JP4975601B2 - Reagent kit for HCV antibody measurement and HCV antibody measurement method - Google Patents

Description

  The present invention relates to a reagent kit for measuring hepatitis C virus (HCV) antibody and a measuring method.

Hepatitis C is an infectious disease caused by hepatitis C virus (HCV).
HCV is a virus having a single-stranded (+ strand) RNA genome having a total length of about 9.5 kb, and its genomic structure consists of an untranslated region on the 5 ′ end side, a translated region, and an untranslated region on the 3 ′ end side. Become.
The translation region consisting of about 3000 amino acid residues includes a region corresponding to the core protein and envelope protein (E1, E2) as a structural protein, and a region corresponding to NS2, NS3, NS4, NS5 as non-structural proteins Exists.

  As a method for detecting HCV, an immunological method for detecting an antibody against an HCV antigen (HCV antibody) in a specimen is known. This method is usually performed by detecting an antigen-antibody complex formed by binding of an HCV antigen in a reagent and an HCV antibody in a specimen. For immunological detection of HCV antibodies, HCV antigens having sequences corresponding to various HCV proteins are used so that a plurality of types of HCV antibodies recognizing different types of HCV proteins can be detected. HCV antigens are known to be isolated from nature, or those produced by gene recombination methods or chemical synthesis methods. For example, JP-A-5-508219 (Patent Document 1), Patent No. 2995216 (Patent Document 2), Japanese Patent No. 3219409 (Patent Document 3), and the like.

A conventional reagent kit for immunologically detecting HCV antibodies provides a plurality of types of HCV antigens immobilized on a solid phase. These antigens are reacted with HCV antibodies in a specimen to react with the immobilized HCV antibodies. An HCV antibody is measured by forming an antigen-antibody complex on the phase and detecting the complex (Patent Documents 1 to 3).
Japanese National Patent Publication No. 5-508219 Japanese Patent No. 2995216 Japanese Patent No. 3219409

  In the process of studying various HCV synthetic peptide antigens and HCV antibody measurement reagents using HCV gene recombinant antigens that can be used for detection of HCV antibodies, the present inventors have studied the stability and reactivity of reagents containing these antigens. As a result, it was found that stability and reactivity differ between the HCV synthetic peptide antigen and the HCV gene recombinant antigen.

  Therefore, an object of the present invention is to provide an HCV antibody measurement reagent kit containing an HCV antigen that is more stable than conventional HCV antibody measurement reagents and can detect HCV antibodies with high sensitivity.

The present invention comprises a first reagent comprising a hepatitis C virus (HCV) synthetic peptide antigen to which a solid phase binding site has been added;
An HCV antibody measurement reagent kit comprising an HCV recombinant antigen and a second reagent containing a solid phase on which a binding substance capable of binding to the solid phase binding site is immobilized.

The present invention also provides a solid phase in which a specimen, a first reagent containing an HCV synthetic peptide antigen added with a solid phase binding site, an HCV gene recombinant antigen and a binding substance capable of binding to the solid phase binding site are immobilized. And a second complex of the HCV synthetic peptide antigen and the HCV antibody and / or a second complex of the HCV gene recombinant antigen and the HCV antibody formed on the solid phase. It is also a method for measuring HCV antibody contained in a specimen, which comprises a step of detecting.

  In the present invention, the first reagent contains an HCV synthetic peptide antigen in a free state. As used herein, “free state” means that the HCV synthetic peptide antigen is not immobilized on any solid phase.

  The reagent kit for HCV antibody measurement of the present invention makes it possible to carry out good measurement over a long period of time because various HCV antigens used for measurement have excellent storage stability and good reactivity with antibodies. . In addition, by using the reagent kit for HCV antibody measurement of the present invention, it is possible to prevent a decrease in measurement sensitivity due to a decrease in storage stability of HCV antigens, so that more accurate measurement can be performed.

Hepatitis C virus (HCV) has a genome encoding a protein consisting of about 3000 amino acid residues. This protein consisting of about 3000 amino acid residues is composed of a structural protein domain (core protein, envelope protein (E1 and E2)) and a non-structural protein domain (NS2, NS3, NS4 and NS5). Are known to be antigens having at least one epitope. Examples of the protein encoded by the HCV genome and the sequence of each domain contained in the protein are described in, for example, JP-T-5-508219.
Since HCV is a highly mutated virus, the sequence of the protein encoded by its genome cannot be uniquely represented. As used herein, “antigen” includes both antigenic proteins or peptides encoded by the HCV genome and variants thereof having antigenicity.

  The reagent kit for HCV antibody measurement of this embodiment immobilizes a first reagent containing an HCV synthetic peptide antigen to which a solid phase binding site has been added, an HCV gene recombinant antigen, and a binding substance that can bind to the solid phase binding site. And a second reagent containing an immobilized solid phase.

The principle of the reagent kit for HCV antibody measurement will be described next. The HCV antibody that can be contained in the specimen can bind to the HCV synthetic peptide antigen in the first reagent and / or the HCV gene recombinant antigen in the second reagent. Further, the solid phase binding site added to the HCV synthetic peptide antigen in the first reagent can bind to the binding substance immobilized on the solid phase in the second reagent. Here, when the specimen, the first reagent, and the second reagent are brought into contact, the HCV antibody in the specimen can be bound to the HCV recombinant antigen immobilized on the solid phase, and the HCV antibody in the specimen is bound to the HCV synthetic peptide antigen. In addition, the binding substance immobilized on the solid phase can bind to an HCV synthetic peptide antigen to which a solid-phase binding site has been added (whether or not bound to an HCV antibody).
Therefore, the HCV antibody is captured on the solid phase. The HCV antibody thus captured on the solid phase can be detected using a known method in the art.

The HCV synthetic peptide antigen is a peptide having an amino acid sequence containing at least one epitope of a protein encoded by the genome of HCV, isolated from a natural protein and cleaved or synthesized by a chemical synthesis method. The HCV synthetic peptide antigen contains at least 8 HCV protein amino acid residues, preferably contains no more than 60 HCV protein amino acid residues, contains at least 10 HCV protein amino acid residues, and 50 More preferably, the amino acid residues of the following HCV proteins are included. Further, the HCV synthetic peptide antigen preferably has a molecular weight in the range of 900 to 7500, more preferably 1000 to 6000.
The HCV synthetic peptide antigen can be obtained by a conventionally known chemical synthesis method such as a solid phase synthesis method, a fragment condensation method, or a solution synthesis method. More preferably, the synthetic peptide antigen is synthesized using a solid phase synthesis method as described in Merrifield, J. Am. Chem. Soc., 85, p.2149, 1963.

  The HCV synthetic peptide antigen is preferably at least one selected from the group consisting of a synthetic peptide NS4 antigen, a synthetic peptide NS5 antigen, and a synthetic peptide core antigen. Two or more of these synthetic peptide antigens can be used. The sequences of these antigens are described, for example, in JP-T-5-508219, but are not limited to these specific sequences as long as they have antigenicity, and may contain mutations. In the present specification, for example, “synthetic peptide core antigen” means a synthetic peptide antigen having an amino acid sequence derived from the amino acid sequence of the core domain of the HCV protein.

The HCV gene recombinant antigen is a protein having an amino acid sequence containing at least one epitope of a protein encoded by the genome of HCV produced by a gene recombination method. The HCV genetically modified antigen preferably has at least 80 amino acid residues, and more preferably has at least 90 amino acid residues. The HCV gene recombinant antigen preferably has a molecular weight of 9000 or more, and more preferably 10,000 or more.
The HCV gene recombinant antigen can be prepared by a conventionally known gene recombination method. For example, a DNA encoding the amino acid sequence of a desired antigen can be constructed, this DNA can be cloned into an appropriate expression vector, and an appropriate host cell can be transformed with the vector to produce an HCV gene recombinant antigen. . The HCV gene recombinant antigen can be prepared, for example, using a gene recombination method described in JP-T-5-508219.

  The HCV gene recombinant antigen is preferably at least one selected from the group consisting of gene recombinant NS3 antigen, gene recombinant NS4 antigen, gene recombinant NS5 antigen and gene recombinant core antigen. The sequences of these antigens are described, for example, in JP-T-5-508219, but are not limited to these specific sequences as long as they have antigenicity, and may have mutations. In the present specification, for example, “genetically modified NS3 antigen” means a gene recombinant antigen having an amino acid sequence derived from the amino acid sequence of NS3 domain of HCV protein.

  The solid phase binding site and the binding substance are not particularly limited as long as they are a combination of substances that can specifically bind when contacted under conditions using a reagent kit. Examples of these combinations include biotin and avidin, hapten and anti-hapten antibody, nickel and histidine tag, glutathione and glutathione-S-transferase, and the like. Examples of the hapten and anti-hapten antibody include DNP and anti-DNP antibody. Preferably, it is a combination of biotin and avidins. More preferably, the solid phase binding site contains biotin and the binding substance is avidin. Either of the substances in the combination of the solid phase binding site and the binding substance may be immobilized on the second solid phase, and is not particularly limited. In the present specification, “avidins” means including avidin and streptavidin.

In the present specification, “adding a solid phase binding site” to the HCV synthetic peptide antigen means that the HCV synthetic peptide antigen is reacted so as to have a solid phase binding site. The reaction may be appropriately selected depending on the type of the solid phase binding site, and examples thereof include chemical bonds such as covalent bonds.
Methods for adding a solid phase binding site to an HCV synthetic peptide antigen are known. For example, when the solid phase binding site contains biotin, for example, biotin can be bound to the antigen via a group reactive with an amino group, a thiol group or the like in the HCV synthetic peptide antigen. Examples of the group reactive with an amino group include an NHS group, and examples of the group reactive with a thiol group include a maleimide group.

Methods for immobilizing the above binding substance on a solid phase are known. The immobilization can be performed by, for example, a physical adsorption method, a covalent bond method, an ionic bond method, or a combination thereof.
When the binding substance is an avidin, the avidin can be immobilized on the solid phase by, for example, a method of binding the avidin to a substance capable of binding to the avidin, for example, a solid phase to which biotin is bound. Alternatively, the avidin can be immobilized on the solid phase by a method of binding the avidin to a solid phase to which avidin can be bound, for example, biotin. Avidins can also be immobilized on a solid phase by the method described in JP-A-2006-226689.
Further, the solid phase to which avidins are bound can be purchased from, for example, JSR Corporation or Dynal Biotech.

A method for immobilizing HCV gene recombinant antigen on a solid phase is known. The immobilization can be performed by, for example, a physical adsorption method, a covalent bond method, an ionic bond method, or a combination thereof.
In addition, the HCV recombinant antigen can be immobilized on the solid phase through the binding of biotin and avidins. The combination of substances that mediate the immobilization of the HCV gene recombinant antigen to the solid phase includes hapten and anti-hapten antibody, nickel and histidine tag, glutathione and glutathione-S-transferase other than the above biotin and avidin. Etc. Examples of the hapten and anti-hapten antibody include DNP and anti-DNP antibody.
The means for immobilizing the HCV gene recombinant antigen on the solid phase may be the same as or different from the means for immobilizing the binding substance on the solid phase.

The solid phase material is not particularly limited as long as it is a normal solid phase material used for immunoassay. For example, latex, rubber, polyethylene, polypropylene, polystyrene, styrene-butadiene copolymer, polyvinyl chloride , Polyvinyl acetate, polyacrylamide, polymethacrylate, styrene-methacrylate copolymer, polyglycidyl methacrylate, acrolein-ethylene glycol dimethacrylate copolymer, polyvinylidene difluoride (PVDF), silicone and other polymer materials; agarose; gelatin Red blood cells; inorganic materials such as silica gel, glass, inert alumina, and magnetic materials. You may combine these 1 type, or 2 or more types.
The shape of the solid phase is not particularly limited as long as it is a normal solid phase shape used for immunoassay, and examples thereof include microtiter plates, test tubes, beads, particles, and nanoparticles. Examples of the particles include magnetic particles, hydrophobic particles such as polystyrene latex, copolymer latex particles having hydrophilic groups such as amino groups and carboxyl groups on the particle surface, erythrocytes, and gelatin particles.

More preferably, the solid phase is a magnetic particle, latex particle or microtiter plate well. In the reagent kit for HCV antibody measurement, the solid phase is intended to mean an independent particle or an independent well.
The magnetic particles are particles containing a magnetic material as a base material. Such magnetic particles are known in the art, and those using, for example, Fe ₂ O ₃ and / or Fe ₃ O ₄ , cobalt, nickel, ferrite, magnetite or the like as a substrate are known. For the purpose of binding proteins or the like to the surface of the magnetic particles, a substrate whose surface is coated with a polymer or the like is more preferable.
As the microtiter plate, those usually used in enzyme-linked immunosorbent assay (ELISA) can be suitably used, and those made of materials such as polystyrene and polypropylene are preferred.

  As described above, the first reagent contains the HCV synthetic peptide antigen in a free state. The first reagent is used in the form of a liquid in which the HCV synthetic peptide antigen is dissolved in a buffer solution that does not affect the stability of the HCV synthetic peptide antigen and the reactivity with the HCV antibody, or at the time of use by adding a liquid such as water. Therefore, it may be in the form of a solid obtained by lyophilizing the HCV synthetic peptide antigen. The first reagent is preferably in the form of a solution in which the HCV synthetic peptide antigen is dissolved in a buffer solution, from the viewpoint of ease of use of the reagent kit.

  When the first reagent is in the form of a solution and contains a plurality of types of HCV synthetic peptide antigens, the first reagent may contain a plurality of types of HCV synthetic peptide antigens in the same buffer solution, or one type of HCV A plurality of buffers containing a synthetic peptide antigen may be included. Preferably, the first reagent contains a plurality of types of HCV synthetic peptide antigens in the same buffer.

  When the first reagent is in the form of a solution, the concentration of the HCV synthetic peptide antigen can be appropriately selected according to the type and number of HCV synthetic peptide antigens.

  The above buffer solution preferably has a pH of about 6.5 to 8.0. For example, phosphate buffered saline (PBS), triethanolamine hydrochloride buffer (TEA), Tris-HCl buffer (Tris-HCl), etc. Can be used. The buffer may contain a known additive such as a surfactant, a preservative, and serum protein.

  The second reagent is provided in a form in which the solid phase on which the HCV gene recombinant antigen and the binding substance are immobilized is suspended or contacted in an appropriate buffer. Examples of the form in which the solid phase is suspended in the buffer include a form in which particles such as magnetic particles and latex particles as the solid phase are suspended in the buffer. The form in which the solid phase is in contact with the buffer solution includes, for example, a form in which the buffer solution is added to the well of the microtiter plate as the solid phase.

On the above solid phase, there is an HCV genetically modified antigen in an amount sufficient for binding of an HCV antibody that can be contained in a specimen, and an HCV synthetic peptide antigen to which a solid phase binding site contained in the first reagent is added. It is sufficient that a sufficient amount of the binding substance is present for binding. The quantity ratio of the HCV recombinant antigen immobilized on the solid phase and the binding substance is the kind and quantity of the HCV synthetic peptide antigen in the first reagent, the kind of HCV recombinant antigen, the kind of binding substance, etc. It can be appropriately selected depending on the situation. For example, the number of molecules of the HCV recombinant antigen and the binding substance immobilized on the solid phase may be in the range of HCV recombinant antigen: binding substance = 1: 10 to 10: 1. When the HCV gene recombinant antigen is gene recombinant NS3 antigen and the binding substance is avidin, for example, gene recombinant NS3 antigen: avidins = 1: 2-8.
As said buffer solution, the thing similar to what can be used for a 1st reagent can be used.

The above reagent kit for measuring HCV antibody preferably further includes a third reagent containing a substance capable of binding to the HCV antibody labeled with the labeling substance.
Substances that can bind to HCV antibodies include antibodies against human HCV antibodies, HCV antigens, and the like. The antibodies exemplified here include antibody fragments and derivatives thereof. Specific examples include Fab fragments, F (ab ′) fragments, F (ab) ₂ fragments, and sFv fragments. Antibody classes can include, but are not limited to, IgG, IgM, and the like.
The labeling substance is not particularly limited as long as it is a labeling substance that can be used in a normal immunoassay, and examples thereof include enzymes, fluorescent substances, and radioisotopes. Examples of the enzyme include alkaline phosphatase, peroxidase, glucose oxidase, tyrosinase, and acid phosphatase. Examples of the fluorescent substance include fluorescein isothiocyanate (FITC), green fluorescent protein (GFP), and luciferin. Examples of the radioisotope include ¹²⁵ I, ¹⁴ C, and ³² P.

When the labeling substance is an enzyme, the HCV antibody measurement reagent kit preferably further includes a fourth reagent containing a substrate for the enzyme.
As the above substrate, a substrate known in the art for the above enzyme can be used. When alkaline phosphatase is used as the above enzyme, a luminescent substrate, a chromogenic substrate and the like known in the art can be used. For example, CDP-star® (4-chloro-3- (methoxyspiro {1,2- Dioxetane-3,2 ′-(5′-chloro) tricyclo [3.3.1.1 ^3,7 ] decan} -4-yl) phenyl phosphate disodium), CSPD® (3- (4 Chemiluminescence of -methoxyspiro {1,2-dioxetane-3,2- (5'-chloro) tricyclo [3.3.1.1 ^3,7 ] decan} -4-yl) phenyl phosphate disodium) Substrate: p-nitrophenyl phosphate, 5-bromo-4-chloro-3-indolyl-phosphate (BCIP), 4-nitroblue tetrazolium chloride (NBT), iodonitrotetrazo Examples include chromogenic substrates such as lithium (INT).

  The fourth reagent is preferably in the form of a solution in which the substrate is dissolved in an appropriate buffer. The concentration of the substrate can be appropriately selected depending on the type of substrate. The buffer that can be used for the fourth reagent can be appropriately selected depending on the type of substrate, and those known in the art can be used.

  The reagent kit for HCV antibody measurement comprises a first reagent containing an HCV synthetic peptide antigen to which a solid phase binding site is added, a solid phase on which an HCV gene recombinant antigen and a binding substance capable of binding to the solid phase binding site are immobilized. A second reagent containing. In the reagent kit for HCV antibody measurement, the first reagent and the second reagent are packaged separately. FIG. 1 is an example of a reagent kit when the first reagent is in the form of a solution and the second reagent is in the form of a solid phase suspended in a buffer solution. In FIG. 1, the first reagent is accommodated in the first reagent container 1, and the second reagent is accommodated in the second reagent container 2. The HCV antibody measurement reagent kit may be provided with another reagent (for example, the above-described third reagent or fourth reagent) housed in another reagent container, if desired. Furthermore, the reagent kit for HCV antibody measurement may include one or more buffer solutions for diluting one or more of the reagents, instructions for use, containers that can be used for the reaction, and the like, if desired.

The method for measuring an HCV antibody of the present invention comprises a specimen, a first reagent containing an HCV synthetic peptide antigen to which a solid phase binding site is added, an HCV gene recombinant antigen, and a binding substance capable of binding to the solid phase binding site. A step (contact step) of contacting a second reagent containing a solid phase on which is immobilized,
Detecting the first complex of the HCV synthetic peptide antigen and the HCV antibody and / or the second complex of the HCV gene recombinant antigen and the HCV antibody formed on the solid phase (detection step). Including.

  The specimen is a biological sample collected from mammals including humans, and includes blood, serum, and plasma.

  In the contact step, the second reagent may be contacted after the sample and the first reagent are contacted first, or the first reagent is contacted after the sample and the second reagent are contacted first. Alternatively, the specimen may be contacted after the first reagent and the second reagent are first contacted. It is more preferable that the specimen and the first reagent are contacted first and then the second reagent is contacted in that the binding between the free HCV antibody in the specimen and the free HCV antigen in the reagent is likely to proceed. . Here, “contact” includes mixing the specimen, the first reagent, and the second reagent. For example, when the first reagent is in the form of a solution and the second reagent is in a form in which the solid phase is suspended in a buffer solution, in the above contact step, the sample and the first reagent are first mixed and then the second reagent is mixed. The reagent may be mixed, the sample and the second reagent may be mixed first, then the first reagent may be mixed, and the first reagent and second reagent may be mixed first and then the sample mixed You may let them.

In the above contacting step, the ratio of the amount of the first reagent to the second reagent is such that there is a sufficient amount of binding substance for the solid phase binding site added to the HCV synthetic peptide antigen in the first reagent. If it is, it will not specifically limit, It can select suitably.
The ratio of the amount of the specimen to the first reagent and the second reagent is not particularly limited as long as a sufficient amount of each HCV antigen is present to detect HCV antibodies that can be contained in the specimen.

The contact step is preferably performed at a temperature at which the reaction between the HCV antibody in the specimen and the HCV antigen in the first reagent and the second reagent is favorably performed, and a temperature of about 30 to 45 ° C. is more preferable.
The contact time can be appropriately selected depending on the type of HCV synthetic peptide antigen, HCV gene recombinant antigen, solid phase binding site, binding substance used, and the like.

  The detection step can be performed according to an immunological detection method known in the art, and is preferably performed using a substance that can bind to the HCV antibody labeled with the labeling substance.

In the detection step, preferably, a substance capable of binding to the HCV antibody labeled with the labeling substance is contacted with the first complex and / or the second complex, and the labeling substance is detected. Is done.
When the labeling substance is an enzyme, the labeling substance can be detected by measuring the light, color, etc. generated by reacting the substrate with the enzyme as described above using an appropriate apparatus. Examples of the apparatus include a spectrophotometer and a luminometer.

A method for preparing each HCV antigen used in Examples and / or Reference Examples described below will be described.
(1) Preparation of recombinant NS3 antigen As an HCV recombinant antigen, a recombinant NS3 antigen having an amino acid sequence of 1192 to 1457 of the amino acid sequence described in FIG. 1 of JP-T-5-508219, It was prepared as described in Example 1 of JP-T-5-508219.
(2) Preparation of Synthetic Peptide NS4 Antigen Synthetic peptide NS4 antigen having the amino acid sequence from 1688 to 1707 of the amino acid sequence described in FIG. Was prepared according to the peptide synthesis method described in the example of Japanese Patent No. 2995216. Then, the synthetic peptide NS4 antigen was biotinylated using Biotinylation Kit (Sulfo-OSu) (Dojindo Laboratories).

(3) Preparation of Synthetic Peptide Core Antigen As a synthetic peptide antigen, a synthetic peptide core antigen having the 7th to 26th amino acid sequence of the amino acid sequence described in FIG. 1 of Japanese Patent No. 2995216 (Peptide II of Japanese Patent No. 2995216) Synthetic peptide core antigens having amino acid sequences 13 to 32 (Peptide III of Patent No. 2995216) and synthetic peptide core antigens having amino acid sequences of 49 to 68 (Peptide V of Patent No. 2995216) are patented. It was prepared according to the peptide synthesis method described in the Example of No. 2995216. Then, the synthetic peptide core antigen was biotinylated using Biotinylation Kit (Sulfo-OSu) (Dojindo Laboratories).

(4) Preparation of recombinant NS3 antigen-immobilized magnetic particles Commercially available magnetic particles (average particle size 2 μm) are suspended in 20 mM sodium phosphate buffer (pH 7.5) so as to have a magnetic particle size of about 5 mg / mL. A particle suspension was prepared. The recombinant NS3 antigen prepared in (1) above was dissolved in 20 mM sodium phosphate buffer (pH 7.5) so as to be 1 mg / mL to prepare an NS3 antigen solution. The recombinant NS3 antigen was immobilized on the magnetic particles by mixing 10 mL of the magnetic particle suspension and 0.1 mL of the NS3 antigen solution, and incubating at 37 ° C. for 30 minutes. Next, blocking treatment was performed by mixing magnetic particles with NS3 antigen immobilized thereon and a blocking solution (1% BSA, 20 mM sodium phosphate buffer, pH 7.5) and incubating at 37 ° C. for 30 minutes. The magnetic particles thus obtained are hereinafter abbreviated as NS3 antigen magnetic particles.

(5) Preparation of genetically modified NS3 antigen and streptavidin-immobilized magnetic particles Commercially available streptavidin-coated magnetic particles (hereinafter referred to as “ST magnetic particles”) are added to a 20 mM sodium phosphate buffer solution at about 5 mg / mL. The suspension was suspended in (pH 7.5) to prepare an ST magnetic particle suspension. The recombinant NS3 antigen prepared in (1) above was dissolved in 20 mM sodium phosphate buffer (pH 7.5) to a concentration of 1 mg / mL to prepare an NS3 antigen solution. The recombinant NS3 antigen was immobilized on ST magnetic particles by mixing 10 mL of ST magnetic particle suspension and 0.06 mL of NS3 antigen solution and incubating at 37 ° C. for 30 minutes. Since the recombinant NS3 antigen used here is not biotinylated, the immobilization of the recombinant NS3 antigen to ST magnetic particles is based on physical adsorption. And the genetically modified NS3 antigen and streptavidin are respectively immobilized on the magnetic particles prepared here.
Next, the magnetic particles on which NS3 antigen and streptavidin are immobilized and the above blocking solution (1% BSA, 20 mM sodium phosphate buffer, pH 7.5) are mixed and incubated at 37 ° C. for 30 minutes. Went. The magnetic particles thus obtained are hereinafter abbreviated as NS3 antigen / ST magnetic particles.

(6) Preparation of synthetic peptide NS4 antigen-immobilized magnetic particles Commercially available ST magnetic particles are suspended in 20 mM sodium phosphate buffer (pH 7.5) so as to be about 5 mg / mL, and ST magnetic particle suspension is obtained. Was prepared. The synthetic peptide NS4 antigen prepared in (2) above was dissolved in 20 mM sodium phosphate buffer (pH 7.5) to a concentration of 1 mg / mL to prepare an NS4 antigen solution. The synthetic peptide NS4 antigen was immobilized on the ST magnetic particles by mixing 10 mL of the ST magnetic particle suspension and 0.01 mL of the NS4 antigen solution and incubating at 37 ° C. for 30 minutes. Next, blocking treatment was performed by mixing magnetic particles with NS4 antigen immobilized thereon and the blocking solution (1% BSA, 20 mM sodium phosphate buffer, pH 7.5) and incubating at 37 ° C. for 30 minutes. . The magnetic particles thus obtained are hereinafter abbreviated as NS4 antigen magnetic particles.

(7) Preparation of Synthetic Peptide Core Antigen-Immobilized Magnetic Particles Commercially available ST magnetic particles are suspended in 20 mM sodium phosphate buffer (pH 7.5) so as to have a concentration of about 1 mg / mL. Prepared. Each synthetic peptide core antigen prepared in (3) above was mixed and dissolved in 20 mM sodium phosphate buffer (pH 7.5) to prepare a core antigen solution. In addition, the core antigen solution was prepared so that the density | concentration of each synthetic peptide core antigen contained might be about 1 mg / mL. The synthetic peptide core antigen was immobilized on the ST magnetic particles by mixing 10 mL of the ST magnetic particle suspension and 0.01 mL of the core antigen solution and incubating at 37 ° C. for 30 minutes. Next, blocking treatment was performed by mixing magnetic particles with immobilized core antigen and a blocking solution (1% BSA, 20 mM sodium phosphate buffer, pH 7.5) and incubating at 37 ° C. for 30 minutes. The magnetic particles thus obtained are hereinafter abbreviated as core antigen magnetic particles.

Reference example 1
First, regarding the recombinant NS3 antigen, the difference in reactivity between when immobilized on magnetic particles and when released in solution without being immobilized was examined.

Here, biotinylated gene recombinant NS3 antigen obtained by biotinylation treatment on NS3 antigen prepared in (1) above was used. Biotinylation Kit (Sulfo-OSu) (Dojindo Laboratories Co., Ltd.) was used for biotinylation treatment for NS3 antigen.
The method for immobilizing biotinylated NS3 antigen on ST magnetic particles is the same as (4) above except that biotinylated NS3 antigen is used as HCV antigen and ST magnetic particles are used as magnetic particles.

The composition of the reagent used in this example is as follows.
(8) First reagent (A)
20 mM sodium phosphate buffer (pH 7.5)
(9) First reagent (B-1)
20 mM sodium phosphate buffer (pH 7.5)
2.5 μg / mL biotinylated NS3 antigen (10) first reagent (B-2)
A solution obtained by diluting the first reagent (B-1) 10 times with 20 mM sodium phosphate buffer (pH 7.5) is defined as a first reagent (B-2).
(11) First reagent (B-3)
A solution obtained by diluting the first reagent (B-1) 50-fold with 20 mM sodium phosphate buffer (pH 7.5) is defined as a first reagent (B-3).
(12) Second reagent (A)
20 mM sodium phosphate buffer (pH 7.5)
3 mg / mL biotinylated NS3 antigen-binding ST magnetic particles 2 mg / mL ST magnetic particles (13) Second reagent (B)
20 mM sodium phosphate buffer (pH 7.5)
5 mg / mL ST magnetic particles (14) Third reagent 20 mM sodium phosphate buffer (pH 7.5)
0.1 U / mL alkaline phosphatase labeled mouse anti-human IgG antibody (ALKALINE PHOSPHASE-MOUSE, Zymed Laboratories)
(15) Fourth reagent CDP-star (registered trademark) with Sapphire II (trademark) (Applied Biosystems)

The measuring method is as follows.
(16) Reactivity of recombinant NS3 antigen when immobilized on magnetic particles First, 10 μL of HCV positive serum and 120 μL of the first reagent (A) were mixed and incubated at 42 ° C. for about 2 minutes. Subsequently, 30 μL of the second reagent (A) was added to the mixture and incubated at 45 ° C. for about 1 minute, and the HCV antibody in serum and the NS3 antigen on the magnetic particles in the second reagent (A) Was reacted. The magnetic particles were washed with a washing solution (0.1% Tween 20,
After washing with 0 mM sodium phosphate buffer (pH 7.5), 100 μL of the third reagent was added and incubated at room temperature for 5 minutes. After washing the magnetic particles with the above washing solution, 100 μL of the fourth reagent was added, and the luminescence intensity was measured using LUMI-COUNTER 700 (Microtech Nichion Co., Ltd.).
Two types of HCV positive sera (positive serum 1 and positive serum 2) were used as specimens. Furthermore, as a negative control, measurement was performed using 20 mM sodium phosphate buffer (pH 7.5) instead of HCV positive serum. The same specimen was measured three times, and the average emission intensity was determined. The results are shown in FIG.

(17) Reactivity of recombinant NS3 antigen when released in solution Using the first reagent (B-1), (B-2) or (B-3) as the first reagent, the second reagent The measurement was performed in the same manner as in (16) except that the second reagent (B) was used. The first reagent (B-1) contains free NS3 antigen so as to have the same concentration as the immobilized NS3 antigen in the second reagent A. The results are shown in FIG.

In FIG. 2, 1 shows the results when the recombinant NS3 antigen is immobilized on magnetic particles, and 2-4 shows the results when the recombinant NS3 antigen is released in a solution. In addition, the vertical axis represents the emission intensity obtained by the measurement. N is the result of the negative control.
As can be seen from FIG. 2, in any HCV positive serum, a higher luminescence intensity was obtained when the recombinant NS3 antigen was immobilized on the magnetic particles than when the recombinant NS3 antigen was released into the solution. I found out that From the above, it was found that the HCV recombinant antigen was more reactive with the HCV antibody when immobilized on magnetic particles than when it was released in solution.

Reference example 2
Next, regarding the recombinant NS3 antigen, the difference in storage stability between when immobilized on magnetic particles and when released in solution without being immobilized was examined.
In this example, an accelerated test was carried out using the second reagent (A) (including immobilized NS3 antigen) and the first reagent (B-2) (including free NS3 antigen) used in Reference Example 1, The storage stability of was examined. In the accelerated test, the second reagent (A) and the first reagent (B-2) were stored in the dark at 37 ° C. for 0, 1 or 5 days. HCV antibodies were measured using each reagent subjected to the acceleration test.

The measuring method is as follows.
(18) Stability of genetically modified NS3 antigen when immobilized on magnetic particles (16) described in Reference Example 1 except that the second reagent (A) subjected to an accelerated test was used as the second reagent Measurement was carried out in the same manner. Two types of HCV positive sera (positive serum 1 and positive serum 2) used in Reference Example 1 were used as specimens. The results are shown in FIGS.

(19) Stability of genetically modified NS3 antigen when released in solution (17) described in Reference Example 1 except that the first reagent (B-2) subjected to the acceleration test was used as the first reagent (17 The measurement was performed in the same manner as in (1). Two types of HCV positive sera (positive serum 1 and positive serum 2) used in Reference Example 1 were used as specimens. The results are shown in FIGS.

FIG. 3 shows the results when positive serum 1 is used as a specimen, and FIG. 4 shows the results when positive serum 2 is used as a specimen. 3 and 4, 1 shows the result when the recombinant NS3 antigen is immobilized on magnetic particles, and 2 shows the result when the recombinant NS3 antigen is released in a solution. The vertical axis indicates the percentage of the average emission intensity for each storage day, with the average emission intensity obtained using the reagent before the acceleration test (day 0) being 100%, and the horizontal axis is the acceleration. Shows the number of days the test is stored.
From the results shown in FIG. 3 and FIG. 4, when the recombinant NS3 antigen is stored in a state of being released in a solution, the stability is poor, and the reactivity rapidly decreases even after 1 day in the accelerated test. I understood it. It was also found that when the recombinant NS3 antigen was stored in a state of being immobilized on magnetic particles, the stability was good and high reactivity was maintained even after 5 days in the accelerated test. From the above, it was found that the HCV gene recombinant antigen was more stable when immobilized on magnetic particles than when it was released in solution.

Reference example 3
Next, regarding the synthetic peptide NS4 antigen and the synthetic peptide core antigen, the difference in storage stability between when immobilized on magnetic particles and when released in solution without being immobilized was examined.
Here, the synthetic peptide NS4 antigen prepared in (2) above and the synthetic peptide core antigen prepared in (3) above were used as free HCV antigens. In addition, NS3 antigen magnetic particles prepared in (4) above, NS4 antigen magnetic particles prepared in (6) above, and core antigen magnetic particles prepared in (7) above were used as immobilized HCV antigens.

The composition of the reagent used in this example is as follows. The third reagent and the fourth reagent used were those used in Reference Example 1.
(20) First reagent (C)
20 mM sodium phosphate buffer (pH 7.5)
1 μg / mL synthetic peptide NS4 antigen 10 μg / mL each synthetic peptide core antigen (21) first reagent (D)
20 mM sodium phosphate buffer (pH 7.5)
(22) Second reagent (C)
20 mM sodium phosphate buffer (pH 7.5)
3 mg / mL NS3 antigen magnetic particles 2 mg / mL ST magnetic particles (23) Second reagent (D)
20 mM sodium phosphate buffer (pH 7.5)
3 mg / mL NS3 antigen magnetic particles 1 mg / mL NS4 antigen magnetic particles 1 mg / mL core antigen magnetic particles

  In this example, an accelerated test is carried out using the first reagent (C) (including free NS4 antigen and free core antigen) and the second reagent (D) (including immobilized NS4 antigen and immobilized core antigen), The storage stability of the reagent was examined. In the acceleration test, the first reagent (C) and the second reagent (D) were stored in the dark at 45 ° C. for 0 or 1 day. HCV antibodies were measured using each reagent subjected to the acceleration test.

The measuring method is as follows.
(24) the solution synthetic peptide NS 4 antigen and an acceleration test using the first reagent (C) which was performed as stability first reagent synthetic peptide core antigen, the second reagent as the second reagent when liberated during The measurement was performed in the same manner as (16) described in Reference Example 1 except that (C) was used. As samples, one type of HCV negative serum (negative serum 1) and four types of HCV positive sera (positive sera 3 to 6) were used. The results are shown in Table 1.

(25) using the first reagent (D) as the stability first reagent synthetic peptide NS 4 antigen and synthetic peptide core antigen in the case of fixing of the magnetic particles, the second was an accelerated test as the second reagent The measurement was performed in the same manner as (16) described in Reference Example 1 except that the reagent (D) was used. As samples, one type of HCV negative serum (negative serum 1) and four types of HCV positive sera (positive sera 3 to 6) were used. The results are shown in Table 1.

In Table 1, the average luminescence intensity obtained using the reagent before the acceleration test (day 0) and on the first day of the acceleration test is shown. Furthermore, the average luminescence intensity obtained using the reagent on the 0th day was taken as 100%, and the percentage of the average luminescence intensity obtained using the reagent on the 1st day was shown.
According to Table 1, when the synthetic peptide NS4 antigen and the synthetic peptide core antigen are stored in a state of being immobilized on magnetic particles, the stability is poor, and all positive sera (positive sera 3) even after one day has passed in the accelerated test. It was found that the reactivity of ˜6) sharply decreased. Moreover, when the synthetic peptide NS4 antigen and the synthetic peptide core antigen are stored in a state of being released in a solution, the stability is good and high reactivity is maintained in all positive sera even after 5 days in the accelerated test. I found out that From the above, it was found that HCV synthetic peptide antigens were more storage-stable when released in solution than when immobilized on a solid phase.

Example 1
An accelerated test was performed using a first reagent containing a synthetic peptide NS4 antigen and a synthetic peptide core antigen released, and a second reagent containing a recombinant NS3 antigen and streptavidin immobilized on magnetic particles. The storage stability of the reagent was examined. In the accelerated test, the first reagent and the second reagent were stored at 37 ° C. in the dark for 0, 4 or 7 days. HCV antibodies were measured using each reagent subjected to the acceleration test. In this example, the first reagent (C) used in Reference Example 3 was used as the first reagent.
In addition, NS3 antigen / ST magnetic particles prepared in (5) above were used as magnetic particles in which recombinant NS3 antigen and streptavidin were immobilized on magnetic particles.
That is, the second reagent used in this example is as follows.
(26) Second reagent (E)
20 mM sodium phosphate buffer (pH 7.5)
5mg / mL NS3 antigen / ST magnetic particles

The measurement method is as follows.
The same as (16) described in Reference Example 1 except that the first reagent (C) subjected to the acceleration test was used as the first reagent and the second reagent (E) subjected to the acceleration test was used as the second reagent. Measurement was performed by the method. The specimen used was one kind of HCV negative serum (negative serum 1) and four kinds of HCV positive sera (positive sera 7 to 10). The positive serum 7 is a serum containing an HCV antibody against the NS3 antigen, an HCV antibody against the NS4 antigen, and an HCV antibody against the core antigen. Positive serum 8 is a serum containing HCV antibody against NS3 antigen, positive serum 9 is a serum containing HCV antibody against core antigen, and positive serum 10 contains HCV antibody against NS3 antigen and HCV antibody against NS4 antigen. It's serum. The results are shown in Table 2.

  In Table 2, the average luminescence intensity obtained using the reagent before the acceleration test (day 0) and on the fourth and seventh days of the acceleration test is shown. Furthermore, for the positive sera 7 to 10, the average luminescence intensity obtained using the reagent on the 0th day is taken as 100%, and the percentage of the average luminescence intensity obtained using the reagents on the 4th and 7th days is shown in FIG. It was shown in 5-8. FIG. 5 shows the results when measuring positive serum 7, FIG. 6 shows the results when measuring positive serum 8, FIG. 7 shows the results when measuring positive serum 9, and FIG. 8 shows positive results. The result when serum 10 is measured is shown. 5 to 8, the vertical axis represents the percentage of the average luminescence intensity in each storage day, with the luminescence intensity obtained by measurement using the reagent before the acceleration test (day 0) as 100%. Yes, the horizontal axis shows the number of days of storage for accelerated tests.

  From the results shown in Table 2 and FIGS. 5 to 8, a reagent kit containing a first reagent containing HCV synthetic peptide antigen in a released state and a second reagent containing HCV recombinant antigen immobilized on magnetic particles is used. Thus, it was found that various samples including antibodies that react with various sites of HCV protein can be detected well. It was also found that the storage stability of the antigen in the reagent kit is very excellent.

  From the above, the HCV synthetic peptide antigen is more stable in the buffer solution than the HCV synthetic peptide antigen while the storage stability and reactivity are better when immobilized on the solid phase in the buffer solution. It was found that the storage stability was better in a state where the phase was not immobilized (free state).

1 is a schematic diagram showing an embodiment of a reagent kit for HCV antibody measurement of the present invention. 6 is a graph showing the results of Reference Example 1. It is a graph which shows the result obtained by measuring the positive serum 1 in the reference example 2. It is a graph which shows the result obtained by measuring the positive serum 2 in Reference Example 2. 2 is a graph showing the results obtained by measuring positive serum 7 in Example 1. 2 is a graph showing results obtained by measuring positive serum 8 in Example 1. FIG. 2 is a graph showing results obtained by measuring positive serum 9 in Example 1. FIG. 2 is a graph showing the results obtained by measuring positive serum 10 in Example 1.

Explanation of symbols

1 1st reagent container 2 2nd reagent container

Claims (10)

A first reagent comprising a hepatitis C virus (HCV) synthetic peptide antigen to which a solid phase binding site has been added;
A reagent kit for measuring an HCV antibody, comprising an HCV gene recombinant antigen and a second reagent containing a solid phase on which a binding substance capable of binding to the solid phase binding site is immobilized.   2. The HCV according to claim 1, wherein the HCV recombinant antigen is at least one selected from the group consisting of a recombinant NS3 antigen, a recombinant NS4 antigen, a recombinant NS5 antigen, and a recombinant core antigen. Reagent kit for antibody measurement.   The reagent kit for HCV antibody measurement according to claim 1 or 2, wherein the HCV synthetic peptide antigen is at least one selected from the group consisting of a synthetic peptide NS4 antigen, a synthetic peptide NS5 antigen and a synthetic peptide core antigen.   The reagent kit for HCV antibody measurement according to any one of claims 1 to 3, wherein the solid phase is a magnetic particle, latex particle, or well of a microtiter plate.   The reagent kit for HCV antibody measurement according to any one of claims 1 to 4, wherein the solid-phase binding site contains biotin, and the binding substance is avidin.   The reagent kit for HCV antibody measurement according to any one of claims 1 to 5, further comprising a third reagent containing a substance capable of binding to the HCV antibody labeled with a labeling substance.   The reagent kit for measuring an HCV antibody according to claim 6, wherein the substance capable of binding to the HCV antibody is an antibody capable of binding to the HCV antibody. The label is an enzyme;
The reagent kit for HCV antibody measurement according to claim 6 or 7, further comprising a fourth reagent containing a substrate for the enzyme. A specimen, a first reagent containing an HCV synthetic peptide antigen added with a solid phase binding site, and a second reagent containing a solid phase on which a binding substance capable of binding to the HCV gene recombinant antigen and the solid phase binding site is immobilized And a step of detecting a first complex of HCV synthetic peptide antigen and HCV antibody and / or a second complex of HCV gene recombinant antigen and HCV antibody formed on the solid phase. A method for measuring HCV antibody contained in a specimen.   The method according to claim 9, wherein the contacting step is performed by contacting the second reagent after contacting the specimen and the first reagent.

Images