JP3193146B2 - Immunological agglutination reagent for diagnosis of hepatitis C - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunological agglutination reagent for diagnosis of hepatitis C for detecting an antibody against hepatitis C virus (hereinafter abbreviated as HCV) which is a cause of hepatitis C.

[0002]

BACKGROUND ART Hepatitis C is caused by HCV, and 90% or more of non-A non-B chronic hepatitis after blood transfusion is HC.
It is said to be hepatitis caused by V infection. H
A part of the gene of CV is disclosed in European Patent EP 0318216 (198
9) and European Patent EP 0388232 (published 1990).

According to previous studies, HCV is considered to be an RNA virus having a total gene sequence length of about 10 kb (about 10,000 nucleotides). Among the proteins produced from the HCV gene, a polypeptide having high antigen activity is
Hepatitis diagnostic reagents have been developed. For example, in European Patent 0318216, a part of a gene encoding a nonstructural protein region is inserted into a yeast expression vector, this gene is expressed, and hepatitis C diagnosis using an HCV antigen active polypeptide called C100 as an antigen is performed. Is one of them.

At present, hepatitis C is diagnosed by enzyme immunoassay (hereinafter abbreviated as EIA) and passive agglutination reaction (hereinafter abbreviated as PA) using one or two HCV antigen-active polypeptides as antigens. Inspection method is used. The above diagnostic agents are effective in preventing hepatitis C during blood transfusion. However, since one or two HCV active polypeptides are used as antigens, there are currently many false negatives and false positives, and there is a problem in the sensitivity of diagnosis at the early stage of HCV infection.

[0005]

SUMMARY OF THE INVENTION Currently used C
Although the hepatitis C diagnostic agent has a great effect in preventing hepatitis C virus infection during blood transfusion, the development of a diagnostic agent having higher sensitivity and specificity and capable of diagnosing in the early stage of HCV infection is desired. The present inventors have conducted intensive studies to solve this problem, and as a result, by using a heated HCV antigen-active polypeptide derived from the hepatitis C virus gene as an antigen, the type C virus with excellent diagnostic sensitivity and specificity was obtained. The present inventors have found that an immunological agglutination reagent for hepatitis diagnosis can be obtained, and have completed the present invention.

[0006]

SUMMARY OF THE INVENTION The present invention provides an immunological agglutination reagent for diagnosing hepatitis C, which comprises using, as an antigen, a heat-treated HCV antigen-active polypeptide of hepatitis C virus-derived gene. It is in. The HCV antigen-active polypeptide is a polypeptide comprising the amino acid sequence of SEQ ID NO: 1
A CV antigen active polypeptide, an HCV antigen active polypeptide comprising the amino acid sequence of SEQ ID NO: 2, an HCV antigen active polypeptide comprising the amino acid sequence of SEQ ID NO: 3, and a HCV antigen active polypeptide comprising the amino acid sequence of SEQ ID NO: 4 .

Further, the present invention relates to an HCV antigen active polypeptide comprising the amino acid sequence of SEQ ID NO: 1, an HCV antigen active polypeptide comprising the amino acid sequence of SEQ ID NO: 2, and an HCV antigen active polypeptide comprising the amino acid sequence of SEQ ID NO: 3. And an HCV antigen-active polypeptide comprising the amino acid sequence of SEQ ID NO: 4 on insoluble carrier particles.

Hereinafter, the present invention will be described in detail. The heat treatment in the present invention is performed by heating a mixture of polypeptides to a predetermined temperature. It is effective that the heating temperature is 20 ° C. or more and 80 ° C. or less. Preferably 25 ° C or higher
The temperature is 60 ° C or lower, more preferably 35 ° C or higher and 50 ° C or lower. The heat treatment of the polypeptide is performed in a buffer solution having a buffering action, and any type may be used. For example, phosphate buffer, glycine buffer, Tris buffer, acetate buffer and the like. The pH may be any, but a neutral region is desirable. Preferably, a phosphate buffer, pH 6.0 to 8.0 is desirable. The treatment time may be any time as long as it is 10 minutes or more, preferably 10 minutes to 5 hours, more preferably 30 minutes to 2 hours.

The hepatitis C virus gene referred to in the present invention is:
For example, Proc. Natl. Acad. Sci. USA, Vol. 87, pp. 9524-95
28 (1990).
It is a kb (about 10,000 nucleotides) RNA. HCV is an RNA virus, and cDNA produced by reverse transcriptase from HCV-derived RNA also corresponds to the hepatitis C virus gene.

[0010] The hepatitis C virus gene is non-A
It can be obtained from a cDNA library prepared by separating viral genes from the serum of non-B hepatitis patients. For example, hepatitis C virus is first separated from patient serum by ultracentrifugation, then gene RNA is prepared from the virus, cDNA is synthesized from the RNA using reverse transcriptase, and then the cDNA fragment is A cDNA library is prepared by inserting into a plasmid vector or phage vector. Next, the cDNA library is subjected to immunoscreening using the serum of a non-A non-B hepatitis patient after blood transfusion (serum containing an anti-HCV antibody) to obtain a target gene. Also known HC
A DNA library may be synthesized based on the base sequence of the V gene, and a cDNA library may be screened by DNA / DNA hybridization. Another alternative is Proceedings of the Japan Academy, V
ol. 65, Ser. B, No. 9, pp. 219-223 (1989), that is, RT- which combines reverse transcriptase and PCR.
A method of amplifying a target region by PCR and cloning the amplified gene fragment is also effective.

The HCV antigen active polypeptide of the present invention has immunological reactivity with anti-HCV antibodies contained in serum and plasma of hepatitis C patients. That is, it has an epitope site for an anti-HCV antibody, and has a property of specifically binding to an anti-HCV antibody in serum and plasma of a hepatitis C patient by an antigen-antibody reaction. The HCV antigen active polypeptide is C
It can be used as an antigen of a hepatitis diagnosis reagent.

The HCV antigen-active polypeptide is a polypeptide produced by the HCV gene. Further H
The length of the CV antigen-active polypeptide may be any, and is preferably 3 to 3000 amino acid residues, more preferably 3 to 2000 amino acid residues. The HCV antigen-active polypeptide can be prepared by a known gene expression system, that is, Escherichia coli host vector system, Bacillus subtilis host vector system, yeast host vector system, insect cell or insect host vector system. The expression can be carried out by using an animal cell host / vector system or the like. Among them, Escherichia coli can be suitably used. To express the HCV antigen-active polypeptide using Escherichia coli, first, a HCV gene is inserted into a vector that can be expressed in Escherichia coli to prepare a recombinant vector. The vector is not particularly limited, and any vector can be used as long as it is a vector usually used as a vector for Escherichia coli. Particularly, a vector in which gene expression occurs at a high frequency is suitably used. For example, a series of pUC vectors (Takara Shuzo Co., Ltd. products), a series of pTV vectors (Takara Shuzo Co., Ltd. products), a series of pTZ vectors (Toyobo Co., Ltd. products), a series of p
ET (shown in Methods in enzymology, Vol. 185) and the like can be used. Further, if a series of pUEX vectors (Amersham Japan Co., Ltd. product) and a series of pEX vectors (Boehringer Mannheim Yamanouchi Co., Ltd. product) are used, HCV antigen-active polypeptide can be used as a fusion polypeptide with β-galactosidase. Can be expressed. A vector that can be expressed in E. coli usually has a promoter for gene expression that works in E. coli and an operator that controls it. A recombinant vector is prepared by inserting an HCV gene using an appropriate restriction enzyme site downstream of the promoter of such a vector. Escherichia coli is transformed with the recombinant vector, and the transformed Escherichia coli is cultured to express the inserted HCV gene, thereby producing an HCV antigen-active polypeptide.

When gene expression is carried out using a recombinant vector, a plurality of amino acids having a random sequence may be added to the N-terminal or C-terminal of the polypeptide. However, since a plurality of amino acids added to the N-terminal or C-terminal are random amino acids,
It is irrelevant to CV antigen activity and does not affect antigen activity measurements.

The HCV antigen-active polypeptide is obtained by culturing the cells obtained by culturing the above-mentioned transformed Escherichia coli by sonication or the like, and isolated from the crushed cells by a known method. The HCV antigen-active polypeptide can be purified by any known method, such as salting out, ion exchange resin adsorption, gel filtration and the like. Preferably, a combination of the above methods is effective. Further, the purified HCV antigen-active polypeptide may be dispersed in any solution,
It is preferably dispersed in a 0.87% aqueous sodium chloride solution (hereinafter abbreviated as physiological saline) or a 20 mM phosphate buffer containing 0.87% sodium chloride, pH 7.2 (hereinafter abbreviated as PBS). The higher the purification purity, the better. Preferably, the HCV antigen-active polypeptide accounts for at least 80% of the total protein.

The first HCV antigen-active polypeptide referred to in the present invention is a polypeptide containing the amino acid sequence shown in SEQ ID NO: 1 (hereinafter, also abbreviated as Core antigen) essential for excellent antigen activity. A plurality of extra amino acids may be added to the N-terminus or C-terminus. The sequence is 1 to 1 from the N-terminus of Japanese type HCV.
It corresponds to the amino acid sequence of the core protein up to the 68th.

The second HCV antigen-active polypeptide referred to in the present invention is a polypeptide (hereinafter abbreviated as NS-3 antigen) containing the amino acid sequence shown in SEQ ID NO: 2 and essential for excellent antigen activity. Alternatively, a plurality of extra amino acids may be added to the N-terminus or C-terminus. The 1st to 211st of this sequence are Japanese type HC
N from the 1323th to 1533th, counting from the N-terminal of V
It corresponds to the amino acid of S-3 protein.

The third HCV antigen-active polypeptide referred to in the present invention is a polypeptide containing the amino acid sequence shown in SEQ ID NO: 3 (hereinafter abbreviated as NS-4 antigen) essential for excellent antigen activity. Alternatively, a plurality of extra amino acids may be added to the N-terminus or C-terminus. The first to 194th positions of this sequence are Japanese type HC
N from the 1605th to the 1798th, counting from the N-terminal of V
It corresponds to the amino acid of S-4 protein.

The fourth HCV antigen-active polypeptide referred to in the present invention is a polypeptide containing the amino acid sequence shown in SEQ ID NO: 4 (hereinafter abbreviated as NS-5 antigen) essential for excellent antigen activity. Alternatively, a plurality of extra amino acids may be added to the N-terminus or C-terminus. The 1st to 160th positions of this sequence are Japanese type HC
N from the 2111st to the 2270th, counting from the N-terminal of V
It corresponds to the amino acid of S-5 protein.

As the insoluble carrier particles in the present invention, any carrier can be used as long as it can be used as a diagnostic agent for a known agglutination method. For example, high specific gravity composite particles obtained by coating a core with an inorganic compound serving as a dye (Japanese Patent Application Laid-Open No. 62-115366,
HDP), sheep red blood cells, polystyrene particles,
Gelatin particles and the like. Preferably HDP, sheep red blood cells,
Polystyrene is used. HDP is more preferred. In addition, the particle size of the insoluble carrier used in the present invention may be any as long as it is within the range used as a diagnostic reagent for agglutination method, preferably from 0.01 μm to 20 μm, more preferably from 0.01 μm to 3 μm belongs to. Further, the specific gravity of the insoluble carrier may be any one, and is preferably from 1.0 to 2.5.

In the present invention, the term "support" means that HC is added to an insoluble carrier.
Any known method of adsorbing the V antigen-active polypeptide may be used, such as a physical adsorption method, a chemical adsorption method, or the like. For example, hydrophobic adsorption, chromium chloride method and the like. Preferably, a hydrophobic adsorption method is desirable. The loading is performed in a buffer solution having a buffering action, and any type may be used. For example, phosphate buffer, glycine buffer, Tris buffer, acetate buffer and the like. The pH may be any, but a neutral region is desirable. Preferably, a phosphate buffer, pH 6.0 to 8.0 is desirable.

When the HCV antigen-active polypeptide is carried on a carrier, the protein concentration is not particularly limited, but is preferably 0.1 μg / ml or more. The time and temperature for supporting the particles on the insoluble carrier particles are not particularly limited, but the temperature is preferably 1 ° C. or more and 80 ° C. or less, and the time is preferably 30 minutes or more, which can be suitably used. The immunological agglutination reagent for diagnosing hepatitis C of the present invention is used in the form of an aqueous suspension, but it is preferable to freeze-dry it for long-term storage. After the freeze-drying, the reagent for agglutination reaction of the present invention exhibits excellent performance without lowering the stability during storage and the breakage of the agglutination image during the reaction, even as an aqueous suspension again. The freeze-drying method is not limited, and may be performed by an ordinary method. For example, a method and conditions adopted for a freeze-drying method of sensitized red blood cells are used. Preferably, a method of rapidly pre-freezing and then vacuum freeze-drying is employed. The rapid preliminary freezing is achieved by immersing a container such as a vial or an ampoule containing the aqueous suspension in liquid nitrogen, dry ice-methanol, dry ice-acetone, fluorocarbon, or the like.

The vacuum freeze-drying method is generally performed by rapidly pre-freezing a vial or the like containing a suspension of the sensitizing carrier, and then cooling the vial in a freeze-dryer chamber which has been cooled to -40 to -60 ° C in advance. The method of gradually raising the temperature over 24 to 72 hours and freeze-drying in a vacuum is preferred. At this time, the pressure in the chamber is preferably 50 to 200 µHg, and the final drying temperature is suitably 20 to 50 ° C. Then, it may be sealed and stored in a vacuum state or filled with an inactivating gas. However, the vacuum freeze-drying method is not limited to the above method.

As the agglutination reagent of the present invention, the agglutination method usually used for diagnosis can be applied without any limitation. For example, a plate method for qualitative diagnosis, a microtiter method for semi-quantitative diagnosis, a turbidimetric method for quantitative diagnosis, a particle counting method, and the like. Among them, the effect of the present invention is particularly remarkable when applied to a microtiter method. The immunological agglutination reagent for diagnosing hepatitis C referred to in the present invention is to diagnose hepatitis C by detecting an anti-HCV antibody present in the serum or plasma of a hepatitis C patient by immunological agglutination. It is a diagnostic reagent. The agglutination method usually used for diagnosis is applied without any limitation. For example, a plate method for qualitative diagnosis, a microtiter method for semi-quantitative diagnosis, a turbidimetric method for quantitative diagnosis, a particle counting method, and the like. Among them, the effect of the present invention is particularly remarkable when applied to a microtiter method.

[0024]

The immunological agglutination reagent for diagnosing hepatitis C according to the present invention has remarkably excellent detection sensitivity and specificity as compared with the conventional product, and can be judged in a short time. In addition, an aggregate formed by an antigen-antibody reaction, which is a criterion for determining the agglutination reaction, (hereinafter, also abbreviated as a tube bottom agglutination image) is very clearly formed. Therefore, the immunological agglutination reagent for diagnosing hepatitis C of the present invention is a diagnostic reagent for hepatitis C which is extremely superior to the conventional reagent.

[0025]

The present invention will be described more specifically below with reference to examples and comparative examples. However, these examples do not limit the technical scope of the present invention. In this example, unless otherwise specified, the genetic engineering experiment was performed according to the method of Sambrook et al. [Sambrook, J., Fritsch, EF,
Maniatis, T., Molecular Cloning, 2nd ed., Cold Spr
ing Harbor Laboratory Press, New York (1989)]. The restriction enzyme used was Takara Shuzo Co., Ltd. product.

Example 1 Production of Core Antigen (1-1) Preparation of Recombinant Plasmid pGC03 (Preparation of RNA) After transfusion, 3000 ml of serum from a non-A non-B hepatitis patient was transfused.
After ultracentrifugation at 19,000 rpm for 16 hours, a precipitate was obtained. The precipitate was dissolved in 100 ml of a GITC solution (4 M guanidium isothiocyanate (manufactured by Fluka), 25 mM sodium citrate, 0.5% sarkosyl, 0.1 M mercaptoethanol), and 100 ml of the lysate was dissolved in 100 ml. Phenol-chloroform (1: 1) was added and shaken at room temperature for 15 minutes.
Centrifuged for 5 minutes. The aqueous layer of the reaction solution was taken out, 100 ml of isopropyl alcohol was added, the mixture was allowed to stand at -20 ° C for 3 hours, and then centrifuged at 3000 rpm for 15 minutes to obtain a precipitate.

To the precipitate was added 10 ml of a GITC solution to prepare a solution. To the solution, 10 ml of phenol-chloroform (1: 1) was added, and the mixture was shaken at room temperature for 10 minutes and then centrifuged at 3000 rpm for 15 minutes. The aqueous layer of the reaction solution was taken out, 20 ml of chloroform was added, and the mixture was shaken for 5 minutes. After shaking, the mixture was centrifuged at 3000 rpm for 5 minutes to recover 10 ml of an aqueous layer. 0.4 ml of a 5M NaCl solution was added to 10 ml of the aqueous layer.

Thereafter, 30 ml of ice-cold ethanol was added,
It was left at −20 ° C. for 12 hours. After standing, the mixture was centrifuged at 3000 rpm for 15 minutes to obtain a precipitate. The precipitate was washed with 75% ethanol, dried, and dissolved in 200 μl of distilled water to obtain an RNA solution. (Construction of cDNA library) cDNA synthesis is performed by BRL
A synthetic kit from the company was used. The method is described in the cDNA synthesis manual [BRL / Cosmo Bio Inc. Instruction Manua
1, Cat. No8267SA]. In this embodiment, (RN
Preparation of A), random primer solution (100 μM) was added to 5 μl of single-stranded RNA solution prepared from non-A, non-B patient serum.
5 µl of [Takara Shuzo Co., Ltd. product catalog number 3810]
In addition, a reverse transcriptase reaction was performed to obtain an RNA / DNA double strand. Next, Escherichia coli DNA polymerase I and Escherichia coli RNase H were added to obtain a DNA / DNA double strand.

Next, EcoRI linkers were bonded to both ends of the double-stranded DNA thus obtained. In this treatment, an enzyme of Takara Shuzo was used, and the reaction was carried out under the reaction conditions attached to the enzyme of Takara Shuzo. First, about 1 μg of double-stranded DNA was treated with EcoRI methylase, and then an EcoRI linker (dGGAATTCC) was bound by a T4 DNA ligase reaction. The reaction solution obtained at the end is cut with EcoRI, and EcoRI is cut.
The fragments were recovered.

Finally, this EcoRI fragment was inserted into the EcoRI site of λgt11 to prepare a recombinant λgt11 phage, using a GIGAPACKII GOLD kit from Stratagene, and the method described in the manual [Protocol / Ins
truction Manual Cat. # 200214, 200215, 200216, Dece
mber 6, 1989]. First, Ec was added to the EcoRI site of λgt11.
The oRI fragment was inserted and ligated with T4 DNA ligase. The obtained recombinant phage DNA solution was used for GIGAPA
The phage was returned using the CKII GOLD In Vitro Packaging Kit. The titer at this time was titrated to 1.0 ×
10 was ⁶ . This titer value indicates the number of independent clones. (Immunoscreening) cDNA inserted into λgt11
When the frames match, β embedded in λgt11
The amino acid sequence encoded by the cDNA is expressed as a fusion protein with galactosidase. This fusion protein was screened with non-A, non-B hepatitis patient serum absorbed into E. coli cells. The indicator bacterium used was E. coli Y1090. 15cm diameter L-bottom plate (Ba per 1 liter of water
cto-tryptone 10g, NaCl 5g, Yeastextract 5g, Bacto-
Agar (15 g) was added, and a phage solution prepared so that the number of plaques per plate was about 40,000 and Y1090 was incubated at 37 ° C for 15 minutes in an autoclave. Then, 2.5 ml of 0.7% L-top agarose warmed to 45 ° C. was mixed, spread on an L-bottom plate, solidified, and incubated at 42 ° C. for 3.5 hours. On the other hand, a nitrocellulose filter was passed through a 10 mM isopropylthio-β-D-galactoside (IP
TG) After dipping in the solution for several minutes, it was dried at room temperature. The filter was placed on the plate and incubated at 37 ° C. overnight. After incubation, remove the filter,
NT buffer (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1 mM
05% Tween20), rinsed well. Again, it was soaked for 30 minutes while shaking in fresh TNT buffer. The filter was further incubated with a blocking buffer (TNT buffer containing 20% fetal calf serum) for 30 minutes. Next, the filter was reacted with a primary antibody solution (a pool serum of a non-A, non-B hepatitis patient absorbed with a sonicated solution of Y1090) diluted 150-fold with a blocking buffer at room temperature for 4 hours with gentle shaking. Then, the filter was washed with TNT buffer containing 0.1% bovine serum albumin (BSA) and 0.1% BS.
A + 10% NP-40-containing TNT buffer and 0.1% BSA-containing TNT buffer were sequentially washed for 10 minutes. Next, 10μ
l of horseradish peroxidase labeled anti-human IgG goat IgG (Kirkeguard & Perry Lab)
Dip the filter in the blocking buffer of
After reacting for 0.1 hour, 0.1% BSA-containing TNT buffer was added.
The plate was washed with a TNT buffer containing 1% BSA + 0.1% NP-40 for 10 minutes. The filter was further filtered with 10 mM Tris-HCl (pH 7.
5) After washing with 150 mM NaCl for 1 minute, stain solution [20 ml of methanol containing 60 mg of 4-chloro-naphthol was used immediately before use.
% H ₂ O 10 mM Tris -HCl containing ₂ of 60μl (pH7.5), 150
mixed with 100 ml of mM NaCl solution] at room temperature for 15 minutes, and washed twice with distilled water to obtain a purple-colored positive plaque.

A phage DNA was prepared from the recombinant phage, treated with EcoRI, and a cDNA fragment was recovered from the agarose electrophoresis gel, and the plasmid vector pUC18 was prepared.
At the EcoRI site. The plasmid was named pGC03, and its nucleotide sequence was determined. This cDNA fragment contains HC
It was revealed that the core region of the structural protein gene of V was included. (1-2) Preparation of E. coli HB101 [pHCX01] After digesting pGC03 with HinfI, DNA polymerase I Kleno
The ends were blunt-ended with w fragment. This DNA and BamH
I linker (dCGGATCCG, manufactured by Takara Shuzo Co., Ltd.) with T4 DN
A ligation reaction was carried out with A ligase, further digested with BamHI, and 0.56 kb including the core region from agarose electrophoresis gel.
The fragments were recovered. This 0.56 kb fragment is converted into a plasmid vector
Insert into the BamHI site of pUC19 and insert the plasmid into BspH
I (New England Biolabs) digested with T4 DN
The ends were blunted with A polymerase. This DNA
After digestion with BamHI, a 0.51 kb core region DNA fragment excluding the 5 ′ untranslated region was recovered from the agarose electrophoresis gel. This 0.51 kb fragment was ligated with the plasmid vector pUEX2 (Amer
sham) to obtain the recombinant vector pHCX01. For the obtained pHCX01, the nucleotide sequence was determined by the plasmid method [Hattori et al., Anal. Bioc.
hem., Vol. 152, pp. 232-238 (1986). This recombinant vector pHCX01 contains a nucleotide sequence encoding the 1st to 168th amino acid sequence from the N-terminus of HCV, and its nucleotide sequence is as shown in SEQ ID NO: 5. Next, host E. coli HB101 was transformed with the recombinant vector pHCX01 to obtain recombinant E. coli HB101 [pHCX01]. Recombinant Escherichia coli HB101 [pHCX01] has been deposited as Microorganism Bacterial Deposit No. 13056 with the Microbial Engineering Research Institute, Ministry of International Trade and Industry, 1-3-1 Higashi, Tsukuba, Ibaraki Prefecture.
This recombinant Escherichia coli HB101 [pHCX01] was added to an LB + Amp medium [Bacto tryptone 1.0%, Yeast extract 0.5%, NaCl 0.5
%, Ampicillin (Amp) 50 µg / ml] at 30 ° C overnight, and glycerin was added to a final concentration of 15%, followed by cryopreservation at -80 ° C. (1-3) Production of Core antigen Core antigen is produced as a fusion polypeptide with β-galactosidase by culturing recombinant Escherichia coli HB101 [pHCX01] and performing gene expression. Recombinant E. coli HB
1 ml of cryopreserved cells of [pHCX01]
B + Amp medium was inoculated and cultured at 30 ° C. overnight. The culture was then inoculated into 20 liters of LB + Amp medium.
Incubate at 30 ° C until the OD540 reaches 1.5, and increase the culture temperature to 42.
The temperature was raised to 0 ° C. and the culture was continued for 3 hours. After the culture, the cells were collected by centrifugation to obtain 57 g of wet cells. The cells were placed in 2 liters of TNE buffer containing 0.6 M urea (50 mM Tris-HCl (pH
8.3), 100 mM NaCl, 1 mM EDTA) and disrupted by sonication. The crushed cells were centrifuged at 10,000 g for 20 minutes to recover insoluble granules containing the Core antigen in a precipitate fraction. The precipitate was suspended again in 2 liters of TNE buffer containing 0.6 M urea to wash the insoluble granules, and the precipitate was recovered by centrifugation. In addition, this precipitate
Suspend in 2 liters of TNE buffer containing 3M urea and at room temperature
After insoluble granules were sufficiently washed by stirring for 30 minutes, the insoluble granules were collected in a precipitate fraction by centrifugation. To the precipitate of the insoluble granules, 200 ml of a TNE buffer containing 8 M urea was added to solubilize the precipitate. 16,000g of this
The supernatant was collected by centrifugation for 20 minutes and dialyzed against TNE buffer. After dialysis, the supernatant was collected by centrifugation at 16,000 g for 20 minutes to obtain a Core antigen. 980 mg of Core antigen was obtained from 20 liters of culture. The molecular weight of the obtained core antigen was examined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and it was confirmed that the obtained antigen coincided with the molecular weight (137 kd) calculated from the amino acid sequence.

(Example 2) Production of NS-3 antigen (2-1) Preparation of recombinant plasmid pHCV7 NS3 of HCV expected to exhibit excellent antigen activity
With respect to the gene fragment in the region, gene amplification was performed by RT-PCR using primers each having 20 bases on both sides of the fragment. Primers were synthesized using a model 340A machine manufactured by Applied Biosystems.
The base sequence of the 5 ′ upstream primer is (5 ′) C
CGACGGTGGATGCTCCGGGG (3 ′),
The nucleotide sequence of the 3 ′ downstream primer is (5 ′) CTGG
AGCCAATCCAACGCCC (3 ').

First, 4 μl of the RNA solution obtained in Example 1 was used.
l) Reverse transcriptase reaction solution [250 mM Tris-HCl (pH8.3), 3
75 mM KCl, 50 mM DTT, 15 mM MgCl2] 2 μl, 3 ′ downstream antisense strand primer solution (25 ng / μl) 1 μl, 4
Kinds of deoxynucleotides [dATP, dGTP, dCTP, dTT
P, 15 mM each) was added in an amount of 0.5 μl to make 9 μl of the solution. Mineral oil was added to this, heated at 70 ° C for 2 minutes, then cooled to 37 ° C, and reverse transcriptase 1 µl (BR
L company product) and reacted at 37 ° C. for 60 minutes. This reaction solution
(10 μl) and a PCR reaction solution [400 mM Tris-HCl (pH 8.
8), 100mM ammonium sulfate, 40mM magnesium chloride, 60mM mercaptoethanol, 0.1% BSA] 8.3μ
l, 4 types of deoxynucleotides [dATP, dGTP, dCT
P, dTTP, 15 mM each] was added in an amount of 5 μl each. Next, a primer solution of 20 bases having the base sequence of the 5 ′ upstream sense strand (100 ng
/ Μl) 5 μl and a 5 μl (100 ng / μl) primer solution of 20 bases having the base sequence of the 3 ′ downstream antisense strand
l) was added, and finally 0.7 μl of water was added to make a total volume of 49 μl. This solution was treated at 92 ° C. for 5 minutes, cooled to room temperature and 1 μl of Taq polymerase (2 units, New England Biola
bs products). Below, annealing (55 ° C, 45 seconds)
, Polymerization (72 ° C, 2 minutes), Denaturation (90 ° C,
1 minute) was repeated 35 times to amplify the DNA.

The H7 fragment of the gene product amplified by the RT-PCR method was electrophoresed on an agarose gel (2%) to recover a DNA of a desired length. Next, Klenow fra
gment Enzyme treatment to make the ends of the DNA smooth,
4 The 5 'end was phosphorylated by polynucleotide kinase. This was inserted into the HincII site of the plasmid vector pTZ19R, and the gene was cloned. Thus, a recombinant plasmid pHCV7 was obtained.

Escherichia coli transformed with the recombinant plasmid pHCV7 is referred to as E. coli HCV7 and deposited with the Microorganisms Industrial Research Institute of the Ministry of International Trade and Industry of the Ministry of International Trade and Industry as Microtechnical Laboratory No. 11831. (2-2) Preparation of Escherichia coli HB101 [pCI07] pHCV7 was digested with EcoRI and StuI.
An 8 bp fragment was obtained. This 338 bp fragment was further partially digested with HinfI and then blunt-ended with DNA polymerase I Klenow fragment to obtain a 263 bp fragment. PHCV7 was replaced by StuI
After digestion with CIP, and digestion with PstI, a 400 bp fragment on the 3 ′ side of cDNA was obtained. On the other hand, pUEX1 (manufactured by Amersham)
Was digested with SmaI and PstI and treated with CIP. This pUEX1 and c
263 bp fragment on the 5 'side of DNA, 400 bp fragment on the 3' side of cDNA
A ligation reaction of the p fragment is performed, and the recombinant vector pC
I got I07. The nucleotide sequence was determined in the same manner as in Example 1,
This recombinant vector pCI07 contains a nucleotide sequence encoding the amino acid sequence at positions 1323 to 1533 as counted from the N-terminus of HCV, and the nucleotide sequence is as shown in SEQ ID NO: 6. Next, host E. coli HB101 is transformed with the recombinant vector pCI07, and the recombinant E. coli HB101 [pCI07] is transformed.
I got Recombinant E. coli HB101 [pCI07] was added to LB + Amp
The cells were cultured overnight at 30 ° C in a medium, and glycerin was added to a final concentration of 15%, followed by cryopreservation at -80 ° C. (2-3) Production of NS-3 Antigen NS-3 antigen is produced as a fusion polypeptide with β-galactosidase by culturing recombinant Escherichia coli HB101 [pCI07] and performing gene expression. Example 1 (1)
-3) In the same manner as in the production of the Core antigen,
Culture of HB101 [pCI07], disruption of cells, and separation and purification of the fusion polypeptide were performed. After culturing in 20 liters of LB + Amp medium, 1,000 mg of NS-3 antigen was obtained. The obtained NS-3 antigen was examined for its molecular weight by SDS-PAGE, and the molecular weight calculated from its amino acid sequence (14
1kd).

Example 3 Production of NS-4 Antigen (3-1) Preparation of Recombinant Plasmid pHCV10 The cDNA library obtained in Example 1 was screened by plaque hybridization. First, using Escherichia coli Y1090 as a host, 10 plates with a diameter of 15 cm,
Recombinant λgt11 phage 5 × 105 of cDNA library
Appeared worthy. The obtained plaque was copied to nitrocellulose and hybridized. Thus, six clones having the HCV gene fragment were selected. Then, phage DNA was recovered from this clone, and then digested with EcoRI. Six types of HCV gene fragments, H1, H5, H10, H13, H20, and H21 fragments were recovered from the agarose electrophoresis gel. Among them, the H10 fragment containing a nucleotide sequence encoding an amino acid sequence essential for excellent antigen activity was converted into a plasmid vector pTZ19R
To obtain a recombinant plasmid pHCV10.

Escherichia coli transformed with the recombinant plasmid pHCV10 is referred to as E. coli HCV10 and has been deposited with the Microorganisms Industrial Research Institute of the Ministry of International Trade and Industry of the Ministry of International Trade and Industry under No. 11834. (3-2) Preparation of Escherichia coli HB101 [pCI10] After digesting pHCV10 with AvaII, DNA polymerase I Kleno
The end was blunt-ended with w fragment and further digested with BamHI to isolate a 583 bp fragment. On the other hand, pUEX3 (manufactured by Amersham) was digested with SmaI, treated with CIP, and further digested with BamHI. Thereafter, electrophoresis was performed to separate the target fragment.
These were ligated to produce a recombinant vector pCI10. The nucleotide sequence was determined in the same manner as in Example 1. This recombinant vector pCI10 contains the nucleotide sequence encoding the amino acid sequence at positions 1605 to 1798, counted from the N-terminus of HCV. As shown in SEQ ID NO: 7. Next, the host E. coli HB was transformed with the recombinant vector pCI10.
Was transformed to obtain recombinant Escherichia coli HB101 [pCI10]. Recombinant Escherichia coli HB101 [pCI10] was cultured overnight in LB + Amp medium at 30 ° C., glycerin was added to a final concentration of 15%, and stored frozen at −80 ° C. (3-3) Production of NS-4 Antigen NS-4 antigen is produced as a fusion polypeptide with β-galactosidase by culturing recombinant Escherichia coli HB101 [pCI10] and performing gene expression. Example 1 (1)
-3) In the same manner as in the production of the Core antigen,
Culture of HB101 [pCI10], disruption of cells, and separation and purification of the fusion polypeptide were performed. After culturing in 20 liters of LB + Amp medium, 720 mg of NS-4 antigen was obtained. The molecular weight of the obtained NS-4 antigen was determined by SDS-PAGE, and the molecular weight calculated from the amino acid sequence (141 k
d) was confirmed to match.

Example 4 Production of NS-5 Antigen (4-1) Preparation of Recombinant Plasmid pHCV14 Amplified by RT-PCR in the same manner as in Example 2.
The H14 fragment of the gene product of the NS5 region of HCV was electrophoresed on an agarose gel (2%) to recover the DNA of the desired length. The base sequence of the 5 ′ upstream primer is
(5 ') CGGGCATGACCACTGACAAC
(3 ′) The base sequence of the 3 ′ downstream primer is (5 ′)
CCGCTCTCTAGGACGCTTTTG (3 ′). This is then treated with Klenow fragment enzyme, and DN
The ends of A were made blunt, and the 5 'end was phosphorylated with T4 polynucleotide kinase. This was inserted into the HincII site of the plasmid vector pTZ19R to obtain a recombinant plasmid pHCV14. Escherichia coli transformed with the recombinant plasmid pHCV14 is referred to as E. coli HCV14 and submitted to
Deposited as No. 8. (4-2) Preparation of Escherichia coli HB101 [pCI14] After digesting pHCV14 with PstI and XbaI, the ends were blunted using a blunting kit, and a fragment containing 484 bp was isolated. On the other hand, pUEX2
Was digested with SmaI and treated with CIP. Thereafter, electrophoresis was performed to separate the target fragment. These were ligated to produce a recombinant vector pCI14. The nucleotide sequence was determined in the same manner as in Example 1. This recombinant vector pCI14 contains the nucleotide sequence encoding the 2111st to 2270th amino acid sequence counted from the N-terminus of HCV. As shown in SEQ ID NO: 8. Next, host E. coli HB101 was transformed with the recombinant vector pCI14 to obtain recombinant E. coli HB101 [pCI14]. Recombinant E. coli HB101
[PCI14] was cultured in LB + Amp medium at 30 ° C. overnight, and glycerin was added to a final concentration of 15% at −80 ° C.
And stored frozen. (4-3) Production of NS-5 antigen The NS-5 antigen is produced as a fusion polypeptide with β-galactosidase by culturing recombinant Escherichia coli HB101 [pCI14] and performing gene expression. Example 1 (1)
-3) In the same manner as in the production of the Core antigen,
Culture of HB101 [pCI14], disruption of cells, and separation and purification of the fusion polypeptide were performed. After culturing in 20 liters of LB + Amp medium, 750 mg of NS-5 antigen was obtained. With respect to the obtained NS-5 antigen, the molecular weight of the obtained NS-4 antigen was examined by SDS-PAGE, and it was confirmed that the obtained molecular weight coincided with the molecular weight (135 kd) calculated from the amino acid sequence.

(Example 5) Immunological agglutination reagent for diagnosing hepatitis C using HCV antigen-active polypeptide (heating operation) Core antigen, NS-3 antigen, NS-4
100 μg / ml of each of the four HCV antigen-active polypeptides of the antigen and the NS-5 antigen are dispersed in PBS in equal amounts to form a mixed antigen solution. The antigen mixed solution is heated at 35 ° C. for 30 minutes to obtain a sensitized antigen solution. (Sensitization) HDP (product of Tokuyama Soda Co., Ltd.) having a diameter of 1.8 μm is adjusted to 5% (w / w) with PBS. To give an HDP suspension. 1 ml of the above HDP suspension and 1 ml of the sensitizing antigen solution subjected to the heating operation were mixed in a test tube, and allowed to stand at room temperature for 1 hour.
The antigen-active polypeptide was adsorbed hydrophobically (hereinafter, this adsorption operation is also abbreviated as sensitization).

(Washing operation) Thereafter, in order to remove excess HCV antigen-active polypeptide, 2,500
After centrifugation at 5 rpm for 5 minutes, the centrifugal supernatant was removed.
To the centrifuged precipitate, 2 ml of PBS was added for washing, the suspension was centrifuged at 2,500 rpm for 5 minutes, and the supernatant was removed.
/ vol)% denatured rabbit serum in PBS (hereinafter abbreviated as solution A) so as to be 0.5 (w / vol)%. The above-described HDP (hereinafter, also abbreviated as sensitized particles) adsorbed with the HCV polypeptide was used as an immunological agglutination reagent for hepatitis C diagnosis (hereinafter, abbreviated as solution B).

(Measurement operation) On the other hand, the sample
The solution was diluted by a factor of more than 2 and diluted by a factor of 8192. Next, a diluted solution of the sample was dropped in a 96-well micro-titer-plate in an amount of 25 μl each from 1 to 12 holes. Then, the solution B prepared above was dropped in 25 μl of each well. After dropping, plate mixer (plat
After shaking with an e-mixer for 30 minutes, the tube bottom aggregation image was observed. Among the tube bottom agglutination images, those in which the sensitizing particles spread to the bottom of the microplate due to the occurrence of the antigen-antibody reaction were defined as positive images. Was set as a negative image. In general, the detection sensitivity of a microtiter reagent is indicated by the highest dilution ratio of serum and plasma in which a positive image is observed (hereinafter, also abbreviated as titer).
The sensitivity of the immunological agglutination reagent for diagnosing hepatitis B is indicated by the titer. In addition, it is said that this titer is lower in a healthy subject sample and higher as the patient sample is higher.

(Results) Next, a commercial product A, which is an ELISA reagent, was compared with the immunological agglutination reagent for hepatitis C diagnosis prepared in Example 5. Five healthy subject samples and five patient serum samples were used for the study. The commercial product A and the immunological agglutination reagent for diagnosing hepatitis C of the present invention showed a good correlation. Among them, only one sample showed a positive result only with the immunological agglutination reagent for diagnosing hepatitis C. . The judgment of positive and negative is 32
Those showing a positive image from the double dilution were regarded as positive (see Table 1).

Thirty additional normal and patient samples were measured (see Table 2). Two samples were determined to be positive for the immunological agglutination reagent for hepatitis C diagnosis of Example 5 and negative for the commercial product A.

[0044]

[Table 1]

[0045]

[Table 2]

Comparative Example 1 Immunological agglutination reagent for diagnosing hepatitis C using HCV antigen-active polypeptide not subjected to heat treatment (mixing operation) Core antigen, NS-3 antigen, NS-4 antigen and NS-5 100 µg / ml of each of the four HCV antigen-active polypeptides of the antigen is dispersed in PBS in an equal amount, and a mixed antigen solution is prepared as a sensitive antigen solution.

(Sensitization) HDP having a diameter of 1.8 μm (manufactured by Tokuyama Soda Co., Ltd.) was suspended in PBS so as to have a concentration of 5 (w / w)% to prepare an HDP suspension. 1 ml of the sensitizing antigen solution was placed in a test tube, mixed with the HDP suspension, and left at room temperature for 1 hour to sensitize the HCV antigen-active polypeptide mixed with the HDP surface.

(Washing operation) Excess HCV antigen-active polypeptide was removed by the same operation as in Example 5 and suspended in Solution A so that the particle concentration became 0.5 (w / vol)%. The above-mentioned sensitized particles are used for immunological agglutination reagent for hepatitis C diagnosis (hereinafter referred to as C
Liquid). (Measurement procedure) As in Example 5, the sample to be used for the test was diluted by a factor of 2 more with solution A, diluted to 8192 times with solution C and D
After the solution, the solution E, and the solution F were dropped, a tube bottom aggregation image was observed.

(Results) In the examination, a specimen having a titer of 8192 times (hereinafter, referred to as the immunological agglutination reagent for hepatitis C diagnosis in Example 5) was used.
A sample with a titer of 8 times (hereinafter abbreviated as a healthy subject sample 1) was used. HC above
The immunological agglutination reagent for diagnosis of hepatitis C sensitized with the V antigen-active polypeptide showed a lower titer than the immunological agglutination reagent for diagnosis of hepatitis C prepared in Example 5 (Table 3).
reference).

[0050]

[Table 3]

Comparative Example 2 Immunological agglutination reagent for hepatitis C diagnosis using one kind of HCV antigen-active polypeptide (heat treatment) Core antigen, NS-3 antigen, NS-4 antigen and NS-5 antigen Each of the four HCV antigen-active polypeptides was dispersed in PBS so as to have a concentration of 100 μg / ml, and heat-treated at 35 ° C. for 30 minutes to give a sensitive Core antigen solution, a sensitive NS-3 antigen solution, and a sensitive NS antigen solution. -4 antigen solution and sensitized NS-5 antigen solution were prepared respectively.

(Sensitization) HDP having a diameter of 1.8 μm (manufactured by Tokuyama Soda Co., Ltd.) was suspended in PBS to a concentration of 5 (w / w)% to give an HDP suspension. The above-mentioned sensitive Core antigen solution,
1 ml each of the sensitized NS-3 antigen solution, the sensitized NS-4 antigen solution and the sensitized NS-5 antigen solution are placed in separate test tubes, mixed with the HDP suspension, and left at room temperature for 1 hour to obtain HD.
Each HCV antigen active polypeptide was sensitized to the P surface.

(Washing operation) Excess HCV antigen-active polypeptide was removed by the same operation as in Example 5, and suspended in Solution A so that the particle concentration became 0.5 (w / vol)%. The above-mentioned sensitized particles are used as an immunological agglutination reagent for hepatitis C diagnosis (hereinafter, referred to as "hepatitis C").
D, E, F and G solutions). (Measurement procedure) In the same manner as in Example 5, the sample to be used for the test was diluted by a factor of 2 with solution A, diluted to 8192 times, and then diluted with solution D and E.
After the solution, the solution F and the solution G were dropped, a tube bottom aggregation image was observed.

(Results) Patient 1 and healthy subject 1 were used for the examination. The immunological agglutination reagent for diagnosing hepatitis C sensitized with the above HCV antigen-active polypeptide showed a lower titer than the immunological agglutination reagent for diagnosing hepatitis C prepared in Example 5 (Table 4). reference).

[0055]

[Table 4]

Comparative Example 3 Immunological agglutination reagent for diagnosing hepatitis C using two kinds of HCV antigen-active polypeptides (heat treatment) Core antigen and NS-3 antigen, Core antigen and NS-4 antigen, Core antigen And NS-5 antigen, NS
HCV of two types each of -3 antigen and NS-4 antigen, NS-3 antigen and NS-5 antigen, and NS-4 antigen and NS-5 antigen
50 μg / ml each of antigen-active polypeptides in PBS
And heat-treated at 35 ° C. for 30 minutes to obtain sensitized antigen solutions 1, 2, 3, 4, 5, and 6, respectively.

(Sensitization) HDP having a diameter of 1.8 μm (manufactured by Tokuyama Soda Co., Ltd.) was suspended in PBS to a concentration of 5 (w / w)% to give an HDP suspension. 1 ml of the above HDP suspension and 1 ml of the sensitizing antigen solution were mixed in a test tube and allowed to stand at room temperature for 1 hour to sensitize the HDP surface with two types of HCV antigen-active polypeptides.

(Washing operation) Excess HCV antigen-active polypeptide was removed by the same operation as in Example 5, and suspended in Solution A so that the particle concentration became 0.5 (w / vol)%. The above-mentioned sensitized particles are used as an immunological agglutination reagent for hepatitis C diagnosis (hereinafter, referred to as "hepatitis C").
H, I, J, K, L and M solutions).

(Measurement procedure) In the same manner as in Example 5, the patient sample 1 and the healthy subject sample 1 used for the test were diluted by a factor of 2 with the solution A, and diluted by a factor of 8192 to H, I, J, K, and L. After dropping solution M and solution M, a tube bottom aggregation image was observed. (Results) Both the immunological agglutination reagents for diagnosing hepatitis C sensitized with two types of HCV antigen-active polypeptides each had a lower titer than the immunological agglutination reagent for diagnosing hepatitis C prepared in Example 5. (See Table 5).

[0060]

[Table 5]

Comparative Example 4 Immunological agglutination reagent for hepatitis C diagnosis using three types of HCV antigen-active polypeptides (heat treatment) Core antigen, NS-3 antigen and NS-4 antigen, Core antigen and NS- 3 antigen and NS-5 antigen, Co
three types of HCV: re antigen, NS-4 antigen and NS-5 antigen, and NS-3 antigen, NS-4 antigen and NS-5 antigen
33 μg / ml of each of the antigen-active polypeptides was added to PB
S are dispersed in equal amounts and subjected to heat treatment at 35 ° C. for 30 minutes to obtain sensitized antigen solutions 7, 8, 9 and 10.

(Sensitization) HDP having a diameter of 1.8 μm (manufactured by Tokuyama Soda Co., Ltd.) was suspended in PBS to a concentration of 5 (w / w)% to give an HDP suspension. 1 ml of the above HDP suspension and 1 ml of each sensitizing antigen solution were mixed in a test tube and allowed to stand at room temperature for 1 hour to sensitize the HDP surface with three types of HCV antigen-active polypeptides.

(Washing Operation) Excess HCV antigen-active polypeptide was removed by the same operation as in Example 5, and suspended in Solution A so that the particle concentration became 0.5 (w / vol)%. The above-mentioned sensitized particles are used as an immunological agglutination reagent for hepatitis C diagnosis (hereinafter, referred to as "hepatitis C").
N, O, P and Q liquids). (Measurement operation) Patient sample 1 used for examination as in Example 5
Then, the healthy subject sample 1 was diluted with the solution A by a factor of 2 or more, diluted to 8192 times, and the N, 0, P, and Q solutions were dropped, and the tube bottom aggregation image was observed.

(Results) The immunological agglutination reagents for diagnosing hepatitis C sensitized with three types of HCV antigen-active polypeptides were lower than the immunological agglutination reagents for diagnosing hepatitis C prepared in Example 5. The titers are shown (see Table 6).

[0065]

[Table 6]

(Example 6) Immunological agglutination reagent for diagnosis of hepatitis C using a sheep red blood cell carrier (fixation of sheep red blood cells) 100 ml of sheep red blood cells, 100 m of Orseba liquid
After the mixture was subjected to gauze filtration and the blood cell concentration was measured, the mixture was washed with physiological saline. The above blood cells were fixed in formalin (Methods in Immunology and Immunochemistory,
vol, pp33-34 (1977) (Williams, C, Hase ed.Academic
Press New York)) (Heat treatment) Core antigen, NS-3 as in Example 5.
A solution in which four HCV antigen-active polypeptides of the antigen, NS-4 antigen and NS-5 antigen were mixed in PBS in equal amounts of 100 μg / ml each was heated at 35 ° C. for 30 minutes (hereinafter, also referred to as R solution). Abbreviated).

(Sensitization) 3 (vol / vol) of washed red blood cells
After adding a% formalin-physiological saline solution and stirring at 10 ° C for 24 hours, a 40 (vol / vol)% formalin-physiological saline solution was further added and stirred for 24 hours. After washing with physiological saline, the cells were suspended at a concentration of 2.5 (vol / vol)% to obtain fixed sheep erythrocytes.

1 ml of the above solution Q and 1 ml of a fixed sheep erythrocyte solution diluted to 5% (w / w) with PBS were reacted at 37 ° C. for 1 hour with stirring. By this operation, the four kinds of HCV antigen-active polypeptides were sensitized to the fixed sheep erythrocytes. (Washing operation) Next, a washing operation was performed in the same manner as in Example 5,
Excess HCV antigen-active polypeptide was removed, and the suspension was suspended in solution A to a particle concentration of 0.5 (w / vol)%. The above-mentioned sensitized particles are used as an immunological agglutination reagent for hepatitis C diagnosis (hereinafter, referred to as "hepatitis C").
S solution).

(Measurement procedure) In the same manner as in Example 5, the patient sample 1 and the healthy subject sample 1 to be used for the test were diluted by a factor of 2 with the solution A, diluted by a factor of 8 to 8192 times, and the solution R was added dropwise. The image was observed. (Results) As in Example 5, the commercially available product A was compared with the immunological agglutination reagent for hepatitis C diagnosis prepared in Example 6.
Five healthy subject samples and five patient serum samples were used for the study. The commercially available product A and the immunological agglutination reagent for diagnosing hepatitis C showed a good correlation. Among them, only one sample showed a positive result only with the immunological agglutination reagent for diagnosing hepatitis C. In addition, the thing which showed a positive image from the 32-fold dilution was judged to be positive (see Table 7).

[0070]

[Table 7]

Comparative Example 5 Comparison of ELISA Reagent and Immunological Agglutination Reagent (Heat Treatment) Core antigen, NS-3 antigen, NS-4 antigen and NS-5 antigen were adjusted to 100 μg / ml by PB.
After dispersing with S, a heat treatment was performed at 35 ° C. for 30 minutes.
These are referred to as heat-treated Core antigen, heat-treated NS-3 antigen, heat-treated NS-4 antigen, and heat-treated NS-5 antigen, respectively. Further, after the Core antigen, NS-3 antigen, NS-4 antigen and NS-5 antigen are mixed in equal amounts of 25 μg / ml, heat treatment is performed at 35 ° C. for 30 minutes to obtain a heat-treated antigen solution.

(Adsorption to Microtiter Plate) Heat-treated Core antigen, heat-treated NS-3 antigen, heat-treated NS-4 antigen and heat-treated NS-5 antigen were added to a microtiter plate (product of Nunc) at 50 μl per well. Was dispensed. Similarly, the heat-treated antigen solution was dispensed into a microtiter plate at 50 μl per well. The microtiter plate was adsorbed at 37 ° C. for 1 hour. After the adsorption, the plate was washed three times with 200 μl of PBS.

(Blocking operation) PBS containing 1% bovine blood albumin (hereinafter referred to as BS) was placed on the microtiter plate.
A solution) is dispensed into each well in an amount of 50 μl and
Time blocked. After blocking, the BSA solution was removed. (Primary antibody reaction) Patient sample 1 and healthy subject sample 1 10μ each
After 10-fold dilution with BSA solution, dispensed into each well
The reaction was performed at 7 ° C. for 1 hour.

(Washing operation) After the completion of the primary antibody reaction, the diluent of each sample was removed. The plate was washed three times with 200 μl of a PBS solution containing 0.5% tween 80 (hereinafter abbreviated as a washing solution). (Secondary antibody reaction) 100 parts of peroxidase-labeled anti-human IgG (manufactured by Capel) diluted 20,000-fold with BSA solution
Each μl was dispensed into each well and reacted at 37 ° C. for 1 hour.

(Washing operation) After the completion of the primary antibody reaction, the diluent of each sample was removed. The plate was washed three times with 200 μl of a PBS solution containing 0.5% Tween 80 (hereinafter abbreviated as a washing solution). (Coloring operation) Hydrogen peroxide aqueous solution (Kappel product) and AB
After adding 100 μl of a mixed solution of an equal volume of TS solution (manufactured by Capel), the mixture was reacted at room temperature for 30 minutes. After the reaction, 100 μl of a 10% sodium dodecyl sulfate solution was added to stop the reaction, and the absorbance (wavelength: 414 nm) of the reaction solution was measured.

(Results) Core antigen, NS-3 antigen, N
The hole in which the S-4 antigen and the NS-5 antigen were respectively adsorbed was compared with the hole in which the four kinds of HCV antigen-active polypeptides were mixed, and the absorbance was always the same, and the sensitivity was as high as that seen with the agglutination reagent. Hepatitis C diagnostic reagent could not be prepared (see Table 8)

[0077]

[Table 8]

[0078]

[Sequence list]

1. SEQ ID NO: 1 (1) Sequence length: 168 (2) Sequence type: amino acid (3) Topology: linear (4) Sequence type: protein (5) Origin Organism name: HCV (hepatitis C virus) 2. SEQ ID NO: 2 (1) Sequence length: 211 (2) Sequence type: amino acid (3) Topology: linear (4) Sequence type: protein (5) Origin Organism name: HCV (hepatitis C virus) 3. SEQ ID NO: 3 (1) Sequence length: 194 (2) Sequence type: amino acid (3) Topology: linear (4) Sequence type: protein (5) Origin Organism name: HCV (hepatitis C virus) 4. SEQ ID NO: 4 (1) Sequence length: 160 (2) Sequence type: amino acid (3) Topology: linear (4) Sequence type: protein (5) Origin Organism name: HCV (hepatitis C virus) 5. SEQ ID NO: 5 (1) Sequence length: 504 (2) Sequence type: nucleic acid (3) Number of chains: double-stranded (4) Topology: linear (5) Sequence type: cDNA to genomic RNA ( 6) Origin Organism name: HCV (hepatitis C virus) (7) Sequence characteristics: Symbol indicating characteristics: peptide Location: 1..504 Method for determining characteristics: E 6. SEQ ID NO: 6 (1) Sequence length: 633 (2) Sequence type: nucleic acid (3) Number of chains: double-stranded (4) Topology: linear (5) Sequence type: cDNA to genomic RNA ( 6) Origin Organism name: HCV (hepatitis C virus) (7) Sequence characteristics: Symbol indicating characteristics: peptide Location: 1..633 Method for determining characteristics: E 7. SEQ ID NO: 7 (1) Sequence length: 582 (2) Sequence type: nucleic acid (3) Number of strands: double-stranded (4) Topology: linear (5) Sequence type: cDNA to genomic RNA ( 6) Origin Organism name: HCV (hepatitis C virus) (7) Sequence characteristics: Symbol indicating characteristics: peptide Location: 1..582 Method for determining characteristics: E 8. SEQ ID NO: 8 (1) Sequence length: 480 (2) Sequence type: nucleic acid (3) Number of chains: double-stranded (4) Topology: linear (5) Sequence type: cDNA to genomic RNA ( 6) Origin Organism name: HCV (hepatitis C virus) (7) Sequence characteristics: Symbol indicating characteristics: peptide Location: 1..480 Method for determining characteristics: E

Continuation of the front page (58) Field surveyed (Int. Cl. ⁷ , DB name) G01N 33/576 BIOSIS (DIALOG) WPI (DIALOG)

Claims (3)

(57) [Claims] 1. An immunological agglutination reagent for diagnosing hepatitis C, which comprises using, as an antigen, an HCV antigen-active polypeptide of a gene derived from hepatitis C virus, which has been heat-treated at 35 ° C. or higher and 50 ° C. or lower. 2. An HCV antigen-active polypeptide, wherein the HCV antigen-active polypeptide comprises the amino acid sequence of SEQ ID NO: 1.
HCV antigen active polypeptide comprising the amino acid sequence of SEQ ID NO: 2, HCV antigen active polypeptide comprising the amino acid sequence of SEQ ID NO: 3 and H comprising the amino acid sequence of SEQ ID NO: 4
The immunological agglutination reagent for diagnosing hepatitis C according to claim 1, comprising a CV antigen-active polypeptide. 3. An HCV comprising the amino acid sequence of SEQ ID NO: 1.
Antigen active polypeptide, HCV antigen active polypeptide comprising the amino acid sequence of SEQ ID NO: 2, HCV antigen active polypeptide comprising the amino acid sequence of SEQ ID NO: 3 and SEQ ID NO: 4
3. An HCV antigen-active polypeptide comprising the amino acid sequence of claim 1 supported on insoluble carrier particles.
The immunological agglutination reagent for hepatitis C diagnosis according to the above.