JPH05262792A - Peptide and its use - Google Patents

Abstract

PURPOSE:To obtain a peptide, having a specific amino acid sequence and the ability to specifically bind to an antibody having specificity for a non-A non-B type hepatitis-related antigen and useful as a detecting agent for an anti-non-A non-B type hepatitis virus antibody in diagnosing, etc., of the non-A non-B type hepatitis. CONSTITUTION:A granular resin of a styrene-divinylbenzene copolymer having 4[Nalpha-(t-butoxycarbonyl)-Nepsilon(2-chlorobenzyloxycarbonyl)-L- lysyloxymethyl]phenylacetylamidomethyl group is used as a solid-phase support and an amino acid having protected alpha-amino group and functional group in the side chain is bound from the C-terminal side according to an amino acid sequence of the peptide by a solid-phase synthetic method and operation to remove the alpha-amino protecting group is successively repeated to synthesize a protected peptide chain on the resin. The resultant peptide chain is subsequently treated with hydrogen fluoride and the peptide is released from the resin. All the protecting groups are removed to afford the objective peptide containing amino acid sequences of formulas I, II and III.

Description

Detailed Description of the Invention

[0001]

This invention relates to peptides and their uses. The peptides provided by the present invention are non-A
Since it has a high specific binding ability to an antibody (hereinafter, abbreviated as anti-HCV antibody) having specificity for a non-hepatitis B-related antigen (hereinafter, abbreviated as HCV related antigen), It can be used to measure anti-HCV antibodies.

The assay reagent for anti-HCV antibody provided by the present invention has the ability to detect anti-HCV antibody in serum or plasma with high sensitivity, and is useful for assaying anti-HCV antibody.

[0003]

2. Description of the Related Art There are currently five known pathogenic viruses of viral hepatitis, which account for the majority of liver diseases. They are called hepatitis A, B, C, D and E viruses, respectively. There is. Among viral hepatitis, hepatitis A and hepatitis E are oral infections, and transient infection alone does not cause chronic infection, but hepatitis B and hepatitis C become chronic due to persistent infection, and there is a high probability that they will become chronic. It becomes a big problem because it shifts to cirrhosis or liver cancer. The causative viruses of hepatitis A, hepatitis B and hepatitis D have been found, and immunological diagnosis is now possible. E
It has been reported that the gene of hepatitis B virus was recently isolated.

Post-transfusion non-A non-B hepatitis (hereinafter referred to as PTN
The etiological virus (abbreviated as ANBH) has long been unknown despite research by many researchers.
In 1988, a research group of Chiron Corporation in the United States isolated and identified the gene of PTNANBH virus from plasma of chimpanzee infected with PTNANBH [Science, Vol. 244, p. 359].
(1988) and Science, 244, 362 (19)
1988)], hepatitis C virus (hereinafter referred to as HCV). Some of the putative nucleotide sequences of the gene have already been clarified [see European Patent 0318216], and it became possible to detect anti-HCV antibodies, and serological diagnosis of HCV infection was made possible. It is possible.

It has been reported that a ribonucleic acid presumed to be a gene of a virus causing PTNANBH was isolated from the serum of a PTNANBH patient and identified by several persons including one of the present inventors. [Gastroenterologia Japonica, Vol. 24, No. 5, p. 540 (1989); Gastroenterologia Japonica;
Japonica, Vol. 24, No. 5, 545 (1989) and Internal Medicine, Vol. 64, No. 6, 1022 (1989)].

So far, the required cDNA from a cDNA library
As a means for screening NA, it has been generally performed to determine positive or negative of anti-HCV antibody by an antigen-antibody reaction using λ phage, but the immunoscreening method described above is not quantitative and E. coli. Reaction with non-specific antigen components in the expression product of Escherichia coli may occur. Currently, the enzyme immunoassay is carried out using an antigen protein cloned from the HCV gene, integrated into a phage and expressed in yeast as a host. Development of a test drug for anti-HCV antibodies is being considered [Internal Medicine, Vol. 64, No. 6, page 1027 (1989).
Year)]. Furthermore, a particle agglutination method utilizing the property that gelatin particles sensitized by a virus or its antigen component aggregate in the presence of an anti-virus antibody or a bead method in which enzyme immunoassay is performed using beads coated with the virus or its antigen component. Is being developed.

[0007]

[Problems to be Solved by the Invention] In conventional enzyme immunoassays utilizing HCV-related antigens, the measurement target is clinically
Even when PTNANBH is diagnosed, the anti-HCV antibody positive rate is about 75%, and the anti-HCV antibody-negative PTNANBH is contained at about 25%. In the enzyme immunoassay described above, when the measurement target is a healthy person, the positive rate is about 1%.
Therefore, about 2% of anti-HCV antibody-positive specimens are overlooked because the statistical HCV prevalence rate is about 3%. Therefore, in HCV carrier screening in blood donors, it is unavoidable that carriers are overlooked, and the rate of blocking transfusion of non-A non-B hepatitis virus-infected blood is not necessarily high.

On the other hand, since the antigenic protein produced by cloning the HCV gene and incorporating it into a phage and expressing it in yeast as a host contains various nonspecific antigenic components,
When the anti-HCV antibody is measured using the antigen protein as a reagent, the reagent also recognizes other non-specific antibody components other than the anti-HCV antibody in the sample, and the measurement result is not always It does not always represent the presence of HCV antibodies. As described above, according to the conventional enzyme immunoassays using HCV-related antigens, it is the current situation that the existence of anti-HCV antibodies cannot be accurately known.

One object of the present invention is to provide peptides having the ability to specifically bind anti-HCV antibodies. Another object of the present invention is to provide a reagent for measuring anti-HCV antibody.

[0010]

[Means for Solving the Problems] The present inventors have found that PTNANBH
From the peptide fragments selected from the polypeptides encoded by the related genes, a specific peptide having the ability to specifically bind to an antibody having specificity for non-A non-B hepatitis-related antigen was found, and the present invention It came to completion.

According to the present invention, the above objects are achieved by Lys Arg.
Ser Thr Asn (SEQ ID NO: 2), Arg Arg Tyr Lys Glu
Lys Glu Lys (SEQ ID NO: 4), or Ala Ile Ile Pr
It is achieved by providing a peptide containing the amino acid sequence of Asp Arg Glu Val Leu Tyr (SEQ ID NO: 6) and having the ability to specifically bind to an antibody with specificity for an HCV-related antigen.

Specifically, the formula (I): Lys Asp Arg
Thr Gln Gln Arg Lys Thr Lys ArgSer Thr Asn Arg Arg
Arg Ser Lys Asn Glu Lys Lys Lys Lys (SEQ ID NO:
A peptide comprising a peptide having the amino acid sequence shown in 1) or a fragment thereof, wherein the peptide is Lys Ar
A peptide having the amino acid sequence of g Ser Thr Asn (SEQ ID NO: 2) and capable of specifically binding to an antibody having specificity for an HCV-related antigen, (II) Formula (2): Gl
u Lys Lys Gly Glu Ala Ser Asn Gly Glu Ala Glu Asn
Asp Thr His Lys Lys Gln Arg Arg Tyr Lys Glu Lys Gl
u Lys Thr Ala Thr Asn Asn Pro Gly Lys Asn Lys Lys
A peptide comprising a peptide having an amino acid sequence represented by Pro Arg (SEQ ID NO: 3) or a fragment thereof, wherein the peptide is ArgArg Tyr Lys Glu Lys Glu Lys
A peptide having the amino acid sequence of (SEQ ID NO: 4) and having the ability to specifically bind to an antibody having specificity for an HCV-related antigen, (III) Formula (3): Arg Val Val Leu Se
r Gly Lys Pro Ala Ile Ile Pro Asp Arg Glu Val Leu
Tyr Arg Glu Phe Asp Glu Met Glu Glu Cys Ser Gln Hi
s Leu Pro Tyr Ile Glu Gln Gly Met Met (SEQ ID NO:
A peptide comprising the peptide having the amino acid sequence shown in 5) or a fragment thereof, wherein the peptide is Ala Il
eIle Pro Asp Arg Glu Val Leu Tyr (SEQ ID NO: 6)
A peptide having the amino acid sequence of and having the ability to specifically bind to an antibody having specificity for an HCV-related antigen.

It is also achieved by providing an anti-HCV antibody measuring reagent comprising the above peptide.

In the present specification, various amino acid residues are described by the following abbreviations. Ala: L-alanine residue Arg: L-arginine residue Asn: L-asparagine residue Asp: L-aspartic acid residue Cys: L-cysteine residue Gln: L-glutamine residue Glu: L-glutamic acid residue Gly: Glycine residue His: L-histidine residue Ile: L-isoleucine residue Leu: L-leucine residue Lys: L-lysine residue Met: L-methionine residue Phe: L-phenylalanine residue Pro: L -Proline residue Ser: L-serine residue Thr: L-threonine residue Trp: L-tryptophan residue Tyr: L-tyrosine residue Val: L-valine residue In this specification, an amino acid is used according to a conventional method. The sequence is written so that the N-terminal amino acid residue is located on the left and the C-terminal amino acid residue is located on the right.

The peptide of the present invention is a peptide having the ability to specifically bind to an antibody having specificity for an HCV-related antigen, and contains at least Lys Arg Ser.
ThrAsn (SEQ ID NO: 2), Arg Arg Tyr Lys Glu Lys G
lu Lys (SEQ ID NO: 4) or Ala Ile Ile Pro Asp
It has the amino acid sequence of Arg Glu Val Leu Tyr (SEQ ID NO: 6).

Of these partial amino acid sequences,
The peptide having the Lys Arg Ser Thr Asn sequence (SEQ ID NO: 2) has high antigenicity and is preferably used in the measurement of an antibody having specificity for an HCV-related antigen.

The peptide of the present invention is not particularly limited as long as it has the above-mentioned partial amino acid sequence, but it is usually a peptide consisting of 10 to 40 amino acids.

Specific examples include peptides consisting of the following peptides or fragments thereof. That is, the peptide of the present invention is a peptide comprising a peptide having the amino acid sequence represented by the above formula (1) or a fragment thereof, wherein the peptide is the amino acid sequence of Lys Arg Ser Thr Asn (SEQ ID NO: 2). And a peptide having the ability to specifically bind to an antibody having specificity for an HCV-related antigen. Examples of the peptide consisting of the fragment include formula (1-a), but the peptide is not limited thereto. Formula (1-a): Thr Lys Arg Ser Thr Asn Arg Arg Arg Ser (SEQ ID NO: 7)

The peptide of the present invention is a peptide comprising the peptide having the amino acid sequence represented by the above formula (2) or a fragment thereof, wherein the peptide is Ar.
Examples include peptides having the amino acid sequence of g Arg Tyr Lys Glu Lys Glu Lys (SEQ ID NO: 4) and having the ability to specifically bind to an antibody having specificity for an HCV-related antigen. Examples of the peptide composed of the fragments include, but are not limited to, formula (2-a), formula (2-b), and formula (2-c). Formula (2-a): Arg Arg Tyr Lys Glu Lys Glu Lys Thr Ala Thr Asn Asn Pro Gly Lys Asn Lys Lys Pro Arg (SEQ ID NO: 8) Formula (2-b): Thr His Lys Lys Gln Arg Arg Tyr Lys Glu Lys Glu Lys (SEQ ID NO: 9) Formula (2-c): Arg Arg Tyr Lys Glu Lys Glu Lys Thr Ala (SEQ ID NO: 10)

The peptide of the present invention is a peptide comprising a peptide having the amino acid sequence represented by the above formula (3) or a fragment thereof, wherein the peptide is Al.
a Ile Ile Pro Asp Arg Glu Val Leu Tyr (SEQ ID NO:
Peptides having the amino acid sequence of 6) and having the ability to specifically bind to an antibody having specificity for an HCV-related antigen can be mentioned. Examples of the peptide composed of a fragment include, for example, formula (3-a), formula (3-b) and formula (3
-C) is exemplified, but not limited thereto. Formula (3-a): Ala Ile Ile Pro Asp Arg Glu Val Leu Tyr Arg Glu Phe Asp Glu Met Glu Glu Cys Ser Gln His Leu Pro Tyr Ile Glu Gln Gly Met Met (SEQ ID NO: 11) Formula (3-b) : Arg Val Val Leu Ser Gly Lys Pro Ala Ile Ile Pro Asp Arg Glu Val Leu Tyr (SEQ ID NO: 12) Formula (3-c): Ala Ile Ile Pro Asp Arg Glu Val Leu Tyr (SEQ ID NO: 13)

As the peptide of the present invention, various peptides can be exemplified as described above, but the peptide of the formula (1-a) is preferable from the viewpoint of reaction specificity. The peptide of the present invention has the ability to specifically bind to an antibody having specificity for such an HCV-related antigen.

The peptide of the present invention is synthesized by a method usually used in peptide synthesis, for example, a solid phase synthesis method or a liquid phase synthesis method such as a stepwise extension method or a fragment condensation method. It is easy to operate by the method [for example, the Journal of the American Chemical Society (Journal of the America
n Chemical Society), Vol. 85, pp. 2149-2154 (1963)
); "Biochemistry Experiment Course 1 Protein Chemistry IV Chemical Modification and Peptide Synthesis," edited by the Japanese Biochemical Society (November 15, 1972)
Nihon Tokyo Kagaku Doujin), pp. 207-495; "Biochemistry Chemistry Lecture 2 Protein Chemistry (2)" edited by The Biochemical Society of Japan (May 20, 1987, Tokyo Kagaku Doujin), No. See pages 641-694, etc.].

The peptide of the present invention can be produced by a solid phase synthesis method, for example, a polymer which is insoluble in a reaction solvent such as a styrene-divinylbenzene copolymer, an amino acid corresponding to the C-terminal of the peptide of interest, or the amino acid corresponding thereto. Α-COO-group or α-CONH obtained by removing a hydrogen atom from an α-COOH group or an α-CONH ₂ group having an amide, respectively.
-A group other than an α-COOH group other than the α-COOH group carried by the amino acid or peptide fragment in the direction of the N-terminus of the peptide of interest to the amino acid or its amide. An operation in which a functional group such as an amino group is protected and then condensed and bonded, and α in the bonded amino acid or peptide fragment
-By sequentially repeating the operation of removing the protecting group carried by the amino group forming a peptide bond such as an amino group,
Peptide of interest by elongating the peptide chain to form a peptide chain corresponding to the desired peptide, then removing the peptide chain from the polymer, and removing the protecting group from the protected functional group Is obtained and then purified. Here, the elimination of the peptide chain from the polymer and the removal of the protective group are preferably carried out simultaneously using hydrogen fluoride, from the viewpoint of suppressing side reactions. Further, it is effective to purify the obtained peptide by reverse phase liquid chromatography.

Since the peptide of the present invention has the ability to specifically bind anti-HCV antibody, the anti-HCV antibody produced by HCV infection has a
It is effective as a measuring reagent for the detection of HCV antibodies. That is, the measuring reagent of the present invention comprises the peptide of the present invention, and the peptide of the present invention as described above may be used alone or in combination of two or more kinds.

The anti-HCV antibody using the peptide of the present invention is measured by using any one of a fluorescent immunoassay method, a passive hemagglutination method, a radioimmunoassay method and an enzyme immunoassay method. Although all of these methods are known, the case of using an enzyme immunoassay will be described below.

The constituent elements of the whole measuring system are a carrier, the peptide of the present invention as a measuring reagent, a blocking agent, a test sample,
It consists of a labeling antibody, an enzyme and a color former. The carrier is coated with the peptide of the present invention, the blocking agent is allowed to act on the peptide-coated carrier to block non-specific protein binding sites on the carrier, and the test sample is added to the peptide-coated carrier and incubated. An enzyme-labeled antibody is contacted and incubated, then a color-developing agent is added to the carrier treated in this way, and the mixture is incubated, and the production amount of the reaction product of the reaction between the enzyme and the color-developing agent is measured using an absorptiometer. The peptide of the present invention used for coating may be one type or two or more types. It is preferable to use an enzyme immunoassay cup or beads made of glass or resin as the carrier.

Prior to the measurement, the peptide of the present invention was added to 0.01M.
After dissolving in a carbonate buffer and adding the solution to a polystyrene enzyme immunoassay cup, for example, at 4 ° C
The surface of the carrier is coated with the peptide of the present invention by standing overnight at room temperature or at room temperature for 3 hours. As a blocking agent for blocking the non-specific protein binding site on the carrier, for example, bovine serum albumin, casein,
Skim milk powder, serum of an immunogenic animal for obtaining anti-human IgG antibody or anti-human IgM antibody, gelatin, etc. are used.
Examples of the labeling antibody include anti-human IgG antibody and anti-human Ig antibody.
M antibody or the like is used. Examples of the enzyme include alkaline phosphatase, glucose oxidase, peroxidase, beta-galactosidase and the like. Prior to the measurement, a compound having two or more functional groups such as glutaraldehyde is used to bind an enzyme to the labeling antibody to form a conjugate, which is prepared in advance as a part of the components of the entire measurement system. Preferably.
The color former may be appropriately used depending on the selected enzyme. For example, when peroxidase is selected as the enzyme, it is preferable to use o-phenylenediamine or the like.

As described above, according to the present invention, a peptide having an ability to specifically bind to an anti-HCV antibody is provided.
This peptide has made it possible to provide an anti-HCV antibody measuring reagent having sensitivity and specificity superior to that of existing anti-HCV antibody measuring reagents.

[0029]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

Example 1 Formula (1): Lys Asp Arg Thr Gln Gln Arg Lys Thr Lys
Arg Ser Thr Asn ArgArg Arg Ser Lys Asn Glu Lys Lys
Lys Lys (SEQ ID NO: 1) was used as a peptide automatic synthesizer [Model 431A (Model 431A, manufactured by Applied Biosystems, USA).
A)] was used for the solid phase synthesis.

That is, 4- [Nα- (t-butoxycarbonyl) -Nε- (2-chlorobenzyloxycarbonyl) -L-lysyloxymethyl] phenylacetylamidomethyl group

[Chemical 1] Having a ratio of 0.65 mmol / g (resin)
Granular resin consisting of divinylbenzene copolymer [styrene / divinylbenzene constituent molar ratio: 99: 1] [PAM Lysine (Applied Biosystems, USA)
, T-Boc-L-Lys (Cl-Z)] was used in an amount of 760 mg according to the series of operations shown in Table 1, and the corresponding L-arginine and L-asparagine were turned toward the N-terminal direction of the target peptide. , L-aspartic acid, L-glutamine, L-glutamic acid, L-lysine, L-serine, and L-threonine were sequentially bound. In the condensation reaction, the above amino acids are Nα- (t-butoxycarbonyl) -Nγ- (mesitylene-2-sulfonyl) -L-arginine and N-, respectively.
(T-Butoxycarbonyl) -L-asparagine, N-
(T-Butoxycarbonyl) -L-aspartic acid-β
-Benzyl ester, N- (t-butoxycarbonyl)
-L-glutamine, N- (t-butoxycarbonyl)-
L-glutamic acid-γ-benzyl ester, Nα- (t
-Butoxycarbonyl) -Nε- (2-chlorobenzyloxycarbonyl) -L-lysine, N- (t-butoxycarbonyl) -o-benzyl-L-serine, N- (t-
Butoxycarbonyl) -o-benzyl-L-threonine, and the amount used was about 4 times the molar amount of the substrate.

[Table 1]

The condensation reaction was carried out at room temperature. The reaction time including all steps required to combine one amino acid residue is 1
It was within the range of 00 to 110 minutes. After the reaction procedure for all amino acids was completed, the obtained resin was washed successively with dichloromethane and methanol on a glass filter and then vacuum dried to obtain 2.58 g of dried resin. Next, a reaction vessel made of polytrifluoromonochloroethylene (manufactured by Peptide Institute Co., Ltd., HF-
0.7 g of the obtained dry resin was mixed with 1.05 ml of anisole and 0.175 ml of ethyl methyl sulfide in a reactor type I), 7.0 ml of hydrogen fluoride was added to the mixture at a temperature of -20 ° C, and the mixture was stirred at the same temperature for 30 minutes. It was stirred for 1 minute and then for 30 minutes at a temperature of 0 ° C. Hydrogen fluoride, anisole and ethyl methyl sulfide were removed from the resulting reaction mixture under reduced pressure, and the residue was thoroughly washed on a glass filter with diethyl ether and dichloromethane. The obtained residue was extracted with a 2N aqueous acetic acid solution, and the extract was freeze-dried to obtain 200 mg of a crude peptide product.

The obtained crude product was subjected to preparative reverse phase high performance liquid chromatography [column: octadecylated silica gel].
(Particle size: 15 μm) Packed column (inner diameter: 50 mm, length: 300 m
m), manufactured by Japan Waters, μBONDASPHERE 15 μC18
-100; Mobile phase: Mixed solvent of acetonitrile and water containing 0.05% by volume of trifluoroacetic acid (concentration of acetonitrile was gradually changed from 10% by volume to 20% by volume in 30 minutes); Flow rate: 5 ml / Min; detection method: absorbance at a wavelength of 210 nm] to obtain 80 mg of a purified product of the target peptide. The obtained purified product was analyzed by reversed-phase high performance liquid chromatography [column: octadecylated silica gel (particle size: 5 μm) packed column (inner diameter: 4 mm,
Length: 150mm), Tosoh Corporation, TSK-gel ODS-80T
M; Mobile phase: mixed solvent of acetonitrile and water containing 0.05% by volume of trifluoroacetic acid (concentration of acetonitrile was gradually changed from 5% by volume to 50% by volume in 30 minutes); Flow rate: 1 ml / Min; detection method: absorbance at a wavelength of 210 nm], a single sharp peak was shown at 15.0 minutes. The molecular weight of the purified product determined by the fast atom bombardment method (hereinafter referred to as FAB method) mass spectrum was 3188 (theoretical value: 3187.53).

Example 2 By carrying out solid-phase synthesis and purification of a peptide in the same manner as in Example 1, Formula (1-a): Thr Lys Ar
A peptide represented by g Ser Thr Asn Arg Arg Arg Ser (SEQ ID NO: 7) was obtained. The resulting peptide was subjected to analytical reverse phase high performance liquid chromatography (same as above) and showed a single sharp peak at 14.2 minutes. The molecular weight of the peptide determined by FAB mass spectrometry is 1243.
(Theoretical value: 1243.33).

Example 3 By carrying out solid-phase synthesis and purification of a peptide in the same manner as in Example 1, the formula (2-a): Arg Arg Ty was obtained.
r Lys Glu Lys Glu Lys Thr Ala Thr Asn AsnPro Gly L
A peptide represented by ys Asn Lys Lys Pro Arg (SEQ ID NO: 8) was obtained. When the obtained peptide was subjected to analytical reverse phase high performance liquid chromatography (the same as above),
A single sharp peak was shown at 22.0 minutes. The molecular weight of the peptide determined by FAB mass spectrometry was 2542 (theoretical value: 2541.85).

Example 4 By carrying out solid-phase synthesis and purification of a peptide by the same method as in Example 1, Formula (2-b): Thr His Ly
A peptide represented by s Lys Gln Arg Arg Tyr Lys Glu Lys Glu Lys (SEQ ID NO: 9) was obtained. The resulting peptide was subjected to analytical reverse phase high performance liquid chromatography (same as above) and showed a single sharp peak at 14.3 minutes. The molecular weight of the peptide determined by FAB mass spectrometry was 1758 (theoretical value: 1758.01).

Example 5 Solid-phase peptide synthesis and purification were carried out in the same manner as in Example 1 to give the compound of formula (2-c): Arg Arg Ty.
A peptide represented by r Lys Glu Lys Glu Lys Thr Ala (SEQ ID NO: 10) was obtained. The resulting peptide was subjected to analytical reverse phase high performance liquid chromatography (as above) and showed a single sharp peak at 14.5 minutes. The molecular weight of the peptide determined by FAB method mass spectrum is 13
It was 06 (theoretical value: 1306.49).

Example 6 By carrying out solid phase synthesis and purification of a peptide by the same method as in Example 1, the compound of formula (3-a): Ala Ile Il
e Pro Asp Arg Glu Val Leu Tyr Arg Glu PheAsp Glu M
et Glu Glu Cys Ser Gln His Leu Pro Tyr Ile Glu Gln
A peptide represented by Gly Met Met (SEQ ID NO: 11) was obtained. The resulting peptide was subjected to analytical reverse phase high performance liquid chromatography (as above) and showed a single sharp peak at 29.8 minutes. The molecular weight of the peptide determined by FAB mass spectrometry was 3785 (theoretical value: 3785.19).

Example 7 By carrying out solid-phase synthesis and purification of a peptide by the same method as in Example 1, Formula (3-b): Arg Val Va
l Leu Ser Gly Lys Pro Ala Ile Ile Pro AspArg Glu V
A peptide represented by al Leu Tyr (SEQ ID NO: 12) was obtained. The resulting peptide was subjected to analytical reverse phase high performance liquid chromatography (as above) and showed a single sharp peak at 28.7 minutes. The molecular weight of the peptide determined by FAB mass spectrometry was 1953 (theoretical value: 1953.15).

Example 8 By carrying out solid-phase synthesis and purification of a peptide by the same method as in Example 1, the compound of formula (3-c): Ala Ile Il
A peptide represented by e Pro Asp Arg Glu Val Leu Tyr (SEQ ID NO: 13) was obtained. The resulting peptide was subjected to analytical reverse phase high performance liquid chromatography (as above) and showed a single sharp peak at 26.9 minutes. The molecular weight of the peptide determined by FAB mass spectrum is 12
It was 03 (theoretical value: 1203.34).

Reference Example 1 Among the peptides having the amino acid sequence represented by the formula (1), Lys Arg SerThr Asn was obtained by carrying out solid phase synthesis and purification of the peptide by the same method as in Example 1.
A peptide represented by the formula: Lys Asp ArgThr Gln Gln Arg Lys Thr Lys (SEQ ID NO: 14) having no amino acid sequence represented by (SEQ ID NO: 2) was obtained. The resulting peptide was subjected to analytical reverse phase high performance liquid chromatography (as above) and showed a single sharp peak at 11.9 minutes. The molecular weight of the peptide determined by FAB mass spectrometry was 1290 (theoretical value: 1290.39).

Reference Example 2 Among the peptides having the amino acid sequence represented by the formula (1), Lys Arg SerThr Asn is prepared by carrying out solid phase synthesis and purification of the peptide by the same method as in Example 1.
Formula having no amino acid sequence represented by (SEQ ID NO: 2): Arg Ser ThrAsn Arg Arg Arg Ser Lys Asn Glu Lys
Lys Lys Lys (SEQ ID NO: 15) was obtained as the peptide. The resulting peptide was subjected to analytical reverse phase high performance liquid chromatography (as above) and showed a single sharp peak at 13.6 minutes. The molecular weight of the peptide determined by FAB mass spectrum was 1915 (theoretical value: 1915.16).

Reference Example 3 By performing solid phase synthesis and purification of a peptide in the same manner as in Example 1, the formula: Glu Gln Asp Gln Ile
Lys Thr Lys Asp Arg Thr Gln Gln Arg Lys Thr Lys Ar
g Ser Thr Asn Arg Arg Arg Ser Lys Asn Glu Lys Lys
Lys Lys (SEQ ID NO: 16) was obtained as the peptide. The resulting peptide was subjected to analytical reverse phase high performance liquid chromatography (as above) and showed a single sharp peak at 24.6 minutes. The molecular weight of the peptide determined by FAB mass spectrometry was 4031 (theoretical value:
4031.38).

Reference Example 4 Arg Arg TyrLys Glu L among peptides having the amino acid sequence represented by the formula (2) was obtained by carrying out solid phase synthesis and purification of peptides by the same method as in Example 1.
Formula having no amino acid sequence represented by ys Glu Lys (SEQ ID NO: 4): Glu Lys Lys Gly Glu Ala Ser Asn Gly
A peptide represented by Glu Ala Glu Asn Asp (SEQ ID NO: 17) was obtained. The obtained peptide was subjected to analytical reverse-phase high performance liquid chromatography (same as above).
A single sharp peak was shown at 4.9 minutes. The molecular weight of the peptide determined by FAB mass spectrometry was 1473 (theoretical value: 1473.47).

Reference Example 5 Among the peptides having the amino acid sequence represented by the formula (2), solid-phase synthesis and purification of the peptide were carried out in the same manner as in Example 1, Arg Arg TyrLys Glu L.
Formula having no amino acid sequence represented by ys Glu Lys (SEQ ID NO: 4): Thr Asn Asn Pro Gly Lys Asn Lys Lys
A peptide represented by Pro Arg (SEQ ID NO: 18) was obtained. The resulting peptide was subjected to analytical reverse phase high performance liquid chromatography (as above) and showed a single sharp peak at 12.6 minutes. The molecular weight of the peptide determined by FAB mass spectrum was 1253 (theoretical value:
1253.38).

Reference Example 6 By carrying out solid phase synthesis and purification of a peptide by the same method as in Example 1, among the peptides having the amino acid sequence represented by the formula (2), Arg Arg TyrLys Glu L
Formula having no amino acid sequence represented by ys Glu Lys (SEQ ID NO: 4): Val Gly Arg Ile Lys Asn Trp Asn Arg
Glu Gly Arg Lys Asp Ala Tyr Gln IleArg Lys Arg
A peptide represented by (SEQ ID NO: 19) was obtained. The resulting peptide was subjected to analytical reverse phase high performance liquid chromatography (same as above) and showed a single sharp peak at 19.4 minutes. The molecular weight of the peptide determined by FAB mass spectrometry was 2644 (theoretical value: 2643.9
2).

Reference Example 7 Among the peptides having the amino acid sequence represented by the formula (3), Ala Ile IlePro Asp A was obtained by carrying out solid phase synthesis and purification of the peptides by the same method as in Example 1.
Formula having no amino acid sequence represented by rg Glu Val Leu Tyr (SEQ ID NO: 6): Arg Val Val Leu Ser Gly Lys
A peptide represented by Pro Ala Ile Ile (SEQ ID NO: 20) was obtained. The resulting peptide was subjected to analytical reverse phase high performance liquid chromatography (as above) and showed a single sharp peak at 26.7 minutes. The molecular weight of the peptide determined by FAB mass spectrometry was 1081 (theoretical value: 1081.20).

Reference Example 8 Test Sample GPT>200IU; HBsHg (-) Serum: 65 Specimens Normal Human Serum: 10 Specimens Assay by Enzyme-Linked Immunosorbent Assay Anti-HC for each serum specimen by the following enzyme-linked immunosorbent assay
The presence or absence of V antibody was assayed.

Thus, E. coli Y1090 was added to 100 μl of a solution containing phage λgt11 carrying # 8, # 14 and # 18 clones cloned from ribonucleic acid isolated by several persons including one of the present inventors. Stock (Escher
ichia coli Y1090) as a supporting bacterium, and then mix at 37 ℃
After incubation for 15 minutes, the phage was infected with E. coli. Subsequently, the above mixed solution was inoculated into an agar medium containing 50 μg / ml of ampicillin and cultured at 43 ° C. for 3 hours. Next, the nitrocellulose membrane obtained by soaking in a 10 mM IPTG aqueous solution for 2 hours and then air-dried was placed on the above agar medium and incubated at 37 ° C. for 3 hours. The nitrocellulose membrane thus obtained was treated with 10 mM containing 150 mM NaCl.
Wash three times with Tris-HCl (pH7.5) (hereinafter, abbreviated as TS Buffer), then shake overnight in room temperature at room temperature in 20 mM Tris-HCl (pH7.5) containing 500 mM NaCl and 3% gelatin. Non-specific protein binding sites on the membrane were blocked. Subsequently, the membrane was washed by shaking in TS Buffer for 2 minutes. The nitrocellulose membrane obtained above was immersed in a solution obtained by diluting a serum sample with TS Buffer containing 1% gelatin, and shaken at room temperature for 3 hours. The nitrocellulose membrane thus obtained was shaken in a TS Buffer containing 0.05% Tween 20 (hereinafter, abbreviated as TS-T Buffer) at room temperature for 5 minutes, and this operation was repeated 5 times. Then, the nitrocellulose membrane was immersed in a goat anti-human IgG antibody-peroxidase conjugate (diluted with TS Buffer containing 1% gelatin to an optimum concentration) and shaken at room temperature for 1.5 hours. The nitrocellulose membrane thus obtained was used as a TS-T Buff.
shaken for 5 minutes at room temperature in an er, and this operation was repeated 5 times.

Next, TS Buffe containing 0.05% HRP-color (manufactured by Bio-Rad), 0.05% H ₂ O ₂ and 17% methanol.
The nitrocellulose membrane was shaken at r for color development reaction, and then shaken in distilled water at room temperature for 5 minutes, and this operation was repeated 5 times. After air drying, the presence or absence of color development was assayed for the presence or absence of anti-HCV antibody in the serum used.

The nucleotide sequence of the # 8 clone (SEQ ID NO: 21) is as follows. GAATTCCAAA AAGAGCAAAA CAAACCGCCG AAGAAAAAAC TAATAAGAGA AGAAAAGGCG 60 AAGAGACACA GGAAAAAAAA AACAGAGACG AAGGTCAGAT AGAAAAAAAG CAAGGAATTC 120 # 14 The nucleotide sequence of the clone (SEQ ID NO: 22) is as follows. GAATTCCGAG AACAAGACCA GATAAAAACC AAAGACAGAA CACAACAGAG AAAGACGAAA 60 AGAAGCACCA ATCGCAGGCG AAGCAAAAAC GAAAAAAAAA AAAAAAAGGA ATTC 114 # 18 The nucleotide sequence (clone number: 23) is as follows. GAATTCCAAG AAAAAAAGGG AGAAGCCAGC AATGGAGAAG CCGAAAACGA CACACACAAG 60 AAACAAAGGA GGTACAAAGA AAAAGAAAAA ACGGCAACAA ATAACCCAGG AAAGAACAAA 120 AAGCCAAGAG TGGGCAGAAT AAAAAACTGG AACCGGGAGG GAAGGAAGGA CGCATATCAG 180 AT

Results Table 2 shows the test results. As a result, the test sample G
It was found that 65 specimens of PT> 200 IU; HBsHg (-) serum can be classified into 3 groups. That is, the group A which was determined to be positive in two of the # 14 clone and the # 18 clone, the group B which was determined to be positive in three of the # 14 clone and the # 18 clone, and the # 8 clone, # 14 It is three groups, group C, which was determined to be negative in both the clone and the # 18 clone.

[Table 2]

Example 9 Test Sample Each serum classified as a result of Reference Example 8 was used. GPT> 200IU; HBsAg (-) serum A: 30 samples GPT> 200IU; HBsAg (-) serum B: 15 samples GPT> 200IU; HBsAg (-) serum C: 20 samples normal human serum D: 10 samples

Assay by enzyme immunoassay For each serum sample, the absorbance was measured by the following enzyme immunoassay to assay the presence or absence of anti-HCV antibody.

That is, the peptides obtained in Example 1, Example 2, Reference Example 1, Reference Example 2 and Reference Example 3 were each dissolved in 0.01 M carbonate buffer (pH 9.5) as an antigen substance,
100 μl of each of the obtained peptide solutions was placed in a polystyrene enzyme immunoassay cup (Dynatech).
After adding each of them, the peptide was coated by allowing to stand at 4 ° C. for 12 hours. Then, after removing the peptide solution contained in the obtained assay cups, the cups containing 20% by volume of normal goat serum were added.
150 μl of 01M phosphate buffered saline (hereinafter abbreviated as PBS) was added, and the mixture was allowed to stand at room temperature for 3 hours to block nonspecific protein binding sites. Then, PBS containing 20% by volume of normal goat serum used for blocking was removed, and then each assay cup was dried.

100 μl of PBS containing 10% by volume of normal goat serum as a serum dilution solution was added to each of the above assay cups.
After adding each, each test serum (GPT>200IU; HBsAg (-)
Serum A30 sample, GPT>200IU; HBsAg (-) serum B15 sample, GPT>200IU; HBsAg (-) serum C20 sample and normal human serum D10 sample), the ratio of serum dilution solution and test serum was 20 to 1 ( (Volume ratio). After incubation for 1 hour at 37 ° C, the cups were
The plate was washed 3 times with PBS containing Tween20.

To each of the obtained assay cups, 100 μl of goat anti-human IgG antibody-peroxidase conjugate (diluted with PBS containing 10% by volume of normal goat serum to an optimum concentration) was added. After incubating at 37 ° C for 30 minutes, the cups are added to PBS containing 0.05% by volume Tween20.
It was washed 3 times with. Subsequently, a color former (o-phenylenediamine was dissolved in 0.1M citric acid-phosphate buffer pH 5.6 containing 0.02% by volume of hydrogen peroxide so as to be 0.3% by weight) in each of the obtained assay cups. 100 μl) was added. After standing at room temperature for 15 minutes, 100 μl of 2N sulfuric acid was added to stop the reaction, and the absorbance OD ₄₉₂ value at ₄₉₂ nm of the reaction solution was measured.

Results The measurement results are shown in Table 3, Table 4, Table 5 and Table 6. Table 3,
Tables 4, 5, and 6 show Examples 1 and 2 and Reference Examples 1 to 1.
The respective measurement results when serum A, serum B, serum C, and serum D were measured by the enzyme immunoassay using the peptide obtained in 3 are shown. Furthermore, normal human serum D10
A cutoff value was set based on the OD ₄₉₂ value of the sample, and anti-HCV antibody positive / negative was determined. The cutoff value was calculated by the formula ((average value of OD ₄₉₂ values of normal human serum) + 2SD).

The cutoff values calculated based on Table 3, Table 4, Table 5 and Table 6 were used for Examples 1-2 and Reference Examples 1--2.
The distribution of OD ₄₉₂ values of each of the 3 peptides is shown in FIGS.
Shown in. Table 7, Table 4, Table 5, Table 6 and the positive rate calculated from the above cut-off values are shown in Table 7. From Table 7, when the enzyme immunoassay using the peptide obtained in Example 1 was carried out, 93.3%, 93.3%, 10.0% and 0% positive positivity was obtained for serum A, serum B, serum C and normal human serum D, respectively. Showed the rate. When the enzyme immunoassay using the peptide obtained in Example 2 was carried out, 96.7%, 93.3%, 0% and 0 were obtained for serum A, serum B, serum C and normal human serum D, respectively.
It was found that the enzyme immunoassay method using these peptides showed a high correlation with the immunoscreening method shown in Reference Example 8.

When the enzyme immunoassays using the peptides obtained in Reference Example 1 and Reference Example 2 were carried out, about 10% of serum A, serum B, serum C and normal human serum D were obtained.
The positive rate was 10.0%, and it was found that the enzyme immunoassay using these peptides had poor sensitivity and specificity. Also isolated by several people, including one of the inventors,
When the enzyme immunoassay using the peptide obtained in Reference Example 3, which is a peptide consisting of the entire amino acid sequence of a peptide obtained by translating one clone selected from cloned clones into amino acids, was performed, serum A, serum B and serum C showed positive rates of 93.3%, 93.3%, and 0%, respectively, whereas normal human serum D showed a positive rate of 10.0%, indicating that false positives occurred. It was found that the specificity and sensitivity were poorer as compared with the enzyme immunoassay method using the peptide obtained in Example 1 or Example 2. From the above results, it was shown that the presence or absence of an effective anti-HCV antibody can be determined by using the peptides obtained in Example 1 and Example 2.

[Table 3]

[Table 4]

[Table 5]

[Table 6]

[Table 7]

Example 10 In the same manner as in Example 9, the peptides obtained in Example 3, Example 4, Example 5, Reference Example 4, Reference Example 5 and Reference Example 6 were used as the antigenic substance. Assay by enzyme immunoassay was performed.

The measurement results are shown in Table 8, Table 9, Table 10 and Table 1.
Shown in 1. The positive rate is shown in Table 12. Moreover, each _OD492 value is shown in FIGS. From Table 12, when the enzyme immunoassay using the peptide obtained in Example 3 or Example 5 was carried out, the positive rates of serum A and serum B were 96.7% and 100%, respectively, and serum C and normal human serum were shown. D shows 0% or very low positive rate,
It was found that the enzyme immunoassay using these peptides has high sensitivity and specificity. Further, when the enzyme immunoassay using the peptide obtained in Example 4 was carried out, 36.7% was obtained for each of serum A, serum B, serum C and serum D,
It showed positive rates of 53.3%, 5.0% and 0.0%, and it was revealed that the peptide used had an antigenicity different from that of the peptides obtained in Examples 3 and 5.

Furthermore, when the enzyme immunoassay using the peptides obtained in Reference Examples 4 to 6 was carried out, serum A, serum B, serum C and serum D showed 0% or a very low positive rate. The enzyme immunoassay using peptides was found to have poor sensitivity and specificity. From the above results, it was shown that the presence or absence of an effective anti-HCV antibody can be determined by using the peptides obtained in Examples 3, 4 and 5.

[Table 8]

[Table 9]

[Table 10]

[Table 11]

[Table 12]

Example 11 In the same manner as in Example 9, the peptides obtained in Examples 6, 7 and 8 and Reference Example 7 were used as the antigenic substance and assayed by the enzyme immunoassay. It was

The measurement results are shown in Table 13, Table 14, Table 15 and Table 16. The positive rate is shown in Table 17. The respective OD ₄₉₂ values are shown in FIGS. 12 to 15. From Table 17, when the enzyme immunoassay using the peptide obtained in Example 6 was carried out, 33.3%, 40.0%, 0% and 0% positivity was obtained for serum A, serum B, serum C and normal human serum D, respectively. Showed the rate. When an enzyme-linked immunosorbent assay with the peptide obtained in Example 7 was carried out, 50.0%, 66.7% and 5.0 of serum A, serum B, serum C and normal human serum D, respectively.
% And 0% were positive. Furthermore, when the enzyme immunoassay using the peptide obtained in Example 8 was carried out, serum A, serum B, serum C and normal human serum D were each obtained.
The positive rates were 23.3%, 93.3%, 5.0% and 10.0%.
Enzyme-linked immunosorbent assays using these peptides include cloned # 8, # 14 and # 8 clones from ribonucleic acid isolated by several people including one of the inventors.
It was found that there is a high correlation with the immunoscreening method shown in Reference Example 8 using 18 clones.

When the enzyme immunoassay using the peptide obtained in Reference Example 7 was carried out, serum A, serum B, serum C and serum D were 13.3%, 13.3% and 5.0%, respectively.
It showed a positive rate of 0.0%, and it was revealed that the peptide used had an antigenicity different from the antigenicity of the peptides obtained in Examples 6, 7 and 8. From the above results, it was shown that the presence or absence of an effective anti-HCV antibody can be determined by using the peptides obtained in Examples 6, 7 and 8.

[Table 13]

[Table 14]

[Table 15]

[Table 16]

[Table 17]

Example 12 Test Samples Table 18 shows the disease names and the number of samples derived from the serum samples used.

[Table 18]

Assay by enzyme immunoassay For each serum sample shown in Table 18, the absorbance was measured by the following enzyme immunoassay to assay the presence or absence of anti-HCV antibody.

That is, in the same manner as in Example 9, the peptides obtained in Example 1, Example 2, Reference Example 1, Reference Example 2 and Reference Example 3 were coated on an assay microcup as an antigen substance, The reaction with each serum sample shown in Table 18 was carried out, and the absorbance OD ₄₉₂ value at ₄₉₂ nm of the reaction solution containing the dye produced from the color former was measured.

Results The measurement results are shown in Tables 19 and 20. Table 19 shows the respective measurement results when the serum samples shown in Table 18 were assayed by the enzyme immunoassay using the peptides obtained in Examples 1 and 2 and Reference Examples 1 to 3. Furthermore, a cutoff value was set based on the OD ₄₉₂ value of 10 normal serum samples and
HCV antibody positive / negative was determined. The cutoff value is
((Average value of OD ₄₉₂ value of serum of normal person) + 2SD).

Tables 19 and 20 show the results of determining the distinction between positive and negative on the basis of the cut-off values calculated based on the measured values of normal human serum samples in Tables 19 and 20. From Table 19, when the enzyme immunoassay using the peptide obtained in Example 1 was carried out, sporadic non-A non-B acute hepatitis / convalescent and chronic hepatitis, and post-transfusion non-A
Patient sera with non-B acute hepatitis / extreme and chronic hepatitis
The HCV antibody was determined to be positive, and the anti-HCV antibody was determined to be negative in the serum of alcoholic hepatitis, the serum of hepatitis B and the serum of normal subjects. When the enzyme immunoassay using the peptide obtained in Example 2 was performed, anti-HCV antibody was determined to be positive in the serum of non-A non-B chronic hepatitis patients, and the alcoholic hepatitis patient serum, hepatitis B patient serum and normal The anti-HCV antibody was determined to be negative in the human serum. From the above, it was found that the enzyme immunoassay using the peptides obtained in Examples 1 and 2 is useful for early diagnosis of non-A non-B hepatitis.

Further, from Table 20, when the enzyme immunoassay using the peptides obtained in Reference Example 1, Reference Example 2 and Reference Example 3 was carried out, the anti-HCV antibody was negative in the sera of patients with non-A non-B hepatitis. In some cases, the anti-HCV antibody was determined to be positive in the sera of hepatitis B patients and sera of normal subjects, and the specificity and sensitivity were poor. That is, the determination results by the enzyme immunoassay using the peptides obtained in Reference Example 1, Reference Example 2 and Reference Example 3 include false negative and false positive, and the diagnosis of non-A non-B hepatitis using this peptide is made. The efficiency turned out to be poor. From the above results, it was shown that the presence or absence of an effective anti-HCV antibody can be determined by using the peptides obtained in Example 1 and Example 2.

[Table 19]

[Table 20]

Example 13 In the same manner as in Example 12, each of the serum samples shown in Table 18 was used as an antigenic substance in Example 3, Example 4, Example 5, Reference Example 4, Reference Example 5, and Reference. Absorbance was measured by the enzyme immunoassay using the peptide obtained in Example 6, and the presence or absence of anti-HCV antibody was assayed.

The measurement results are shown in Tables 21 and 22. Table 21 shows the measurement results when each serum sample shown in Table 18 was assayed by the enzyme immunoassay using the peptides obtained in Examples 3 to 5 and Reference Examples 4 to 6. Furthermore, a cut-off value was set based on the OD ₄₉₂ value of 10 normal human sera, and anti-HCV antibody positive / negative was determined. The cut-off value is ((average OD ₄₉₂ value of normal serum) + 2
SD) was calculated by the formula.

Tables 21 and 22 show the results of determining the distinction between positive and negative on the basis of the cutoff values calculated based on the measured values of the serum samples of normal subjects in Tables 21 and 22. From Table 21, when the enzyme immunoassay using the peptides obtained in Example 3 and Example 5 was carried out, non-A
Anti-HCV antibody was determined to be positive in the sera of patients with acute and convalescent non-B hepatitis and the sera of patients with non-A non-B chronic hepatitis. Furthermore, anti-HCV antibody was determined to be negative in serum of alcoholic hepatitis patients, serum of patients with hepatitis B, and serum of normal subjects, and enzyme immunoassays using these peptides were found to be useful in the diagnosis of the above patients. It was In addition, when the enzyme immunoassay using the peptide obtained in Example 4 was carried out, the anti-specific immunoassay was obtained only in the serum of patients with non-A non-B chronic hepatitis.
The HCV antibody was determined to be positive, and it was found to be useful for the diagnosis of the above patients.

Furthermore, when the enzyme immunoassay using the peptides obtained in Reference Examples 4 to 6 from Table 22 was carried out, Table 1
The sera of patients with 8 diseases may have negative anti-HCV antibodies, and the sera of normal individuals may also have positive anti-HCV antibodies. The determination results by the immunoassay method included false positives and false negatives, and it was found that the diagnostic efficiency of non-A non-B hepatitis using these peptides was poor.
From the above results, effective anti-HCV can be obtained by using the peptides obtained in Example 3, Example 4 and Example 5.
It was shown that the presence or absence of the antibody can be determined.

[Table 21]

[Table 22]

Example 14 In the same manner as in Example 12, the peptides obtained in Example 6, Example 7, Example 8 and Reference Example 7 were used as the antigenic substances for each serum sample shown in Table 18. Absorbance was measured by the enzyme immunoassay method, and the presence or absence of anti-HCV antibody was assayed.

Table 23 shows the measurement results. Table 23 shows the respective measurement results when the serum samples shown in Table 18 were assayed by the enzyme immunoassay using the peptides obtained in Examples 6 to 8 and Reference Example 7. Furthermore, normal person serum 10
A cutoff value was set based on the OD ₄₉₂ value of the sample, and anti-HCV antibody positive / negative was determined. The cut-off value was calculated by the formula ((average value of OD ₄₉₂ value of normal human serum) + 2SD).

Table 23 shows the results of judging the distinction between positive and negative on the basis of the cutoff values calculated based on the measured values of the serum samples from normal subjects in Table 23. From Table 23, when the enzyme-linked immunosorbent assay with the peptide obtained in Example 6 was carried out, anti-HC was observed in the sera of patients with sporadic non-A non-B chronic hepatitis.
V antibody was determined to be positive, and PTNANBH acute / convalescent and chronic hepatitis were all determined to be positive. Furthermore, anti-HCV antibody was determined to be negative in the sera of alcoholic hepatitis patients, the sera of patients with hepatitis B, and the sera of normal subjects. From the above, it was found that the enzyme immunoassay using the peptide obtained in Example 6 is useful for early diagnosis of non-A non-B hepatitis. Further, when the enzyme immunoassay using the peptides obtained in Examples 7 and 8 was carried out, anti-HCV antibody was specifically positive in the sera of non-A non-B chronic hepatitis convalescent patients and chronic hepatitis patients. Therefore, it was found to be useful for the diagnosis of the above patients.

When the enzyme immunoassay using the peptide obtained in Reference Example 7 was carried out, all the sera of non-A non-B hepatitis patients were determined to be negative, and the sera of alcoholic hepatitis and hepatitis B patients were also determined. In some cases, it was determined to be positive.
That is, the determination result by the enzyme immunoassay using the peptide obtained in Reference Example 7 includes false negative and false positive,
It was found that the diagnostic efficiency of non-A non-B hepatitis using this peptide was poor. From the above results, it was shown that the presence or absence of an effective anti-HCV antibody can be determined by using the peptides obtained in Examples 6, 7 and 8.

[Table 23]

Example 15 Test sample Blood donor serum: 2476 samples Assay by enzyme immunoassay

The absorbance of each serum sample was measured by the following enzyme immunoassay method, and the presence or absence of anti-HCV antibody was sampled.

That is, each of the peptides obtained in Example 1 as an antigenic substance was dissolved in 0.01 M carbonate buffer (pH 9.5) and the obtained peptide solution was used as a polystyrene enzyme immunoassay cup (Dynatech). In each 1
After adding 00 μl each, the mixture was allowed to stand at 4 ° C. for 12 hours to perform peptide coating. The cups were then washed 3 times with PBS containing 0.05% Tween 20 by volume. Subsequently, 150 μl of PBS containing 20% by volume of normal goat serum was added to the cups and left at room temperature for 3 hours to block nonspecific protein binding sites. Then
P containing 20% by volume of normal goat serum used for blocking P
After removing BS, each assay cup was dried.

100 μl of PBS containing 10% by volume of normal goat serum as a serum dilution solution was added to each of the above assay cups.
After that, each test serum was added so that the ratio of the serum-diluting solution and the test serum was 20: 1 (volume ratio). 37 ° C
After incubation for 1 hour at 0.05
The cells were washed 3 times with PBS containing Tween 20 at a volume of 3.

To each of the obtained assay cups, 100 μl of goat anti-human IgG antibody-peroxidase conjugate (diluted to an optimum concentration with PBS containing 10% by volume of normal goat serum) was added. After incubating at 37 ° C for 30 minutes, the cups are added to PBS containing 0.05% by volume Tween20.
It was washed 3 times with. Subsequently, a color former (o-phenylenediamine was dissolved in 0.1M citric acid-phosphate buffer pH 5.6 containing 0.02% by volume of hydrogen peroxide so as to be 0.3% by weight) in each of the obtained assay cups. 100 μl) was added. After standing at room temperature for 15 minutes, 100 μl of 2N sulfuric acid was added to stop the reaction, and the absorbance OD ₄₉₂ value at ₄₉₂ nm of the reaction solution was measured.

Results As a result of the inhibition test of the anti-HCV antibody by the peptide obtained in Example 1, the cutoff value was set. Based on the above measurement results, those having an OD ₄₉₂ value of 0.5 or more were determined to be anti-HCV antibody positive, and those having an OD ₄₉₂ value of less than 0.5 were determined to be negative. In addition, Table 24 also shows the results of the determination by the EIA method and the RIBA method using the commercially available anti-HCV antibody measuring reagent (manufactured by Ortho) for the above-mentioned 2476 blood donor sera.

[Table 24]

As is clear from Table 24, commercially available anti-HCV
Of the 2462 samples that were determined to be negative by the antibody measurement reagent
35 samples were anti-HC measured with the peptide obtained in Example 1.
V antibody was determined to be positive. In addition, commercially available anti-HC
The 14 samples that were determined to be positive by the V antibody measurement reagent were determined to be positive by the determination of anti-HCV antibody measured with the peptide obtained in Example 1.

[0088]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 25 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Lys Asp Arg Thr Gln Gln Arg Lys Thr Lys Arg Ser Thr Asn Arg Arg 1 5 10 15 Arg Ser Lys Asn Glu Lys Lys Lys Lys 20 25

SEQ ID NO: 2 Sequence length: 5 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 3 Sequence length: 40 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Glu Lys Lys Gly Glu Ala Ser Asn Gly Glu Ala Glu Asn Asp Thr His 1 5 10 15 Lys Lys Gln Arg Arg Tyr Lys Glu Lys Glu Lys Thr Ala Thr Asn Asn 20 25 30 Pro Gly Lys Asn Lys Lys Pro Arg 35 40

SEQ ID NO: 4 Sequence length: 8 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 5 Sequence length: 39 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Arg Val Val Leu Ser Gly Lys Pro Ala Ile Ile Pro Asp Arg Glu Val 1 5 10 15 Leu Tyr Arg Glu Phe Asp Glu Met Glu Glu Cys Ser Gln His Leu Pro 20 25 30 Tyr Ile Glu Gln Gly Met Met 35

SEQ ID NO: 6 Sequence length: 10 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 7 Sequence length: 10 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 8 Sequence length: 21 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Arg Arg Tyr Lys Glu Lys Glu Lys Thr Ala Thr Asn Asn Pro Gly Lys 1 5 10 15 Asn Lys Lys Pro Arg 20

SEQ ID NO: 9 Sequence length: 13 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Thr His Lys Lys Gln Arg Arg Tyr Lys Glu Lys Glu Lys 1 5 10

SEQ ID NO: 10 Sequence length: 10 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 11 Sequence length: 31 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ala Ile Ile Pro Asp Arg Glu Val Leu Tyr Arg Glu Phe Asp Glu Met 1 5 10 15 Glu Glu Cys Ser Gln His Leu Pro Tyr Ile Glu Gln Gly Met Met 20 25 30

SEQ ID NO: 12 Sequence length: 18 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Arg Val Val Leu Ser Gly Lys Pro Ala Ile Ile Pro Asp Arg Glu Val 1 5 10 15 Leu Tyr

SEQ ID NO: 13 Sequence length: 10 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 14 Sequence length: 10 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 15 Sequence length: 15 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Arg Ser Thr Asn Arg Arg Arg Ser Lys Asn Glu Lys Lys Lys Lys 1 5 10 15

SEQ ID NO: 16 Sequence length: 32 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Glu Gln Asp Gln Ile Lys Thr Lys Asp Arg Thr Gln Gln Arg Lys Thr 1 5 10 15 Lys Arg Ser Thr Asn Arg Arg Arg Ser Lys Asn Glu Lys Lys Lys Lys 20 25 30

SEQ ID NO: 17 Sequence length: 14 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Glu Lys Lys Gly Glu Ala Ser Asn Gly Glu Ala Glu Asn Asp 1 5 10

SEQ ID NO: 18 Sequence length: 11 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 19 Sequence length: 21 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Val Gly Arg Ile Lys Asn Trp Asn Arg Glu Gly Arg Lys Asp Ala Tyr 1 5 10 15 Gln Ile Arg Lys Arg 20

SEQ ID NO: 20 Sequence length: 11 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 21 Sequence length: 120 Sequence type: Nucleic acid Number of strands: Unknown Topology: Unknown Sequence type: cDNA to genomic RNA Fragment type: Intermediate fragment Origin Solid / isolated clone name: # 8 Clone Tissue type: Human serum Sequence characteristics Characteristic symbol: CDS Location: 1. ． 120 Method of characterizing: E sequence GAATTCCAAA AAGAGCAAAA CAAACCGCCG AAGAAAAAAC TAATAAGAGA AGAAAAGGCG 60 AAGAGACACA GGAAAAAAAA AACAGAGACG AAGGTCAGAT AGAAAAAAAG CAAGGAATTC 120

SEQ ID NO: 22 Sequence length: 114 Sequence type: Nucleic acid Number of strands: Unknown Topology: Unknown Sequence type: cDNA to genomic RNA Fragment type: Intermediate fragment Origin Solid / isolated clone name: # 14 Clone Tissue type: Human serum Sequence characteristics Characteristic symbol: CDS Location: 1. ． 114 Method of characterizing: E sequence GAATTCCGAG AACAAGACCA GATAAAAACC AAAGACAGAA CACAACAGAG AAAGACGAAA 60 AGAAGCACCA ATCGCAGGCG AAGCAAAAAC GAAAAAAAAA AAAAAAAGGA ATTC 114

SEQ ID NO: 23 Sequence length: 201 Sequence type: Nucleic acid Number of strands: Unknown Topology: Unknown Sequence type: cDNA to genomic RNA Fragment type: Intermediate fragment Origin Solid / isolated clone name: # 18 Clone Tissue type: Human serum Sequence characteristics Characteristic symbol: CDS Location: 1. ． 201 Method of characterizing: E sequence GAATTCCAAG AAAAAAAGGG AGAAGCCAGC AATGGAGAAG CCGAAAACGA CACACACACAAG 60 AAACAAAGGA GGTACAAAGA AAAAGAAAAA ACGGCAACAA ATAACCCAGG AAAGAACAAA 120 AAGCCAGAGAGAAAGAGAGAGAGAGGAACCAGA

[Brief description of drawings]

FIG. 1 is a distribution chart of OD ₄₉₂ values given by each serum sample measured by the method described in Example 9 using the peptide obtained in Example 1.

FIG. 2 is a distribution chart of OD ₄₉₂ values given by each serum sample measured by the method described in Example 9 using the peptide obtained in Example 2.

FIG. 3 is a distribution chart of OD ₄₉₂ values given by each serum sample measured by the method described in Example 9 using the peptide obtained in Reference Example 1.

FIG. 4 is a distribution chart of OD ₄₉₂ values given by each serum sample measured by the method described in Example 9 using the peptide obtained in Reference Example 2.

FIG. 5 is a distribution chart of OD ₄₉₂ values given by each serum sample measured by the method described in Example 9 using the peptide obtained in Reference Example 3.

FIG. 6 is a distribution chart of OD ₄₉₂ values given by each serum sample measured by the method described in Example 9 using the peptide obtained in Example 3.

FIG. 7 is a distribution chart of OD ₄₉₂ values given by each serum sample measured by the method described in Example 9 using the peptide obtained in Example 4.

FIG. 8 is a distribution chart of OD ₄₉₂ values given by each serum sample measured by the method described in Example 9 using the peptide obtained in Example 5.

FIG. 9 is a distribution chart of OD ₄₉₂ values given by each serum sample measured by the method described in Example 9 using the peptide obtained in Reference Example 4.

FIG. 10 is a distribution chart of OD ₄₉₂ values given by each serum sample measured by the method described in Example 9 using the peptide obtained in Reference Example 5.

FIG. 11 is a distribution chart of OD ₄₉₂ values given by each serum sample measured by the method described in Example 9 using the peptide obtained in Reference Example 6.

FIG. 12 is a distribution chart of OD ₄₉₂ values given by each serum sample measured by the method described in Example 9 using the peptide obtained in Example 6.

FIG. 13 is a distribution chart of OD ₄₉₂ values given by each serum sample measured by the method described in Example 9 using the peptide obtained in Example 7.

FIG. 14 is a distribution chart of OD ₄₉₂ values given by each serum sample measured by the method described in Example 9 using the peptide obtained in Example 8.

FIG. 15 is a distribution chart of OD ₄₉₂ values given by each serum sample measured by the method described in Example 9 using the peptide obtained in Reference Example 7. In addition, in these figures, each symbol shows the following.

[Explanation of symbols]

●: GPT>200IU; OD ₄₉₂ value given by HBsAg (-) serum A ○: GPT>200IU; OD ₄₉₂ value given by HBsAg (-) serum B ×: GPT>200IU; HBsAg (-) serum C given OD ₄₉₂ value

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location // A61K 39/29 8413-4C G01N 33/569 L 9015-2J C07K 99:00 (31) Priority Claim No. Japanese Patent Application No. 2-296899 (32) Priority Day 2 (1990) October 31, (33) Country of priority Japan (JP) (72) Inventor Toshihiko Namba 1660 Sakata, Kurashiki City, Okayama Prefecture (72) Inventor Masao Tsuji 1-1 Mizue, Kurashiki City, Okayama Prefecture

Claims (13)

[Claims] 1. A Lys Arg Ser Thr Asn (SEQ ID NO:
2), Arg Arg Tyr LysGlu Lys Glu Lys (SEQ ID NO:
4), or Ala Ile Ile Pro Asp Arg Glu ValLeu Tyr
A peptide comprising the amino acid sequence of (SEQ ID NO: 6) and having the ability to specifically bind to an antibody having specificity for a non-A non-B hepatitis-related antigen. 2. Formula (1): Lys Asp Arg Thr Gln Gln Ar
g Lys Thr Lys ArgSer Thr Asn Arg Arg Arg Ser Lys A
sn Glu Lys Lys Lys Lys (SEQ ID NO: 1), which is a peptide consisting of the peptide according to claim 1 or a fragment thereof, wherein the peptide is Lys A.
The peptide according to claim 1, which has the amino acid sequence of rg Ser Thr Asn (SEQ ID NO: 2) and has the ability to specifically bind to an antibody having specificity for a non-A non-B hepatitis-related antigen. 3. Formula (2): Glu Lys Lys Gly Glu Ala Se
r Asn Gly Glu AlaGlu Asn Asp Thr His Lys Lys Gln A
rg Arg Tyr Lys Glu Lys Glu Lys Thr AlaThr Asn Asn
Pro Gly Lys Asn Lys Lys Pro Arg (SEQ ID NO: 3), which is a peptide comprising the peptide or a fragment thereof according to claim 1 having the amino acid sequence represented by Arg Arg Tyr Lys Glu Lys Glu Lys (Sequence Number: 4)
The peptide according to claim 1, which has the amino acid sequence of and has the ability to specifically bind to an antibody having specificity for a non-A non-B hepatitis-related antigen. 4. Formula (3): Arg Val Val Leu Ser Gly Ly
s Pro Ala Ile IlePro Asp Arg Glu Val Leu Tyr Arg G
lu Phe Asp Glu Met Glu Glu Cys Ser GlnHis Leu Pro
A peptide comprising the peptide or a fragment thereof according to claim 1 having the amino acid sequence represented by Tyr Ile Glu Gln Gly Met Met (SEQ ID NO: 5), wherein the peptide is Al.
a Ile Ile Pro Asp Arg Glu Val Leu Tyr (SEQ ID NO:
The peptide according to claim 1, which has the amino acid sequence of 6) and has the ability to specifically bind to an antibody having specificity for a non-A non-B hepatitis-related antigen. 5. Formula (1): Lys Asp Arg Thr Gln Gln Ar
g Lys Thr Lys ArgSer Thr Asn Arg Arg Arg Ser Lys A
The peptide according to claim 2, which is represented by sn Glu Lys Lys Lys Lys (SEQ ID NO: 1). 6. Formula (1-a): Thr Lys Arg Ser Thr As
The peptide according to claim 2, which is represented by n Arg Arg Arg Ser (SEQ ID NO: 7). 7. Formula (2-a): Arg Arg Tyr Lys Glu Ly
s Glu Lys Thr AlaThr Asn Asn Pro Gly Lys Asn Lys L
The peptide according to claim 3, which is represented by ys Pro Arg (SEQ ID NO: 8). 8. The formula (2-b): Thr His Lys Lys Gln Ar.
The peptide according to claim 3, which is represented by g Arg Tyr Lys GluLys Glu Lys (SEQ ID NO: 9). 9. Formula (2-c): Arg Arg Tyr Lys Glu Ly
The peptide according to claim 3, which is represented by s Glu Lys Thr Ala (SEQ ID NO: 10). 10. Formula (3-a): Ala Ile Ile Pro Asp
Arg Glu Val Leu Tyr Arg Glu Phe Asp Glu Met Glu Gl
u Cys Ser Gln His Leu Pro Tyr Ile Glu Gln Gly Met
The peptide according to claim 4, which is represented by Met (SEQ ID NO: 11). 11. Formula (3-b): Arg Val Val Leu Ser
Gly Lys Pro Ala Ile Ile Pro Asp Arg Glu Val Leu Ty
The peptide according to claim 4, which is represented by r (SEQ ID NO: 12). 12. Formula (3-c): Ala Ile Ile Pro Asp
The peptide according to claim 4, which is represented by Arg Glu Val Leu Tyr (SEQ ID NO: 13). 13. A reagent for measuring an antibody having specificity for a non-A non-B hepatitis-related antigen, which comprises the peptide according to any one of claims 1 to 12.