JPH07179493A - Antigen peptide compound and immunoassay - Google Patents

Abstract

PURPOSE:To obtain a new antigen peptide compound having a specific amino acid sequence and capable of specifically bonding with hepatitis C virus antibody, determining the serotype of the specimen while suppressing the cross- reaction and non-specific reaction and accurately estimating the effect of interferon therapy. CONSTITUTION:This new antigen peptide compound contains a sequence consisting of at least 6 consecutive amino acids and included in the amino acid sequence of formula, etc., and is useful for the determination of HCV antibody in a specimen by immunoassay taking advantage of its specific bonding property with hepatitis C virus (HCV) antibody and capable of accurately estimating the effect of interferon therapy. The compound can be produced by concentrating viral particles from the plasma of chimpanzee affected by non-A non-B hepatitis, extracting RNA from the virus, synthesizing a cDNA using the RNA as a template, preparing a cDNA library by conventional method, selecting a positive clone by immuno-screening, etc., recovering a DNA from the clone and carrying out a genetic engineering procedure (recombinant method) using the recovered DNA.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an antigenic peptide compound useful for measuring HCV (hepatitis C virus) related antibody and an immunological measuring method.

[0002]

2. Description of the Related Art There are various viruses on the earth, some of which are pathogens. For example, the hepatitis virus, which causes viral hepatitis, is known to be transmitted during blood transfusion, injection, or childbirth.

[0003] Blood transfusion is an important means for sustaining human life, and many cases of viral hepatitis caused by this blood transfusion have been reported to date. This viral hepatitis is generally classified into A type, B type and non-A non-B type.

In the past, hepatitis B virus (HB
V) was also known as a cause of viral hepatitis at the time of blood transfusion. In recent years, a test reagent for detecting the presence of HBV has been developed, and it has become possible to easily confirm the presence of HBV before blood transfusion. On the other hand, hepatitis (non-A non-B type; non-A non-B type) caused by hepatitis A virus (HAV), which has been considered as a cause of hepatitis until now, is neither viral type A nor HBV type Although its existence has been known, it has been conventionally difficult to confirm the source (virus).

In 1989, Choo, QL. Et al. (Science,
244 , 359-362, 1989) and Kuo, G. et al. (Science, 2
44 , 362-364, 1989) revealed the presence of non-A non-B hepatitis virus. This hepatitis virus is HC
Although named V (hepatitis C virus), it has been previously reported that this HCV infects only humans and chimpanzees.

The above-mentioned literature describes the following experiments. That is, non-A non-B when administered to a patient
The same preparation as the blood coagulation factor VIII preparation that caused hepatitis C was administered to chimpanzee-No. 1 to cause non-A non-B hepatitis. Further, the extract from the liver of the chimpanzee was administered to chimpanzee No. 2 to cause non-A non-B hepatitis in the same manner, and then the plasma of chimpanzee No. 2 was extracted. In addition, the plasma of this chimpanzee-No.
It was administered to No. 3 and the onset of non-A non-B hepatitis was confirmed. Assuming that the target virus is flavinovirus, plasma particle concentration operation for plasma confirmed to have this onset
(Bradley, DW et al .: Gastroenterology, 88 : 773-779,
1989) to extract viral RNA. CDNA was synthesized based on the RNA thus obtained, and λgt
11 libraries were prepared. This λgt11 library was subjected to immunoscreening using a chimpanzee serum in the convalescent stage or a serum of a non-A non-B chronic hepatitis patient to select a clone to react with. As a result, it was confirmed that only the clone called 5-1-1 was a cDNA fragment derived from HCV.

The above-mentioned operation is performed according to Japanese Patent Publication No. 2-500880.
It is also described in the publication, and the gene sequence is clarified. In addition, a report on the gene sequence of HCV has been made in Japan (Journal of virology, 65 (3) , 11 ) .
05 ~ 1113, 1991).

[0008]

[Problems to be Solved by the Invention] At the Japan Society of Hepatology in June 1990, the gene sequence of the HCV structural region was clarified by Okamoto et al. Of Jichi Medical University (26th Annual Meeting of the Japan Society of Hepatology, 1990). . At the Japan Cancer Society in July 1990, the gene sequence of the structural region of HCV was clarified as in Okamoto et al. (49th Annual Meeting of the Japan Cancer Society, 1990). Comparing these presentations, HC
There are few gene mutations in the V core region,
Regarding the gene sequence of the HCV envelope region, some differences are observed between the two.

Then, the core antigen of the HCV structural region and HC
An antibody test in combination with the antigenic site of the V non-structural region has been proposed (clinical pathology, 40 (12) , 1245-1251, 1992), and such an antibody test almost completely screens HCV antibody-positive individuals. It has become possible.

On the other hand, the treatment of hepatitis C with interferon has come to be performed in parallel with the HCV test.
It has been found that the effect of interferon treatment differs depending on the genotype of HCV. This geno
Type classification is Okamoto H .: Hepatobiliary and pancreas, 24 , 7-14 (1992)
Type I (Proc. Natl. Aca
d. Sci. USA, 88, 2451-2455, 1991), Type II (Journal
of virology, 65 (3) , 1105-1113, 1991), type III (Journal of General Virology, 72 , 2697-2704,199
1) and type IV (Virology, 188 , 331-341, 1992). At the Japan Society of Hepatology held in June 1993, genotype type I and type II (interferon treatment: 20% effective) have lower therapeutic effect of interferon than genotype type III and type IV (interferon treatment: 80% effective). It was reported (29th Annual Meeting of the Japanese Society of Hepatology, page 55, 1993).

Since the administration of interferon is often accompanied by strong side effects such as hair loss and fever, which imposes a heavy burden on patients, determination of HCV genotype before treatment is extremely important for predicting the interferon administration effect. However,
According to the conventional genotype determination method, “HC in patient sample is
V RNA extraction, corresponding cDNA synthesis, c
Amplification of DNA by PCR (polymerase chain reaction) method, confirmation of band of amplified cDNA using electrophoretic method "
After a complicated process that usually lasts over 48 hours
Since the genotype was decided, long time and high cost were required.

On the other hand, using an antigen (consisting of about 300 amino acids) produced by the method of gene recombination,
It has been reported that HCV antibodies in patient serum can be classified into two types (hepato-biliary-pancreatic, 22 , 883-889,
(1991), but this classification was not always sufficient in terms of correspondence with the genotype (which is important for determining interferon treatment) described above.

As described above, administration of interferon costs a large amount of money, but there is a drawback in that determination of the genotype, which is a prerequisite for administration of interferon, also costs a large amount of money. If the genotype of HCV can be easily determined before treatment, the effect of interferon administration can be easily predicted, and the interferon administration method and treatment guideline can be systematically formulated. A simple HCV genotype that can reduce the mental, physical and financial burden on the patient.
The decision was longed for.

The object of the present invention is therefore the HCV geno.
HC that enables easy measurement of HCV antibody capable of distinguishing between type I and II types and genotype III and IV types
To provide an antigen for measuring V antibody.

Another object of the present invention is HCV genotype I.
It is an object of the present invention to provide an antigen for HCV antibody measurement, which is capable of distinguishing between the type II and type II and the genotype type III and type IV, and which is capable of measuring an HCV antibody with suppressed cross-reaction or non-specific reaction.

Still another object of the present invention is HCV genoty.
An object of the present invention is to provide a simple method for measuring an HCV antibody capable of distinguishing between pe type I and type II and genotype type III and type IV.

Still another object of the present invention is HCV genoty.
It is an object of the present invention to provide a method for measuring an HCV antibody capable of distinguishing pe type I and type II from genotype type III and type IV and suppressing cross-reaction or non-specific reaction.

Still another object of the present invention is HCV genoty.
An object of the present invention is to provide a low-cost method for measuring an HCV antibody capable of distinguishing pe type I and type II from genotype type III and type IV.

[0019]

As a result of earnest research, the present inventor determines the HCV antibody type (serotype) using a specific amino acid sequence (a sequence containing at least 6 amino acids) based on the HCV antigen site. That is the HCV genotype
Achievement of the above-mentioned object by not only enabling simple distinction between type I and type II and genotype type III and type IV, but also effectively suppressing cross-reaction or non-specific reaction associated with antigen-antibody reaction It was found to be extremely effective for.

The antigenic peptide compound of the present invention is based on the above findings, and more specifically, it is an amino acid sequence contained in the sequence represented by the following formula (1), which is a sequence consisting of at least 6 consecutive amino acids. It is characterized by including.

Leu- Ser- Gly- Arg- Pro- Ala- Ile- Val- Pro-Asp- Arg- Glu- Val- Leu- Tyr- Gln- Glu- Phe-Asp- Glu ... 1) According to the present invention, further, an amino acid sequence contained in the sequence represented by the following formula (2), which has at least 6 consecutive amino acid sequences
There is provided an antigenic peptide compound characterized in that it comprises a sequence of amino acids.

Val- Asn- Gln- Arg- Ala- Val- Val- Ala- Pro-Asp- Lys- Glu- Val- Leu- Tyr- Glu- Ala- Phe-Asp- Glu ... .. (2) According to the present invention, further, at least 6 consecutive amino acid sequences contained in the sequence represented by the following formula (3):
There is provided an antigenic peptide compound characterized in that it comprises a sequence of amino acids.

Cys- Thr- Thr- His- His- Val- Ser- Pro- Asp-Ala- Asp- Leu- Ile- Glu- Ala- Asn- Leu- Leu-Trp- Arg ... .. (3) According to the present invention, further, an amino acid sequence included in the sequence represented by the following formula (4), which has at least 6 consecutive amino acids.
There is provided an antigenic peptide compound characterized in that it comprises a sequence of amino acids.

Cys- Thr- Thr- His- Gly- Lys- Ala- Tyr- Asp-Val- Asp- Met- Val- Asp- Ala- Asn- Leu- Phe-Met- Gly ... (4) According to the present invention, further, at least 6 consecutive amino acid sequences contained in the sequence represented by the following formula (5):
There is provided an antigenic peptide compound characterized in that it comprises a sequence of amino acids.

Gln- Glu- Phe- Asp- Glu- Met- Glu- Glu- Cys-Ala- Ser- His- Leu- Pro- Tyr- Ile- Glu- Gln-Gly- Met ... (5) According to the present invention, further, an amino acid sequence contained in the sequence represented by the following formula (6), which has at least 6 consecutive amino acid sequences:
There is provided an antigenic peptide compound characterized in that it comprises a sequence of amino acids.

Glu- Ala- Phe- Asp- Glu- Met- Glu- Glu- Cys-Ala- Ser- Arg- Ala- Ala- Leu- Ile- Glu- Glu-Gly- Gln ... (6) According to the present invention, further, at least 6 consecutive amino acid sequences contained in the sequence represented by the following formula (7):
There is provided an antigenic peptide compound characterized in that it comprises a sequence of amino acids.

Gly- Arg- Arg- Gln- Pro- Ile- Pro- Lys- Ala-Arg- Arg- Pro- Glu- Gly- Arg- Thr- Trp- Ala-Gln- Pro ... (7) According to the present invention, further, at least 6 consecutive amino acid sequences contained in the sequence represented by the following formula (8):
There is provided an antigenic peptide compound characterized in that it comprises a sequence of amino acids.

Gly- Arg- Arg- Gln- Pro- Ile- Pro- Lys- Asp-Arg- Arg- Ser- Thr- Gly- Lys- Ser- Trp- Gly-Lys- Pro ... (8) According to the present invention, further, at least 6 consecutive amino acid sequences contained in the sequence represented by the following formula (9):
There is provided an antigenic peptide compound characterized in that it comprises a sequence of amino acids.

Ile- Ile- Leu- Ser- Gly- Arg- Pro- Ala- Ile-Val- Pro- Asp- Arg- Glu- Leu- Leu- Tyr- Gln-Glu- Phe- Asp-Glu- Glu- -Glu- Cys- Ala-Ser- His- Leu- Pro- Tyr- Ile- Glu- Gln- Gly- Met- Gln- Leu- Ala ... (9) (ckk-n1 (40)) (One-letter code: IILSG RPAIV PDREL LYQEF DEMEE CASHL PYIEQ GMQLA) According to the present invention, further, an amino acid sequence contained in the sequence represented by the following formula (10), which comprises at least 6 consecutive amino acids: There is provided an antigenic peptide compound comprising a sequence consisting of:

Leu-His- Val- Asn- Gln- Arg- Ala- Val- Val- Ala- Pro- Asp- Lys- Glu- Val- Leu- Tyr- Glu-Ala- Phe- Asp- Glu- Glu- -Glu- Cys- Ala-Ser- Arg- Ala- Ala- Leu- Ile- Glu- Glu- Gly- Gln- Arg- Ile- Ala ... (10) (ckk-n2 (40)) (One-letter code: LHVNQ RAVVA PDKEV LYEAF DEMEE CASRA ALIEE GQRIA) According to the present invention, the antigen peptide according to any one of the above formulas (1) to (10) is further bound to a carrier to form an antigen-antibody. The reaction is used to bind the HCV antibody in the sample to the peptide, and the antigen-antibody reaction is used to bind the labeled ligand to the HCV antibody. To allow the HC in the sample to
An immunological assay method for HCV antibodies, which comprises measuring V antibodies is provided.

Here, the term "ligand" means an antigen and / or
Alternatively, it refers to a substance having specific binding properties to an antibody (for example, an antibody and / or an antigen).

[0032]

In the conventional genotype determination described above, the HC
HCV typing is performed using the V gene sequence itself, but in the present invention, HCV typing is performed using the antibody type (serotype) to the antigenic site unique to HCV. Such antibody type determination is based on conventional geno
Type determination (HCV RNA extraction → cDNA synthesis → c
According to the present invention, it can be performed easily and at low cost because it can be performed in a significantly shorter time than that of (amplification of DNA by PCR method → confirmation of amplified band using electrophoretic method) and the working process is simple. A method for determining the HCV type (genotype) can be provided.

Furthermore, in comparison with the above-mentioned conventional serotyping method (using an antigen consisting of about 300 amino acids), in the present invention, the cross-reaction or non-specific reaction associated with the antigen-antibody reaction is effective. Is suppressed.

The present invention will be described in detail below. In the present specification, in the notation of amino acid sequence, it is described from N terminus (left side) to C terminus (right side) (for example, in the following amino acid sequence (1), Leu is the N terminus and G
lu is the C-terminus). In addition, in the present specification, the following amino acid notation is used.

(One- and Three-Letter Amino Acid Codes) <Abbreviations><Name><Abbreviations><Name> Asp D Aspartic Acid Val V Valine Asn N Asparagine Met M Methionine Thr T Threonine Ile I Isoleucine Ser S Serine Leu L Leucine Glu E Glutamate Tyr Y Tyrosine Gln Q Glutamine Phe F Phenylalanine Pro P Proline Trp W Tryptophan Gly G Glycine Lys K Lys Ala A Alanine His H Histidine Cys C Glycine Antigen The following peptide (antigens of the invention) His peptide His peptide Cys C cysteine Argin R A sequence contained in the sequence (20 amino acids) represented by the formula (1) or (2), which has a sequence consisting of at least 6 consecutive amino acids. It is a peptide compound.

Leu- Ser- Gly- Arg- Pro- Ala- Ile- Val- Pro-Asp- Arg- Glu- Val- Leu- Tyr- Gln- Glu- Phe-Asp- Glu ... 1) (corresponding to NS4L2-1, NS4 region, NS4L2-1, NS4 region) (single letter notation: LSGRPAIVPDREVLYQEFDE) Val- Asn- Gln- Asp- Arg- Ala- Val- Val- Ala- Pro- Asp- Lys- Glu- Leu-Tyr- Glu- Ala-Phe- Asp (2) (ckk-n6; NS4L2-2, corresponding to NS4 region) (One-letter code: VNQDR AVVAPDKEVLYEAFD) Findings by the present inventor According to the above, amino acid sequences of various lengths constituting the sequence consisting of 20 amino acids represented by the above formula (1) or (2), In the present invention, the sequence containing 6 consecutive amino acids is the peptide (1).
And / or like (2), it can be suitably used as an antigen.

Examples of such amino acid sequences of various lengths (6 to 20 amino acids) include the following sequences.

The amino acid contained in the above peptide compound (1)
Acid sequence

[0039]

[Table 1]

The amino acid contained in the above peptide compound (2)
Acid sequence

[0041]

[Table 2]

According to the knowledge of the present inventor, the above (1)
Alternatively, a peptide containing the sequence of (2) (comprising 21 or more amino acids) can also be suitably used as an antigen, as in the above (1) or (2). The number of amino acids constituting such a peptide is preferably 40 or less when chemically synthesized, and preferably 100 or less when obtained by the recombinant method.

The method for obtaining the above-mentioned antigenic peptide compound (a peptide compound containing at least 6 amino acids) is not particularly limited. The peptide compound may be chemically synthesized, or may be produced by using a genetic engineering technique (for example, a gene recombination technique, that is, a recombinant method). When using a chemical method, it is preferable to link amino acids by a solid phase method from the viewpoint of easy purification of intermediate products, and further, an automatic peptide synthesizer (for example, 430A peptide manufactured by Applied Biosystems) is used. It is preferable to synthesize using a synthesizer.

(Confirmation of Antigenicity of Antigenic Peptide Compound) The sequences of the peptide compounds (1) and (2) described above are H
It was discovered based on the extraction of the antigenic site from the amino acid sequence of the NS4 region of CV (see Example 1 below). The present inventor has further conducted an enzyme-linked immunosorbent assay (ELIS) using these peptide compounds (1) and (2).
HCV genotype inspection by A to EIA),
The specificity was confirmed using a sample of a patient whose HCV genotype was separately determined (see Examples 4 and 5 described below).

(Other Antigen Peptide Compounds) The above-mentioned peptide compounds (1) and (2) (both HCV NS
It is possible to determine the serotype as described later by using the sequence corresponding to 4 regions).
From the viewpoint of improving the accuracy of determination or expanding the range of application, it is preferable to combine the peptide compounds (1) and / or (2) with other antigenic peptide compounds. Examples of such "another antigenic peptide compound" include the following peptides (3) to (8).

Cys- Thr- Thr- His- His- Val- Ser- Pro- Asp-Ala- Asp- Leu- Ile- Glu- Ala- Asn- Leu- Leu-Trp- Arg ... .. (3) (ckk-n3; NS5L2-1, corresponding to NS5 region) (One-letter code: CTTHHVSPDADLIEANLLWR) Cys- Thr- Thr- His- Gly- Lys- Ala- Tyr- Asp- Val- Asp-Met- -Asp- Ala- Asn- Leu- Phe- Met- Gly ... (4) (ckk-n4; NS5L2-2, NS5 region supported) (One-letter code: CTTHGKAYDVDMVDANLFMMG) Gln- Glu- Phe- Asp- Glu- Met- Glu- Glu- Cys-Ala- Ser- His- Leu- Pro- Tyr- Ile- Gl -Gln-Gly- Met (5) (corresponding to ckk-n1; NS4L3-1, NS4 area) (One letter notation: QEFDEMEECA SHLPYIEQGM) Glu- Ala- Phe- Asp- Glu -Met- Glu- Glu- Cys-Ala- Ser- Arg- Ala- Ala- Leu- Ile- Glu- Glu-Gly- Gln ... (6) (ckk-n2; NS4L3) -2, corresponding to the NS4 region) (One-letter code: EAFDEMEECASRAALIEEGQ) Gly- Arg- Arg- Gln- Pro- Ile- Pro- Lys- Ala-Arg- Arg- Pro- Glu- Gly- Arg- Thr- a-Thr- ap-Thr- ap- Thr- Gln-Pro (7) (corresponds to ckk-c1; core-1, core area) (One-letter code: GRRQPIPKARRRPEGRT WAQP) Gly- Arg- Arg- Gln- Pro- Ile- Pro- Lys- Asp-Arg- Arg- Ser- Thr- Gly- Lys- Ser- Trp- Gly-Lys- Pro ... .. (8) (corresponds to crk-c2; core-2, core area) (single letter notation: GRRQPIPKDRRSTGKSWGKP) Ile-Ile-Leu-Ser-Gly-Arg-Pro-Ala-Ile-Val-Pro-Asp-Arg -Glu- Leu- Leu- Tyr- Gln-Glu- Phe- Asp- Glu- Met- Glu- Glu- Cys- Ala-Ser- His- Leu- Pro- Tyr- Ile- Glu- Gln- Gln- Gln- Gln- Gln- Gln- -Leu- Ala (9) (corresponding to ckk-n1 (40), NS4 area) (One-letter code: IILSG RPAIV DREL LYQEF DEMEE CASHL PYIEQ GMQLA) Leu-His- Val- Asn- Gln- Arg- Ala- Val- Val- Ala- Pro- Asp- Ly- -Met- Glu- Glu- Cys- Ala-Ser- Arg- Ala- Ala- Leu- Ile- Glu- Glu- Gly-Gln- Arg- Ile- Ala ... (10) (Corresponds to ckk-n2 (40), NS4 region) (One-letter code: LHVNQ RAVVA PDKEV LYEAF DEMEE CASRA ALIEE GQRIA) The peptides (3) to (10) also correspond to the NS4 region (1) and (2). Confirmation of antigenicity, production, etc. can be carried out in the same manner as in the above case.

According to the knowledge of the present inventor, the amino acid sequences of various lengths constituting the sequence consisting of 20 amino acids represented by the above formulas (3) to (10), and having at least 6 amino acid sequences In the present invention, a sequence containing consecutive amino acids is a peptide (3), (4), (5), (6),
Like any of (7), (8), (9), and / or (10), it can be preferably used as an antigen peptide.

Examples of such amino acid sequences of various lengths (6 to 20 amino acids) include the following sequences.

The amino acid contained in the above peptide compound (3)
Acid sequence

[0050]

[Table 3]

The amino acid contained in the above peptide compound (4)
Acid sequence

[0052]

[Table 4]

The amino acid contained in the above peptide compound (5)
Acid sequence

[0054]

[Table 5]

The amino acid contained in the above peptide compound (6)
Acid sequence

[0056]

[Table 6]

According to the knowledge of the present inventor, the above (3)
A peptide containing any of the sequences (10) to (10) (comprising 21 or more amino acids) can also be suitably used as an antigen, as with the peptide having the sequences (3) to (10). The number of amino acids constituting such a peptide is preferably 40 or less when chemically synthesized, and is 1 when obtained by the recombinant method.
It is preferable that the number is 00 or less.

According to the knowledge of the present inventor, when each of the above peptide compounds (1) to (10) is used alone as an antigen, the preferred order as an antigen is as follows from the viewpoint of accuracy of serotype determination ( In the description in this column,
Collectively "(1) and / or (2)" into "NS4-
1 "," (3) and / or (4) "are collectively represented as" NS5 "," (5) and / or (6) "are collectively represented as" NS4-2 ", and" (9 ) And / or (10) ”are collectively referred to as“ NS4-40 ”,
"(7) and / or (8)" are collectively "core"
Represents).

Preferred order: NS4-1>NS5> NS
4-2> core On the other hand, the above peptide compounds NS4-1, NS5, NS4
-2, and when any two types of core are used as an antigen in combination, in terms of accuracy of serotype determination, the preferred order as the antigen is as follows: Preferred order: (NS4-1) + (NS5)> (NS4-
2) + (NS5)> (NS4-1) + (core)>
(NS4-2) + (core)> (NS5) + (cor
e) In addition, the above peptide compounds NS4-1, NS5, NS4
-2, and when any three kinds of cores are used as an antigen in combination, in terms of accuracy of serotype determination, the preferable order of the antigens is as follows: (NS4-1) + (NS5) + (Cor
e)> (NS4-2) + (NS5) + (core) The peptide compound “NS4-40” (amino acid number = 4)
0) can be preferably used in place of “NS4-1” in the above combination. In this way, "NS4-4
When “0” is used, a higher determination rate (= (number of samples that can be determined) / (total number of samples)) can be obtained compared to when “NS4-1” is used.

(Method for measuring HCV antibody) The HCV antibody in a sample or specimen can be immunologically measured by utilizing the specific binding property of the above-mentioned antigenic peptide compound with the HCV antibody. The immunological assay method used in the present invention is not particularly limited, but, for example, a known immunoassay method can be used without particular limitation. As such an immunoassay method, for example, an enzyme immunoassay method, a radioimmunoassay method (RIA), a fluorescence immunoassay method (FIA) or the like can be used. In this immunoassay, it is possible to use known measurement techniques (for example, competitive method, two antibody method, sandwich method, etc.) without particular limitation. For such known measurement techniques, see the literature (EI
Regarding A, for example, Eiji Ishikawa “Enzyme Immunoassay”
(3rd edition), p. 180, Ikusho Shoin).

According to the present invention, by immunologically measuring an HCV antibody using the above-mentioned antigenic peptide compound,
The type (serotype) of the HCV antibody in the sample can be determined (for example, as described later, the serotype of HCV is gr
It can be determined whether it is oup I or group II). As will be described later, HCV serotype group I is HCVgenoty
Corresponds to pe I and II, HCV serotype group II is H
Simple HC to support CV genotype III and IV
Vgenotype can be determined.

In the above immunoassay, the above-mentioned antigen peptide compound is used as a carrier (or support), if necessary.
It is possible to determine the type of antibody against the HCV antigen (that is, the serotype of HCV) by utilizing the specific binding property with the HCV antibody by binding to or immobilizing on. As the carrier or support in this case, bovine serum albumin (BSA), preferably a polypeptide having a molecular weight of about 50,000 to 100,000, a well of a microplate, preferably a polystyrene box having a diameter of about 0.1 μm to 6 mm. (Or polystyrene beads) are preferably used.

The above peptides (1) to (8) can be preferably used for the measurement of HCV antibody as described above,
Further, it can be used as an antigen or an immunogen for producing a vaccine against HCV, for example. The vaccine in this case can be produced, for example, using a genetic engineering method (recombinant method).

Hereinafter, the present invention will be described more specifically based on examples.

[0065]

[Example] Example 1 (Identification of antigenic site by Hydrophilicity Value) Geoffr
ey RS, Nature, 302 , 490-495 (1983), the sequence of the peptide having antigenicity and the gene sequence of HCV (26th Annual Meeting of the Japanese Society of Hepatology, 1990; 49th Annual Meeting of the Japanese Cancer Society) Shu, 1990; Proc. Natl. Acad. Sc
i. USA, 88, 2451-2455, 1991; Journal of virology,
65 (3) , 1105-1113, 1991; Journal of General Viro
logy, 72 , 2697-2704,1991; Virology, 188 , 331-34
1, 1992) and the amino acid sequences of the nonstructural and structural regions of HCV were determined.

Based on the amino acid sequence thus obtained,
The antigenic site is Hopp TP, Proc. Natl. Acad. Sci. 78 ,
Calculated by the method of 3824-3828 (1981). More specifically, it was performed as follows using the Hydrophobicity Value of each amino acid.

Based on the amino acid sequence constituting HCV obtained above, Hyrdrophi consisting of 6 consecutive amino acids
The licity Value is summed. This calculation was performed for the entire region of the HCV amino acid sequence, and the hydrophilicity and hydrophobicity were calculated. The highly hydrophilic portion was defined as the site showing antigenicity.

At this time, Jack K., Mol. Biol. 157, 105
(1982) Hydropathy Value and Alan M
Antige shown in S., Science, 227, 429 (1985).
It was possible to calculate the nicityValue and the like in the same manner as the Hydrophilicity Value. Above Hydrophilicity Val
The results obtained by calculation using ue are shown in the graphs of FIG. 1 (core region), FIG. 2 (NS4 region) and FIG. 3 (NS5 region) (Hydropathy Value and Antigenici above).
The results using ty Value are also shown).

Based on the results obtained in FIGS. 1 to 3 above, a region which can be an antigen of hepatitis C was extracted, and further, among the regions thus obtained, genotype I and genotype I
A region having a common amino acid sequence in type II and genotype
A region having an amino acid sequence common to III and genotype IV was determined. As a result, as the amino acid sequence of the antigen satisfying such conditions, the
ckk-n5 and ckk-n6 were sought.

Leu- Ser- Gly- Arg- Pro- Ala- Ile- Val- Pro-Asp- Arg- Glu- Val- Leu- Tyr- Gln- Glu- Phe-Asp- Glu ... ckk-n5) Val- Asn- Gln- Arg- Ala- Val- Val- Ala- Pro- Asp- Lys- Glu- Val- Leu- Tyr- Glu- Ala- Phe- Asp- Glu ... ... (ckk-n6) NS5 was used as the amino acid sequence of the antigen by the same method.
The following ckk-n3 and ckk-n4 were determined for the region.

Cys- Thr- Thr- His- His- His- Val- Ser- Pro- Asp-Ala- Asp- Leu- Ile- Glu- Ala- Asn- Leu- Leu-Trp- Arg ... -(Ckk-n3) Cys-Thr-Thr- His- Gly- Lys- Ala- Tyr- Asp- Val- Asp- Met- Val- Asp- Ala- Asn- Leu- Phe- Met-Gly ... ..... (ckk-n4) NS4 was used as the amino acid sequence of the antigen by the same method.
The following ckk-n1 and ckk-n2 were determined for the region.

Gln- Glu- Phe- Asp- Glu- Met- Glu- Glu- Cys-Ala- Ser- His- Leu- Pro- Tyr- Ile- Glu- Gln-Gly- Met ... ... (ckk-n1) Glu- Ala- Phe- Asp- Glu- Met- Glu- Glu- Cys-Ala- Ser- Arg- Ala- Ala- Leu- Ile- Glu- Glu-Gly- Gln ...・ ・ ・ ・ ・ ・ ・ ・ (Ckk-n2) By the same method, as the amino acid sequence of the antigen, cor
The following ckk-c1 and ckk-c2 were obtained for the e region.

Gly- Arg- Arg- Gln- Pro- Ile- Pro- Lys- Ala-Arg- Arg- Pro- Glu- Gly- Arg- Thr- Trp- Ala-Gln- Pro ... ... (ckk-c1) Gly- Arg- Arg- Gln- Pro- Ile- Pro- Lys- Asp-Arg- Arg- Ser- Thr- Gly- Lys- Ser- Trp- Gly- Lys- Pro ... ... (ckk-c2) Example 2 (Preparation of antigen for hepatitis C) The above-mentioned HCV antigenic peptide compounds (ckk-n5) and (ckk-n6) were synthesized by the method shown below. did.

Peptide synthesis was performed using an automatic peptide synthesizer Ap.
Using plied Biosystems 430A Peptide Synthesizer,
Performed using a symmetrical anhydride of the t-Boc amino acid as the reagent. The obtained synthetic peptide was dissolved in anisole / dimethyl sulfide / parathiocresol, and then reacted in the presence of hydrofluoric acid at 0 to 5 ° C for 1 hour for deprotection (S. Sakakibara). , Bull. Chem. Soc. Jpn. 4
0 , 2164 (1967)).

The crude crystals obtained by deprotection were dissolved in 2N acetic acid, extracted with ether, and the extract was freeze-dried. The lyophilizate was purified using HPLC (High Performance Liquid Chromatography).

This HPLC purification was carried out using a column (SISEIDO Capsule Pack C18SG120, diameter 46 mm, length 25.
0 mm), a solution containing 0.1% trifluoroacetic acid (TFA) and 5% acetonitrile (CH ₃ CN) in pure water, and 0.1% in pure water.
It was performed by using TFA and a solution containing 50% acetonitrile and applying a gradient to the mobile phase at a flow rate of 12 ml / min.

The chromatogram obtained at this time is shown in FIG. The main peak shown in FIG. 4 is the synthetic antigen peptide ckk-n5 of this example.

Synthetic antigenic peptide ckk-n6 was prepared as described above.
Was purified using HPLC. The chromatogram obtained at this time is shown in FIG. The main peak shown in FIG. 5 is the synthetic antigen peptide ckk-n6 of this example.

In the same manner as above, the above 6 kinds of synthetic antigen peptides ckk-n3, ckk-n4, ckk-n1, ckk-n2, ckk-c.
1 and ckk-c2 were synthesized.

HPLC chromatograms at the time of purification are shown in FIG. 6 (ckk-n3), FIG. 7 (ckk-n4), and FIG. 8 (ckk-n).
1), FIG. 9 (ckk-n2), FIG. 10 (ckk-c1), and FIG.
1 (ckk-c2).

In the same manner as above, the following two types of antigen peptides ckk-n1 (40) and ckk-n2 (40) were synthesized.

Ile- Ile- Leu- Ser- Gly- Arg- Pro- Ala- Ile-Val- Pro- Asp- Arg- Glu- Leu- Leu- Tyr- Gln-Glu- Phe- Asp-Glu- Glu- Phe- Asp-Glu- -Glu- Cys- Ala-Ser- His- Leu- Pro- Tyr- Ile- Glu- Gln- Gly- Met- Gln- Leu- Ala ... (9) (ckk-n1 (40)) Leu- His- Val- Asn- Gln- Arg- Ala- Val- Val- Ala- Pro- Asp- Lys- Glu- Val- Leu- Tyr- Glu-Ala- Phe- Msp-et- Glu- Glu- Glu- Cys- Ala-Ser- Arg- Ala- Ala- Leu- Ile- Glu- Glu- Gly-Gln- Arg- Ile- Ala ... ( 10) (ckk-n2 (40)) The HPLC chromatograms at the time of purification are shown in FIG.
(Ckk-n1 (40)) and FIG. 13 (ckk-n2 (40)).

Example 3 (Preparation of plate for ELISA) The antigen peptide ckk-n5 obtained in Example 2 was treated with 0.15M NaCl-0.10M Na ₂ H.
PO ₄ -NaH ₂ PO ₄・ 2H ₂ O buffer (PBS)
It was dissolved in pH 7.0 to a concentration of 0.04 g / ml. This peptide solution was added to a 96-well microplate (trade name: ELISA PLATE 68667, manufactured by NUNC).
Dispense 100 μl per well into each well and incubate at 37 ° C for 60
The above antigen peptide was immobilized on a microplate by allowing to stand for a minute.

After the excess peptide solution was removed, the microplate was washed with 0.01MPBS (pH 7.0) for 3 minutes.
Washed twice. After removing the washing solution, a 0.01 M PBS (pH 7.0) solution of gelatin having a concentration of 0.1% was dispensed in an amount of 300 μl per well, and the mixture was left standing at 37 ° C. for 60 minutes to coat the plate with gelatin. After removing the excess gelatin solution, Tween with a concentration of 0.05%
0.01M PBS solution containing n20 (pH 7.0)
It was washed 3 times.

The gelatin-coated plate was dried at 25 ° C. for 6 hours and then stored at 4 ° C. until the time of sample analysis.

Example 4 (Analysis of Specimens) Specimens (about 100 kinds) were prepared in advance in SRL Hachioji Laboratory Co., Ltd. by the PC described in the above-mentioned reference (Okamoto H., Hepatobiliary-Pancreatic, 24 , 7-14 (1992)).
Method using R HCV genotype inspection
Obtained genotype.

In this way, HCVgeno was obtained by the PCR method.
The specificity of the antigenic peptide obtained in Example 2 was confirmed as follows using the sample for which the type was determined. In this examination, the antigen peptide-immobilized microplate prepared in Example 3 was used, and analysis was carried out according to the following procedure.

Each of the above-mentioned samples was subjected to 0.01% BS.
0.01 containing A and 0.05% Tween 20
Dilute 50 times with MPBS (pH 7.0), dispense 100 μl into each well, and mix with the above antigen peptide at 37 ° C.
For 60 minutes (antigen peptide-HCV antibody reaction).

After the reaction, the microplate was washed with 0.05% T.
5 with 0.01MPBS (pH 7.0) containing ween 20
After washing twice and removing the washing solution, the concentration is 0.1 μg / 20
ml of peroxidase-labeled anti-human IgG antibody (KPL
100 μl) was injected into each well, and the mixture was added at 37 ° C for 6
Reaction was carried out for 0 minutes (HCV antibody-POD-labeled anti-human Ig
Reaction of G antibody).

After the reaction, the microplate was washed with 0.05% T.
5 with 0.01MPBS (pH 7.0) containing ween 20
After washing twice and removing the washing solution, add 0.023 to each well.
% Hydrogen peroxide, and 0.1M citric acid-Na ₂ H containing 0.005% o-phenylenediamine (OPD).
100 μl each of PO ₄ buffer was dispensed and reacted at 37 ° C. for 30 minutes (peroxidase activity measurement reaction). After the reaction, add 50 μl of 5N sulfuric acid to stop the reaction,
The absorbance (ABS or OD) at a wavelength of 491 nm was measured with a spectrophotometer (trade name: Pre-Treader, NJ-2001 manufactured by Nippon Intermed Co., Ltd.).

Example 5 (Measurement of Sample Using Antigen Peptide ckk-n2) The antigen peptide ckk-n6 obtained in Example 2 was used instead of the antigen peptide ckk-n5 used in Example 3. A microplate on which the antigenic peptide ckk-n6 was immobilized was prepared in the same manner as in Example 3. In the same manner as in Example 4 except that the thus obtained microplate was used, samples (78 types) whose HCV genotype was determined in advance were analyzed.

The results obtained in Examples 4 and 5 above are summarized in the following (Table 7).

[0093]

[Table 7]

In Table 7 above, the first column shows the sample number, the second column shows the absorbance (OD value) of the sample when the antigen peptide ckk-n5 (NS4L2-1) was used, and the third column is The absorbance of the sample when using the antigen peptide ckk-n6 (NS4L2-2) is shown, the fourth column shows the determination result (genotype) by the PCR method previously obtained, and the fifth column shows the method of the present invention. Indicates the judgment result (serotype).

In the serotype determination by the method of the present invention, the absorbance of the first column (ckk-n5) and the absorbance of the second column (ck
k-n6), if either value is 1.5 times or more of the other value, the type with the higher absorbance (serotype1, ckk-for the case of ckk-n5) If you are n6
serotype 2).

The serotypes obtained in Examples 4 and 5 above
The following correspondences were obtained when the judgment results were summarized and compared with the genotype judgment results (Hepatology, 16 (4)
, 886, 1992).

[0097] (In the table, "genotype V" means that type determination is not possible or unknown (including mixed) in genotype determination by PCR method.
Shows the thing. Example 6 (Measurement of Specimen Using Antigen Peptides ckk-n3 and ckk-n4) Instead of the antigen peptide ckk-n1 used in Example 3, the NS5 region antigen peptide ckk-n3 obtained in Example 2 was used. And ck
A microplate on which each antigenic peptide was immobilized was prepared in the same manner as in Example 3 except that k-n4 was used. In the same manner as in Example 4 except that the thus obtained microplate was used, samples (48 types) whose HCV genotype was determined in advance were analyzed.

The results obtained in this example are summarized below (Table 8).

[0099]

[Table 8]

In the serotype determination by the method of the present example, the absorbance (ckk-n3;
NS5L2-1) and the absorbance in the second column (ckk-n2; NS5L2-2) are compared, and when either value is 1.5 times or more of the other value, the absorbance is high. Type (serotype 1 for ckk-n3, serotype for ckk-n4)
2).

When the results of serotype determination obtained in this example were summarized and compared with the results of genotype determination, similar to the results of Examples 4 and 5, the following correspondences were obtained.

[0102] Example 7 (Measurement of Specimen Using Antigen Peptides ckk-n1 and ckk-n2) Instead of the antigen peptide ckk-n5 used in Example 3, the antigen peptide ckk-n1 of the NS4 region obtained in Example 2 and ck
Microplates on which the respective antigenic peptides were immobilized were prepared in the same manner as in Example 3 except that k-n2 was used. In the same manner as in Example 4 except that the thus obtained microplate was used, samples (45 types) whose HCV genotype was determined in advance were analyzed.

The results obtained in this example are summarized in the following (Table 9).

[0104]

[Table 9]

In the serotype determination by the method of the present example, the absorbance (ckk-n1;
NS4L3-1) and the absorbance in the second column (ckk-n2; NS4L3-2) are compared, and when either value is 1.5 times or more of the other value, the absorbance is high. Type of one (serotype 1 for ckk-n1 and serotype for ckk-n2)
2).

When the results of serotype determination obtained in this example were summarized and compared with the results of genotype determination, similar to the results of Examples 4 and 5, the following correspondences were obtained.

[0107] Example 8 (Measurement of specimen using antigen peptides ckk-c1 and ckk-c2) Instead of the antigen peptide ckk-n5 used in Example 3, the antigen peptides ckk-c1 and crk-c1 in the core region obtained in Example 2 were obtained.
A microplate on which each antigenic peptide was immobilized was prepared in the same manner as in Example 3 except that each of ckk-c2 was used. HCV genotype was prepared in the same manner as in Example 4 except that the microplate thus obtained was used.
The samples (42 types) for which the above determination was made were analyzed.

The results obtained in this example are summarized in Table 10 below.

[0109]

[Table 10]

In the serotype determination by the method of the present example, the absorbance (ckk-c1;
core-1) and the absorbance in the second column (ckk-c2; core-2) are compared, and when either value is 1.5 times or more of the other value, the absorbance is high. The type (serotype 1 for ckk-c1 and serotype 2 for ckk-c2).

When the results of serotype determination obtained in this Example were summarized and compared with the results of genotype determination, similar to the results of Examples 4 and 5, the following correspondences were obtained.

[0112] The results of the serotype determination obtained in the above Examples 4 to 8 are shown together in FIG.

In FIG. 14, the numbers in the circles indicating the respective sets indicate the number of specimens that could be identified when the antigen peptides in the respective regions were used. The common part of each set is
The number of specimens that can be commonly identified when the antigen peptides in each region are used is shown.

As mentioned above, genotype I and genot
ype II correlates well with serotype I, while genotype I
II and genotype IV correlated well with serotype II.

The NS4 region described above (NS4-1 and N4)
S4-2), NS5 region, and core
When the serotype determination results using the antigenic peptide compounds in the region were combined, 8 out of the samples that could be typed by genotype
6% was able to judge the serotype by the method of the present invention.

Example 9 (Measurement of specimen using antigen peptides ckk-n1 (40) and ckk-n2 (40)) Instead of the antigen peptide ckk-n5 used in Example 3, it was obtained in Example 2. NS4 domain antigenic peptide ckk-
A microplate on which each antigenic peptide was immobilized was prepared in the same manner as in Example 3 except that n1 (40) and ckk-n2 (40) (having 40 amino acids) were used.

In the same manner as in Example 4 except that the thus obtained microplate was used, Example 4 (using the antigen peptide ckk-n5) and Example 5 (antigen peptide) were prepared in advance.
Samples (100 types) whose HCV serotype was determined by the method of using ckk-n6) were analyzed. The breakdown of the 100 kinds of specimens is 83 kinds of specimens of CAH (chronic active hepatitis) patients,
And 17 kinds of CPH (chronic persistent hepatitis) patient specimens.

The results obtained in this example are summarized in the following (Table 11). In Table 11, the method of Examples 4 and 5 (the number of amino acids is 2 for the NS4 antigen peptide)
0 is used for ckk-n5 and ckk-n6)
S4 (20) ", and the determination result by the method of this example (using ckk-n1 (40) and ckk-n2 (40) having 40 amino acids as the NS4 antigen peptide) is referred to as" NS4 (4) ".
0) ”.

[0119]

[Table 11]

In the above serotype determination by the method of the present example, as in the case of Table 1, the absorbance when using ckk-n1 (40) and the absorbance when using ckk-n2 (40) were used. When one of the values is 1.5 times or more of the other value, the type with the higher absorbance (serotype1, ckk-n2 (for the case of ckk-n1 (40) If you are 40) serotoy
pe2).

As shown in Table 11 above, in the case of CAH samples (83 types), the judgment rate in the case of NS4 (20) was 62.
What was% (= 52/83) was improved to the judgment rate of 83% (= 69/83) in the case of NS4 (40). On the other hand, in the case of CPH specimens (17 types), the judgment rate in the case of NS4 (20) was 75% (= 13 ÷ 17)
The determination rate in the case of S4 (40) was improved to 88% (= 15 ÷ 17).

Example 10 CH2A (Chronic active hepatitis CAH, C
48 types of patient specimens that have not progressed from H2B,
CH2B (chronic active hepatitis CAH,
36 types of patient specimens and CP
Using 17 types of specimens of H (chronic persistent hepatitis) patients, se
Rotype determination (performed in the same manner as in Example 9 and shown as "TYPE" in the table below), genotype (performed in the same manner as in Example 4 and shown as "SUBTYPE" in the following table), and interferon (IFN ) Effectiveness (in the table below, N = no effect, P (partially ef
fective) = effective, and C (completely effective)
== significant effect) was determined.

The results are shown below (Table 12).

[0124]

[Table 12]

Furthermore, the same samples as those shown in Table 12 above were analyzed by the method using the NS5 region antigen peptide (the method of Example 6) and the method using the core region antigen peptide (the method of Example 8). Serotype judgment was also performed. The results are shown below (Table 13) (the determination result when using the above "NS4 region antigen peptides" ckk-n1 (40) and ckk-n2 (40) in this Example was shown as "NS4". ).

[0126]

[Table 13]

In Table 13, the total number of CH2A (40 samples) and CH2B (29 samples) corresponds to the NS4 (40) judgment number (69 samples) in Table 11 above.

The results of serotype determination obtained in Examples 9 to 10 are summarized in FIG. 15 (CPH sample) and FIG. 16 (C).
AH2A sample) and FIG. 17 (CAH2B sample). In FIGS. 13 to 15, the determination results when using “NS4 region antigenic peptides” ckk-n1 (40) and ckk-n2 (40) are shown as “NS4”.

In FIGS. 15 to 17, the numbers in the circles indicating the respective sets indicate the number of specimens that could be identified when the antigen peptides in the respective regions were used. The common part of each set indicates the number of specimens that can be commonly identified when the antigenic peptides in the respective regions are used.

[0130]

As described above, according to the present invention, the HCV
There is provided an antigenic peptide compound having a specific amino acid sequence unique to the above and an HCV antibody measuring method using the same.

According to the present invention, it is possible to easily and accurately determine the serotype of a sample while suppressing cross reaction or non-specific reaction. Therefore, according to the present invention, for example, the effect of interferon treatment can be predicted in advance based on accurate and simple serotype determination.

Furthermore, based on the HCV antibody titer measurement according to the present invention, it becomes possible to easily observe the healing or treatment course of hepatitis C.

[Brief description of drawings]

FIG. 1 shows the Hydrophilicity Value, Hydropathy Value, and An in the core region of HCV in Example 1.
It is a graph which shows the result of having calculated tigenicity Value.

FIG. 2 relates to the NS4 region of HCV in Example 1.
Hydrophilicity Value, Hydropathy Value, and Anti
It is a graph which shows the result of having calculated the genicity Value.

FIG. 3 relates to the NS5 region of HCV in Example 1.
Hydrophilicity Value, Hydropathy Value, and Anti
It is a graph which shows the result of having calculated the genicity Value.

FIG. 4 is a chromatogram showing the results of HPLC analysis of the antigenic peptide compound (1) (ckk-n5) obtained in Example 2.

FIG. 5 is a chromatogram showing the HPLC analysis results of the antigenic peptide compound (2) (ckk-n6) obtained in Example 2.

FIG. 6 is a chromatogram showing the HPLC analysis result of the antigenic peptide compound (3) (ckk-n3) obtained in Example 2.

FIG. 7 is a chromatogram showing the results of HPLC analysis of the antigenic peptide compound (4) (ckk-n4) obtained in Example 2.

FIG. 8 is a chromatogram showing the results of HPLC analysis of the antigenic peptide compound (5) (ckk-n1) obtained in Example 2.

FIG. 9 is a chromatogram showing the results of HPLC analysis of the antigenic peptide compound (6) (ckk-n2) obtained in Example 2.

FIG. 10 is a chromatogram showing the HPLC analysis result of the antigenic peptide compound (7) (ckk-c1) obtained in Example 2.

FIG. 11 is a chromatogram showing the HPLC analysis result of the antigenic peptide compound (8) (ckk-c2) obtained in Example 2.

FIG. 12 is a chromatogram showing the HPLC analysis result of the antigenic peptide compound (9) (ckk-n1 (40)) obtained in Example 2.

FIG. 13 is a chromatogram showing the results of HPLC analysis of the antigenic peptide compound (10) (ckk-n2 (40)) obtained in Example 2.

FIG. 14 is a diagram showing a set in which serotype determination results obtained in Examples 4 to 8 are summarized.

FIG. 15 is a diagram showing a set of serotype determination results regarding CPH samples obtained in Examples 9 to 10.

FIG. 16 is a diagram showing a set of serotype determination results regarding CAH2A samples obtained in Examples 9 to 10.

FIG. 17 is a diagram showing a set of serotype determination results regarding CAH2B samples obtained in Examples 9 to 10.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C12P 21/08 9161-4B C12Q 1/70 9453-4B G01N 33/53 D 33/576 Z // A61K 39/395 D

Claims (18)

[Claims] 1. An antigenic peptide compound comprising an amino acid sequence contained in the sequence represented by the following formula (1), the sequence comprising at least 6 contiguous amino acids. Leu- Ser- Gly- Arg- Pro- Ala- Ile- Val- Pro- Asp- Arg- Glu- Val- Leu- Tyr- Gln- Glu- Phe-Asp- Glu ... (1) ( ckk-n5) 2. An antigenic peptide compound comprising an amino acid sequence contained in the sequence represented by the following formula (2), which comprises a sequence consisting of at least 6 consecutive amino acids. Val- Asn- Gln- Arg- Ala- Val- Val- Ala- Pro- Asp- Lys- Glu- Val- Leu- Tyr- Glu- Ala- Phe-Asp- Glu ... 2) (ckk-n6) 3. An antigen peptide compound comprising an amino acid sequence contained in the sequence represented by the following formula (3), the sequence comprising at least 6 contiguous amino acids. Cys- Thr- Thr- His- His- Val- Ser- Pro- Asp-Ala- Asp- Leu- Ile- Glu- Ala- Asn- Leu- Leu-Trp- Arg ... ( 3) (ckk-n3) 4. An antigenic peptide compound comprising an amino acid sequence contained in the sequence represented by the following formula (4), which comprises a sequence consisting of at least 6 consecutive amino acids. Cys- Thr- Thr- His- Gly- Lys- Ala- Tyr- Asp-Val- Asp- Met- Val- Asp- Ala- Asn- Leu- Phe-Met- Gly ... (4) (ckk-n4) 5. An antigenic peptide compound, which is an amino acid sequence contained in the sequence represented by the following formula (5) and comprises a sequence consisting of at least 6 consecutive amino acids. Gln- Glu- Phe- Asp- Glu- Met- Glu- Glu- Cys-Ala- Ser- His- Leu- Pro- Tyr- Ile- Glu- Gln-Gly- Met ... (5) (ckk-n1) 6. An antigen peptide compound comprising an amino acid sequence contained in the sequence represented by the following formula (6), the sequence comprising at least 6 consecutive amino acids. Glu- Ala- Phe- Asp- Glu- Met- Glu- Glu- Cys-Ala- Ser- Arg- Ala- Ala- Leu- Ile- Glu- Glu-Gly- Gln ... (6) (ckk-n2) 7. An antigenic peptide compound comprising an amino acid sequence contained in the sequence represented by the following formula (7), the sequence comprising at least 6 consecutive amino acids. Gly- Arg- Arg- Gln- Pro- Ile- Pro- Lys- Ala-Arg- Arg- Pro- Glu- Gly- Arg- Thr- Trp- Ala-Gln- Pro ... (7) 8. An antigenic peptide compound comprising an amino acid sequence contained in the sequence represented by the following formula (8), the sequence comprising at least 6 consecutive amino acids. Gly- Arg- Arg- Gln- Pro- Ile- Pro- Lys- Asp-Arg- Arg- Ser- Thr- Gly- Lys- Ser- Trp- Gly-Lys- Pro ... (8) 9. An antigen peptide compound comprising an amino acid sequence contained in the sequence represented by the following formula (9), the sequence comprising at least 6 consecutive amino acids. Ile- Ile- Leu- Ser- Gly- Arg- Pro- Ala- Ile-Val- Pro- Asp- Arg- Glu- Leu- Leu- Tyr- Gln-Glu- Phe- Asp- Glu- Glu- Met- Cys- Ala-Ser- His- Leu- Pro- Tyr- Ile- Glu- Gln- Gly-Met- Gln- Leu- Ala ... (9) (ckk-n1 (40) ) 10. An antigen peptide compound comprising an amino acid sequence contained in the sequence represented by the following formula (10), the sequence comprising at least 6 consecutive amino acids. Leu- His- Val- Asn- Gln- Arg- Ala- Val- Val- Ala- Pro- Asp- Lys- Glu- Val- Leu- Tyr- Glu-Ala- Phe- Asp- Glu- Glu- Met- Cys- Ala-Ser- Arg- Ala- Ala- Leu- Ile- Glu- Glu- Gly-Gln- Arg- Ile- Ala ... (10) (ckk-n2 (40) ) 11. The method according to claim 1, which is manufactured by using a chemical method.
11. The antigenic peptide compound according to any one of 1 to 10. 12. The antigenic peptide compound according to claim 1, which is produced by using a genetic engineering technique. 13. The antigen peptide according to any one of claims 1 to 10 is bound to a carrier, and an HCV antibody in a sample is bound to the peptide by utilizing an antigen-antibody reaction, and further an antigen-antibody reaction is performed. An immunological assay method for an HCV antibody, which comprises utilizing the labeled ligand to bind to the HCV antibody to measure the HCV antibody in the sample. 14. The immunological assay method for an HCV antibody according to claim 13, wherein a ligand labeled with an enzyme is used as the labeled ligand. 15. The immunological assay method for an HCV antibody according to claim 13, wherein a ligand labeled with a radioactive substance is used as the labeled ligand. 16. The immunological assay method for an HCV antibody according to claim 13, wherein a ligand labeled with a fluorescent substance is used as the labeled ligand. 17. The HCV antibody according to claim 13, wherein the serotype of HCV is determined using any two or more antigenic peptides selected from the antigenic peptides according to any one of claims 1 to 10. Immunological assay. 18. Using two or more antigen peptides corresponding to different regions selected from the antigen peptides corresponding to the NS4 region, NS5 region and core region of HCV according to any one of claims 1 to 10. HC of claim 13, wherein the serotype of HCV is determined.
Immunoassay for V antibody.