JPH07285994A - Synthetic peptide highly reactive with serum of patient infected with hepatitis c virus(hcv) and diagnosis using the same - Google Patents

Abstract

PURPOSE:To obtain a new synthetic peptide having a specified amino acid sequence, highly reactive with serum of a patient infected with hepatitis C virus(HCV), effective for, e.g. detection of HCV antibody and useful for high- accurate diagnosis of infection with HCV. CONSTITUTION:This new synthetic peptide is represented by formulae I to V, etc., highly reactive with serum of a patient infected with hepatitis C virus(HCV), capable of specifying B cell epitope highly reactive with serum of a patient infected with hepatitis C virus(HCV) in the regions of core, NS3 and NS4 considered effective for detection of HCV antibody, excellent in stability and useful for, e.g. high-sensitive and accurate diagnosis of infection with HCV. This synthetic peptide is obtained by conducting synthesis according to the multi-pin peptide synthesis method while shifting a peptide composed of 10 amino acid residues by three amino acid residues in the amino terminal side half region of the core, the whole region of NS3 and a front half region of NS4, respectively considered effective for detection of HCV antibody.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a peptide which is expected to have a high detection rate effective for diagnosing infection with HCV, and a method for diagnosing HCV infection using the peptide.

[0002]

BACKGROUND OF THE INVENTION HCV is said to be a pathogen that causes more than 90% of the causes of non-A non-B hepatitis that occurs after blood transfusion.
It is said that most people who develop the infection with this virus follow the course of acute hepatitis to chronic hepatitis, and develop cirrhosis or liver cancer after a long time. From patients with liver cirrhosis or liver cancer, HCV was detected at a high rate by RT-PCR.
Many of these diseases have H
It is believed that CV is deeply involved. Since HCV is deeply involved in many liver diseases in this way, H
Diagnosis of CV infection has become an important issue. Further, recently, a number of cases in which a therapeutic method using recombinant interferon has been developed and cured have been reported, and therefore the importance of early diagnosis of HCV infection is increasing.

Infection of HCV is considered to occur via blood or body fluid, and most of them are considered to be due to medical procedures such as blood transfusion. Therefore, it is important to transfuse blood or use blood free of HCV in blood products in order to cut off the infection route and eradicate HCV.

HCV has a very poor growth in vivo, and its substance has remained unclear for a long time because there is no efficient in vitro growth system. However, in 1989, Choo et al. Cloned the gene of the virus causing non-A non-B hepatitis for the first time. Since then, the nucleotide sequences of the genomes of many virus strains have been reported up to the present, and the nucleotide sequences of some of them have been determined over the entire length of the viral genome. Furthermore, by using the expressed protein of the gene as an antigen, anti-HCV
Possibility of diagnosis of infection by detection of antibody was shown (Scienc
e, 21, 359-361, 1989). HCV is currently classified into five subtypes based on the comparison of the determined nucleotide sequences.
Of these, those detected in Japan are type I to I
There are four subtypes of V type, and among them, type II is 55-55.
It is detected in 78% and is considered to be the major epidemic type in Japan.

Under these circumstances, efforts have been made to develop and improve a system for detecting the anti-HCV antibody or the HCV genome itself, and the detection rate has now exceeded 90%.

The initial antigen used in the anti-HCV antibody detection system was an expressed protein in a region that straddled a part of NS3 and NS4, but since an antibody against this antigen was not detected in the acute stage of infection, There is a problem that it is not suitable for early diagnosis, and there is a non-negligible proportion of HCV patients that are negatively determined by this detection system. These problems have been greatly improved by using the antigen in the core region.

The B cell epitope on the antigen side recognized by an antibody is usually said to consist of 5-6 amino acid residues. When an immune response occurs in vivo, it is known that antibodies are induced not to all parts of the amino acid sequence of an antigen protein but to some specific parts. This means that there is a certain limitation on the area of the solid phase on which the antigen is adsorbed, so it is expected that a more reactive detection system will be constructed by using only the epitope part effective for antibody detection as the antigen. . In addition, the NS3 region of HCV contains a portion encoding a protease and helicase having high homology with other viruses such as teng virus and Japanese encephalitis virus, which contributes to non-specific reaction in detecting anti-HCV antibody. It is thought to leave the possibility of becoming. Furthermore, when the expressed protein is used as an antigen, the process of purifying the target antigen is indispensable because it is necessary to remove the nonspecific reaction, which is a major problem in constructing an antibody detection system. These problems can be expected to be solved by using a synthetic peptide of the epitope part effective for detecting an antibody specific to HCV. In addition, by connecting a gene encoding a target protein to a gene encoding a known protein,
Purification methods using a chimeric protein and an antibody against the known protein have been developed. At that time, it is considered necessary to identify an epitope effective for antibody detection.

[0008] There is also a document (Japanese Patent Publication No. 4-504715) which discloses a peptide presumed to have an HCV epitope used for a diagnostic drug or vaccine for HCV. However, of the many peptides disclosed herein, only a few have been shown to be actually reactive with HCV patient sera. In addition, these are all about 100 amino acid residues or more in length except for one case because of the analysis using the expressed protein, and it is considered that a large number of unnecessary sequences are included in the detection of anti-HCV antibody. To be Moreover, the other specified peptides are those obtained by computer analysis of the hydrophilicity / hydrophobicity profile of the deduced amino acid sequence encoded by the nucleotide sequence of HCV and the antigen index, and whether or not they can be epitopes is as described above. So it is just beyond the estimation range.

[0009]

Under the current circumstances of the prior art as described above, the present inventor has identified a shorter epitope region that actually reacts with the serum of HCV patients, and is able to identify a diagnostic agent for HCV and The present invention has been accomplished for the purpose of providing a synthetic peptide useful as a vaccine.

That is, the present invention identifies B cell epitopes highly reactive with HCV patient serum in the core, NS3, and NS4 regions that are effective for detecting anti-HCV antibodies, and synthesizes them with constant quality. It was made for the purpose of providing it as a peptide.

Another object of the present invention is to provide a diagnostic method capable of highly sensitively measuring anti-HCV antibody in human serum by using a peptide highly reactive with HCV patient serum by an immunological method. The purpose is to provide.

[0012]

In order to achieve the above-mentioned object, the present inventors based on the gene sequence of HCV-J strain belonging to type II (type Ib), which is a major isolated virus type in Japan. , The amino terminal half region of the core (1-100
Amino acid residues), NS3 whole region (1007-1615 amino acid residues) and NS4 first half region (1688-1).
715 amino acid residues), peptides of 10 amino acid residues were synthesized by shifting the peptides by 3 each by the multipin peptide synthesis method. The reason for adopting such a method in the present invention is that, as described above, since B cell epitopes usually consist of 5 to 6 amino acid residues, it was judged that the epitope region could be detected without exception by this synthetic method.

[0013] Then, the sample was subjected to H-
Seven HCV patient sera with positive CV genome and two normal human sera as negative controls were used, and these samples were subjected to the enzyme-antibody method (ELISA) using the synthetic peptide as an antigen to obtain patient sera. It is intended to provide peptides having high reactivity with HCV patient sera shown in SEQ ID NOS: 1 to 13 below, by identifying a peptide region that reacts with, that is, an epitope region recognized by an antibody induced by HCV infection. This is one of the features of the invention.

Another feature of the present invention is as follows. That is, when an antigen enters the living body, an immune response is triggered, and as one of the results, an antibody against the antigen is produced. The easiness of producing this antibody differs depending on the epitope. The antigens used to detect early HCV antibodies as described above are not suitable for detecting the acute phase of infection. Moreover, individual differences in the repertoire of antibodies to individual epitopes produced may be considered, and diagnosis by antibody detection should be performed by summing the reactions of each antibody to individual epitopes. From these facts, when a peptide containing a specific epitope is used alone as an antigen, there is a high possibility that the detection rate and the reaction strength will decrease.

Therefore, in the present invention, the above SEQ ID NO: 1 is used.
~ 13, at least two or more, and the maximum is SEQ ID NOS: 1 to 1
It is intended to provide a diagnostic method for measuring an anti-human HCV antibody in human serum by an immunological method by using all of 3 as an antigen. However, when using the peptide of SEQ ID NO: 13, it is not necessary to use the peptides of SEQ ID NO: 11 and 12.

As the immunological method, in addition to the enzyme-antibody method (ELISA) described later, a passive hemagglutination reaction (PHA), a latex agglutination reaction, and a highly sensitive antibody using an antibody labeled with acridinium, etc. can be used. A chemiluminescence immunoassay method (chemiluminescence method) or the like can be used.

[0017]

EXAMPLES The present invention will be described below based on examples, but it goes without saying that the present invention is not limited to these examples.

(1) Peptide Synthesis Peptides were synthesized according to the protocol of the multi-pin peptide synthesis kit purchased from Chiron Mimotopes Pty Ltd. This synthesis method uses a solid-phase peptide synthesis method in which Fmoc (9-fluorenylmethoxycarbonyl) amino acid activated ester is condensed, and a pin attached to a supporting block so as to fit a 96-well microplate is used. Peptide is synthesized on the tip of.

The peptide is based on the amino acid sequence reported for the HCV-J strain, in the range of 1-100 amino acid residues for the core region, 1007-1516 amino acid residues of the entire region for the NS3 region, Then, the NS4 region was synthesized in a range of 1678-1715 amino acid residues by shifting the length of 10 mer by 3 amino acids. The types of peptides synthesized by this method are 31 types for the core region and 2 types for the NS3 region.
There were 01 types and 7 types of NS4 areas.

(2) ELISA As test sera, 7 human sera from HCV patients and 2 normal human sera were used. Prior to the reaction, add 200 μl of 3% skim milk / PBS to each pin to prevent nonspecific adsorption of serum.
It was immersed in the solution at room temperature for 1 hour, washed 3 times with PBS, and reacted with 100 μl of test serum diluted 100-fold with 3% skim milk / 0.1% Tween 20 / PBS at room temperature for 1 hour. After washing three times with PBS, and peroxidase-labeled anti-human immunoglobulin antibody 100μl diluted 500-fold with the above buffer, at room temperature, allowed to react for 1 hour, washed with PBS, and color former of ABTS hydrogen peroxide to substrate As, the antibody bound to the peptide antigen was visualized and the absorbance at OD ₄₁₅ was measured.

After measuring the absorbance, the pin was regenerated and repeatedly used. Pin regeneration is performed with sodium dodecyl sulfate and 2-
This was performed by a method of dissociating the bound antibody by using a buffer solution containing mercaptoethanol.

1 to 6 show the results of ELISA performed using a 10-mer peptide synthesized as an antigen based on the amino acid sequence of the HCV-J strain as an antigen. Those showing a reactivity higher than the horizontal line in these figures were determined to be positive. As can be seen, while these antigens hardly reacted with normal human serum, HCV patient serum reacted with a specific peptide antigen. However, the degree of reaction and the type of peptide antigen that reacts with
It was different depending on the individual patient. For example, the synthetic peptide with amino acid Nos. 19-28 in the core region reacted with the serum of 6 out of 7 patients, although the reaction intensity was different. On the other hand, the synthetic peptide of amino acid Nos. 43-52 in the same core region is No. 2 Reacts strongly with patient sera but not with other patient sera.

FIG. 7 shows the epitope region that reacted with the patient's serum as described above on the hydropathic plot of the amino acid sequence of the HCV-J strain. As can be seen from this figure, all the epitope regions other than the 1283-1298th epitope region of the NS3 region were hydrophilic regions. Further, none of the identified epitope regions was in agreement with the region presumed to be the active domain of protease and helicase on NS3.

Further, Tables 1 to 6 show the presence or absence of reactivity with each synthetic peptide for each patient serum. Based on this, the region of the B cell epitope of the HCV-J strain was identified.

[0025]

[Table 1]

[0026]

[Table 2]

[0027]

[Table 3]

[0028]

[Table 4]

[0029]

[Table 5]

[0030]

[Table 6]

There are three epitope regions in the analyzed core region, and amino acid numbers 1-19 and 16-3, respectively.
It was 1, 49-61. Seven regions have been identified in the NS3 region, which are amino acid numbers 1043-1058,
1112-1124, 1208-1223, 1283-
1298, 1424-1433, 1475-1487,
1568-1583. Then, in the analyzed NS4 region, amino acid numbers 1694-1706, 1700-1
It was found that there are two epitope regions of 712.

(3) Examination of peptide for detecting antibody to NS4 region The peptides shown in SEQ ID NO: 11 and SEQ ID NO: 12 are:
Seven amino acids of Val to Asp overlap. In such a case, instead of separately synthesizing these peptides, it is considered that the peptides in this region are collectively synthesized and used for detection. This is advantageous, for example, in improving the productivity and reducing the cost when manufacturing the diagnostic agent. Therefore, for this purpose, the following two peptides, A and B, were synthesized by the F-moc method using a peptide synthesizer 430A manufactured by Apladivos System and examined.

[0033]

[Chemical l]

[0034]

[Chemical 2]

The above-mentioned peptide A is a peptide containing all of the sequences of SEQ ID NO: 11 and SEQ ID NO: 12, and E
Three glycines were added to the amino terminus in the hope that the efficiency of immobilization on the LISA plate would increase.

Another peptide B is a peptide A in which 2 amino acid residues are removed from both ends of the glycine-free portion. This peptide B was designed as follows assuming that the epitope length is 6 amino acid residues. That is, Table 6 showing the results of examining the presence or absence of reactivity of the NS4 region between the above-mentioned synthetic peptide and patient serum
Since the second peptide of 2 is not reactive, it can be seen that the 7 amino acids at the amino terminal of the peptide cannot form an epitope. Therefore, considering this,
The site of the epitope from the most amino terminus that can exist in peptide A with an epitope of 6 amino acid residues is 3-8.
Position. From the same idea, two amino acid residues can be removed from the carboxyl group terminal side. So peptide A
15mer peptide B with 4 amino acid residues less than
(However, as described above, 3 glycines were added to the amino terminus to improve the efficiency of immobilization on an ELISA plate), and if this peptide B shows the same reactivity as peptide A, It can be considered that the peptide B has an epitope region and is effective as an anti-HCV antibody equivalent to SEQ ID NOs: 11 and 12.

Then, first, for the purpose of obtaining patient sera positive for peptide A, 14 plates of HCV-RNA positive patient sera were subjected to EL using a plate on which peptide A was immobilized.
As a result of ISA, 9 cases were positive. This positive rate (64%) was almost equal to the positive rate 4/7 (57%) shown above.

Next, the reactivity to the two peptides A and B was examined by ELISA using these 9 positive patient sera and 2 negative patient sera.

That is, 0.25 mg of each of the two kinds of peptides was added with 0.05M carbonate buffer (pH 9.6).
/ Ml and 0.06mg / ml, 50 per 1 hole
μl was dispensed and left overnight at 4 ° C. to immobilize on a plate. After washing 3 times with a washing solution (8% NaCl, 0.5% Tween 80) and filling the plate with 3% skim milk / PBS and blocking at 37 ° C. for 1 hour, the diluted solution (0.2% Teen 20, 3% skim milk) was used. / PBS)
Add 50 μl of patient serum diluted 100 times with 37 ° C
And reacted for 45 minutes. After washing three times, 50 μl of alkaline phosphatase-labeled antibody human immunoglobulin antibody (Tago 4503) diluted 500 times with a diluting solution was added, and the mixture was reacted at 37 ° C. for 45 minutes. After 3 washes, chromogenic substrate solution (0.8% ABTS, 0.003% H 2 0 2, 0.
1M citric acid, 0.2M disodium phosphate, pH 4.
50 μl of 0) was added and reacted at room temperature for 15 minutes.
Absorbance at 5 nm was measured.

As a result, as shown in FIG. 8, no difference in reactivity was observed between the two peptides A and B at the two concentrations, and this peptide B had an epitope region,
It was confirmed that the anti-HCV antibody has the same efficacy as SEQ ID NOS: 11 and 12.

Therefore, in the NS4 region which is a synthetic peptide having high reactivity with HCV patient serum, three glycines on the amino terminal side of peptide B were removed in place of the two SEQ ID NOs: 11 and 12. It was found that the 15-mer peptide of SEQ ID NO: 13 can be effectively used.

[0042]

INDUSTRIAL APPLICABILITY According to the present invention, B cell epitope regions on the N-terminal side of the core region of the HCV-J strain, the entire NS3 region, and the N-terminal side of the NS4 region are identified and used as the diagnostic antigen for HCV infection. There is an effect that a possible synthetic peptide can be provided.

Further, the reactivity of these epitope regions with the patient's serum was unique in both the positive rate and the strength of the reaction. Differences are also observed in the strength of the reaction in the epitope region that reacted with all patient sera. Therefore, rather than using one of these epitope regions alone, using a plurality of them as in the present invention has the effect of establishing a more sensitive diagnosis.

Although the antigens that have been used so far for the diagnosis of HCV have been mainly expressed proteins, the use of synthetic peptides has the effect of enabling the supply of antigens of constant quality.

[0045]

[Sequence list]

SEQ ID NO: 1 Sequence length: 19 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: N-terminal fragment Origin Biological name: Hepatitis C virus Strain name: HCV- J strain Sequence features Characteristic symbols: CDS Location: 1. ． 19 Method of characterizing: E sequence Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr 1 5 10 15 Asn Arg Arg Pro SEQ ID NO: 2 Sequence length: 16 Sequence type: Amino acid Topology: Direct Chain Type of sequence: Peptide Fragment type: Intermediate fragment Origin Biological name: Hepatitis C virus Strain name: HCV-J strain Sequence features Characteristic symbols: CDS Life position: 16. ． 31 Method of characterizing: E sequence Asn Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val 1 5 10 15 SEQ ID NO: 3 Sequence length: 13 Sequence type: Amino acid Topology: Linear sequence Type: Peptide Fragment type: Intermediate fragment Origin Biological name: Hepatitis C virus Strain name: HCV-J strain Sequence features Characteristic symbols: CDS Location: 49. ． 61 Characterization Method: E Sequence Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg 1 5 10 SEQ ID NO: 4 Sequence Length: 16 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Fragment type: Intermediate fragment Origin Biological name: Hepatitis C virus Strain name: HCV-J strain Sequence features Characteristic symbols: CDS Location: 1043. ． 1058 Method of characterizing: E sequence Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln Val Asp Gly Glu 1 5 10 15 SEQ ID NO: 5 Sequence length: 13 Sequence type: Amino acid Topology: Linear sequence Type: peptide fragment type: middle fragment origin organism name: hepatitis C virus (Hepatitis C)
virus) Strain name: HCV-J strain Features of sequence Characteristic symbol: CDS Location: 1112. ． 1124 Method of characterizing: E sequence Pro Ala Pro Pro Gly Ala Arg Ser Met Thr Pro Cys Thr 1 5 10 SEQ ID NO: 6 Sequence length: 16 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Origin organism name: Hepatitis C virus Strain name: HCV-J strain Sequence features Characteristic symbols: CDS Location: 1208. ． 1223 Method of characterizing: E sequence Pro Val Phe Thr Asp Asn Ser Ser Pro Pro Ala Val Pro Gln Thr Phe 1 5 10 15 SEQ ID NO: 7 Sequence length: 16 Sequence type: Amino acid Topology: Linear sequence Type: Peptide Fragment type: Intermediate fragment Origin Biological name: Hepatitis C virus Strain name: HCV-J strain Sequence features Characteristic symbols: CDS Location: 1283. ． 1298 Method of characterizing: E sequence Arg Thr Ile Thr Thr Gly Gly Pro Ile Thr Tyr Ser Thr Tyr Cys Lys 1 5 10 15 SEQ ID NO: 8 Sequence length: 10 Sequence type: Amino acid Topology: Linear sequence Type: Peptide Fragment type: Intermediate fragment Origin Biological name: Hepatitis C virus Strain name: HCV-J strain Sequence features Characteristic symbols: CDS Location: 1424. ． 1433 Method by which the characteristics were determined: E SEQ ID NO: 9 Sequence length: 13 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Origin Biological name: Hepatitis C virus Strain name: HCV- J strain Sequence features Characteristic symbol: CDS Location: 1475. ． 1487 Method of characterizing: E sequence Thr Thr Leu Pro Gln Asp Ala Val Ser Arg Ala Gln Arg 1 5 10 SEQ ID NO: 10 Sequence length: 16 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Origin Biological name: Hepatitis C virus Strain name: HCV-J strain Sequence features Characteristic symbols: CDS Location: 1568. ． 1583 Method of characterizing: E sequence Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln Ala Gly Asp Asn Leu 1 5 10 15 SEQ ID NO: 11 Sequence length: 13 Sequence type: Amino acid Topology: Linear sequence Type: Peptide Fragment type: Intermediate fragment Origin Biological name: Hepatitis C virus Strain name: HCV-J strain Sequence features Characteristic symbols: CDS Location: 1694. ． 1706 Method of characterizing: E sequence Val Ile Pro Asp Arg Glu Val Leu Tyr Gln Glu Phe Asp 1 5 10 SEQ ID NO: 12 Sequence length: 13 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Origin Biological name: Hepatitis C virus Strain name: HCV-J strain Sequence features Characteristic symbols: CDS Location: 1700. ． 1712 Method of characterizing: E sequence Val Leu Tyr Gln Glu Phe Asp Glu Met Glu Glu Cys Ala 1 5 10 SEQ ID NO: 13 Sequence length: 15 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Origin Biological name: Hepatitis C virus Strain name: HCV-J strain Sequence features Characteristic symbols: CDS Location: 1700. ． 1712 Method of characterizing: E sequence Pro Asp Arg Glu Val Leu Tyr Gln Glu Phe Asp Glu Met Glu Glu 1 5 10 15

[Brief description of drawings]

FIG. 1 is a diagram showing the reactivity of 10 mer peptides synthesized in the core region of 1 to 100 amino acid residues with each serum in ELISA.

FIG. 2 is an EL of each 10-mer peptide synthesized in the NS3 region 1007-1163 amino acid residues and each serum.
The figure which showed the reactivity by ISA.

FIG. 3 is an EL of each 10-mer peptide synthesized in the NS3 region 1157-1313 amino acid residues and each serum.
The figure which showed the reactivity by ISA.

FIG. 4 is an EL of each serum and each 10-mer peptide synthesized in the NS3 region 1307-1463 amino acid residues.
The figure which showed the reactivity by ISA.

FIG. 5: EL of each serum and each 10-mer peptide synthesized in the NS3 region 1457-1613 amino acid residue range
The figure which showed the reactivity by ISA.

FIG. 6 shows EL of each 10meI peptide synthesized in the NS4 region in the range of 1688-1715 amino acid residues and each serum.
The figure which showed the reactivity by ISA.

FIG. 7 is a diagram showing the correspondence on the hydropathy plot of the region of the identified B cell epitope.

FIG. 8 shows the reactivity of peptides A and B with patient serum
The figure which showed the result investigated by ISA.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Keiji Mitamura 3-16-11 Motoamuma, Suginami-ku, Tokyo

Claims (15)

[Claims] 1. A synthetic peptide having high reactivity with HCV patient serum, which is composed of the amino acid sequence shown in SEQ ID NO: 1. 2. A synthetic peptide having a high reactivity with HCV patient serum, which is composed of the amino acid sequence shown in SEQ ID NO: 2. 3. A synthetic peptide having a high reactivity with HCV patient serum, which is composed of the amino acid sequence shown in SEQ ID NO: 3. 4. A synthetic peptide having a high reactivity with HCV patient serum, which is composed of the amino acid sequence shown in SEQ ID NO: 4. 5. A synthetic peptide having a high reactivity with HCV patient serum, which is composed of the amino acid sequence shown in SEQ ID NO: 5. 6. A synthetic peptide having high reactivity with HCV patient serum, which is composed of the amino acid sequence shown in SEQ ID NO: 6. 7. A synthetic peptide having a high reactivity with HCV patient serum, which is composed of the amino acid sequence shown in SEQ ID NO: 7. 8. A synthetic peptide having a high reactivity with HCV patient serum, which is composed of the amino acid sequence shown in SEQ ID NO: 8. 9. A synthetic peptide having a high reactivity with HCV patient serum, which is composed of the amino acid sequence shown in SEQ ID NO: 9. 10. A synthetic peptide having high reactivity with HCV patient serum, which is characterized by being constituted by the amino acid sequence shown in SEQ ID NO: 10. 11. A synthetic peptide having a high reactivity with HCV patient serum, which is composed of the amino acid sequence shown in SEQ ID NO: 11. 12. A synthetic peptide having a high reactivity with HCV patient serum, which is composed of the amino acid sequence shown in SEQ ID NO: 12. 13. A synthetic peptide having a high reactivity with HCV patient serum, characterized by being constituted by the amino acid sequence shown in SEQ ID NO: 13. 14. An immunological assay for anti-human HCV antibody in human serum using at least two peptides selected from the peptides of SEQ ID NOS: 1 to 13 shown in claims 1 to 13. A diagnostic method for HCV infection characterized by: 15. In human serum, using at least two or more peptides selected from the peptides of SEQ ID NOS: 1 to 10 shown in claims 1 to 10 and the peptide of SEQ ID NO: 13 shown in claim 13. A method for diagnosing HCV infection, which comprises immunologically measuring the anti-human HCV antibody.