JP2001124779A - Method for detecting or determining hcv core antigen and detecting or determining reagent used therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for simply detecting or determining an HCV with high sensitivity and a reagent for detecting or determining the HCV used therefor. SOLUTION: An immunoassay detects or determines an HCV core antigen by forming an immune complex by conducting an antigen-antibody reaction of an alkaline phosphatase labeled anti-HCV core protein antibody with the HCV core antigen in a specimen in an aqueous solution containing a salt of a high concentration and measuring alkaline phosphatase activity in the complex. The reagent detects or determines the HCV core antigen containing the alkaline phosphatase labeled anti-HCV core protein antibody, 0.5M to saturated concentration of a salt and the reagent for measuring the alkaline phosphatase activity.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for detecting or quantifying hepatitis C virus (hereinafter abbreviated as HCV) in a specimen and a reagent for detecting or quantifying HCV.

[0002]

2. Description of the Related Art Inspection for viruses that cause infectious diseases
Antibody tests, antigen tests, and genetic tests are performed by various methods. Antibody tests for viruses and antigen tests for viruses themselves are mainly immunoassays, and are widely used for diagnosis of virus infection, virus screening tests for blood for transfusion, and monitors for virus therapy. In addition, genetic testing is based on the polymerase chain react, which amplifies genes.
ion: PCR method, ligase chain reaction (LCR) method, Nucleic Acid Sequence Based Amplification (nucleic acid sequence based amplificatio)
n: NASBA) method and branched
The DNA (branched DNA: bDNA) method and the like have been developed, and high-sensitivity measurement is possible.

[0003] Genetic tests have been used for the purpose of definitive diagnosis, as well as in tests in a sensitive region that cannot be detected by the above-described antibody test and antigen test, and in evaluation of virus exclusion in therapy and the like. Recently, the gene amplification method has been applied to a screening test of transfused blood at an early stage of virus infection, which cannot be detected sensitively by an antibody test or an antigen test.

[0004] However, the genetic test is complicated in operation, requires special equipment, requires technical skill, has high cost, has poor reproducibility and measurement accuracy, and is not suitable for processing a large number of samples. There are also problems such as false negatives caused by inhibitors in the sample, instability of the nucleic acid itself in the sample as compared to proteins, and inconstant amplification efficiency of the gene. It has been reported that when the virus-derived nucleic acid is RNA, it is more unstable, and the value greatly fluctuates depending on the operation process and the storage state of the test sample.

[0005] The measurement of antibody against virus is a method for detecting an antibody against virus generated by an in vivo immune mechanism, and is a method for observing the influence of virus infection. Therefore, the production of an antibody against an antigen differs depending on the individual, and there are individuals exhibiting a high antibody titer and individuals producing no antibody at all. Further, since the amount of antibody does not always correlate with the amount of virus, it is difficult to predict the actual disease state and the amount of virus in the body. In addition, antibodies are not produced in the early stage of virus infection, and detection is not possible in the early stages of infection. Further, non-specific reaction of antibodies is also observed, so that accurate determination cannot be performed.

[0006] The antigen test is a method for detecting a virus-derived antigen, and various virus-derived antigen measurement systems have been put on the market. Antigen tests mainly focus on immunoassays using antibodies that recognize virus-derived antigens, but their sensitivity is insufficient compared to gene amplification methods. Therefore, there is a problem that detection cannot be sufficiently performed when the amount of virus is small. In addition, in the body, an antibody against the virus coexists with the virus, and the coexisting antibody competes with the antibody which is a material for the immunoassay, so that the antigen may not be measured with high sensitivity. Although there is an antigen measurement method for HBV, other hepatitis viruses, HIV and the like, the sensitivity is inferior to gene amplification methods such as PCR, and it cannot be said that it is a sufficient marker for diagnosis or treatment.

Recently, HC, an internal antigen of HCV,
A V-core protein measurement system has been developed (Japanese Unexamined Patent Publication No. Hei 8-29).
427). This method is a kit that can be measured more easily and stably than a method for measuring a gene, and can be used for definitive diagnosis of HCV, prediction of treatment effect, and judgment of treatment effect. However, the sensitivity is insufficient compared with the PCR quantification method,
There is a problem that diagnosis and treatment monitoring cannot be performed only by measuring core protein. In fact, the detection limit of this method is 8 pg / mL (10 ⁴ in terms of PCR titer).
~ A ⁵ viral particles / mL), the detection limit compared to PCR higher than 10 times. Therefore, when judging the therapeutic effect, a sample having a concentration of 8 pg / mL or less is judged to be negative, and it is not possible to determine the virus amount at a low value.

It is generally known that the use of a high concentration of a salt in the reaction between an antigen and an antibody inhibits the reaction between the antigen and the antibody and dissociates the antigen from the antigen-antibody reactant. For example, Chapter 9 of an ultrasensitive enzyme immunoassay [Eiji Ishikawa, Gakkai Shuppan Center, (1993)] describes as follows. "Since the non-specific adsorption of proteins to the solid phase increases or decreases under various conditions, serum interference should be reduced under conditions that reduce non-specific adsorption. Reducing the incubation concentration of the insolubilized polystyrene balls and serum and reducing the time reduces serum interference (Table IX-14) (Ruan et al., 198).
6). In addition, when the concentration of the inorganic salt is increased, nonspecific adsorption of the protein to the solid phase is reduced, so that serum interference can be reduced. (Table IX-15, Figures IX-18 to IX
-21) (Hashida et al., 1983). Although the effective concentration differs depending on the type of inorganic salt, an excessively high concentration inhibits the immune response itself. 0.3-0.4mo with salt
1 / l will be the upper limit. "

[0009]

SUMMARY OF THE INVENTION An object of the present invention is to provide a simple and highly sensitive method for detecting or quantifying an HCV-positive sample, which is suitable for treating a large number of samples, for example, for screening purposes in the blood business or health examination. An object of the present invention is to provide an HCV antigen detection or quantification method and a detection or quantification reagent used therefor.

[0010]

The present inventors have surprisingly found that the use of an alkaline phosphatase-labeled anti-HCV core protein antibody allows the antigen-antibody reaction with the HCV core protein in an aqueous solution containing a high concentration of salt. When performed, anti-H labeled with a labeling substance other than alkaline phosphatase
Unlike the case where a CV core protein antibody is used, the present inventors have found that the antigen can be detected with higher sensitivity without inhibiting the immune reaction, and completed the present invention.

The present invention relates to the following (1) to (29). (1) An alkaline-phosphatase-labeled anti-HCV core protein antibody and an HCV core antigen in a sample are subjected to an antigen-antibody reaction in an aqueous solution containing a high concentration of salt to form an immune complex. An immunoassay for detecting or quantifying an HCV core antigen in a sample, comprising measuring alkaline phosphatase activity of the sample. (2) The salt is an alkali metal halide (1)
The described method.

(3) The salt is sodium chloride (1)
The described method. (4) The salt concentration is 0.5M to the saturation concentration (1) to
The method according to any one of (3). (5) The anti-HCV core protein antibody is an antibody that recognizes at least 5 consecutive amino acid sequences among the 11th to 50th amino acid sequences from the N-terminal of the HCV core protein (1) to (4). The method described in.

(6) The anti-HCV core protein antibody is KTM
-145, KTM-153, KTM-157, KTM-
The method according to any one of (1) to (4), which is an antibody selected from the group consisting of 163 and KTM-167. (7) The sample has been treated with a chaotropic substance and / or an alkaline agent before the antigen-antibody reaction (1)
The method according to any one of (1) to (6).

(8) The method according to (7), wherein the chaotropic substance is a guanidine salt. (9) The method according to (7) or (8), wherein the alkali agent is an alkali metal hydroxide salt. (10) The method according to any one of (7) to (9), wherein the chaotropic substance has a concentration of 1 M to a saturation concentration.

(11) The treatment with an alkali agent is carried out at 45 to 5
The method according to any one of (7) to (10), which is performed at a temperature of 5 ° C.

(12) A reagent for detecting or quantifying an HCV core antigen, comprising an alkaline phosphatase-labeled anti-HCV core protein antibody, a salt having a concentration of 0.5 M to saturation concentration and a reagent for measuring alkaline phosphatase activity. (13) The salt is an alkali metal halide salt (1
2) The reagent according to the above.

(14) The salt is sodium chloride (1
The reagent according to 2) or (13). (15) The anti-HCV core protein antibody is an antibody that recognizes at least 5 consecutive amino acid sequences among the 11th to 50th amino acid sequences from the N-terminus of the HCV core protein (12) to (14). The reagent according to 1. (16) the anti-HCV core protein antibody is KTM-145;
The reagent according to any one of (12) to (14), which is an antibody selected from the group consisting of KTM-153, KTM-157, KTM-163 and KTM-167.

(17) A reagent kit for detecting or quantifying an HCV core antigen, comprising the reagent according to any one of (12) to (16) and a sample processing reagent containing a chaotropic substance and / or an alkaline agent. (18) The kit according to (17), wherein the chaotropic substance is a guanidine salt.

(19) The kit according to (17) or (18), wherein the alkaline agent is an alkali metal hydroxide. (20) The kit according to any one of (17) to (19), wherein the chaotropic substance has a concentration of 1 M to a saturation concentration.

(21) 1 from the N-terminus of the HCV core protein
A monoclonal antibody that recognizes at least five consecutive amino acid sequence sites in the amino acid sequences of Nos. 1 to 50. (22) A monoclonal antibody that recognizes the 41st to 50th amino acid sequence from the N-terminus of an HCV core protein. (23) Hybridoma KTM-145 (FERM
BP-6838). (24) A monoclonal antibody that recognizes the 11th to 30th amino acid sequence from the N-terminus of an HCV core protein. (25) Hybridoma KTM-153 (FERM
(24) produced by BP-6839). (26) A monoclonal antibody that recognizes an amino acid sequence at positions 31 to 50 from the N-terminus of an HCV core protein. (27) Hybridoma KTM-157 (FERM
BP-6840) produced by (26). (28) A monoclonal antibody which recognizes the 21st to 40th amino acid sequence from the N-terminal of the HCV core protein. (29) Hybridoma KTM-163 (FERM
BP-6841) or hybridoma KTM-167
(28) The monoclonal antibody according to (28), which is produced by (FERM BP-6842).

[0021]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. The alkaline phosphatase-labeled anti-H of the present invention
As alkaline phosphatase in the CV core protein antibody, alkaline phosphatase of any origin can be used. Examples include alkaline phosphatase derived from microorganisms such as Escherichia coli and animals such as cattle.

The anti-HCV core protein antibody in the alkaline phosphatase-labeled anti-HCV core protein antibody of the present invention is described in JP-A-7-145194 or Hepatolog.
Any antibody that reacts with the HCV core protein described in y, 16 , 886 (1992) can be used. The HCV core protein is disclosed in JP-A-7-145194 and JP-A-8-294.
No. 27 and Hepatology, 16 , 886 (1992). The antibody can be produced by using the above-described recombinant HCV core protein as an immunogen according to a known method for producing a monoclonal antibody. The monoclonal antibody can be produced by a method described in, for example, "Sakuji Toyama, Monoclonal Antibody Experimental Manual, Kodansha".

Animals to which the immunogen thus obtained is administered include mice, rats, hamsters, rabbits, guinea pigs, goats, sheep, chickens, and the like. Mice and rats are used for preparing monoclonal antibodies. preferable. As an immunization method, for example, a method described in [Nishimichi, Toshima, Shinsei Chemical Laboratory Course, 1 , 389 (1990), Tokyo Kagaku Dojin] and the like can be used. For example, the immunogen is emulsified in Freund's complete or incomplete adjuvant and administered intraperitoneally, subcutaneously, or intramuscularly. For example, immunization can be completed by administering 2 or more times, preferably 2 to 4 times at regular intervals of 7 to 30 days, preferably 12 to 16 days.

Sources for collecting antibody-producing cells include the spleen, lymph nodes, and peripheral blood of the immunized animal. Also, so-called in vitro immunization (Arai, Ota, Experimental Medicine, etc.), in which antibody-producing cells are extracted from the spleen, lymph nodes, peripheral blood, etc. of non-immunized animals, and these cells are directly immunized to produce antibody-producing cells 6 , 43 (1988)].

The myeloma cells used for cell fusion between the antibody-producing cells and the myeloma cells are not particularly limited, but it is preferable to use animal-derived cell lines of the same species as the antibody-producing cells. In order to efficiently select only cells that have been appropriately subjected to cell fusion, myeloma cells having a specific drug marker are preferable. For example, 8-azaguanine-resistant myeloma cells cannot grow in a medium containing hypoxanthine, aminopterin and thymidine (hereinafter referred to as HAT medium), but cells in which these cells and normal cells are fused are in HAT medium. It is preferably used because it can grow in it and can be distinguished from unfused myeloma cells. Specifically, P3 × 63-Ag. 8.653 [Journal of Immunol., 123 , 1548 (197
9)] and P3 × 63-Ag. 8. U1 [Curr. Topics. Mic
robiol. Immunol., 81 , 1 (1978)] (hereinafter simply referred to as P3U
Abbreviated as 1. ), Sp / O-Ag14 [Nature, 276 , 269 (1978)] and the like.

For the cell fusion, a method developed by Koehler and Milstein [Nature, 256 , 495 (1975)] and an improved method thereof can be applied. As a frequently used method, antibody-producing cells and myeloma cells
This is a method of mixing at a ratio of 3: 1 and treating the mixture with 30 to 50% of polyethylene glycol (average molecular weight of 1,500 to 6,000) as a fusing agent. Fusion can also be achieved by electric pulses [Okochi et al., Experimental Medicine, 6 , 50 (1
988)].

The cells after cell fusion are suspended in a selection medium (for example, HAT medium), and only the fused cells are grown using a culture vessel such as a 96-well culture plate, which is advantageous for the subsequent selection of target cells. At the stage where only the fused cells have selectively grown, only cells producing antibodies against the HCV core protein are selected. In this selection, the presence or absence of the target antibody in the culture supernatant of the fused cells is examined using a method such as enzyme immunoassay or radioimmunoassay. The selected cells are cloned using a method such as the limiting dilution method or the soft agar medium method, and a hybridoma cell line producing an HCV core protein-specific monoclonal antibody is established.

The monoclonal antibody is obtained by culturing the established hybridoma cell line in an appropriate medium and collecting the culture, or transplanting the cell line into the peritoneal cavity of an animal and growing it in ascites to collect ascites. It can be obtained from the obtained culture solution or ascites. The antibody in the culture solution or ascites can be purified and used as needed. Purification methods include, for example, salting-out fractionation using ammonium sulfate, ion-exchange chromatography, gel filtration column chromatography, affinity column chromatography using protein A or protein G, and a gel on which an antigen is immobilized. Various methods such as an affinity column chromatography method to be used or a method combining these methods can be mentioned. The antibodies used in the present invention include Fab and Fa obtained by treating the antibody obtained by the above method with an enzyme such as pepsin and / or a reduction treatment.
Antibody fragments such as b 'and F (ab) ₂ may be used.

The antibody of the present invention is preferably an antibody that recognizes at least five consecutive amino acid sequences among the 11th to 50th amino acid sequences from the N-terminal of the HCV core protein. Specific examples of the hybridoma producing the monoclonal antibody of the present invention include hybridoma K
TM-145, hybridoma KTM-153, hybridoma KTM-157, hybridoma KTM-16
3 and hybridoma KTM-167.

Hybridoma KTM-145, Hybridoma KTM-153, Hybridoma KTM-15
7. Hybridoma KTM-163 and Hybridoma KTM-167 were produced on August 12, 1999, 1-3 1-3 Higashi, Tsukuba, Ibaraki, Japan (postal code 305-
8566) FERM BP-6838 and FERM BP-68 were added to the Institute of Biotechnology and Industrial Technology, the Ministry of International Trade and Industry.
39, FERM BP-6840, FERM BP-6
841 and FERM BP-6842, respectively. Hybridoma KTM-145, Hybridoma KTM-153, Hybridoma KTM-1
57, a monoclonal antibody produced by hybridoma KTM-163 and hybridoma KTM-167,
Hereinafter, KTM-145, KTM-153, K
Designated as TM-157, KTM-163 and KTM-167.

The alkaline phosphatase-labeled anti-HCV core protein antibody of the present invention is bound by utilizing a method of forming a covalent bond between the anti-HCV core protein antibody and the alkaline phosphatase. Examples of the method include a glutaraldehyde method, a periodate method, a maleimide method, a pyridyl disulfide method, and a method using a known crosslinking agent (for example, Eiji Ishikawa, “Enzyme Immunoassay”, Medical Shoin). Issue).

As a specific preparation method, for example, an antibody sulfhydrylated with iminothiolane and the like,
(Succinimidyl 4- [N-maleimidomethyl] -cyclohexane-1
-carboxylate) can be prepared by mixing alkaline phosphatase maleimidated with -carboxylate).

The aqueous solution of the present invention containing a high concentration of salt is an aqueous solution containing 0.5 M or more of an inorganic salt. Examples of the inorganic salts include alkali metal halide salts, such as sodium chloride, potassium chloride, and lithium chloride. The concentration of the inorganic salt is preferably 0.5M to saturation concentration, more preferably 1 to 3M. The solution containing the salt at a high concentration may optionally contain a buffer, a salt other than sodium chloride,
It may contain a surfactant, serum, preservative, non-specific adsorption inhibitor in an immune reaction, and the like.

The buffer is not particularly restricted but includes Tris buffer and the like. Examples of the surfactant include Triton X-100, Triton X-705, and Tween 2.
0 and the like. Preservatives include sodium azide and the like. Serum includes normal mouse serum and the like. Salts other than sodium chloride include zinc chloride, magnesium chloride and the like. Non-specific adsorption inhibitors in the immune reaction include, for example, proteins such as bovine serum albumin and casein, and commercially available products include Block Ace (manufactured by Dainippon Pharmaceutical Co., Ltd.).

Samples to be used in the present invention include, for example, whole blood, serum, plasma, urine, lymph and the like. H
The sample for which the presence or absence of the CV antigen is to be detected may be used as it is, or a sample that has been concentrated using a virus fractionating agent or the like can be used. Specific examples of the fractionating agent include polyethylene glycol (PEG) reagents and reagents for density gradient such as Percoll. The molecular weight and use concentration of PEG are not particularly limited, but the molecular weight is 1,000 to 2
000 is preferable, and the use concentration is preferably in the range of 3 to 10%.

The sample is preferably treated with a reagent having a protein denaturing action, a protein solubilizing action, and a protein dispersing action. Examples of the reagent include a chaotropic substance, an alkali agent, an acid agent and a surfactant, and a chaotropic substance or an alkali agent is preferable.
Urea, guanidine hydrochloride as chaotropic substances,
Guanidium thiocyanate and the like can be mentioned, and urea and guanidine hydrochloride are preferable. Guanidine hydrochloride concentration is 1
~ 8M is preferred.

The alkali agent may be any alkali agent which shows an alkali having a pH of 10 or more in an aqueous solution. Examples thereof include alkali metal hydroxide salts, and preferably a sodium hydroxide solution. Examples of the acidic agent include hydrochloric acid, sulfuric acid, acetic acid, trifluoroacetic acid, and trichloroacetic acid. Surfactants include nonionic, anionic, cationic and amphoteric surfactants.

As the treatment of the specimen, a combination treatment with a chaotropic substance and an alkali agent is preferable. The treatment with an alkali agent is preferably performed at a temperature of 45 to 55 ° C. It is preferable that a sample treated with an acidic agent or an alkaline agent is subjected to an antigen-antibody reaction after neutralization. For the neutralization, an alkali is used when an acid agent is used, and an acid agent is used when an alkali agent is used. In each case, a solution having a high buffer capacity can be used.

The immunoassay method of the present invention is not particularly limited, and examples thereof include a widely used immunoassay method such as a sandwich method. Specifically, the immunoassay method of the present invention is carried out by a well-known sandwich method such as a one-step method, a delay one-step method, or a two-step method using an antibody-bound solid phase and an HCV core protein antibody labeled with alkaline phosphatase. Comes out. As the sandwich method, a two-step method is preferable. For example, after incubating an antibody-bound solid phase and an analyte containing an antigen in an aqueous solution, the solid phase is washed with a washing solution. Next, an aqueous solution containing a labeled antibody and a high concentration of salt is added, and after further incubation, the solid phase is washed again. Next, the alkaline phosphatase activity of the immune complex formed on the solid phase is measured.

Various solid phases used in conventional immunoassays can be used as the solid phase. Examples of the solid phase include plastic test tubes, microplate wells, glass beads, plastic beads, various membranes, magnetic particles, and the like (for example, see Eiji Ishikawa, “Enzyme Immunoassay” published by Medical Shoin). Examples of the antibody to be bound to the solid phase include the aforementioned anti-HCV core protein monoclonal antibody, and it is preferable to use an antibody that recognizes an epitope different from the antibody labeled with alkaline phosphatase. The binding between the solid phase and the antibody can be performed by a known method such as physical adsorption and chemical bonding.

The alkaline phosphatase activity can be measured using a chromogen, a fluorescent reagent, a luminescent reagent and the like by a conventional method (supervised by Bunji Maruo, Enzyme Handbook, Asakura Shoten). Various chromogens are known and any of them can be used, but an enzyme cycling reaction reagent capable of high-sensitivity detection, specifically, an enzyme that cycles oxidation-reduction of coenzyme NAD And an enzyme cycling reaction reagent [AmpliQ, manufactured by DAKO]. Examples of the fluorescent reagent include a 4-methylumbelliferone derivative and a 2-hydroxybiphenyl derivative.

As a luminescent reagent, a derivative having a dioxetane structure, specifically, AMPPD [3- (2′-spiroadamantane) -4-methoxy-4- (3 ″ -phosphoryloxy) phenyl-1,2-dioxetane ], CDP-
Star [4-chloro-3- (methoxyspiro @ 1,2-
Dioxetane-3,2 '-(5'-chloro) tricyclo [3.3.1.1 ^3,7 ] decane {-4-yl) phenyl disodium phosphate], CSPD [3- (4-methoxyspiro} 1,2-dioxetane-3,2 ′-(5′-chloro) tricyclo [3.3.1.1 ^3,7 ] decane｝ -4
-Yl) phenyl disodium phosphate]. Lumigen without dioxetane structure (Lumigen
gen) derivative APS compound ([10-methyl-9 (10
H) -Acridinylidene] phenoxymethyl phosphate disodium salt).

The quantification reagent of the present invention comprises an alkaline phosphatase-labeled anti-HCV core protein antibody, a salt having a concentration of 0.5 M to saturation concentration, and a reagent for measuring alkaline phosphatase activity. The quantification reagent also includes a reagent containing a solid phase on which an anti-HCV core protein antibody is immobilized, a reagent containing an HCV core protein standard, and the like. In the quantitative reagent of the present invention, each of the aforementioned reagents may be configured in the form of a kit.

The kit may further incorporate a reagent containing a sample concentrating reagent, a reagent containing a sample processing reagent, and the like. Examples of the sample concentration reagent include the above-described fractionating agents. Examples of the sample processing reagent include reagents having the above-described protein denaturing action, protein solubilizing action, and protein dispersing action. Examples of the alkaline phosphatase quantitative reagent include a reagent containing the aforementioned chromogen, fluorescent reagent, luminescent reagent, or the like. Examples of the anti-HCV core protein antibody include the aforementioned monoclonal antibodies.

The following examples illustrate the invention but do not limit the scope of the invention. Serum samples were collected from Uniglove.
be) or Boston Biomedica
dica, Inc. : BBI) was used.

[0046]

EXAMPLES Reference Example 1 Preparation of HCV Core Protein According to the method described in JP-A-8-29427,
A recombinant HCV core protein having 125 amino acid residues from the N-terminal of the V core protein was prepared and purified. The protein concentration of the purified recombinant HCV core protein was calculated using a Bio-Rad protein quantification kit (Bio-Rad) and a BCA protein quantification kit (Pierce).

Reference Example 2 Preparation of Anti-HCV Core Protein Antibody 25 to 100 μg of HCV core protein obtained in Reference Example 1
Is emulsified with Freund's complete adjuvant and B
ALB / C mice or SD rats were primed. After 2-3 weeks, HCV core protein 25-100 μg
Is emulsified with incomplete Freund's adjuvant,
A boost was performed. After confirming the increase in the antibody titer, 25 to 100 μg of the HCV core protein was administered intravenously, and 3 to 4 g
One day later, the spleen was removed from the mouse to prepare spleen cells. Next, mouse myeloma cells (P3U1) and spleen cells previously cultured in the RPMI-1640 medium were mixed at a ratio of 1: 2 to 1: 5, and cell fusion was performed using polyethylene glycol. After the fused cells are suspended in HAT medium,
The mixture was dispensed into a 96-well culture plate and cultured in a 37 ° C. carbon dioxide incubator. Screening for anti-HCV core protein antibody was performed as follows.

The HCV core protein was suspended at 2 μg / mL in 8 M guanidine hydrochloride solution or 10 M urea solution,
50 μl / well in a 6-well ELISA plate (Nunc)
The HCV core protein was adsorbed on the plate by dispensing wells and allowing to stand at 4 ° C. overnight. After blocking with physiological saline containing phosphate buffer (hereinafter abbreviated as PBS) containing 1% bovine serum albumin (hereinafter abbreviated as BSA), 50 μl of the culture supernatant of the fused cells was added to each well. And reacted at room temperature for 1 hour. After washing, a peroxidase-labeled anti-mouse IgG antibody was added and reacted at room temperature for 1 hour. After washing, a substrate ABTS was added to select wells in which color development was observed, and hybridoma KTM-145 and hybridoma KTM- which produced antibodies recognizing the HCV core protein were selected.
153, hybridoma KTM-157, hybridoma KTM-163 and hybridoma KTM-167
I got The hybridomas were transplanted into the abdominal cavity of a mouse treated with pristane or the like, and monoclonal antibodies produced in ascites were obtained. The monoclonal antibody was purified using a protein A column according to a conventional method (Sakuji Toyama, manual for monoclonal antibody experiment, Kodansha). The obtained antibodies were KTM-145 and KT, respectively.
M-153, KTM-157, KTM-163 and K
Named TM-167.

Reference Example 3 Reaction Specificity of Monoclonal Antibody The reaction specificity of the monoclonal antibody obtained in Reference Example 2 was confirmed using a 96-well microtiter plate to which HCV core protein or Escherichia coli lysate was adsorbed. As a result, KTM-145, KTM-153, KT
M-157, KTM-163 and KTM-167 are H
The specific reaction with the CV core protein was confirmed.
Similarly, the same reactivity was confirmed by Western blot. Furthermore, as a result of analyzing the epitope of the HCV core protein recognized by the antibody using the partial peptide of the HCV core protein, KTM-145 was located at positions 41 to 50 from the N-terminus of the HCV core protein, and KTM-153 was located at positions 11 to 3 from the N-terminal.
0-th, KTM-157 the 31st-50th, K
It was confirmed that TM-163 and KTM-167 recognize the 21st to 40th positions, respectively.

Reference Example 4 Preparation of Alkaline Phosphatase-Labeled Monoclonal Antibody The monoclonal antibody obtained in Reference Example 2 and alkaline phosphatase (Boehringer Mannheim) were prepared according to a conventional method (Eiji Ishikawa, Enzyme Immunoassay, published by Medical Shoin). Coupling was achieved by the maleimide method. 5 mg of the monoclonal antibody was dialyzed against 50 mM Tris buffer (pH 8), and sulfhydrylated with iminothiolane. Free iminothiolane was removed from the iminothiolanated antibody using a Sephadex G-25 column (Pharmacia). Alkaline phosphatase was maleimidated using maleimide reagent SMCC (Pierce) to give G-
Free SMCC was removed on 25 columns.

The above-mentioned sulfhydrylated monoclonal antibody and maleimidated alkaline phosphatase were mixed and reacted at 4 ° C. overnight. A monoclonal antibody labeled with alkaline phosphatase produced after the reaction was applied to a Sephacryl S-200 column (Pharmacia).
Was purified. The purified alkaline phosphatase-labeled antibody was used in a labeled antibody diluent [50 mM Tris buffer (pH
7.6) Aqueous solution containing 10 mM magnesium chloride, 0.1 mM zinc chloride, and 20% block ace (Dainippon Pharmaceutical Co., Ltd.)] to a predetermined concentration, and enzyme-labeled immunoassay (EL).
ISA).

Reference Example 5 Preparation of Solid Phase Immobilized with Anti-HCV Core Protein Antibody The final concentration of the monoclonal antibody obtained in Reference Example 2 was 5 μg.
/ ML containing 0.1% sodium azide
After dilution with BS, 200 μl was added to each well of a 96-well microtiter plate for solid phase. After standing at 4 ° C. overnight, the plate was washed with PBS, 300 μl of PBS containing 0.1% BSA was added, and the plate was allowed to stand at 4 ° C. overnight to block.

Reference Example 6 Preparation of Peroxidase Enzyme-Labeled Monoclonal Antibody The monoclonal antibody obtained in Reference Example 2 and peroxidase (manufactured by Toyobo Co., Ltd.) were subjected to a conventional method (Eiji Ishikawa, Enzyme Immunoassay, published by Medical Shoin). Coupling was achieved by the maleimide method. 5 mg of the monoclonal antibody was dialyzed against 50 mM Tris buffer (pH 8), and sulfhydrylated with iminothiolane. Free iminothiolane was removed from the iminothiolanated antibody using a Sephadex G-25 column (Pharmacia). Peroxidase was maleimidated using a maleimide reagent SMCC (manufactured by Pierce), and free SMCC was removed with a Sephadex G-25 column.

The above-mentioned sulfhydrylated monoclonal antibody and maleimidated peroxidase were mixed and reacted at 4 ° C. overnight to obtain a peroxidase-labeled monoclonal antibody.
Purified with a 00 column (manufactured by Pharmacia). The purified labeled antibody was labeled antibody diluent [50 mM Tris buffer (pH 7.6), 10 mM magnesium chloride, 0.1 mM
M zinc chloride, 20% block ace (Dainippon Pharmaceutical Co., Ltd.)
And an aqueous solution containing the same was used for ELISA.

Example 1 Measurement of Recombinant HCV Core Protein The recombinant HCV core protein obtained in Reference Example 1 was
Alternatively, the cells were diluted with PBS containing 0.1% BSA to 100 pg / mL.

200 μl of the diluted recombinant HCV core protein solution was added to each well on the microplate prepared in Reference Example 5 and reacted at room temperature for 1 hour. After washing with a washing solution (supplied with AMPAK sensitizing reagent, manufactured by Dako), the labeled antibody obtained in Reference Example 4 was added. Labeled antibodies were 50 mM Tris buffer (pH 7.6), 10 mM
After diluting with a solution containing mM magnesium chloride, 0.1 mM zinc chloride, 20% Block Ace (Dainippon Pharmaceutical Co., Ltd.) and 0 to 3 M sodium chloride, use 20 μl per well.
0 μl was added. After stirring for 1 hour at room temperature, washing with a washing solution, 200 μL of an enzyme cycling reaction reagent (manufactured by Dako) as a coloring reagent was added, and the mixture was stirred at room temperature for 2 hours.
The reaction was performed for 0 minutes. Stop solution attached to the reagent
Add 00μl, main wavelength 490nm, sub wavelength 660nm
The absorbance was measured with. In this experiment, KTM-
145 for alkaline phosphatase labeling
-163 was used.

The results are shown in Table 1. In Table 1, the number for each antigen concentration indicates absorbance (Abs).

[0058]

[Table 1]

By increasing the concentration of sodium chloride in the diluent containing the labeled antibody, a test group without antigen (0 pg / m
In L), the absorbance hardly changed even when the concentration of sodium chloride was increased, whereas the absorbance of the test group (10
0 pg / mL), the absorbance obtained by increasing the concentration of sodium chloride increased. In the table, the S / N ratio indicates the ratio of the absorbance in the antigen-existing section (100 pg / mL) to the absorbance in the antigen-free section (0 pg / mL). It can be seen that the addition of sodium chloride increases the S / N ratio and makes it possible to detect antigens with higher sensitivity. That is, when sodium chloride is not used,
This shows that even if the antigen is present, the absorbance is slightly increased compared to the antigen-free sample, and even if the sample cannot be determined to be antigen-positive, it can be determined to be antigen-positive by applying the method of the present invention.

Example 2 Measurement of Recombinant HCV Core Protein The recombinant HCV core protein obtained in Reference Example 1 was
0.1% BSA to achieve a concentration range of ~ 20 pg / mL
And diluted with PBS.

200 μl of the diluted recombinant HCV core protein solution was added to each well of the microplate prepared in Reference Example 5 and reacted at room temperature for 1 hour.
After washing with a washing solution (supplied with AMPAK sensitizing reagent, manufactured by Dako), the labeled antibody obtained in Reference Example 4 was added. Labeled antibody is 50 mM Tris buffer (pH 7.6), 10 mM
Dilute with a solution containing magnesium chloride, 0.1 mM zinc chloride, 20% Block Ace (Dainippon Pharmaceutical Co., Ltd.) and 0 to 2 M sodium chloride, and add 200 μl to each well.
One by one was added. After stirring for 1 hour at room temperature and washing with a washing solution, 200 μL of an enzyme cycling reaction reagent (manufactured by Dako) as a coloring reagent was added, and the mixture was reacted at room temperature with stirring for 40 minutes. 100 μl of stop solution attached to the reagent
Then, the absorbance was measured at a main wavelength of 490 nm and a sub wavelength of 660 nm. In this experiment, KTM-145 was used for the solid phase.
Is used for labeling alkaline phosphatase with KTM-16.
3 was used.

The results are shown in Table 2. In Table 2, the numbers for each antigen concentration indicate absorbance (Abs).

[0063]

[Table 2]

By increasing the concentration of sodium chloride in the diluent containing the labeled antibody, the absorbance hardly changes even when the concentration of sodium chloride is increased in the test group without the antigen, whereas the increase in the concentration of sodium chloride in the test group with the antigen is as follows. Increasing the concentration of sodium chloride increased the absorbance obtained. This means that, when sodium chloride is not used, the increase in absorbance even when an antigen is present is small compared to an antigen-free sample, even in a sample that cannot be determined to be antigen-positive, by applying the method of the present invention. This indicates that the antigen can be determined to be positive.

Example 3 Detection Limit The recombinant HCV core protein obtained in Reference Example 1 was adjusted to 0.1% BS so as to have a concentration range of 0 to 10 pg / mL.
A was diluted with PBS containing A.

200 μl of the diluted recombinant HCV core protein solution was added to each well of the microplate obtained in Reference Example 5, and reacted at room temperature for 1 hour. After washing with a washing solution (supplied with AMPAK sensitizing reagent, manufactured by Dako), the labeled antibody obtained in Reference Example 4 was added. Labeled antibody is 50 mM Tris buffer (pH 7.6), 10 mM
Dilute with a solution containing magnesium chloride, 0.1 mM zinc chloride, 20% Block Ace (Dainippon Pharmaceutical Co., Ltd.) and 0 or 1 M sodium chloride, and use each well for 20 minutes.
0 μl was added. After reacting with stirring at room temperature for 1 hour, washing with a washing solution, 100 μL of an enzyme cycling reaction reagent (manufactured by Dako) as a coloring reagent was added, and the mixture was stirred at room temperature for 3 hours.
The reaction was performed for 0 minutes. Stop solution attached to the reagent
Add 00μl, main wavelength 490nm, sub wavelength 660nm
The absorbance was measured with. In this experiment, KTM-
145 for alkaline phosphatase labeling
-163 was used.

Table 3 shows the results. In Table 3, the numbers for each salt concentration indicate absorbance (Abs).

[0068]

[Table 3]

By increasing the concentration of sodium chloride in the diluent containing the labeled antibody, the concentration of the antigen at which the presence of the antigen can be detected was extremely low. As shown in Table 3, the absorbance of antigen 0 and the absorbance of 1 at 1 pg / mL without sodium chloride were added.
While only a difference of the order of 0 mAbs was observed, the addition of 1 M sodium chloride resulted in a 0. 0 mAbs order.
At 125 pg / mL, a difference on the order of 10 mAbs from the absorbance of antigen 0 was observed, and the detection sensitivity was improved.

Comparative Example 1 The recombinant HCV core protein obtained in Reference Example 1 was diluted with PBS containing 0.1% BSA to a concentration of 0, 10, 100 or 1000 pg / mL.

200 μl of the diluted recombinant HCV core protein solution was added to each well of the microplate prepared in Reference Example 5 and reacted at room temperature for 1 hour.
After washing with a washing solution (supplied with AMPAK sensitizing system reagent, manufactured by Dako), the peroxidase-labeled antibody obtained in Reference Example 6 was added. Labeled antibody is 50 mM Tris buffer (pH
7.2), diluted with a solution containing 0.1% BSA and 0-4 M sodium chloride and used, 200 μl per well
Was added. After stirring at room temperature for 1 hour, washing with a washing solution, TMB (tetramethylbenzidine,
200 μL (product of Intergen) was added and reacted at room temperature with stirring for 30 minutes. 100 μl of 1N sulfuric acid was added to stop the color reaction, and the main wavelength was 450 nm and the sub wavelength was 66.
The absorbance was measured at 0 nm. In this experiment, K
KTM-TM was used for peroxidase labeling.
163 was used.

The results are shown in Table 4. In Table 4, the numbers for each antigen concentration indicate absorbance (Abs).

[0073]

[Table 4]

When a peroxidase-labeled antibody was used,
In a reaction system in which no antigen was present, the addition of sodium chloride reduced the reaction specificity. Further, even when the concentration of sodium chloride was increased in the reaction system in which the antigen was present, no improvement in the reaction specificity was observed.

Example 4 Measurement of Human Specimens Human serum was treated according to the method described in JP-A-8-29427. That is, 20% of PE was added to 200 μl of serum sample.
50 μl of G4000 was added, and the mixture was allowed to stand on ice for 1 hour. 4
After centrifugation at 000 × g for 5 minutes at 4 ° C., the precipitated fraction was suspended in 50 μl of 50 mM Tris buffer (pH 8.0), and 0.5 M sodium hydroxide solution was further added to 50 μl.
The mixture was treated at 37 ° C. for 30 minutes. Thereafter, the mixture was neutralized with 50 μL of a 0.5 M sodium hydrogen phosphate solution containing 0.3% of Triton X-100. Finally 0.1% BSA-P
300 μL of a BS solution was added to dilute it three-fold to obtain a solution for measurement. The measurement target is an anti-HCV antibody-positive serum sample (Un
i-2 and 0401) and a normal human serum sample as a control were tested in the same manner as in Example 1 and the absorbance (Abs) was measured. The results are shown in Table 5.

[0076]

[Table 5]

As shown in Table 5, when a normal human serum sample was used, the absorbance increased only slightly when the concentration of sodium chloride in the solution containing the labeled antibody was increased.
When a V antibody-positive serum sample was used, the absorbance obtained was increased by increasing the concentration of sodium chloride in the solution containing the labeled antibody. This makes it possible to determine antigen positivity even for a sample containing a lower concentration of antigen. From the S / N ratio, the sodium chloride concentration is 0.5 to 3M, especially 1 to 3M.
It can be seen that the sensitivity is significantly increased at M.

Example 5 Guanidine hydrochloride and alkali treatment of a sample 50% of 20% PEG 4000 was added to 200 μl of a serum sample.
l, and left on ice for 1 hour. After centrifugation at 4000 × g for 5 minutes at 4 ° C., the precipitate was washed with 50 mM Tris buffer (pH 8.0) containing 0 to 8 M guanidine hydrochloride.
The suspension was denatured with 0 μl. Further, after adding 50 μl of 0.5 M sodium hydroxide solution and treating at 37 ° C. for 30 minutes,
Neutralized with 0.5M sodium hydrogen phosphate solution. Then, it was diluted three times with a 0.1% BSA-PBS solution to obtain a solution for measurement. The measurement was performed in the same manner as in Example 4 except that the concentration of sodium chloride was changed to 1M. The measurement targets were a normal human serum sample and three anti-HCV antibody positive serum samples (U-1
9, U-21 and U-29) were used. The results are shown in Table 6. In Table 6, the number for each sample is the absorbance (Ab
s).

[0079]

[Table 6]

As shown in Table 6, it can be seen that the absorbance increases depending on the concentration of guanidine hydrochloride.

Example 6 A test was conducted in the same manner as in Example 5 using urea and guanidine hydrochloride. The concentration of urea was 10M and the concentration of guanidine hydrochloride was 8M. Samples were normal human serum samples and anti-HC
Four serum samples positive for V antibody (104, 106, 187 and 197) were used. The results are shown in Table 7. In Table 7,
The numbers for each treatment reagent indicate absorbance (Abs).

[0082]

[Table 7]

As shown in Table 7, no significant difference was observed in the absorbance between the normal human serum and the normal human serum when the urea treatment was performed on the specimens 104 and 197. By processing, it was determined to be positive.

Example 7 Examination of Temperature in Alkaline Treatment A test was conducted in the same manner as in Example 6, except that the temperature conditions in the alkali treatment were changed. Serum sample is 6,
9 and 138, the temperature in the alkaline treatment was 3
7 ° C., 50 ° C., 70 ° C., and 100 ° C. The results are shown in Table 8. In Table 8, the number for each sample is the absorbance (Ab
s).

[0085]

[Table 8]

As shown in Table 8, it is found that the alkaline treatment temperature of the serum is preferably around 50 ° C.

Example 8 Comparison with Various Detection Methods The measurement according to the present invention was performed as follows. Various serum samples 2
50 μl of 20% PEG 4000 was added to 00 μl, and the mixture was allowed to stand on ice for 1 hour. After centrifugation at 4000 × g for 5 minutes at 4 ° C., the precipitate was centrifuged at 50 mM containing 8 M guanidine hydrochloride.
The suspension was denatured with 50 μl of Tris buffer (pH 8.0). 50 μl of 0.5 M sodium hydroxide solution
Add 50% for 30 minutes, then 0.3% Triton X-1
Neutralized with 0.5M sodium hydrogen phosphate solution containing 00. Then, it was diluted three times with a 0.1% BSA-PBS solution to obtain a solution for measurement. The measurement was performed in the same manner as in Example 4 except that 2 M of sodium chloride was used in the labeled antibody solution.

Further, an immunocheck, F-HCVAg core (manufactured by Kokusai Reagents, hereinafter simply referred to as an HCV core protein measurement kit), which is a kit for measuring HCV core protein,
Generation III anti-HC kit for measuring anti-HCV antibodies
Using commercially available reagents of V antibody (manufactured by Ortho, hereinafter, simply referred to as anti-HCV antibody measurement kit) and Amplico qualitative (manufactured by Roche, hereinafter, simply referred to as RNA measurement kit) which is a kit for measuring HCV RNA by PCR method The same sample was measured and the sensitivity was compared with the method of the present invention. The results are shown in Table 9. In Table 9, "-" in the judgment column indicates that detection is not possible, and "+" in the judgment column indicates that detection is possible.

[0089]

[Table 9]

As shown in Table 9, according to the method of the present invention, it is possible to detect even a sample which cannot be detected by the HCV core protein measurement kit (manufactured by Kokusai Reagents). These serum samples excluding normal persons are anti-HCV antibody-positive, and samples tested with an RNA measurement kit (Roche) are determined to be HCV-positive. The sensitivity for serum samples is 10 ⁴ virus / mL with the HCV core protein measurement kit (manufactured by Kokusai Reagents), but using the method of the present invention, 10 ³ virus / mL is equivalent to that of the RNA measurement kit (manufactured by Roche). It becomes detectable.

Example 9 Combination of Antibodies The type of the antibody used for the solid phase and the type of the antibody to be labeled with alkaline phosphatase were changed, and HCV-uninfected serum and HCV-infected serum were used as samples. The test was performed in the same manner as in Example 6 except that the sodium concentration was set to 0 or 1 M, and the absorbance was measured to determine the S / N ratio. The results are shown in Table 10. In Table 10, the numbers for each sodium chloride concentration indicate the S / N ratio.

[0092]

[Table 10]

As shown in Table 10, in any combination of the antibody used for the solid phase and the labeled antibody, by adding sodium chloride to the solution for diluting the labeled antibody,
/ N ratio increased. This indicates that the HCV infected sample can be detected with higher sensitivity.

Example 10 Detection Limit The sample (U14 and U19) solution was made 1-fold, and a sample serially diluted with a normal serum solution was prepared. To 200 μl of serially diluted sample, 50 μl of 20% PEG 4000 was added,
It was left on ice for 1 hour. After centrifugation at 4000 × g for 5 minutes at 4 ° C., the precipitate was centrifuged at 50 m containing 8 M guanidine hydrochloride.
The suspension was denatured with 50 μl of M Tris buffer (pH 8.0). Further, 50 μl of 0.5 M sodium hydroxide solution is added.
After adding, and treating at 50 ° C. for 30 minutes, 0.3% Triton X
Neutralized with 0.5 M sodium hydrogen phosphate solution containing 100. Then, it was diluted three times with a 0.1% BSA-PBS solution to obtain a solution for measurement. Each diluted sample was measured by the method described in Example 4. The concentration of sodium chloride in the labeled antibody solution was 2M.

A calibration curve was prepared using the HCV core protein of known concentration obtained in Reference Example 1 as a standard substance, and the HCV core protein mass was determined from the absorbance (Abs) of each sample. Table 11 shows the results using the U14 sample, and Table 12 shows the results using the U19 sample.

[0096]

[Table 11]

[0097]

[Table 12]

As shown in Tables 11 and 12, the quantification method of the present invention uses HCV core protein of 0.1 pg / m
It is possible to detect up to L orders.

Example 11 Detection of HCV Core Antigen in Seroconversion Panel Sample Commercially available seroconversion panel PHV908 (BBI
Was used as a sample, and the HCV core antigen was measured by the method described in Example 8. HCV core antigen has a normal human absorbance of 1.0
And expressed as a ratio (S / N).

The sample was also measured with an anti-HCV antibody measurement kit (manufactured by Ortho). The antibody titer is represented by a cutoff index (S / CO), and 1.0 or more is determined to be positive. The results are shown in Table 13.

[0101]

[Table 13]

As shown in Table 13, according to the method of the present invention, the core antigen could be detected before the anti-HCV antibody became positive, and the detection was possible in the early stage of infection.

Example 12 Reagent Kit for Quantifying HCV Core Protein A reagent kit comprising the following reagents was constructed. Antibody coated plate (KTM-14 obtained in Reference Example 2)
Sample diluent (aqueous solution containing 0.1% BSA-containing PBS and 1% normal mouse serum) Enzyme-labeled antibody (KTM- obtained in Reference Example 2) 163 prepared in the same manner as in Reference Example 4. Standard antigens 0 pg / mL and 10 pg / mL (HCV core protein obtained in Reference Example 1
% BSA-containing PBS, 0.89 M guanidine hydrochloride,
5.56 mM Tris buffer (pH 8), 0.056 M
A solution containing sodium hydroxide, 0.056 M sodium dihydrogen phosphate and 0.005% Triton X-100 dissolved at 0 or 10 pg / mL) Labeled antibody diluent [50 mM Tris buffer (pH 7 .6) 10 mM
Magnesium chloride, 0.2 mM zinc chloride, 20% block ace, 2M sodium chloride, 0.1% sodium azide, 0.1% Triton X-705 and 1%
Normal mouse serum is contained] Coloring reagent [Coloring reagent contained in enzyme cycling reaction reagent (AmpliQ, Dako)] Stop solution [Stop solution contained in enzyme cycling reaction reagent (AmpliQ, Dako)]

Precipitation reagent (20% PEG 4000) Dispersion reagent [8M guanidine hydrochloride and 50 mM Tris buffer (p
H8)] Alkaline reagent (0.5 M sodium hydroxide solution) Neutralization reagent (0.5 M sodium dihydrogen phosphate and 0.3% Triton X-100)

[0105]

Industrial Applicability The present invention provides an immunoassay method for detecting an antigen derived from HCV which causes hepatitis C with high sensitivity, and a reagent and a kit used for the immunoassay method. ADVANTAGE OF THE INVENTION According to this invention, the sample which cannot be detected by the conventional HCV core antigen measurement system can be detected with high sensitivity, and can be widely utilized for the antigen test of HCV. In addition, an immunoassay having a sensitivity equivalent to a highly sensitive gene measurement method by PCR can be easily constructed, and can contribute widely to the medical field.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/536 G01N 33/536 C 33/577 33/577 B // C12N 15/02 C12P 21/08 C12P 21/08 (C12P 21/08 (C12P 21/08 C12R 1:91) C12R 1:91) C12N 15/00 C (72) Inventor Masanori Fukui 2-1-1 Irifune, Chuo-ku, Tokyo Kyowa Medex Co., Ltd. Kyowa Medex Headquarters (72) Inventor Dai Kondo 600-1, Kamiyama, Minami-Ishiki, Nagaizumi-cho, Sunto-gun, Shizuoka Prefecture 1 Kyowa Medex Research Institute, Kyowa Medex Co., Ltd. (72) Michiho Ohara 3-15 Tabata, Kita-ku, Tokyo 3-408

Claims (29)

[Claims] 1. An anti-C labeled with alkaline phosphatase
Hepatitis B virus (hereinafter, Hepatitis C virus is abbreviated as HCV) core protein antibody and HCV core antigen in the specimen are subjected to an antigen-antibody reaction in an aqueous solution containing a high concentration of salt to form an immune complex. An immunoassay method for detecting or quantifying an HCV core antigen in a sample, comprising forming and measuring alkaline phosphatase activity in the immune complex. 2. The method according to claim 1, wherein the salt is an alkali metal halide. 3. The method according to claim 1, wherein the salt is sodium chloride. 4. The method according to claim 1, wherein the salt concentration is 0.5M to a saturation concentration. 5. The anti-HCV core protein antibody according to any one of claims 1 to 4, wherein the antibody recognizes at least 5 consecutive amino acid sequences among the 11th to 50th amino acid sequences from the N-terminus of the HCV core protein. The method described in. 6. The method according to claim 6, wherein the anti-HCV core protein antibody is KTM-14.
5, KTM-153, KTM-157, KTM-163
The method according to any one of claims 1 to 4, which is an antibody selected from the group consisting of KTM-167 and KTM-167. 7. The method according to claim 1, wherein the specimen has been treated with a chaotropic substance and / or an alkaline agent before the antigen-antibody reaction. 8. The method according to claim 7, wherein the chaotropic substance is a guanidine salt. 9. The method according to claim 7, wherein the alkali agent is an alkali metal hydroxide salt. 10. The method according to claim 7, wherein the chaotropic substance is at a concentration of 1 M to a saturation concentration. 11. The method according to claim 7, wherein the treatment of the alkali agent is performed at a temperature of 45 to 55 ° C. 12. An HC comprising an alkaline phosphatase-labeled anti-HCV core protein antibody, a salt having a concentration of 0.5 M to saturation and a reagent for measuring alkaline phosphatase activity.
V-core antigen detection or quantification reagent. 13. The reagent according to claim 12, wherein the salt is an alkali metal halide. 14. The salt of claim 12, wherein the salt is sodium chloride.
Or the reagent according to 13. 15. The anti-HCV core protein antibody according to any one of claims 12 to 14, wherein the antibody recognizes at least 5 consecutive amino acid sequences among the 11th to 50th amino acid sequences from the N-terminus of the HCV core protein. The reagent according to 1. 16. The method according to claim 16, wherein the anti-HCV core protein antibody is KTM-1.
45, KTM-153, KTM-157, KTM-16
The reagent according to any one of claims 12 to 14, which is an antibody selected from the group consisting of 3 and KTM-167. 17. A reagent kit for detecting or quantifying an HCV core antigen, comprising a reagent according to any one of claims 12 to 16 and a sample processing reagent containing a chaotropic substance and / or an alkaline agent. 18. The kit according to claim 17, wherein the chaotropic substance is a guanidine salt. 19. The kit according to claim 17, wherein the alkaline agent is an alkali metal hydroxide. 20. The kit according to claim 17, wherein the chaotropic substance has a concentration of 1 M to saturation. 21. From the N-terminal of the HCV core protein, 11 to 5
A monoclonal antibody recognizing at least 5 consecutive amino acid sequence sites in the 0th amino acid sequence. 22. From the N-terminus of HCV core protein, 41 to 5
A monoclonal antibody that recognizes the 0th amino acid sequence site. 23. Hybridoma KTM-145 (FE
23. The monoclonal antibody according to claim 22, which is produced by RM BP-6838). 24. From the N-terminus of the HCV core protein,
A monoclonal antibody that recognizes the 0th amino acid sequence site. 25. Hybridoma KTM-153 (FE
25. The monoclonal antibody according to claim 24, which is produced by RM BP-6839). 26. 31-5 from the N-terminus of the HCV core protein
A monoclonal antibody that recognizes the 0th amino acid sequence site. 27. Hybridoma KTM-157 (FE
The monoclonal antibody according to claim 26, which is produced by RM BP-6840). 28. From the N-terminus of HCV core protein, 21 to 4
A monoclonal antibody that recognizes the 0th amino acid sequence site. 29. Hybridoma KTM-163 (FE
RM BP-6841) or hybridoma KTM-
29. The monoclonal antibody according to claim 28 produced by 167 (FERM BP-6842).