JP3241057B2 - Peptides and their uses - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

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

[0002] The reagent for measuring anti-HCV antibodies provided by the present invention has the ability to detect anti-HCV antibodies in serum or plasma with high sensitivity, and is useful for measuring anti-HCV antibodies.

[0003]

2. Description of the Related Art Currently, five types of viral hepatitis viruses are known as the causative viruses of hepatitis, which account for the majority of liver diseases. These viruses are called hepatitis A, B, C, D and E viruses, respectively. I have. Among viral hepatitis, hepatitis A and E are oral infections and do not become chronic due to transient infection alone, but hepatitis B and C become chronic due to persistent infection and have a high probability. This is a major problem because it progresses to cirrhosis or liver cancer. For hepatitis A, B and D, the causative viruses have been found, and immunological diagnosis is now possible. E
It has been reported that the gene for hepatitis B virus was recently isolated.

[0004] Post-transfusion non-A non-B hepatitis (hereinafter referred to as PTN)
(Abbreviated as ANBH) has been unknown for a long time despite many investigators,
In 1988, a research group at Chiron Corporation in the United States isolated and identified the gene for the PTNANBH virus from the plasma of chimpanzees infected with PTNANBH [Science, 244, 359].
(1988) and Science, 244, 362 (19
1988)] and hepatitis C virus (hereinafter abbreviated as HCV). Some of the putative nucleotide sequence of the gene has already been elucidated [see EP 0 318 216], and it has become possible to detect anti-HCV antibodies, and serodiagnosis of HCV infection can be performed. It is possible.

[0005] In addition, several persons including one of the present inventors have 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. [Gastroenterologia Japonica, Vol. 24, No. 5, p. 540 (1989); Gastroenterologia Japonica;
Japonica, Vol. 24, No. 5, p. 545 (1989) and Internal Medicine, Vol. 64, No. 6, p. 1022 (1989)].

[0006] Required cDNA from cDNA libraries so far
As a means of screening for NA, it has been generally performed to determine whether an anti-HCV antibody is positive or negative by an antigen-antibody reaction using λ phage.However, the above immunoscreening method is not quantitative, and May react with non-specific antigen components in the expression product of E. coli.Currently, an HCV gene is cloned, incorporated into a phage, and expressed by an enzyme immunoassay using an antigen protein expressed as a yeast host. The development of testing agents for anti-HCV antibodies is under consideration [Internal Medicine, Vol. 64, No. 6, p. 1027 (1989)
Year)). Furthermore, a particle agglutination method utilizing the property that gelatin particles sensitized by a virus or its antigen component agglutinates in the presence of an antiviral antibody, or a bead method for performing enzyme immunoassay using beads coated with a virus or its antigen component Is being developed.

[0007]

In the conventional enzyme immunoassay using HCV-related antigens, the measurement target is clinically
Even when PTNANBH is diagnosed, the anti-HCV antibody positive rate is about 75%, and about 25% of PTNANBH, which is negative for anti-HCV antibodies, is contained. In the enzyme immunoassay described above, the positive rate is about 1% when the measurement target is a healthy person.
Since the statistical HCV prevalence is about 3%, about 2% of anti-HCV antibody-positive samples are overlooked. For this reason, in HCV carrier screening in blood donors, it is inevitable that carriers will be 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 antigen protein obtained by cloning the HCV gene and incorporating it into phage and expressing it using yeast as a host contains various nonspecific antigen components,
When an anti-HCV antibody is measured using the antigen protein as a reagent, the reagent also recognizes non-specific antibody components other than the anti-HCV antibody in the sample, and the measurement result is not necessarily anti-HCV. It will not always accurately represent the presence of HCV antibodies. As described above, according to the conventional enzyme immunoassay using HCV-related antigens, it is impossible at present to accurately know the presence of anti-HCV antibodies.

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

[0010]

Means for Solving the Problems The present inventors have proposed PTNANBH
Among peptide fragments selected from polypeptides encoded by related genes, a specific peptide having the ability to specifically bind to an antibody having specificity for a non-A non-B hepatitis-related antigen was found, and the present invention It was completed. That is, the present invention relates to (1) Arg Arg Tyr Lys Glu Lys Glu Lys (SEQ ID NO:
4) a non-A, non-B hepatitis-related antibody comprising the amino acid sequence of
Ability to specifically bind to antibodies with specificity for the original
A peptide having 10 to 40 amino acids
Ranaru peptide, (2) (2): Glu Lys Lys Gly Glu Ala Ser Asn Gl
y Glu Ala Glu Asn AspThr His Lys Lys Gln Arg Arg T
yr Lys Glu Lys Glu Lys Thr Ala Thr Asn AsnPro Gly
Lys Asn Lys Lys Pro Arg (SEQ ID NO: 3)
The peptide according to the above (1), which has an amino acid sequence or
A peptide comprising the fragment, wherein the peptide is Arg
Arg Tyr Lys Glu Lys Glu Lys (SEQ ID NO: 4)
No acid sequence, specific for non-A non-B hepatitis-related antigen
Having the ability to specifically bind to an antibody having
(1) the peptide described in (1), (3) Formula (2-a): Arg Arg Tyr Lys Glu Lys Glu Ly
s Thr Ala Thr Asn AsnPro Gly Lys Asn Lys Lys Pro A
rg (SEQ ID NO: 8), Formula (2-b): Thr His Lys Lys Gln Arg Arg Tyr Lys
Glu Lys Glu Lys (SEQ ID NO: 9) or Formula (2-c): Arg Arg Tyr Lys Glu Lys Glu Lys Thr
Ala according to the above (2), which is represented by Ala (SEQ ID NO: 10)
(4) The peptide according to any one of (1) to (3) above.
Specificity for non-A non-B hepatitis-related antigens comprising
A reagent for measuring antibodies.

[0011] According to the present invention, the above object has been achieved by providing 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
o It is achieved by providing a peptide having 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 having specificity for an HCV-related antigen.

Specifically, (I) Formula (1): 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 consisting of a peptide having the amino acid sequence represented by 1) or a fragment thereof, wherein the peptide is Lys Ar
g A peptide having the amino acid sequence of Ser Thr Asn (SEQ ID NO: 2) and having the ability to specifically bind 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 consisting of 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:
5) A peptide comprising the peptide having the amino acid sequence represented by 5) or a fragment thereof, wherein the peptide is Ala Il
eIle Pro Asp Arg Glu Val Leu Tyr (SEQ ID NO: 6)
And has the ability to specifically bind to an antibody having specificity for an HCV-related antigen.

The present invention is also achieved by providing a reagent for measuring an anti-HCV antibody 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, amino acids are used according to a conventional method. The sequence is described such that the N-terminal amino acid residue is on the left and the C-terminal amino acid residue is on the right.

The peptide of the present invention has the ability to specifically bind to an antibody having specificity for an HCV-related antigen, and has 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 sequence of Lys Arg Ser Thr Asn (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 partial amino acid sequence, but is usually a peptide consisting of 10 to 40 amino acids.

Specifically, there may be mentioned peptides comprising the following peptides or fragments thereof. That is, the peptide of the present invention is a peptide comprising the peptide having the amino acid sequence represented by the above formula (1) or a fragment thereof, wherein the peptide has 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. Here, as the peptide composed of a fragment, for example, the formula (1-a) is exemplified, 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
g Arg Tyr Lys Glu Lys A peptide having the amino acid sequence of Glu Lys (SEQ ID NO: 4) and having the ability to specifically bind to an antibody having specificity for an HCV-related antigen. Here, examples of the peptide composed of 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 the 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:
6) The peptide having the amino acid sequence of 6) and having the ability to specifically bind to an antibody having specificity for an HCV-related antigen. Here, as the peptide consisting of a fragment, for example, the formula (3-a), the formula (3-b) and the formula (3)
-C), but is 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 described above, various peptides can be exemplified as the peptide of the present invention. Preferably, the peptide of the formula (1-a) is used in view of the specificity of the reaction. 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 operationally convenient to carry out by the method [for example, Journal of the America Chemical Society (Journal of the America)
n Chemical Society), Vol. 85, pp. 2149-2154 (1963
); Biochemical Experiments Lecture 1, Chemistry of Proteins IV, Chemical Modification and Peptide Synthesis, edited by The Biochemical Society of Japan (November 15, 1977)
(Published by Tokyo Chemical Co., Ltd.), pages 207-495, edited by The Biochemical Society of Japan, “Seizoku Chemistry Experiment Course 2: Chemistry of Proteins (2)” (published by Tokyo Chemical Co., Ltd., May 20, 1987). See pages 641 to 694].

The peptide of the present invention can be produced by a solid-phase synthesis method by, for example, adding a polymer insoluble in a reaction solvent such as a styrene-divinylbenzene copolymer to an amino acid corresponding to the C-terminal of the target peptide or its amino acid. Α-COO-group or α-CONH obtained by removing a hydrogen atom from an α-COOH group or an α-CONH ₂ group which has an amide, respectively.
And then linking the amino acid or amide thereof toward the N-terminus of the peptide of interest with the corresponding amino acid or peptide fragment with an α-COOH group other than the α-COOH group of the amino acid or peptide fragment. 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 of the amino group forming a peptide bond such as an amino group,
Elongating the peptide chain to form a peptide chain corresponding to the peptide of interest, then removing the peptide chain from the polymer, and removing the protecting group from the protected functional group And then purifying it. Here, the elimination of the peptide chain from the polymer and the removal of the protecting group are preferably carried out simultaneously using hydrogen fluoride from the viewpoint of suppressing side reactions. Further, it is effective that the obtained peptide is purified by reversed-phase liquid chromatography.

Since the peptide of the present invention has an ability to specifically bind an anti-HCV antibody, anti-HCV infection-induced
It is effective as a measuring reagent for detecting HCV antibodies. That is, the measurement reagent of the present invention comprises the peptide of the present invention, and the above-described peptide of the present invention is used alone or in combination of two or more.

The measurement of an anti-HCV antibody using the peptide of the present invention is performed by using any one of a fluorescent immunoassay, a passive hemagglutination assay, a radioimmunoassay, and an enzyme immunoassay. All of these methods are known. For example, a case where an enzyme immunoassay is used will be described below.

The components of the entire measurement system are a carrier, the peptide of the present invention as a measurement 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, and then a blocking agent is applied to the peptide-coated carrier to block non-specific protein binding sites on the carrier, and a test sample is added to the peptide-coated carrier and incubated. The enzyme-labeled antibody is allowed to contact and incubate, and then the carrier thus treated is added with a color former and incubated, and the amount of a reaction product produced by the reaction between the enzyme and the color former is measured using an absorbance meter. The peptide of the present invention used for coating may be one kind or two or more kinds. As a carrier, it is preferable to use an enzyme immunoassay cup or glass or resin beads.

Prior to the measurement, the peptide of the present invention was treated with 0.01M
After dissolving in a carbonate buffer and adding the solution to a polystyrene enzyme immunoassay cup, for example,
And overnight at room temperature for 3 hours to coat the carrier surface with the peptide of the present invention. As a blocking agent for blocking non-specific protein binding site on the carrier, for example, bovine serum albumin, casein,
Skim milk powder, serum of an immunogenic animal for obtaining an anti-human IgG antibody or an anti-human IgM antibody, gelatin, and the like are used.
Antibodies for labeling include, for example, anti-human IgG antibodies, anti-human IgG
M antibody and the like are used. Examples of the enzyme include alkaline phosphatase, glucose oxidase, peroxidase, beta-galactosidase and the like. Prior to measurement, using a compound having two or more functional groups, such as glutaraldehyde, an enzyme is bound to the labeling antibody to form a conjugate, which is prepared in advance as a part of components of the entire measurement system. Is preferred.
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.

Thus, according to the present invention, there is provided a peptide capable of specifically binding to an anti-HCV antibody.
With this peptide, it has become possible to provide an anti-HCV antibody measurement reagent having sensitivity and specificity exceeding that of existing anti-HCV antibody measurement reagents.

[0029]

EXAMPLES The present invention will be described below in more detail 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
The peptide represented by Lys Lys (SEQ ID NO: 1) was synthesized with an automatic peptide synthesizer [Applied Biosystems, USA, Model 431A (Model 431A)].
A)] and synthesized by a solid phase synthesis method.

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

Embedded image Having a ratio of 0.65 mmol / g (resin)
Granular resin composed of a divinylbenzene copolymer [constituent molar ratio of styrene and divinylbenzene: 99: 1] [PAM lysine (manufactured by Applied Biosystems, USA)
, T-Boc-L-Lys (Cl-Z)] and the corresponding L-arginine and L-asparagine toward the N-terminus of the target peptide according to a series of operations shown in Table 1. , L-aspartic acid, L-glutamine, L-glutamic acid, L-lysine, L-serine, and L-threonine. In the condensation reaction, the above amino acids are Nα- (t-butoxycarbonyl) -Nγ- (mesitylene-2-sulfonyl) -L-arginine, N-
(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 was used in an amount about 4 times the molar amount of the substrate.

[Table 1]

The condensation reaction was performed at room temperature. The reaction time including all steps required to bind one amino acid residue is 1
It was in the range of 00-110 minutes. After the reaction operation for all amino acids was completed, the obtained resin was washed sequentially on a glass filter using dichloromethane and methanol, and then dried under vacuum to obtain 2.58 g of a dried resin. Next, a reaction vessel made of polytrifluoromonochloroethylene (HF-
In a reaction apparatus I), 0.7 g of the obtained dry resin was mixed with 1.05 ml of anisole and 0.175 ml of ethyl methyl sulfide, and 7.0 ml of hydrogen fluoride was added to the mixture at a temperature of -20 ° C. For 30 minutes then at a temperature of 0 ° C. for 30 minutes. Hydrogen fluoride, anisole and ethyl methyl sulfide were removed from the obtained reaction mixture under reduced pressure, and the residue was sufficiently washed on a glass filter using diethyl ether and dichloromethane. The obtained residue was extracted with a 2N aqueous solution of acetic acid, and the extract was freeze-dried to obtain 200 mg of a crude peptide.

The obtained crude product was subjected to preparative reverse phase high performance liquid chromatography [column: octadecylated silica gel].
(Particle size: 15μm) Packed column (inner diameter: 50mm, length: 300m
m), manufactured by Nippon Waters Co., Ltd., μBONDASPHERE 15μC18
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 is subjected to analytical reversed-phase high-performance liquid chromatography [column: octadecylated silica gel (particle size: 5 μm) packed column (inner diameter: 4 mm,
(Length: 150mm), manufactured by Tosoh Corporation, TSK-gel ODS-80T
M; mobile phase: mixed solvent of acetonitrile and water containing 0.05% by volume of trifluoroacetic acid (the 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], showed a single sharp peak at 15.0 minutes. The molecular weight of the purified product determined by the fast atom bombardment method (hereinafter, abbreviated as FAB method) mass spectrum was 3188 (theoretical value: 3187.53).

Example 2 The solid phase synthesis and purification of the peptide were carried out in the same manner as in Example 1 to obtain the formula (1-a): Thr Lys Ar
g Ser Thr Asn Arg Arg A peptide represented by Arg Ser (SEQ ID NO: 7) was obtained. The resulting peptide was subjected to analytical reverse-phase high-performance liquid chromatography (same as above), showing a single sharp peak at 14.2 minutes. The molecular weight of the peptide determined by the FAB method mass spectrum was 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 compound of the formula (2-a): Arg Arg Ty
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 reversed-phase high-performance liquid chromatography (same as above),
A single sharp peak was shown at 22.0 minutes. The molecular weight of the peptide determined by the FAB method mass spectrum was 2,542 (theoretical value: 2541.85).

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

Example 5 The solid phase synthesis and purification of the peptide were carried out in the same manner as in Example 1 to obtain the compound of the formula (2-c): Arg Arg Ty
r Peptide represented by 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 (same as described above) and showed a single sharp peak at 14.5 minutes. The molecular weight of the peptide determined by the FAB mass spectrum was 13
06 (theoretical: 1306.49).

Example 6 The solid phase synthesis and purification of the peptide were carried out in the same manner as in Example 1 to obtain the compound of the 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 (same as described above) and showed a single sharp peak at 29.8 minutes. The molecular weight of the peptide determined by the FAB method mass spectrum was 3785 (theoretical value: 3785.19).

Example 7 By performing solid phase synthesis and purification of a peptide in the same manner as in Example 1, the compound of the formula (3-b): Arg Val Va
l Leu Ser Gly Lys Pro Ala Ile Ile Pro AspArg Glu V
The 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 (same as above) and showed a single sharp peak at 28.7 minutes. The molecular weight of the peptide determined by the FAB method mass spectrum was 1953 (theoretical value: 1953.15).

Example 8 By carrying out solid phase synthesis and purification of a peptide in the same manner as in Example 1, the compound of the 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 (same as described above) and showed a single sharp peak at 26.9 minutes. The molecular weight of the peptide determined by the FAB mass spectrum was 12
03 (theoretical: 1203.34).

Reference Example 1 Solid-phase synthesis and purification of a peptide were performed in the same manner as in Example 1 to obtain Lys Arg SerThr Asn from the peptide having the amino acid sequence represented by the formula (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 (same as above) and showed a single sharp peak at 11.9 minutes. The molecular weight of the peptide determined by the FAB method mass spectrum was 1290 (theoretical value: 1290.39).

REFERENCE EXAMPLE 2 By performing solid phase synthesis and purification of the peptide in the same manner as in Example 1, among the peptides having the amino acid sequence represented by the formula (1), Lys Arg SerThr Asn
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. The resulting peptide was subjected to analytical reversed-phase high-performance liquid chromatography (same as above) and showed a single sharp peak at 13.6 minutes. The molecular weight of the peptide determined by the FAB method mass spectrum was 1915 (theoretical value: 1915.16).

Reference Example 3 The solid phase synthesis and purification of the peptide were carried out in the same manner as in Example 1 to obtain the compound having 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
A peptide represented by Lys Lys (SEQ ID NO: 16) 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 24.6 minutes. The molecular weight of the peptide determined by the FAB method mass spectrum was 4031 (theoretical value:
4031.38).

Reference Example 4 The peptide having the amino acid sequence represented by the formula (2) was subjected to solid phase synthesis and purification in the same manner as in Example 1 to obtain Arg Arg TyrLys Glu L
Formula having no amino acid sequence represented by ys Glu Lys (SEQ ID NO: 4): Glu Lys Lys Gly Glu Alu 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 reversed-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 the FAB method mass spectrum was 1473 (theoretical value: 1473.47).

Reference Example 5 The peptide having the amino acid sequence represented by the formula (2) was subjected to solid-phase synthesis and purification in the same manner as in Example 1 to obtain 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 (same as above) and showed a single sharp peak at 12.6 minutes. The molecular weight of the peptide determined by the FAB method mass spectrum was 1253 (theoretical value:
1253.38).

Reference Example 6 The peptide having the amino acid sequence represented by the formula (2) was subjected to solid-phase synthesis and purification in the same manner as in Example 1 to obtain 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
(SEQ ID NO: 19) was obtained. The resulting peptide was subjected to analytical reverse phase high performance liquid chromatography (same as described above) and showed a single sharp peak at 19.4 minutes. The molecular weight of the peptide determined by the FAB method mass spectrum was 2,644 (theoretical value: 2643.9)
2).

Reference Example 7 The peptide having the amino acid sequence represented by the formula (3) was subjected to solid-phase synthesis and purification in the same manner as in Example 1, whereby Ala Ile IlePro Asp A
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 (same as above) and showed a single sharp peak at 26.7 minutes. The molecular weight of the peptide determined by the FAB method mass spectrum was 1081 (theoretical value: 1081.20).

Reference Example 8 Test sample GPT> 200 IU; HBsHg (-) Serum: 65 samples Normal human serum: 10 samples Assay by enzyme immunoassay For each serum sample, anti-HC was determined by the following enzyme immunoassay.
The presence or absence of the V antibody was tested.

That is, E. coli Y1090 was added to 100 μl of a solution containing phage λgt11 having # 8 clone, # 14 clone and # 18 clone cloned from ribonucleic acid isolated by several persons including one of the present inventors. Escher
ichia coli Y1090) as a supporting bacterium.
For 15 minutes to infect E. coli with the phage. Subsequently, the above mixture was inoculated on an agar medium containing 50 μg / ml of ampicillin, and cultured at 43 ° C. for 3 hours. Next, the nitrocellulose membrane obtained by immersing in a 10 mM IPTG aqueous solution for 2 hours and air-drying was placed on the agar medium and incubated at 37 ° C. for 3 hours. The nitrocellulose membrane thus obtained was treated with 10 mM containing 150 mM NaCl.
After washing three times with Tris-hydrochloric acid (pH 7.5) (hereinafter abbreviated as TS Buffer), and then shaking overnight at room temperature in 20 mM Tris-hydrochloric acid (pH 7.5) containing 500 mM NaCl and 3% gelatin. Nonspecific 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 at room temperature for 5 minutes in a TS Buffer containing 0.05% Tween20 (hereinafter, abbreviated as TS-T Buffer), and this operation was repeated five times. Subsequently, the nitrocellulose membrane was immersed in a goat anti-human IgG antibody-peroxidase conjugate (diluted to an optimum concentration with TS Buffer containing 1% gelatin) and shaken at room temperature for 1.5 hours. The nitrocellulose membrane thus obtained is transferred to TS-T Buff
This was shaken at room temperature for 5 minutes 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 was used.
After shaking the nitrocellulose membrane at r and performing a color development reaction, the membrane was shaken in distilled water at room temperature for 5 minutes, and this operation was repeated five times. After air drying, the serum used was assayed for the presence or absence of anti-HCV antibodies based on the presence or absence of color development.

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

Results The test results are shown in Table 2. From the results, the test sample G
PT> 200 IU; HBsHg (-) serum 65 samples were found to be classified into three groups. That is, group A determined to be positive in two of the # 14 clone and # 18 clone, group B determined to be positive in three of the # 8 clone, # 14 clone and # 18 clone, and # 8 clone, # 14 Group C, which was determined to be negative for both the clone and the # 18 clone, is three groups.

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

That is, the peptides obtained in Example 1, Example 2, Reference Example 1, Reference Example 2 and Reference Example 3 were dissolved in 0.01 M carbonate buffer (pH 9.5) as antigen substances, respectively.
Transfer 100 μl of the obtained peptide solution to a polystyrene enzyme immunoassay cup (Dynatech).
After each addition, the mixture was allowed to stand at 4 ° C. for 12 hours to perform coating with the peptide. Next, after removing the peptide solution contained in the obtained assay cups, those cups contain 20% by volume of normal goat serum.
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. Next, after removing PBS containing 20% by volume of normal goat serum used for blocking, each assay cup was dried.

As a serum diluting solution, 100 μl of PBS containing 10% by volume of normal goat serum was added to each of the above assay cups.
After each addition, 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) (Volume ratio). After 1 hour incubation at 37 ° C., the cups are
The plate was washed three times with PBS containing Tween20.

100 μl of a goat anti-human IgG antibody-peroxidase conjugate (diluted to an optimal concentration with PBS containing 10% by volume of normal goat serum) was added to each of the assay cups obtained. After incubation at 37 ° C for 30 minutes, the cups are washed with PBS containing 0.05% by volume Tween20.
And washed three times. Subsequently, a coloring agent (o-phenylenediamine was dissolved in 0.1 M citrate-phosphate buffer pH5.6 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 OD ₄₉₂ value of the reaction solution at 492 nm was measured.

Results The measurement results are shown in Tables 3, 4, 5, and 6. Table 3,
Table 4, Table 5 and Table 6 show Examples 1-2 and Reference Examples 1--2.
3 shows the results of measurement of serum A, serum B, serum C, and serum D, respectively, by enzyme immunoassay using the peptide obtained in 3. 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 cut-off value was determined by the formula ((mean value of OD ₄₉₂ value of normal human serum) + 2SD).

The cut-off values calculated based on Tables 3, 4, 5, and 6 give Examples 1 and 2 and Reference Examples 1 and 2.
The distribution of OD ₄₉₂ values for each of the three peptides is shown in FIGS.
Shown in Table 7 shows the positive rates calculated from Table 3, Table 4, Table 5, and Table 6 and the above cut-off value. As shown in Table 7, when the enzyme immunoassay using the peptide obtained in Example 1 was performed, 93.3%, 93.3%, 10.0%, and 0% positive results were obtained in serum A, serum B, serum C, and normal human serum D, respectively. Rate. When the enzyme immunoassay using the peptide obtained in Example 2 was performed, serum A, serum B, serum C and normal human serum D were 96.7%, 93.3%, 0% and 0%, respectively.
%, Indicating that the enzyme immunoassay using these peptides has a high correlation with the immunoscreening method shown in Reference Example 8.

When the enzyme immunoassay using the peptides obtained in Reference Examples 1 and 2 was performed, serum A, serum B, serum C, and normal human serum D
A positive rate of 10.0% was shown, indicating that the enzyme immunoassay using these peptides had poor sensitivity and specificity. It has also been isolated by several individuals, including one of the present inventors,
When an enzyme immunoassay was performed 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, serum A, serum B and serum C showed positive rates of 93.3%, 93.3%, and 0%, respectively, while 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 worse as compared with the enzyme immunoassay 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 was determined by using the peptides obtained in Examples 1 and 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 antigen substances. Assay by enzyme immunoassay was performed.

The measurement results are shown in Table 8, Table 9, Table 10, and Table 1.
It is shown in FIG. Table 12 shows the positive rates. 6 to 11 show the respective OD ₄₉₂ values. According to Table 12, when the enzyme immunoassay using the peptide obtained in Example 3 or Example 5 was performed, the positive rate was 96.7% or 100% for serum A and serum B, respectively, and serum C and normal human serum were obtained. D shows 0% or very low positive rate,
The enzyme immunoassay using these peptides was found to have high sensitivity and specificity. In addition, when the enzyme immunoassay using the peptide obtained in Example 4 was performed, serum A, serum B, serum C, and serum D each showed 36.7%,
Positive rates of 53.3%, 5.0% and 0.0% were shown, indicating that the peptide used had an antigenicity different from that of the peptides obtained in Examples 3 and 5.

Further, when an enzyme immunoassay was carried out using the peptides obtained in Reference Examples 4 to 6, serum A, serum B, serum C and serum D showed 0% or a very low positive rate. The enzyme immunoassay using the peptide 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 was 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, an assay was performed by enzyme immunoassay using the peptides obtained in Examples 6, 7, 8, and 7 as antigenic substances. Was.

The measurement results are shown in Tables 13, 14, 15, and 16. Table 17 shows the positive rates. 12 to 15 show the respective OD ₄₉₂ values. As shown in Table 17, when the enzyme immunoassay using the peptide obtained in Example 6 was performed, 33.3%, 40.0%, 0%, and 0% of positive results were obtained in serum A, serum B, serum C, and normal human serum D, respectively. Rate. When the enzyme immunoassay using the peptide obtained in Example 7 was performed, serum A, serum B, serum C, and normal human serum D were 50.0%, 66.7%, and 5.0%, respectively.
%, 0% positive rate. Further, when an enzyme immunoassay using the peptide obtained in Example 8 was performed, serum A, serum B, serum C, and normal human serum D
Positive rates were 23.3%, 93.3%, 5.0% and 10.0%.
Enzyme-linked immunosorbent assays using these peptides were performed on clones # 8, # 14 and # 14 cloned from ribonucleic acids isolated by several individuals, including one of the present inventors.
It was found that there was 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 performed, serum A, serum B, serum C and serum D were 13.3%, 13.3%, 5.0%,
A positive rate of 0.0% was shown, indicating 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 was 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 names of the diseases derived from the serum samples used and the number of the samples.

[Table 18]

Assay by Enzyme Immunoassay Each serum sample shown in Table 18 was measured for absorbance by the following enzyme immunoassay to test for the presence or absence of anti-HCV antibodies.

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

Results The measurement results are shown in Tables 19 and 20. Table 19 shows the measurement results when each of the serum samples shown in Table 18 was assayed by the enzyme immunoassay using the peptides obtained in Examples 1 and 2 and Tables 1 to 3. Furthermore, a cut-off value was set based on the OD ₄₉₂ value of 10 normal serum samples.
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 discriminating between positive and negative based on the cutoff values calculated based on the measured values of the serum samples of normal subjects in Tables 19 and 20. From Table 19, when the enzyme immunoassay using the peptide obtained in Example 1 was performed, sporadic non-A non-B acute hepatitis / recovery period and chronic hepatitis, and non-A
Serum from patients 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 sera of alcoholic hepatitis, the sera of hepatitis B, and the sera of normal subjects. When the enzyme immunoassay using the peptide obtained in Example 2 was performed, the serum of non-A non-B chronic hepatitis patients was determined to be positive for anti-HCV antibodies, and the serum of alcoholic hepatitis patients, the serum of hepatitis B patients and the normal The serum of the subject was judged negative for anti-HCV antibody. From the above, it was found that the enzyme immunoassay using the peptides obtained in Examples 1 and 2 was useful for early diagnosis of non-A non-B hepatitis.

Further, from Table 20, when the enzyme immunoassay using the peptides obtained in Reference Examples 1, 2 and 3 was carried out, it was determined that the anti-HCV antibody was negative in the serum of a patient with non-A non-B hepatitis. In some cases, the serum was determined to be positive for the anti-HCV antibody in the serum of a hepatitis B patient and the serum of a normal individual, and the specificity and sensitivity were poor. That is, the results of the enzyme immunoassay using the peptides obtained in Reference Example 1, Reference Example 2 and Reference Example 3 include false negatives and false positives, and diagnosis of non-A non-B hepatitis using these peptides. The efficiency was found to be poor. From the above results, it was shown that the presence or absence of an effective anti-HCV antibody was determined by using the peptides obtained in Examples 1 and 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 Examples 3, 4, 4, 5, 4, 5, and 5. Using the peptide obtained in Example 6, the absorbance was measured by an enzyme immunoassay, and the presence or absence of an anti-HCV antibody was tested.

Table 21 and Table 22 show the measurement results. 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, respectively. Furthermore, a cutoff value was set based on the OD ₄₉₂ value of 10 normal serum samples, and positive / negative of anti-HCV antibody was determined. The cut-off value is ((average OD ₄₉₂ value of normal serum) +2
SD).

Tables 21 and 22 show the results of discriminating between positive and negative, based on the cut-off values calculated based on the measured values of the serum samples of normal subjects in Tables 21 and 22. According to Table 21, when the enzyme immunoassay using the peptides obtained in Example 3 and Example 5 was performed, non-A
The anti-HCV antibody was determined to be positive in the sera of patients with acute and convalescent phases of non-B hepatitis and the sera of patients with non-A non-B chronic hepatitis. Furthermore, the serum of alcoholic hepatitis, the serum of hepatitis B, and the serum of normal subjects were determined to be negative for anti-HCV antibodies, and the enzyme immunoassay using these peptides was found to be useful in diagnosing the above patients. Was. In addition, when the enzyme immunoassay using the peptide obtained in Example 4 was carried out, it was found that the anti-specifically specific antiserum was obtained only from the serum of a patient with non-A non-B chronic hepatitis.
The HCV antibody was determined to be positive and proved to be useful for the diagnosis of the above patients.

Further, from Table 22, when an enzyme immunoassay was carried out using the peptides obtained in Reference Examples 4 to 6, Table 1
In some cases, anti-HCV antibodies may be determined to be negative in the sera of patients having the above-mentioned disease 8 and anti-HCV antibodies may also be determined to be positive in the sera of normal subjects. The determination results by the immunoassay method include 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, by using the peptides obtained in Examples 3, 4 and 5, an effective anti-HCV
It was shown that the presence or absence of the antibody was determined.

[Table 21]

[Table 22]

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

Table 23 shows the measurement results. Table 23 shows the respective measurement results when each serum sample shown in Table 18 was assayed by the enzyme immunoassay using the peptides obtained in Examples 6 to 8 and Reference Example 7. Further normal 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 cutoff value was determined by the formula ((average value of OD ₄₉₂ value of normal serum) + 2SD).

Table 23 shows the results of discriminating between positive and negative, based on the cutoff values calculated based on the measured values of the serum samples of normal subjects in Table 23. As shown in Table 23, when the enzyme immunoassay using the peptide obtained in Example 6 was performed, the serum of patients with sporadic non-A non-B chronic hepatitis had anti-HC
V antibody was determined to be positive, and PTNANBH acute and convalescent phases and chronic hepatitis were all positive. Furthermore, antiserum HCV antibodies were determined to be negative in the sera of alcoholic hepatitis patients, the sera of hepatitis B patients, and the sera of normal subjects. From the above, it was found that the enzyme immunoassay using the peptide obtained in Example 6 was useful for early diagnosis of non-A non-B hepatitis. In addition, when the enzyme immunoassay using the peptides obtained in Examples 7 and 8 was carried out, the anti-HCV antibody was specifically positive in the sera of patients with non-A non-B non-B chronic hepatitis recovery and chronic hepatitis. Was determined to be useful for the diagnosis of the above patients.

When an enzyme immunoassay using the peptide obtained in Reference Example 7 was carried out, the serum of non-A non-B hepatitis patients was determined to be negative, and the serum of alcoholic hepatitis and hepatitis B Was determined to be positive in some cases.
That is, the results of the enzyme immunoassay using the peptide obtained in Reference Example 7 include false negatives and false positives,
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 was 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, and the presence or absence of an anti-HCV antibody was sampled.

That is, each of the peptides obtained in Example 1 as antigen substances was dissolved in a 0.01 M carbonate buffer (pH 9.5), and the obtained peptide solutions were used for a polystyrene enzyme immunoassay cup (manufactured by Dynatech). To each one
After adding 00 μl at a time, the mixture was allowed to stand at 4 ° C. for 12 hours to perform coating with the peptide. The cups were then washed three times with PBS containing 0.05% by volume Tween20. Subsequently, 150 μl of PBS containing 20% by volume of normal goat serum was added to the cups, and the mixture was allowed to stand 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
After removing the BS, each assay cup was dried.

As a serum diluting solution, 100 μl of PBS containing 10% by volume of normal goat serum was added to each of the above assay cups.
After each addition, each test serum was added such that the ratio of the serum diluting solution to the test serum was 20: 1 (volume ratio). 37 ℃
After 1 hour incubation at
The plate was washed three times with PBS containing 20% by volume of Tween20.

100 μl of a goat anti-human IgG antibody-peroxidase conjugate (diluted to an optimum concentration with PBS containing 10% by volume of normal goat serum) was added to each of the assay cups obtained. After incubation at 37 ° C for 30 minutes, the cups are washed with PBS containing 0.05% by volume Tween20.
And washed three times. Subsequently, a coloring agent (o-phenylenediamine was dissolved in 0.1 M citrate-phosphate buffer pH5.6 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 OD ₄₉₂ value of the reaction solution at 492 nm was measured.

Results As a result of the inhibition test of the anti-HCV antibody with the peptide obtained in Example 1, a 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. Table 24 also shows the results of the determination of the above blood donor blood serum (2476 samples) by the EIA method and the RIBA method using a commercially available anti-HCV antibody measurement reagent (manufactured by Ortho).

[Table 24]

As is clear from Table 24, commercially available anti-HCV
Out of 2462 samples determined to be negative by the antibody measurement reagent,
Thirty-five specimens were tested for anti-HC using the peptide obtained in Example 1.
V antibody was determined to be positive. Also, commercially available anti-HC
The 14 specimens that were determined to be positive by the V antibody measurement reagent were all determined to be positive in the determination of the anti-HCV antibody measured using 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 1510

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: number of nucleic acid strands: unknown Topology: unknown Sequence type: cDNA to genomic RNA Fragment type: middle fragment Origin Solid / isolated clone name: # 8 Type of clone tissue: human serum Sequence characteristics Symbol indicating characteristics: CDS Location: 1. . 120 Method of Characterization: E Sequence GAATTCCAAA AAGAGCAAAA CAAACCGCCG AAGAAAAAAC TAATAAGAGA AGAAAAGGCG 60 AAGAGACACA GGAAAAAAAAAA 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: middle fragment Origin Solid / isolated clone name: # 14 Type of clone tissue: human serum Sequence characteristics Symbol indicating characteristics: CDS Location: 1. . 114 Method of Characterization: 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: middle fragment Origin Solid / isolated clone name: # 18 Type of clone tissue: human serum Sequence characteristics Symbol indicating characteristics: CDS Location: 1. . 201 Method for determining characteristics: E sequence GAATTCCAAG AAAAAAAGGG AGAAGCCAGC AATGGAGAAG CCGAAAACGA CACACACAAG 60 AAACAAAGGA GGTACAAAGA AAAAGAAAAA ACGGCAACAA ATAACCCAGG AAAGAACAAGA 120 AAGCCAAGAG TGGGCAGAAT AAAAAGAGGATGA CATALOG

[Brief description of the drawings]

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

FIG. 2 is a distribution diagram of the OD ₄₉₂ value 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 diagram of an OD ₄₉₂ value provided 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 diagram of OD ₄₉₂ values given to each serum sample measured by the method described in Example 9 using the peptide obtained in Reference Example 2.

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

FIG. 6 is a distribution diagram of the OD ₄₉₂ value 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 diagram of OD ₄₉₂ values given to each serum sample measured by the method described in Example 9 using the peptide obtained in Example 4.

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

FIG. 9 is a distribution diagram of the OD ₄₉₂ value 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 diagram of the OD ₄₉₂ value 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 diagram of the OD ₄₉₂ value 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 diagram of an OD ₄₉₂ value given to each serum sample measured by the method described in Example 9 using the peptide obtained in Example 6.

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

FIG. 14 is a distribution diagram of the OD ₄₉₂ value 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 diagram of the OD ₄₉₂ value given by each serum sample measured by the method described in Example 9 using the peptide obtained in Reference Example 7. In these figures, each symbol indicates the following.

[Explanation of symbols]

●: GPT>200IU; HBsAg ( -) OD 492 values Serum A gave ○: GPT>200IU; HBsAg ( -) serum B gives the OD ₄₉₂ value ×: GPT>200IU; HBsAg ( -) given serum C is OD ₄₉₂ value

──────────────────────────────────────────────────続 き Continuation of front page (31) Priority claim number Japanese Patent Application No. 2-296899 (32) Priority date October 31, 1990 (Oct. 31, 31) (33) Priority claim country Japan (JP) (72) Inventor Toshihiko Namba 1660 Sazu, Kurashiki City, Okayama Prefecture (72) Inventor Masao Tsuji 1-1 Mizue, Kurashiki City, Okayama Prefecture (56) References JP-A-5-148298 (JP, A) JP-A-4- 253998 (JP, A) Special Table 60-500684 (JP, A) WO 89/4669 (WO, A1) (58) Fields investigated (Int. Cl. ⁷ , DB name) C07K 7/06 A61K 39 / 29 C07K 7/08 C07K 14/18 G01N 33/576 BIOSIS (DIALOG) CA (STN) MEDLINE (STN) REGISTRY (STN) WPIDS (STN)

Claims (4)

(57) [Claims] The ability to specifically bind to an antibody having an amino acid sequence of Arg Arg Tyr Lys Glu Lys Glu Lys (SEQ ID NO: 4) and having specificity to a non-A non-B hepatitis-related antigen A peptide comprising 10 to 40 amino acids. 2. Formula (2): Glu Lys Lys Gly Glu Ala Se
r Asn Gly GluAla Glu Asn Asp Thr His Lys Lys Gln A
rg Arg Tyr Lys Glu Lys Glu Lys ThrAla Thr Asn Asn
2. A peptide comprising the peptide according to claim 1 or a fragment thereof having an amino acid sequence represented by Pro Gly Lys Asn Lys Lys Pro Arg (SEQ ID NO: 3), wherein said peptide is Arg Arg Tyr Lys Glu Lys Glu Lys (sequence). No .: 4)
2. 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. 3. Formula (2-a): Arg Arg Tyr Lys Glu Ly
s Glu Lys ThrAla Thr Asn Asn Pro Gly Lys Asn Lys L
ys Pro Arg (SEQ ID NO: 8), Formula (2-b): Thr His Lys Lys Gln Arg Arg Arg Tyr Lys
Glu Lys Glu Lys (SEQ ID NO: 9) or Formula (2-c): Arg Arg Tyr Lys Glu Lys Glu Lys Thr
The peptide according to claim 2, which is represented by Ala (SEQ ID NO: 10). 4. A reagent for measuring an antibody having specificity to a non-A, non-B hepatitis-related antigen, comprising the peptide according to claim 1.