JP2018124277A - Particle-containing composition, immunoassay reagent, immunoassay method and particle storage method - Google Patents

Abstract

The object of the present invention is to provide a particle-containing composition, an immunoassay reagent, an immunoassay method, and a particle storage method that enhance the stability of the NS3 region of the HCV antigen immobilized on the particle surface and enable continuous and stable measurement. . A particle-containing composition having a pH of 7-9, comprising particles (A) having an HCV antigen NS3 region immobilized on the surface thereof, gelatin (B) and water. The particle-containing composition preferably further contains sugar (C), and the weight ratio of gelatin (B) to sugar (C) [(B)/(C)] is 0.025 to 1.6. is preferred. [Selection figure] None

Description

  The present invention relates to a particle-containing composition, an immunoassay reagent, an immunoassay method, and a particle storage method.

  In an immunoassay reagent, the measurement target substance, a similar substance to the measurement target substance, or a substance that specifically binds to the measurement target substance on the surface of a magnetic particle or glass bead that is a solid phase carrier as a main component, such as an antigen Is fixed. In an immunoassay reagent, it is desirable that a substance immobilized on a solid support maintains a certain activity. If the activity of the substance decreases over time or the activity of the substance changes depending on the storage state, an accurate measurement result cannot be expected, which is a big problem as an immunoassay reagent.

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

JP 9-127114 A JP 2011-241206 A JP-A-60-149972

  An object of the present invention is to increase the stability of the NS3 region of the HCV antigen immobilized on the particle surface and to enable a stable measurement, a particle-containing composition, an immunoassay reagent, an immunoassay method, and a particle storage method Is to provide.

  The inventor of the present invention has arrived at the present invention as a result of intensive investigations to achieve the above object. That is, the present invention comprises a particle-containing composition containing particles (A), gelatin (B) and water having an NS3 region immobilized on the surface thereof, and having a pH of 7 to 9; An immunoassay method for measuring a substance to be measured in a sample using the immunoassay reagent; particles (A) on which NS3 region of HCV antigen is immobilized on the surface, gelatin (B) It is a preservation | save method of particle | grains (A) preserve | saved in the aqueous solution to contain, Comprising: It is a preservation | save method of particle | grains (A) whose pH of aqueous solution is 7-9.

The particle-containing composition and the immunoassay reagent of the present invention have high stability of the NS3 region of the HCV antigen immobilized on the particle surface even after long-term storage. In addition, the particle storage method of the present invention can stably store the NS3 region of the HCV antigen immobilized on the particle surface. Furthermore, the immunoassay method of the present invention enables highly sensitive and accurate clinical tests even after long-term storage.
In the present invention, “the stability of the NS3 region of the HCV antigen on the surface” and “the stability of the immunoassay reagent” refer to the HCV immediately after preparation of the particle-containing composition and the immunoassay reagent and after storage for a certain period of time. It means that there is little change in the activity of the NS3 region of the antigen.

  The particle-containing composition of the present invention is a particle-containing composition having particles (A), gelatin (B), and water with the NS3 region of the HCV antigen immobilized on the surface and having a pH of 7-9.

The particles (A) are not particularly limited as long as they are generally used in the field of immunoassay, and typical examples thereof include glass beads, polystyrene beads, magnetic particles, microplates, and latex.
Of these, magnetic particles are preferable from the viewpoint of shortening the measurement time in immunoassay, and more preferable are magnetic silica particles described in JP-A-2014-210680 and JP-A-2013-019889. It is.
The particle-containing composition containing the particles (A) can be used for an immunoassay reagent, protein purification, and the like. Among these, the use of an immunoassay reagent is most preferable.

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

  The lower limit of the content of superparamagnetic metal oxide in the magnetic silica particles is preferably 60% by weight, more preferably 65% by weight, and 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 obtained magnetic silica particles are sufficiently magnetized, so that it does not take time for the separation operation in actual use. Those having a content of 95% by weight or less are easy to synthesize.

  In the present invention, the NS3 region of the HCV antigen is a nonstructural protein region of hepatitis C virus, which binds to NS4A and functions in viral protein processing and RNA replication.

In the present invention, the method for immobilizing the NS3 region of the HCV antigen on the surface of the particle (A) is not particularly limited as long as it is an immobilization method generally used in the field of immunoassay. The method of letting it be mentioned.
From the viewpoint of immobilization more efficiently, at least one organic compound selected from the group consisting of glutaraldehyde, albumin, carbodiimide, streptavidin, biotin, and an alkylalkoxysilane having a functional group is bound to the particle (A) surface, It is preferable to fix via them.
Of these organic compounds, (A) alkylalkoxysilane having a functional group is more preferable from the viewpoint of bonding to the surface.
Examples of the alkylalkoxysilane having such a functional group include vinyltrimethoxysilane, vinyltriethoxysilane, 2- (3,4-epoxycyclohexyl) ethyltrimethoxysilane, 3-glycidoxypropylmethyldimethoxysilane, 3- Glycidoxypropyltrimethoxysilane, 3-glycidoxypropylmethyldiethoxysilane, 3-glycidoxypropyltriethoxysilane, p-styryltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyl Trimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-acryloxypropyltrimethoxysilane, N-2- (aminoethyl) -3-aminopro Rumethyldimethoxysilane, N-2- (aminoethyl) -3-aminopropyltrimethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-triethoxysilyl-N- (1,3 -Dimethyl-butylidene) propylamine, N-phenyl-3-aminopropyltrimethoxysilane, tris- (trimethoxysilylpropyl) isocyanurate, 3-ureidopropyltrialkoxysilane, 3-mercaptopropylmethyldimethoxysilane, 3-mercapto Examples thereof include propyltrimethoxysilane and 3-isocyanatopropyltriethoxysilane.

  The content of the particles (A) in the particle-containing composition in the present invention is preferably 0.000025 to 0.01% by weight based on the weight of the particle-containing composition from the viewpoint of the sensitivity of the immunoassay reagent, More preferably, it is 0.0005 to 0.003% by weight.

The particle-containing composition of the present invention contains gelatin (B) and water.
The gelatin (B) includes a known gelatin, and there is no limitation on the molecular weight and properties, and the gelatin (B) may be obtained from any animal (such as milk, fish, birds and fish). Examples of gelatin include acid-treated gelatin and alkali-treated gelatin produced by subjecting collagen to chemical treatment with acid or alkali and then heat treatment.
Furthermore, a gelatin derivative chemically modified by introducing a functional group such as amino group, imino group, carboxyl group, mercapto group and hydroxyl group into this gelatin using a known method can also be used.

  The content of gelatin (B) in the particle-containing composition of the present invention is preferably 1 to 8% by weight, more preferably, based on the weight of the particle-containing composition, from the viewpoint of the stability of the immunoassay reagent. 3 to 6% by weight.

The particle-containing composition of the present invention may further contain a saccharide (C).
Examples of the saccharide (C) include monosaccharide (C1), disaccharide (C2), and polysaccharide (C3).
Examples of the monosaccharide (C1) include triose (ketotriose and the like), tetrose (ketotetorose and the like), pentose (ketopentose, aldopentose and deoxysugar and the like), hexose [ketohexose (psicose, fructose, sorbose and tagatose and the like), aldohexose ( Allose, altrose, glucose, mannose, gulose, idose, galactose, talose, etc.) and deoxy sugars (fucose, fucose, rhamnose, etc.)] and heptose (sedheptulose, etc.).

  Examples of the disaccharide (C2) include those in which two molecules of the above monosaccharide are dehydrated and condensed to form a glycosidic bond, and specific examples include sucrose, lactose, trehalose, maltose, and cellobiose.

  Examples of the polysaccharide (C3) include those in which three or more molecules of the above monosaccharides are dehydrated and condensed to form a glycosidic bond. Specifically, amylose, amylopectin, glycogen, cellulose, hyaluronic acid, chondroitin sulfate And heparin.

Saccharides (C) may be used alone or in combination of two or more.
The saccharide (C) is preferably a disaccharide (C2) from the viewpoint of the stability of the immunoassay reagent, more preferably sucrose, lactose and trehalose, and particularly preferably trehalose.

  The content of the saccharide (C) in the particle-containing composition of the present invention is preferably 1 to 40% by weight, more preferably, based on the weight of the particle-containing composition, from the viewpoint of the stability of the reagent for immunoassay. 10 to 20% by weight.

  The weight ratio [(B) / (C)] of gelatin (B) to saccharide (C) in the particle-containing composition is preferably 0.025 to 1.6 from the viewpoint of the stability of the reagent for immunoassay, More preferably, it is 0.15-0.5.

The particle-containing composition of the present invention may further contain an inorganic salt (D).
Inorganic salts (D) include alkali metal salts [halides (such as sodium chloride, potassium chloride, sodium bromide and sodium fluoride), sulfates (such as sodium sulfate and potassium sulfate), nitrates (such as sodium nitrate and potassium nitrate) And phosphates (such as sodium phosphate and potassium phosphate)], alkaline earth metal salts [halides (such as calcium chloride and magnesium chloride) and sulfates (such as magnesium sulfate)], and the like.
Among these, sodium chloride, potassium chloride, magnesium chloride, sodium sulfate, potassium sulfate, sodium nitrate and potassium nitrate are preferable from the viewpoint of the stability of the immunoassay reagent.
An inorganic salt (D) may be used individually by 1 type, or may use 2 or more types together.

  The content of the inorganic salt (D) in the particle-containing composition is preferably 0.1 to 2% by weight, more preferably, based on the weight of the particle-containing composition, from the viewpoint of the stability of the reagent for immunoassay. 0.5 to 1% by weight.

  In addition to the above, the particle-containing composition of the present invention contains particles (A ′) in which at least one region selected from the group consisting of NS4 region, NS5 region and CORE protein region of HCV antigen is immobilized on the particle surface. May be.

The NS4 region, NS5 region, and CORE protein region of the HCV antigen are not particularly limited as long as they are generally used in the field of immunoassay, but a peptide or a recombinant antigen is preferable because it is easy to handle. .
The immobilization method is the same as the NS3 region immobilization method of the HCV antigen.

  The content of particles (A ′) in which at least one selected from the group consisting of NS4 region, NS5 region and CORE protein region of HCV antigen in the particle-containing composition is immobilized is from the viewpoint of the sensitivity of the reagent for immunoassay. Based on the weight of the particle-containing composition, 0.000025 to 0.01% by weight is preferable, and 0.0005 to 0.003% by weight is more preferable.

In addition to the above, the particle-containing composition of the present invention may contain a protein.
The protein is not particularly limited as long as it is generally measured in the field of immunoassay, and examples thereof include bovine serum albumin (BSA), casein, skim milk, and the like.
A protein may be used individually by 1 type, or may use 2 or more types together.
The content of the protein in the particle-containing composition is preferably 0.01 to 5% by weight, more preferably 0.1 to 5% by weight, based on the weight of the particle-containing composition, from the viewpoint of the stability of the immunoassay reagent. 3% by weight.

The pH of the particle-containing composition of the present invention is 7-9. If the pH is less than 7, the stability of the NS3 region of the HCV antigen is poor. If the pH is greater than 9, the background of the measured value increases, adversely affects the measurement result, and a correct measured value cannot be obtained.
The pH is measured at a measurement temperature of 25 ° C. according to JIS K0400-12-10: 2000.

In addition to the above, the particle-containing composition of the present invention may contain a buffer.
The buffer is not particularly limited as long as it is generally used in the field of immunoassay and has a buffering capacity during the above pH. Phosphoric acid, carbonic acid, tris (hydroxymethyl) aminomethane-hydrochloric acid, N , N-bis (2-hydroxyethyl) glycine, 4- (2-hydroxyethyl) -1-piperazinepropanesulfonic acid, N- [tris (hydroxymethyl) methyl] glycine and N- (2-hydroxyethyl) piperazine- N'-2-ethanesulfonic acid etc. are mentioned.
A buffering agent may be used individually by 1 type, or may use 2 or more types together.
The content of the buffering agent in the particle-containing composition is preferably 0.001 to 1M, more preferably 0.01 to 0.1M as the concentration in the particle-containing composition from the viewpoint of pH stability. .

  In the present invention, the particle-containing composition contains particles (A), gelatin, and water, and can be produced without particular limitation as long as the pH is 7 to 9, but from the viewpoint of the stability of the HCV antigen on the particles (A). Therefore, it is preferable to add particles (A) to an aqueous solution containing gelatin and having a pH of 7 to 9 to obtain a particle-containing composition.

The storage conditions for the particle-containing composition of the present invention are not particularly limited as long as they are methods generally used for storage of immunoassay reagents.
The storage temperature is preferably 2 ° C. to 10 ° C., which is a general storage temperature that does not impair the activity of the HCV antigen immobilized on the particles (A).
The container is not particularly limited as long as it is a durable storage container. For example, a cryotube [manufactured by Thermo Fisher Scientific Co., Ltd.], a centrifuge tube [manufactured by Corning International Co., Ltd.], a microtube [Eppendorf] Manufactured by Co., Ltd.], screw tube [manufactured by Maruemu Co., Ltd.], PCR tube [manufactured by Nippon Genetics Co., Ltd.].
Further, the particles in the particle-containing composition during storage may be in a precipitated state or a dispersed state.
The storage period is not particularly limited, but is preferably within 1.5 years from the viewpoint of the stability of the HCV antigen immobilized on the particles (A).

The immunoassay reagent of the present invention is not particularly limited as long as it contains the particle-containing composition, and the particle-containing composition may be used as it is as an immunoassay reagent.
In addition to the particle-containing composition, the immunoassay reagent contains an immune reaction buffer containing a protein, a surfactant, a polymer compound, and the like for the purpose of suppressing nonspecific reactions in immunoassay. Also good.

In addition to the same protein used in the above-mentioned particle-containing composition, the protein in the immune reaction buffer includes animal sera and animal antibodies. From the viewpoint of suppressing non-specific reactions, animal antibodies are Mouse antibodies, rabbit antibodies, sheep antibodies, goat antibodies and horse antibodies are preferred.
The concentration of the animal antibody is preferably 0.0001 to 0.2% by weight, more preferably 0.05 to 0.1% by weight, based on the weight of the particle-containing composition, from the viewpoint of suppressing non-specific reactions. It is.

  Surfactants in the immune reaction buffer include known nonionic surfactants, amphoteric surfactants and anionic surfactants. From the viewpoint of suppressing nonspecific reactions, water-soluble nonionic surfactants are included. Agents are preferred.

  Water-soluble nonionic surfactants include higher alcohol ethylene oxide (ethylene oxide abbreviated as EO) adduct, fatty acid EO adduct, fatty acid amide EO adduct, sorbitan fatty acid ester EO adduct, and polypropylene glycol EO adduct. And the like (described in JP-A-8-261944) can be used.

Surfactant may be used individually by 1 type, or may use 2 or more types together.
The content of the surfactant is preferably 0.01 to 5% by weight, more preferably 0.1 to 3% by weight, based on the weight of the particle-containing composition, from the viewpoint of suppressing nonspecific reactions. .

  The polymer compound in the immune reaction buffer is not particularly limited as long as it is generally used in the field of immunoassay. For example, polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, Blockmaster [manufactured by JSR Corporation] And Lipidure [manufactured by NOF Corporation].

A high molecular compound may be used individually by 1 type, or may use 2 or more types together.
The content of the polymer compound is preferably from 0.001 to 3% by weight based on the weight of the particle-containing composition from the viewpoint of suppressing non-specific reaction, Preferably it is 0.5 to 1 weight%.

  In addition to the particle-containing composition, the immunoassay reagent is specific to an NS3 region of an HCV antigen labeled with a labeling substance or an anti-HCV antibody that specifically binds to an NS3 region of an HCV antigen labeled with a labeling substance. May contain a reagent (hereinafter, may be abbreviated as a labeling reagent) containing a substance (anti-human IgG antibody labeled with a labeling substance) that binds chemically. Further, it may contain NS4 region, NS5 region and CORE protein region of HCV antigen labeled with a labeling substance.

Examples of labeling substances used for labeling include alkaline phosphatase, β-galactosidase, peroxidase (hereinafter abbreviated as POD), microperoxidase, glucose oxidase, and glucose-6-phosphate dehydration used in enzyme immunoassay (EIA). Enzymes such as elementary enzyme, malate dehydrogenase, luciferase, tyrosinase, acid phosphatase, such as ^99m Tc, ¹³¹ I, ¹²⁵ I, ¹⁴ C, ³ H, ³² P, etc. used in radioimmunoassay (RIA) Radioisotopes, such as fluorescein, dansyl, fluorescamine, coumarin, naphthylamine or derivatives thereof used in fluorescence immunoassay (FIA), green fluorescent protein (GFP) and other fluorescent substances such as luciferin, Luminol, Luminescent substances such as minol and bis (2,4,6-trifluorophenyl) oxalate, such as substances having absorption in the ultraviolet region such as phenol, naphthol, anthracene or derivatives thereof, such as 4-amino-2,2, 6,6-tetramethylpiperidine-1-oxyl, 3-amino-2,2,5,5-tetramethylpyrrolidine-1-oxyl, 2,6-di-t-butyl-α- (3,5-di- And a substance having a property as a spin labeling agent typified by a compound having an oxyl group such as -t-butyl-4-oxo-2,5-cyclohexadiene-1-ylidene) -p-trioxyl.
Among these, from the viewpoint of sensitivity and the like, enzymes and fluorescent substances are preferable, alkaline phosphatase, POD and glucose oxidase are more preferable, and POD is particularly preferable.

The labeling reagent includes a buffer other than the substance that specifically binds to the NS3 region of the HCV antigen labeled with the enzyme immunoassay labeling substance or the antibody that specifically binds to the NS3 region of the HCV antigen labeled with the labeling substance. Liquid and the like.
As the buffer solution used for the labeling reagent, a buffer solution generally used for immunoassay can be preferably used, and examples thereof include the same buffers as those used in the above-mentioned particle-containing composition, The concentration and pH of the buffer are the same as those used in the above-mentioned particle-containing composition.

The reagent for immunoassay of the present invention may contain a chemiluminescent reagent in addition to the particle-containing composition and the labeling reagent of the present invention.
The chemiluminescent reagent is selected based on the above-mentioned labeling substance. For example, when the labeling substance is POD, a chemical containing a 2,3-dihydro-1,4-phthalazinedione compound and a chemiluminescence enhancer as essential components. A luminescent reagent first liquid and a chemiluminescent reagent second liquid containing oxidant and water as essential components are included.

Examples of the 2,3-dihydro-1,4-phthalazinedione compound include those disclosed in JP-A-2-291299, JP-A-10-319015, JP-A-2000-279196, and the like. 3-dihydro-1,4-phthalazinedione compounds 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.) are preferable. Further preferred is luminol and its metal salt, and particularly preferred is the sodium salt of luminol.

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 the chemiluminescence enhancing effect and the like, a phenol skeleton-containing compound is preferable, and more preferable are P-iodophenol, 4- (cyanomethylthio) phenol and 4-cyanomethylthio-2-chlorophenol, Preferred is 4- (cyanomethylthio) phenol.

The first chemiluminescent reagent liquid 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 at a measurement temperature of 25 ° C. according to JIS K0400-12-10: 2000.

As the oxidizing agent contained in the second liquid of the chemiluminescent reagent, for example, known oxidizing agents described in JP-A-8-261944 and JP-A-2000-279196 etc. [inorganic peroxide {} peroxidation Examples include aqueous solutions of hydrogen, sodium perborate and potassium perborate, peroxo acid compounds (such as peroxosulfuric acid and peroxophosphoric acid), and organic peroxides (such as dialkyl peroxide and acyl peroxide).
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.

  The immunoassay method of the present invention is an immunoassay method for measuring an anti-HCV antibody (measurement target substance) in a sample using the above-described immunoassay reagent of the present invention.

Specifically, the following methods are included as immunoassay methods.
Sandwiches described in the literature [for example, enzyme immunoassay second edition (edited by Eiji Ishikawa et al., Medical School) 1982], which is generally performed in the field of immunoassay, except that the reagent for immunoassay of the present invention is used. Method, competition method and measurement method described in JP-A-6-130063.

In the sandwich method, for example, a sample containing a substance to be measured is brought into contact with a particle (A) on which the NS3 region of the HCV antigen is immobilized on the surface, and a substance that specifically binds to the substance to be measured on the particle surface and the object to be measured A complex with a substance is formed, and a substance that specifically binds to the labeled measurement target substance is contacted with the complex, and the NS3 region of the HCV antigen immobilized on the particle, the measurement target substance, and the label Forming a complex (labeled complex) with a substance that specifically binds to the measured substance to be measured, separating the labeled complex B / F, measuring the amount of the labeled substance in the labeled complex, What is necessary is just to measure the measuring object substance in a sample based on a result.
In this method, after the substance to be measured is reacted with the particles (A) on which the NS3 region of the HCV antigen is immobilized, a substance that specifically binds to the labeled substance to be measured is reacted. Even after reacting a substance that specifically binds to the labeled substance to be measured and the substance to be measured, and reacting with the particles (A) on which the NS3 region of the HCV antigen is immobilized, these three substances react simultaneously. It does n’t matter.

B / F separation in the sandwich method means separation of the labeled complex from a substance that specifically binds to a labeled measurement target substance that was not involved in the formation of the labeled complex. Are the NS3 region of the HCV antigen immobilized on the particle, the complex of the NS3 region of the HCV antigen immobilized on the particle and the substance to be measured, and the above labeled complex, and other components (the measurement target in the sample). It means separation from components other than substances, and substances that specifically bind to labeled substances to be measured that were not involved in the formation of labeled complexes.
The B / F separation step is an essential step after the formation of the labeled complex, but can also be performed after the complex of the NS3 region of the HCV antigen and the measurement target substance is formed on the particle surface. .

In the immunoassay method of the present invention, as a method of contacting a sample, particles, a substance that specifically binds to a labeled measurement target substance, a measurement target substance labeled with a labeling substance, or a similar substance, immunoassay is possible. The particles may be dispersed by a treatment such as stirring and mixing generally performed in the field.
Although reaction time can be suitably set according to the density | concentration of each reagent, 1 to 20 minutes are preferable, More preferably, it is 3 to 10 minutes.

  B / F separation in the immunoassay method of the present invention is, for example, when the particles are magnetic silica particles, using magnetism, collecting the magnetic silica particles with a magnet or the like from the outside of the reaction tank, etc., and discharging the reaction solution, After adding the cleaning solution, the magnet is removed, the magnetic silica particles are mixed and dispersed, and then washed. The above operation may be repeated 1 to 3 times. The washing solution is not particularly limited as long as it is generally used in the field of immunoassay.

  Examples of methods for measuring the labeling substance or its activity include radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), and chemiluminescence immunoassay (CLIA and CLEIA). From the viewpoint of sensitivity in immunoassay over time, EIA, CLIA and CLEIA are preferred, and CLEIA is more preferred.

The method for preserving the particles (A) of the present invention is a method for preserving the particles (A) in which the particles (A) having the NS3 region of the HCV antigen immobilized on the surface are stored in an aqueous solution containing gelatin (B). The method for preserving particles (A) in which the pH of the aqueous solution is 7-9.
The storage temperature is preferably 2 to 45 ° C., which is a general storage temperature that does not impair the activity of the HCV antigen immobilized on the particles (A).
The container and the storage period are preferably the same as the storage conditions for the particle-containing composition.
In the present invention, even when stored at 20 to 45 ° C. in an aqueous solution containing gelatin (B), the activity of the HCV antigen immobilized on the surface of the solid support can be maintained and the stability can be improved. it can. In this case, the stability can be enhanced by further containing the saccharide (C) and / or the inorganic salt (D) in the aqueous solution.
The storage period for storage at 20 to 45 ° C. is not particularly limited, but is preferably 1 day to 4 weeks from the viewpoint of the stability of the HCV antigen immobilized on the particles (A).

  EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited to this.

<Example 1>
According to the method described below, a particle-containing composition containing magnetic silica particles in which the HCV antigen NS3 region is immobilized (NS3-binding magnetic silica particle reagent), immune reaction buffer, labeling reagent (POD-labeled anti-human IgG antibody reagent), An immunoassay reagent (S-1) of the present invention comprising a luminol luminescence reagent and hydrogen peroxide was obtained.

Production of magnetic silica particles:
A reaction vessel was charged with 2.7 parts of iron (III) chloride hexahydrate, 1.0 part by weight of iron (II) chloride tetrahydrate and 375 parts by weight of water, dissolved, heated to 50 ° C., and stirred. While maintaining the lower temperature at 50 to 55 ° C., a solution prepared by mixing 3.8 parts by weight of a 25% by weight aqueous ammonia solution and 100 parts by weight of water was added dropwise over 1 hour, stirred for 1 hour after the addition, and oleic acid 10. 5 parts by weight was added 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 50 parts by weight of water was performed three times. The obtained magnetite particles adsorbed with oleic acid were charged into a container, and 5.7 parts by weight of decane and 2.2 parts by weight of tetraethoxysilane were added and mixed to prepare dispersion (1).
In a reaction vessel, 39.0 parts by weight of 25% by weight aqueous ammonia solution, 55.4 parts by weight of isopropanol, 2.9 parts by weight of sorbitan monooleate (“Ionet S-80” manufactured by Sanyo Chemical Industries, Ltd.) and polyoxyethylene (addition) 20 moles) alkyl ether (Sanyo Chemical Industries Co., Ltd. “Emalmin 200”) 2.0 parts by weight, mixed using Clearmix (M Technique Co., Ltd.), heated to 50 ° C. The dispersion (1) was added dropwise over 1 hour with stirring at a rotational 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 5 minutes to remove the supernatant containing fine particles. 50 parts by weight of water was added to the obtained solid phase to disperse the particles, followed by centrifugation at 1,000 rpm for 10 minutes, and then the operation of removing the supernatant containing fine particles was performed 10 times. Subsequently, 50 parts by weight of water is added to the obtained solid phase to disperse the particles and centrifuged at 500 rpm for 5 minutes to precipitate particles having a large particle size and contain particles having a target particle size. The supernatant (1) to be collected was collected. After adding 50 parts by weight of water to the remaining solid phase and centrifuging at 500 rpm for 5 minutes, the operation of recovering the supernatant (2) is performed twice to recover particles having the desired particle size in the solid phase. did. Next, for the supernatants (1) and (2), the particles were collected using a magnet, and the collected particles were dried at 80 ° C. for 8 hours to obtain magnetic silica particles.

Preparation of NS3-bound magnetic silica particle reagent:
After adding 40 mg of the above magnetic silica particles to a polyethylene bottle with a lid containing 40 mL of an acetone solution containing 0.1 wt% γ-aminopropyltriethoxysilane, reacting at 25 ° C. for 1 hour, and collecting the magnetic silica particles with a neodymium magnet The liquid was removed by suction with an aspirator. Next, 40 mL of deionized water was added, the cap was capped, and the polystyrene bottle was slowly inverted and stirred twice. After collecting the magnetic silica particles with a neodymium magnet, the liquid was sucked and removed with an aspirator to wash the magnetic silica particles. 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 a 0.2 wt% glutaraldehyde-containing aqueous solution and reacted at 25 ° C. for 1 hour. Then, 40 mL of deionized water was added, the cap was closed, the polystyrene bottle was slowly inverted and stirred twice, and after collecting the magnetic silica particles with a neodymium magnet, the liquid was sucked and removed with an aspirator to wash the magnetic silica particles. . This washing operation was performed 10 times. Further, a lid containing 120 mL of 0.02 M phosphate buffer (pH 8.7) containing the magnetic silica particles after washing at a concentration of 10 μg / mL of NS3 region of HCV antigen [“Recombinant NS3”, Funakoshi Co., Ltd.] In addition to the attached polyethylene bottle, the mixture was reacted at 25 ° C. for 3 hours. After the reaction, the magnetic silica particles were collected with a neodymium magnet, and then the phosphate buffer containing NS3 region of HCV antigen was removed. Next, the magnetic silica particles were added to a polyethylene bottle with a lid containing 40 mL of 0.02M phosphate buffer (pH 7) containing 1% by weight of casein and immersed in a polyethylene bottle at 25 ° C. for 12 hours. After collecting the magnetic flux, the phosphate buffer containing 1% by weight of casein was removed to obtain magnetic silica particles (A-1) having the NS3 region of the HCV antigen immobilized on the surface. The obtained particles (A-1) were mixed with 5% by weight of gelatin [“trade name: Super Gelatin BPG-160” manufactured by Nippi Co., Ltd.], 10% by weight of sucrose [manufactured by Wako Pure Chemical Industries, Ltd.], Using a 0.02M phosphate buffer (pH 7.8) containing 0.85% by weight sodium chloride and 0.01% sodium azide, the concentration of particles (A-1) is 0.2 mg / mL. Then, a particle-containing composition containing magnetic silica particles (A-1) in which the NS3 region of the HCV antigen was immobilized on the surface was prepared and stored in a refrigerator (2 to 10 ° C.). This was used as NS3-bonded magnetic silica particle reagent (T-1).

Preparation of immune reaction buffer:
Skim milk powder [manufactured by Wako Pure Chemical Industries, Ltd.] 0.3% by weight, Emalmin L-90-S [manufactured by Sanyo Chemical Industries] 1% by weight, sodium chloride [manufactured by Wako Pure Chemical Industries, Ltd.] A 0.02M phosphate buffer solution (pH 7) containing 0.85% by weight and 1% by weight of mouse serum [manufactured by Cosmo Bio Co., Ltd.] was prepared and stored in a refrigerator (2 to 10 ° C.).

Preparation of labeling reagent:
Using anti-human IgG polyclonal antibody [manufactured by Dako Japan Co., Ltd.] and horseradish-derived POD [manufactured by Toyobo Co., Ltd.], literature (S Yoshitake, M Imagawa, E Ishikawa, Etol; J. Biochem, Vol. 92, 1982, 1413-1424), a POD-labeled anti-human IgG antibody was prepared. This was diluted with a 0.02M phosphate buffer containing 0.5% by weight of casein hydrolyzate to a concentration of 0.5 nM as a POD-labeled anti-human IgG antibody concentration to prepare a labeling reagent, and refrigerated (2- (10 ° C.).

Luminol reagent preparation:
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. A luminol luminescence reagent was prepared by mixing. It was stored refrigerated (2-10 ° C.) until used for measurement.

Preparation of hydrogen peroxide water:
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 mixed uniformly at 25 ° C. to prepare a hydrogen peroxide solution. It was stored refrigerated (2-10 ° C.) until used for measurement.

<Examples 2 to 21>
In Example 1, “Preparation of NS3-bonded magnetic silica particle reagent”, the concentration of gelatin (B), the type and concentration of saccharide (C), the type and concentration of inorganic salt (D), and pH are listed in Table 1. NS3-bonded magnetic silica particle reagents (T-2) to (T-21) were prepared in the same manner as in Example 1 except that and the same immune reaction buffer solution and labeling reagent as in Example 1 (POD-labeled anti-human IgG) Antibody Reagents), Luminol Luminescent Reagent, and Hydrogen Peroxide Solution were used as immunoassay reagents (S-2) to (S-21).

<Comparative Examples 1-4>
In Example 1, “Preparation of NS3-bonded magnetic silica particle reagent”, the concentration of gelatin (B), the type and concentration of saccharide (C), the type and concentration of inorganic salt (D), and pH are listed in Table 1. The comparative NS3-bonded magnetic silica particle reagents (T′-1) to (T′-4) were prepared in the same manner as in Example 1 except that and the same immune reaction buffer solution and labeling reagent as in Example 1 ( Reagents for immunoassay (S′-1) to (S′-4) were combined with POD-labeled anti-human IgG antibody reagent), luminol luminescence reagent and hydrogen peroxide solution.

  The following immunoassay was performed using the immunoassay reagents (S-1) to (S-21) and (S′-1) to (S′-4) by the following method, and the particle-containing composition (immunoassay) The stability of the reagent for use was evaluated. The stability results are shown in Table 1.

<Immunoassay method>
0.03 mL of NS3-bonded magnetic silica particle reagents (T-1) to (T-21) and (T′-1) to (T′-4) obtained in Examples and Comparative Examples were put in test tubes, respectively. Collect 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, and add 0.2 mL of immune reaction buffer and 0.025 mL of HCV positive serum. The mixture was mixed in a test tube and reacted in the test tube at 37 ° C. for 3 minutes to form a complex of NS3-bound magnetic silica particles / HCV antibody. 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, 0.1 mL of the labeling reagent was injected into the test tube and reacted in the test tube at 37 ° C. for 3 minutes to form an NS3-bound magnetic silica particle / HCV antibody / POD-labeled anti-human IgG antibody complex. 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, 0.1 mL of luminol reagent and 0.1 mL of hydrogen peroxide solution were added simultaneously, and the mixture was allowed to emit light at 37 ° C. After adding the luminol reagent and hydrogen peroxide solution, the average amount of luminescence per second for 40 to 45 seconds Was measured with a luminometer [“Lumat LB9507” manufactured by Bertoled Japan Co., Ltd.].

<Stability evaluation at 4 ° C>
The above immunoassay was performed using an immunoassay reagent stored at 4 ° C. for 0.5 years, and the obtained luminescence cps was defined as the activity (X1) after storage for a certain period. Further, immunoassay was performed immediately after preparation of the reagent for immunoassay, and the obtained light emission amount cps was defined as activity (X2) immediately after preparation. Using the obtained activity measurement value, the stability rate at 4 ° C. was calculated by the following formula.
Stability rate at 4 ° C. (%) = (X1 / X2) × 100

<Stability evaluation at 37 ° C>
The above immunoassay was performed using an immunoassay reagent stored at 37 ° C. for 3 days, and the resulting luminescence cps was defined as the activity (X3) after storage for a certain period. Further, immunoassay was performed immediately after preparation of the reagent for immunoassay, and the obtained light emission amount cps was defined as activity (X2) immediately after preparation. Using the obtained activity measurement value, the stability rate at 37 ° C. was calculated by the following formula.
Stability rate at 37 ° C. (%) = (X3 / X2) × 100

From the comparison between Example 17 and Comparative Example 3 in Table 1 and the comparison between Examples 1, 15 and 16 and Comparative Examples 1 and 2, gelatin was contained and the pH was 7-9, so that it was stable at 4 ° C. It can be seen that the rate increases and the stability of the NS3 region of the HCV antigen on the particle surface increases. In particular, in addition to gelatin, the immunoassay reagents of Examples 1 to 16 and 18 to 21 containing saccharides and inorganic salts have both a 4 ° C. stability rate and a 37 ° C. stability rate of 80% or more, and have a wide range. It can be seen that stability improves with temperature.
Therefore, it can be seen that the immunoassay reagent of the present invention can be subjected to a highly sensitive and highly accurate clinical test even after long-term storage.

  The particle-containing composition, the reagent for immunoassay, the immunoassay method and the particle storage method of the present invention are useful for a test drug using HCV antigen used in the fields of immunoassay and clinical examination.

Claims (12)

  A particle-containing composition comprising particles (A), gelatin (B) and water having an NS3 region of HCV antigen immobilized on the surface, and having a pH of 7-9.   The particle-containing composition according to claim 1, further comprising a saccharide (C).   The particle-containing composition according to claim 2, wherein the content of gelatin (B) in the particle-containing composition is 1 to 8% by weight and the content of saccharide (C) is 1 to 40% by weight.   The particle-containing composition according to claim 2 or 3, wherein a weight ratio [(B) / (C)] of gelatin (B) to saccharide (C) is 0.025 to 1.6.   The particle-containing composition according to any one of claims 2 to 4, wherein the saccharide (C) is a disaccharide (C2).   Furthermore, the particle | grain containing composition of any one of Claims 1-5 containing an inorganic salt (D).   The particle-containing composition according to claim 6, wherein the inorganic salt (D) is at least one selected from the group consisting of sodium chloride, potassium chloride, magnesium chloride, sodium sulfate, potassium sulfate, sodium nitrate and potassium nitrate.   The particle-containing composition according to any one of claims 1 to 7, wherein the particles (A) are magnetic silica particles.   The reagent for immunoassay containing the particle | grain containing composition of any one of Claims 1-8.   An immunoassay method for measuring a substance to be measured in a sample using the immunoassay reagent according to claim 9.   A method for preserving particles (A) in which particles (A) on which NS3 regions of HCV antigen are immobilized are stored in an aqueous solution containing gelatin (B), wherein the pH of the aqueous solution is 7-9 (A) storage method.   The preservation | save method of the particle | grains (A) of Claim 11 preserve | saved at the temperature of 2-45 degreeC.