 4 for Immunoassay Kit of the Present Invention (Measuring Substance 3 (F1): FT4)>
Standard FT4 (F1) solution having a FT4 (F1) concentration of 1 ng / dL prepared with 0.025 mL of solid phase carrier reagent (E-4) containing magnetic silica particles and 0.02 M phosphate buffer (pH 7.0) 0.025 mL and 0.025 mL of the labeling reagent (A-9) are mixed in a test tube, reacted in the test tube at 37 ° C. for 6 minutes, and T4 (F1) / on the magnetic silica particles (B-1). A POD-labeled anti-T4 monoclonal antibody (F3C) complex was formed. After the reaction, collect the magnetic silica particles from the outside of the test tube with a neodymium magnet for 10 seconds, remove the liquid in the test tube with an aspirator, sufficiently separate the neodymium magnet from the side, add 0.5 mL of physiological saline, and add the magnetic silica particles. After collecting and magnetizing, the washing operation of removing the liquid with an aspirator was performed three times. Finally, 0.07 mL of luminol luminescence reagent (K-1) and 0.07 mL of hydrogen peroxide solution (L-1) were added at the same time, and the luminol luminescence reagent (K-1) and luminescence reaction were performed at 37 ° C. for 43 seconds. After adding hydrogen peroxide solution (L-1), the average amount of luminescence for 43 to 45 seconds was measured with a luminometer [“Lumat LB9507” manufactured by Bertoled Japan Co., Ltd.].

The value measured using an immunoassay kit within 5.0 hours after the preparation of the immunoassay kit is the initial luminescence amount, the immunoassay kit is 4 ° C for 1 year (365 days), 40 ° C for 5 days, or 40 ° C. The stability rate was calculated by the following formula, using the value measured using the product after storage for 10 days as the amount of luminescence after storage for 1 year, 5 days or 10 days.
Stability rate (%) = B / A × 100
(A: initial light emission amount, B: light emission amount after storage for 5 days or 10 days for one year)

  Using the obtained immunoassay kit (S-10), the storage stability in the immunoassay was evaluated by the following method. The results are shown in Table 1.

<Stability Evaluation Method for Immunoassay Kit of the Present Invention (Measurement Substance 3 (F1): FT4)>
Standard FT4 (F1) solution having an FT4 (F1) concentration of 1 ng / dL prepared with 0.025 mL of solid phase carrier reagent (E-3) containing magnetic silica particles and 0.02 M phosphate buffer (pH 7.0) 0.025 mL and 0.025 mL of the labeling reagent (A-10) are mixed in a test tube, reacted in the test tube at 37 ° C. for 6 minutes, and anti-T4 antibody (F3) on the magnetic silica particles (B-1). ) / T4 (F1) complex or anti-T4 antibody (F3) / POD-labeled T3 (F2C) complex. After the reaction, collect the magnetic silica particles from the outside of the test tube with a neodymium magnet for 10 seconds, remove the liquid in the test tube with an aspirator, sufficiently separate the neodymium magnet from the side, add 0.5 mL of physiological saline, and add the magnetic silica particles. After collecting and magnetizing, the washing operation of removing the liquid with an aspirator was performed three times. Finally, 0.07 mL of luminol luminescence reagent (K-1) and 0.07 mL of hydrogen peroxide solution (L-1) were added at the same time, and the luminol luminescence reagent (K-1) and luminescence reaction were performed at 37 ° C. for 43 seconds. After adding hydrogen peroxide solution (L-1), the average amount of luminescence for 43 to 45 seconds was measured with a luminometer [“Lumat LB9507” manufactured by Bertoled Japan Co., Ltd.].

The value measured using an immunoassay kit within 5.0 hours after the preparation of the immunoassay kit is the initial luminescence amount, the immunoassay kit is 4 ° C for 1 year (365 days), 40 ° C for 5 days, or 40 ° C. The stability rate was calculated by the following formula, using the value measured using the product after storage for 10 days as the amount of luminescence after storage for 1 year, 5 days or 10 days.
Stability rate (%) = B / A × 100
(A: initial light emission amount, B: light emission amount after storage for 5 days or 10 days for one year)

<Dispersibility test of magnetic silica particles of the immunoassay kit of the present invention>
Disperse 1.0 mg of anti-CEA monoclonal antibody-immobilized magnetic silica particles (B1F3) in 2 mL of ion-exchanged water and place it in a glass container having a mouth inner diameter × body diameter × total height = φ10.3 mm × φ12.0 mm × 35 mm. Put a 1 cm × 1 cm × 1 cm neodymium magnet on the side, collect the magnetic silica particles completely, remove the supernatant, sufficiently separate the neodymium magnet from the side, and add 2 mL of immunoassay reagent (S-1 ~ S-10) or immunoassay reagents (H-1 to H-3) were added to the magnetic silica particles while being sprayed, mixed by pipetting three times, and then scanning electron microscope (model No. JSM) within 10 seconds. -7000F, JEOL Ltd.) at a magnification of 5,000 to 20,000 times, the ratio of the number of aggregated particles to the total number of particles in the image was calculated, and the dispersibility was determined according to the following criteria. .
○: Aggregated particles are less than 5% Δ: Aggregated particles are 5% or more and less than 20% ×: Aggregated particles are 20% or more

From Table 1, the immunoassay kit containing the labeling reagent (A) which is the immunoassay reagent of the present invention of Examples 1 to 10 has a storage stability rate of 92 to 97% after storage at 4 ° C. for 1 year. It is extremely high, and it is 72 to 82% even after storage at 40 ° C. for 5 days, and 48 to 56% even after storage at 40 ° C. for 10 days, which indicates that the storage stability is excellent.
In the immunoassay kit of the present invention, the aggregated particles are less than 5% in the evaluation results of the magnetic silica particle dispersibility, and the antibody is immobilized as compared with the case of using the comparative immunoassay kit. It can be seen that the dispersibility of the solid phase carrier (BF) is excellent.
Therefore, by using the immunoassay kit containing the labeling reagent (A) that is the reagent for immunoassay of the present invention, the solid phase carrier does not aggregate and stable immunoassay can be performed even after long-term storage.

  Since the labeling reagent of the present invention is excellent in storage stability, the immunoassay method using the labeling reagent of the present invention can be widely applied to clinical tests such as enzyme immunoassay. The reagent of the present invention is suitable for use in the above-described measurement method, and can be used as a clinical test agent for enzyme immunoassay.

Claims (7)

  An immunoassay reagent (X) containing a substance (FC) and a hydrophilic protein (D), wherein the substance (FC) is a substance obtained by labeling the substance (F) with an enzyme (C), F) is at least one substance selected from the group consisting of a measurement target substance (F1), a similar substance (F2) of the measurement target substance, and a substance (F3) that specifically binds to the measurement target substance, and is a hydrophilic protein (D) is a protein that dissolves at least 2.0 g in 100 g of water at 25 ° C. and pH 7.0, and the content of the hydrophilic protein (D) in the immunoassay reagent (X) is based on the weight of (X) As an immunoassay reagent (X) of 2 to 6% by weight.   The immunoassay reagent according to claim 1, wherein the hydrophilic protein (D) is a hydrophilic casein protein (D1).   The reagent for immunoassay according to claim 1 or 2, wherein the hydrophilic protein (D) is a casein hydrolyzate.   An immunoassay kit comprising a labeling reagent (A) and a solid phase carrier reagent (E), wherein the labeling reagent (A) is the immunoassay reagent (X) according to any one of claims 1 to 3. A kit for immunoassay, wherein the solid phase carrier reagent (E) contains a solid phase carrier (BF) having the substance (F) immobilized on the surface of the solid phase carrier (B).   The immunoassay kit according to claim 4, further comprising a luminol luminescence reagent (K) and an aqueous peroxide solution (L), wherein the enzyme (C) is peroxidase.   The immunoassay kit according to claim 4 or 5, wherein the solid phase carrier (B) is magnetic silica particles (B1).   The immunoassay method using the kit for immunoassay of any one of Claims 4-6.