HCV peptide antigens and methods for the determination of HCV

Abstract

Novel HCV peptide antigens are described representing partial sequences of the C-100-3 and env/core with C-regions. These peptide antigens are suitable for the determination of HCV antibodies as immunogens for the production of antibodies against HCV and as vaccines for the production of vaccines against HCV.

Description

The invention concerns new HCV peptide antigens, a process for the production of these peptide antigens as well as a method for the determination of HCV using the peptide antigens.

The occurrence of viral hepatitis in the absence of serologic markers of previously known hepatotropic agents (e.g. hepatitis A virus, hepatitis B virus, hepatitis Δ virus, cytomegalovirus and Epstein-Barr virus) is termed non-A, non-B hepatitis (NANB hepatitis). NANB hepatitis is in turn subdivided into parenterally and sporadically transmitted non-a, non-B hepatitis and non-A, non-B hepatitis transmitted by the intestinal route. The causative agent for the parenterally and sporadically transmitted NANB hepatitis, the hepatitis C virus (HCV), has recently been isolated (Choo Q. -L. et al., Science 244 (1989) 359-362 and Kuo, G. et al., Science 244 (1989) 362-364).

HCV is worldwide an important cause of NANB hepatitis and is transmitted by contaminated blood or blood products, by blood transfusions or close personal contact.

The amino acid sequence of the HCV viral proteins is known from EP-A 0 318 216, EP-A 0 363 025, EPA 388 232 and EP-A 0 396 748. The genome of the HCV has a length of 10862 nucleotides. The proteins arising from translation have a total length of ca. 3000 amino acids. The proteins can be divided into structural proteins (envelope and core proteins) and non-structural proteins (NS1-NS5).

It is expedient to carry out the determination of HCV by detecting antibodies against HCV in body fluids using immunological tests. Therefore binding partners for anti-HCV antibodies are necessary for such immunological tests.

Thus it is known that for example the non-structural C 100-3-HCV protein can be used as a binding partner in immunological tests (tests from ABBOTT LABORATORIES, USA and ORTHO DIAGNOSTIC SYSTEMS INC., USA; Science 244 (1989) 359-364; Van der Poel C. L. et al. Lancet 337 (1991) 317; Alter H. J. J. Gastroent. Hepatol. (suppl.) 1990, 78).

A disadvantage of these tests is that a recombinant protein is used as antigen. Proteins are difficult to handle in diagnostic tests because of their susceptibility to denaturation and consequent reduced solubility and function. As a result of the low epitope density on a protein the magnitude of the measurement signal is also less than in a test in which a short-chained peptide antigen is used as the binding partner of the antibody. In addition, when proteins or long-chained peptides are used as antigens in an immunological test there can be an increase in cross-reactivities and unspecific bindings of antibodies. Moreover, reactions with proteins are often diffusion controlled which is an impediment to achieving the desired short times for immunological tests. In addition the production of protein which can be used for diagnostics in sufficient quantity and quality is time-consuming and expensive. Peptides are easily accessible by synthesis and are defined molecules.

Accordingly it is advantageous in an immunological test for anti-HCV antibodies to use peptide antigens which are as short-chained as possible and only represent sections of the total proteins. Such an immunological method is described by Okamoto (Japan J. Exp. Met. 60 (1990) 223 -234). However, it has been shown that the short-chained peptide antigen (sequence 9) described in this publication which is derived from the core region is not sufficiently sensitive to HCV.

The object of the present invention is therefore to provide peptide antigens which are specific for anti-HCV antibodies and are suitable for immunological tests for anti-HCV antibodies.

This object is achieved by the peptide antigens of the sequences

1: SerGlyLysProAlallelleProAspArgGluValLeuTyrArgGluPheAsp (SEQ ID NO. 1)

2: GluCysSerGlnHisLeuProTyrIleGluGInGlyMetMetLeuAlaGluGlnPheLysGInLysGInLysAlaLeuGlyLeuLeuGInThrAlaSerArg- Gln (SEQ ID NO. 2)

3: AlaValGInThrAsnTrpGlnLysLeuGluThrPheTrpAlaLysHisMetTrpAsn (SEQ ID NO. 11) 4: AsnProLysProGlnLysLysAsnLysArgAsnThrAsnArgArg (SEQ ID NO. 12)

5: AsnProLysProGlnArgLysThrLysArgAsnThrAsnArgArg (SEQ ID NO. 15)

6: ProGlnAspValLysPheProGlyGlyGlyGlnlleValGlyGlyVal (SEQ ID NO. 16)

7: ProArgGlySerArgProSerTrpGlyProThrAspProArgArg (SEQ ID NO. 22)

8: GlnLeuPheThrPheSerProArgArgHisTrpThrThrGlnGly CysAsnCysSerlleTyrProGlyHisileThrGlyHisArgMetAlaTrp Asp-MetMetMetAsnTrpSerProThrThrAlaLeuValMetAla (SEQ ID NO. 23)

10: GlnLysLysAlaAlaArgAsnThrAsnArgArg (SEQ ID NO. 29)

11: HisTrpThrThrGlnGlySerAsnSerSerIleTyrProGlyHis (SEQ ID NO. 30)

12: SerSerIleTyrProGlyHisIleThrGlyH isArgMetAlaTrpAspMetMet (SEQ ID NO. 31)

13: ProGluGlyArgThrTrpAlaGlnProGlyTyrProTrpProLeuTyr (SEQ ID NO. 32)

or peptide antigens which represent partial sequences of these peptide antigens with a length of at least four, preferably of at least seven amino acids.

Suitable partial sequences are shown in the sequence protocols and are indicated by letters/number combinations (e.g. 6 a, 2 b).

Particularly preferred partial sequences are:

From sequence 2:

GluCysSerGlnHisLeuProTyrlleGluGlnGlyMetMetLeu (sequence 2a) (SEQ ID NO. 3)

MetMetLeuAlaGluGlnPheLysGlnLysAlaLeuGlyLeuLeuGlnThrAla (sequence 2b) (SEQ ID NO. 4)

MetMetLeuAlaGluGlnPheLysGlnLysAlaLeuGlyLeuLeuGlnThrAlaSerArgGln (sequence 2c) (SEQ ID NO. 5)

HisLeuProTyrIleGlu (sequence 2d) (SEQ ID NO. 6)

Ser Gln His Leu Pro Tyr Ile Glu Gln (sequence 2e) (SEQ ID NO. 7)

Lys Ala Leu Gly Leu Leu Gln (sequence 2f) (SEQ ID NO. 8)

Gln Lys Ala Leu Gly Leu Leu Gln Thr (sequence 2g) (SEQ ID NO. 9)

from sequence 4: Lys Asn Lys Arg Asn Thr Asn Arg Arg (sequence 4a) (SEQ ID NO. 13)

from sequence 6;

ProGlnAspValLysPheProGlyGlyGlyGlnIle (sequence 6a) (SEQ ID NO. 17) Lys Phe Pro Gly Gly Gly Gln Ile Phe (sequence 6b) (SEQ ID NO. 18)

Lys Phe Pro Gly Gly Gly Gln Ile Val (sequence 6d) (SEQ ID NO. 20)

Gln Asp Val Lys Phe Pro Gly Gly Gly (sequence 6e) (SEQ ID NO. 21)

Partial sequences are particularly preferred which have a maximum length of 9 amino acids. These are in particular the sequences 6b, 6d, 6e, 2e, 2f, 2d, 2g, 4a.

The peptide antigens with the sequences 1-3 are contained in the C 100-3 region of the HCV proteins and the peptide antigens with the sequences 4-8, 10-13 are contained in the env/core region of the HCV proteins. The peptide antigens with the sequences 1-8, 10-13 according to the present invention and the peptide antigen 9 of sequence (ArgGlyProArgLeuGlyValArgAlaThrArg LysThrSerGluArgSerGInProArgGlyArgArgGln ProlleProLysAlaArgArgProGluGlyArgThrTrpAlaGlnProGlyTyrProTrpPro, (SEQ ID NO. 25), Okamoto loc. cit) are specified in the sequence protocols SEQ ID NO: 1-32.

An anti-HCV antibody test is carried out according to methods known to one skilled in the art. The invention therefore also concerns a method for the determination of HCV antibodies which is characterized in that the sample is incubated with a combination of at least two peptide antigens from the group of sequences 1-13 or peptide antigens which represent partial sequences of these peptide antigens which have a length of at least 4, preferably of at least 7 amino acids and the amount of anti-HCV antibodies bound to the peptide antigen is determined under conditions which allow the formation of an antibody-antigen complex.

According to the present invention a combination of at least two of the peptide antigens or partial sequences thereof according to the present invention are used. It is particularly preferred that the peptide antigens of sequences 1-3 or partial sequences thereof be combined with at least one peptide antigen from the group of the sequences 4-13 or partial sequences thereof.

Suitable partial sequences of sequence 9 are:

ArgGlyProArgLeuGlyValArgAlaThrArg LysThrSerGluArgSerGlnProArgGly (sequence 9a) (SEQ ID NO. 26)

SerGlnProArgGlyArgArgGlnProlleProLysAlaArgArgProGluGlyArgThr (sequence 9b) (SEQ ID NO. 27)

LysAlaArgArgProGluGlyArgThrTrpAlaGln ProGlyTyr (sequence 9c) (SEQ ID NO. 28)

The combination of the antigens can for example be carried out by using several individual peptide antigens or in that peptide antigens are covalently bound to one another, appropriately by means of an amino acid bridge which differs from the amino acid sequences that naturally occur in HCV proteins or by means of a peptide linker.

The following combinations of antigens are particularly preferred:

Sequence 2b (SEQ ID NO. 4), 4 (SEQ ID NO. 12), and 6 (SEQ ID NO. 16)

Sequence 2b (SEQ ID NO. 4), 2c (SEQ ID NO. 5),4 (SEQ ID NO. 12), and 6 (SEQ ID NO. 16)

Sequence 2a (SEQ ID NO. 3), 2b (SEQ ID NO. 4), 2c (SEQ ID NO. 5), 4 (SEQ ID NO. 12), and 6 (SEQ ID NO. 16)

Sequence 2a (SEQ ID NO. 3), 2b (SEQ ID NO. 4), 2c (SEQ ID NO. 5), 4 (SEQ ID NO. 12), 6 (SEQ ID NO. 16), 9a (SEQ ID NO. 26), and 9b (SEQ ID NO. 27)

Sequence 2a (SEQ ID NO. 3), 2b (SEQ ID NO. 4), 4 (SEQ ID NO. 12), 6 (SEQ ID NO. 16), 9a (SEQ ID NO. 26), and 3 (SEQ ID NO. 11)

Sequence 2a (SEQ ID NO. 3), 2b (SEQ ID NO. 4), 4 (SEQ ID NO. 12), 6 (SEQ ID NO. 16), and 9a (SEQ ID NO. 26)

Sequence 2e (SEQ ID NO. 7), 2g (SEQ ID NO. 9), 4a (SEQ ID NO. 13), 6d (SEQ ID NO. 20), and 6e (SEQ ID NO. 21)

Sequence 2d (SEQ ID NO. 6), 2f (SEQ ID NO. 8), 4a (SEQ ID NO. 13), 6c (SEQ ID NO. 19), and 9c (SEQ ID NO. 28)

Sequence 11 (SEQ ID NO. 30), 12 (SEQ ID NO31), and 8a (SEQ ID NO. 24)

The antigens in the combinations are preferably used in approximately equimolar amounts.

The combination of the antigens of sequences 11, 12, 8a is particularly suitable for detecting patient sera in which a HCV infection has been cured (convalescent sera).

The antigens are preferably used separately without being covalently bound to one another or bound together using a peptide linker.

Since a high sensitivity is necessary for the infection parameter HCV, heterogeneous immunoassays are preferably used for the detection. These heterogeneous tests allow washing steps which considerably reduce the background measurement signal resulting in an increase in sensitivity.

The determination can for example be carried out by means of a radioimmunoassay, enzyme immunoassay or by immunofluorescence. For this the peptide antigen is usually immobilized. The sample which is to be examined for anti-HCV antibodies is added and the antibodies bound to the antigen are determined by means of a labelled anti-human immunoglobulin antibody. The immobilization of the peptide antigen according to the present invention can be carried out adsorptively, covalently or by means of a biological binding pair such as biotin/streptavidin, antibody/antigen or sugar/lectin. In this process the peptide antigen is covalently bound to this partner.

The peptide antigens according to the present invention can preferably be immobilized according to methods familiar to one skilled in the art such as on beads, plastic tubes or microtitre plates (preferably polystyrene or copolymers of polystyrene). This is preferably carried out by adsorbing the peptide antigens unspecifically onto the surface or covalently binding the peptide antigen to functionalized or activated surfaces. The unspecific adsorption can be improved by linking the peptide antigen to a protein to form a conjugate and using this conjugate for the adsorption (cf. e.g. EP-A 0 269 092). The binding can also be carried out via an immobilized antibody. For this the peptide antigen should be modified in such a way that the epitope is not blocked by the antibody binding e.g. by formation of a peptide-protein conjugate.

The conjugation of the peptide antigen to the binding partner is preferably carried out via a spacer. This spacer appropriately contains 10-50, preferably 10-30 atoms and is also preferably an essentially linear molecule. Examples for this are spacers made of alkyl chains, polyether chains or polyamide chains. In a particularly preferred embodiment a peptide antigen with a length of 4 -9 amino acids is bound to the carrier via a linear spacer of 10-30 atoms. If a spacer made of amino acids is to be used, it is appropriate that it consists of amino acids which do not correspond to the sequence in the direct vicinity of the peptide antigen in the HCV gene.

In a preferred embodiment the peptide antigen according to the present invention is covalently bound to biotin whereby the immobilization is carried out by means of an avidin/streptavidin solid phase.

Methods of determination are also suitable in which the detection is not via a labelled antibody but via a labelled additional peptide antigen sequences 1 -13 or partial sequences thereof.

The peptide antigens according to the present invention can be produced according to methods for peptide synthesis familiar to one skilled in the art. The invention therefore also concerns a process for the production of the peptide antigen according to the present invention which comprises binding the amino acid forming the C-terminal end to a carrier, assembling stepwise the peptide antigen starting at the C-terminal end and subsequently cleaving it from the carrier.

The details of this process are that an amino acid is linked, for example via its carboxyl group, to an insoluble polymer which can be easily filtered and then the peptide chain is assembled stepwise starting at the C-terminal end. For this purpose a N-protected amino acid is reacted with a reactive group of the artificial resin. The Nα-protective group is removed from the amino acid which is covalently anchored to the carrier particle and the resulting amino acyl polymer is reacted with the next N-protected amino acid. The Nα-protective group is removed from the dipeptide covalently bound to the carrier resin and the resulting amino acyl polymer is reacted with the next N-protected amino acid. All excess reagents and by-products are removed by simple filtration. As soon as the desired peptide sequence has been prepared in this way, the covalent binding between the C-terminal amino acid and the anchor group of the polymeric carrier is cleaved. The insoluble carrier is removed from the peptide which is now in solution by simple filtration. The peptide is purified by chromatographic methods.

The peptide antigens according to the present invention can for example be prepared according to Merrifield, JACS 85 (1964) 2146. If a biotinylation is necessary this can for example be carried out according to PNAS USA 80 (1983) 4045. A preferred biotinylation agent for this is biotinyl-aminocaproic acid-N-hydroxysuccinimide ester.

A preferred process for the production of biotinylated peptide antigens is to introduce the biotin residue at the N-terminus during a solid phase synthesis of the peptide antigen. This process is preferably used in cases in which the peptide antigen contains several ε-lysine amino groups which are not intended to be biotinylated. This is for example the case when N-α-Fmoc-N-ε-biotinyl-aminocaproyllysine, N-α-Fmoc-N-ε-biotinyllysine is used or when for the biotinylation of the N-terminal amino acids biotin, biotinyl-aminocaproic acid or dimethoxytritylbiotin is used with an activating reagent, such as for example dicyclohexylcarbodiimide, or as an active ester.

In a further preferred embodiment a detection antibody which is for example directed against the Fc part of human IgG is immobilized. A monoclonal antibody is preferably used for this. The peptide antigen is then present in solution. The antibody (analyte) to be detected and also all other antibodies in the sample liquid are then bound by the immobilized antibody. The bound antibody can then bind the analyte which can be detected with a suitable detection system e.g. competitively with a peptide antigen-enzyme conjugate.

It is also possible using the peptide antigens according to the present invention to obtain antibodies by immunization methods familiar to one skilled in the art with which the virus itself can be detected in an immunological test.

The invention therefore also concerns a process for the production of antibodies which is characterized in that a mammal is immunized with a peptide according to the present invention which, if desired, is bound to a carrier and the antibodies are obtained, for example from the serum or the spleen, according to known methods.

In a preferred embodiment B lymphocytes of the immunized animals are fused with a suitable cell line in the presence of transforming agents, the cell line which produces the desired antibodies is cloned and cultured and the monoclonal antibodies are isolated from the cells or from the culture supernatant.

Using this antibody it is possible to directly determined HCV viruses. The invention therefore also concerns a process for the determination of HCV viruses which is characterized in that the sample is incubated with an antibody according to the present invention under conditions which allow the formation of an antigen-antibody complex and the amount of antibody-antigen complex formed is determined.

The invention in addition concerns a process for the production of vaccines using the peptide antigens according to the present invention and a vaccine for treating HCV infections containing a peptide antigen of the sequences 1-8, 10-13 which is carrier-bound if desired or partial sequences thereof or at least two peptide antigens of the sequences 1 -13 or partial sequences thereof as an immunogen in a pharmacologically effective dose and in a pharmaceutically acceptable formulation.

The production of these vaccines can be carried out according to known methods. However, the peptide antigens are preferably first lyophilized and subsequently suspended, if desired with addition of auxiliary substances.

Vaccination with these vaccines or combinations of vaccines according to the present invention can be carried out according to methods familiar to one skilled in the art for example intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously or intranasally.

For the intramuscular or subcutaneous administration, the vaccine can for example be suspended in physiological saline. For an intranasal or intraoccular application, the vaccine can for example be used in the form of a spray or an aqueous solution. For a local, for example an oral administration, it is often necessary to temporarily protect the immunogens against inactivation, for example against proteolytic enzymes in the cavity of at the mouth or in the stomach. Such a temporary protection can for example be achieved by encapsulating the immunogens. This encapsulation can for example be carried out by coating with a protective agent (microencapsulation) or by embedding a multitude of immunogens according to the present invention in a protective carrier (macroencapsulation).

The encapsulation material can be semipermeable or become semipermeable when introduced into the human or animal body. A biological degradable substance is usually used as a carrier for the encapsulation.

The invention is further elucidated by the following examples and sequence protocols.

The sequence protocols denote the following:

Sequence SEQ ID NO
1        1
2        2
2 a      3
2 b      4
2 c      5
2 d      6
2 e      7
2 f      8
2 g      9
2 h      10
3        11
4        12
4 a      13
4 b      14
5        15
6        16
6 a      17
6 b      18
6 c      19
6 d      20
6 e      21
7        22
8        23
8 a      24
9        25
9 a      26
9 b      27
9 c      28
10       29
11       30
12       31
13       32

EXAMPLE 1

Synthesis of H-ProArgGlySerArgProSerTrpGlyProThrAspProArgArg-OH (SEQ ID NO. 22)

The peptide was produced by means of Fmoc(fluorenyloxycarbonyl) solid-phase synthesis. The reactions were carried out on a Labortec (Switzerland) SP 640 peptide synthesizer. The coupling reactions with regard to the Fmoc amino acid derivative were carried out with 2.4 equivalents of dicyclohecylcarbodiimide and 2.2 equivalents of N-hydroxybenzotriazole for 90 minutes. Dimethylformamide was used as the reaction medium. The Fmoc group was cleaved by means of 20 % piperidine in DMF in 10 and 20 minutes. 2.0 equivalents of the following amino acid derivatives were used: Pro, Arg(with PMC(pentamethylchroman) protective group), Gly, Ser(with tert.-butyl protective group), Trp, Thr(with tert.-butyl protective group), Asp(with tert.-butyl ester protective group). The coupling reactions were repeated with half the reagents. The coupling result was checked by means of the Kaiser test (Anal. Biochemistry 34 (1970) 595), the loading of the resin was determined by means of the UV absorbance of the released fulvene group after each piperidine cleavage. The peptide was synthesized on 5 g Wang resin (polystyrene/1% divinylbenzol) loaded with 0.50 mMol/g (JACS, 95 (1973) 1328). After the synthesis the degree of loading was still 0.39 mMol/g.

The peptide was released with 200 ml trifluoroacetic acid, 200 ml dichloromethane, 10 ml ethanedithiol, 10 ml m-cresol, 5 ml ethylmethylsulfide and 5 ml water in 30 minutes at room temperature. The cleavage solution was evaporated several times with toluol and then the peptide was precipitated with diethyl ether.

In order to remove the scavenger and other small molecules, the crude material was purified on a Sephadex G10 column. After lyophilization, 3.2. g material was obtained with a purity of 42% (RP-HPLC). In order to bring the material to a final purity of >95 %, 400 mg peptide was purified on a preparative RP-HPLC column (400 mm×250 mm) filled with C18 material (5 micrometer, 300 Angström) and employing a water/trifluoroacetic acid, acetonitrile/trifluoroacetic acid gradient. After lyophilization 118 mg 96.5 % (HPLC) white material was obtained. The identity of the material was checked by means of FAB-MS.

EXAMPLE 2

In order to biotinylate the peptide antigen from Example 1, a mole equivalent was dissolved as concentrated as possible (the solubility depends on the amino acid sequence) in an argon-saturated potassium phosphate buffer (0.1 mol/, pH 8.0) and 3 equivalents D-biotinyl-ε-aminocaproic acid-N-hydroxysuccinimide ester dissolved in argon-saturated dimethylformamide (solution of 1 μmol reagent in 5 μl DMF) is added.

The reaction mixture was stirred for 2 hours at room temperature under argon while continuously monitoring by means of analytical RP-HPLC. When <5% educt was present the reaction preparation was applied directly to a preparative RP-HPLC column and the product material was purified by means of a 0.1% trifluoroacetic acid/water to 0.1% trifluoroacetic acid/acetonitrile gradient (gradient: 0% to 100% in 90 minutes). The product material was obtained by evaporating and lyophilizing the product fractions. The yields were between 40% and 90%. The purity was analysed by means of HPLC, HPCE and TLC, the identity was determined with FAB-MS (mole peak) and TLC with specific staining reagents (p-dimethyl-aminocinnamic aldehyde on biotin) and the amount was assayed by microanalysis (nitrogen).

EXAMPLE 3

HCV antibodies are determined in a 2-step sandwich immunoassay. Reagents with the following composition are used for the test:

Reagent 1:

0.10 μg/ml (peptide antigens 1, 3, 4, 5, 6) or 0.25 μg/ml (peptide antigens 2, 4, 7) biotinylated peptide antigen or a 1:1 mixture of such peptide antigens.

40 mmol/l phosphate buffer pH 7.0

0.9 % by weight NaCl

10 % by volume bovine serum

Reagent 2:

20 mU/ml of a conjugate of polyclonal antibody against human immunoglobulin (sheep) and peroxidase

40 mmol/l phosphate buffer pH 7.0

0.05 % by weight Tween® 20

0.2 % bovine serum albumin

0.2 % bovine IgG

1 ml reagent 1 and 10 μl sample are incubated for one hour at room temperature in a streptavidin-coated polystyrene tube (produced according to Example 1 of EP-A 0 344 578). Subsequently it is washed three times with tap water and incubated for one hour at room temperature with 1 ml reagent 2. It is subsequently washed three times with tap water. 1 ml ABTS® (2,2′-azino-di[3-ethyl-benzthiazoline sulfate(6)]diammonium salt, 1.9 mmol/l, in 100 mmol/l phosphate-citrate buffer pH 4.4 containing 3.2 mmol/l sodium perborate) is added for the detection reaction. The absorbance at 420 nm is measured photometrically after 60 minutes. The results are shown in Table 1.

	TABLE 1
	Peptide antigens (sequence No)
Serum	9	1	2	3	4	5	6	7	8	1 + 4	3 + 6
1	+	−	+	+	+	+	+	−	+	+	+
2	−	−	−	−	+	+	+	−	−	+	+
3	−	+	−	−	−	−	+	−	−	+	+
4	+	−	+	+	+	−	+	+	+	+	+
5	−	−	+	−	+	−	−	−	+	+	+
6	+	−	+	+	+	+	+	−	+	+	+
7	+	+	+	−	+	−	+	+	+	+	+
Explanatory notes for Table 1:
−/+: negative/positive (The cut-off for a positive signal in the ELISA is defined as the mean absorbance at 420 nm plus 3 standard deviations for a group of 10 negative control sera. The samples were measured at a sample dilution of 1:250).

Serum 1 was negative in the test in the Ortho-HCV antibody ELISA test system of ORTHO DIAGNOSTIC SYSTEMS INC. but positive on the basis of the clinical findings.

The sera 2-5 were identified as positive by the test of Ortho Laboratories, the sera 6 and 7 were identified as positive with the ABBOTT HCV EIA, catalogue No. 3 A53-24, ABBOTT LABORATORIES INC.

The peptide antigens 1 -6 were biotinylated with dimethoxytrityl-biotin on a solid phase at the ε-amino group of an additional lysine introduced at the N-terminus.

The peptide antigen mixtures 1+4 and 3+6 were used at a molar mixing ratio of 1:1.

EXAMPLE 4

Further sera were checked with peptides and peptide mixtures in a two-step sandwich immunoassay on microtitre plates coated with streptavidin.

The determination was largely carried out in an analogous way to Example 3. The following reagents were used for this:

Reagent 1:

50 ng peptide (or the amounts stated in the explanatory notes for the table) in 100 μl incubation buffer (40 mmol/l phosphate buffer, pH 7.0, 0.9 % by weight NaCl, 10 % by volume bovine serum).

Reagent 2:

Conjugate of polyclonal antibody against human immunoglobulin (sheep) and peroxidase (peroxidase activity 20 mU/ml), 40 mmol/l phosphate buffer pH 7.0, 0.05 % by weight Tween® 20, 0.2 % bovine serum albumin, 0.2 % bovine IgG.

Washing solution

40 mmol/l phosphate buffer pH 7.0, 0.9 % by weight sodium chloride, 0.05 % by weight Tween® 20.

Colour reagent

10 mg ABTS®, 80 μl 0.4 % H 2 O 2 in 10 ml citrate phosphate buffer (pH 4.4, 100 mmol/l).

Serum (diluted 1:10 in 50 μl incubation buffer) and 100 μl reagent 1 are added to each well of a microtitre plate coated with streptavidin. It is incubated for one hour at room temperature and subsequently washed five times with 200 μl washing solution each time. 150 μl reagent 2 is added, incubated for one hour at room temperature and washed three times with 200 μl washing solution each time. 150 μl colour reagent is added, incubated for one hour at room temperature and the absorbance is measured photometrically at 420 nm.

The results are shown in Tables II, III, IV, V, VI and VII.

The denotation in the tables is as follows:

Table II

Ortho: relative size of the measurement signal in the Ortho test (cf. Example 3).

blank space: measured value is smaller than twice the blank value or is identical to the blank value (determined with biotinylated peptide which is not reactive with HCV antibodies (nonsense sequence)).

filled circle: measured value is three times the blank value or more with 50 ng peptide per well.

empty circle: measured value is twice the blank value at 50 ng peptide per well

filled square: measured value is three times the blank value or more at 250 ng peptide per well

empty square: measured value is twice the blank value or more at 250 ng peptide per well.

* : negative controls

Table III

blank space: as in Table II

filled circle: measured value is four times the blank value or more at 50 ng peptide per well

empty circle: measured value is three times the blank value or more at 50 ng peptide per well

n.t.: measurement was not carried out

2a, 2b, 3, 4, Instead of a single peptide antigen, 6: a mixture of 10 ng each of the stated peptides was used in reagent 1.

* : negative controls

Table IV

The meaning of the symbols corresponds to the details for Table II.

The peptide mixtures each contained 50 ng of the individual peptides.

EXAMPLE 5

Tables V, VI and VII

The results of immunoassays analogous to Example 3 whereby the following peptide concentrations were used in reagent 1:

When several antigens were used in a combination the amounts used were reduced according to the number of different antigens.

Sequence 2a 50 μg/ml
Sequence 2b 50 μg/ml
Sequence 2d 100 μg/ml
Sequence 2f 100 μg/ml
Sequence 2h 100 μg/ml
Sequence 4  400 μg/ml
Sequence 4a 350 μg/ml
Sequence 4b 250 μg/ml
Sequence 4c 300 μg/ml
Sequence 6  350 μg/ml
Sequence 6a 350 μg/ml
Sequence 6b 350 μg/ml
Sequence 6c 250 μg/ml
Sequence 6d 300 μg/ml
Sequence 8a 900 μg/ml
Sequence 9a 350 μg/ml
Sequence 9c 350 μg/ml
Sequence 11 300 μg/ml
Sequence 12 550 μg/ml

+/−: pos./neg. (The cut-off for a positive signal in the immaunoassay as in Example 3 is defined as the mean absorbance at 420 nm plus 2 standard deviations for a group of 6 negative control sera. The samples are diluted with incubation buffer 1:100)

TABLE II

Peptide

C400.3 Region

core env. Region

Seren

Ortho

1

2a

2c

2b

3

4

6

9a

9b

7

8

Ortho

S1

−

◯

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

4

S2

2

◯

◯

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

◯

4

S3

1

◯

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

4

S4 (*)

−

—

S5

+/−

&Circlesolid;

◯

&Circlesolid;

4

S6

1

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

◯

4

S7 (*)

−

—

S8

+/−

▪

□

—

S9

−

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

—

S10

+/−

□

—

S11

+/−

◯

&Circlesolid;

&Circlesolid;

&Circlesolid;

◯

2

S12

2

◯

&Circlesolid;

◯

2

S13

4

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

—

S14

+/−

&Circlesolid;

&Circlesolid;

◯

0

S15

+/−

&Circlesolid;

&Circlesolid;

◯

&Circlesolid;

&Circlesolid;

&Circlesolid;

0

S16

4

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

◯

&Circlesolid;

&Circlesolid;

&Circlesolid;

0

S17

4

&Circlesolid;

◯

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

0

S18

2

&Circlesolid;

6

S19 (*)

+/−

—

S20

1

□

□

▪

□

—

S21

+/−

&Circlesolid;

&Circlesolid;

◯

&Circlesolid;

4

S22

−

◯

&Circlesolid;

4

S23

3

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

4

S24

4

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

4

S25

1

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

4

D61

◯

&Circlesolid;

&Circlesolid;

&Circlesolid;

◯

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

◯

01-421535

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

56-128431

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

&Circlesolid;

▪

□

19971

&Circlesolid;

◯

&Circlesolid;

&Circlesolid;

19075

&Circlesolid;

◯

&Circlesolid;

&Circlesolid;

◯

◯

▪

□

▪

19575

&Circlesolid;

&Circlesolid;

&Circlesolid;

▪

20004

◯

&Circlesolid;

20069

&Circlesolid;

&Circlesolid;

&Circlesolid;

◯

&Circlesolid;

&Circlesolid;

▪

□

RS (*)

HG (*)

AB (*)

(*) Negative sera

	TABLE III
	Peptide antigens
							2a, 2b,
Serum	2a	2b	3	4	6	9a	3, 4, 6
LL485561*
LL488301	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;
LL491001*
LL493411*
LL496131*
LL504111*
FF194591*
FF206011*
FP200311	&Circlesolid;	◯	◯	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;
FP211511*
FP210051	&Circlesolid;	&Circlesolid;					&Circlesolid;
FP804511*
B1	nt	&Circlesolid;		&Circlesolid;	◯		&Circlesolid;
B2	nt		◯	&Circlesolid;	&Circlesolid;	◯	&Circlesolid;
B3	nt	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;
B4	nt	◯		&Circlesolid;	&Circlesolid;	◯	&Circlesolid;
B5	nt	&Circlesolid;	◯	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;
B6	nt			&Circlesolid;			&Circlesolid;
B7	nt	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;
B8	nt	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;
B9	nt	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;
B10	nt	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;
B11
B12	&Circlesolid;			&Circlesolid;			&Circlesolid;
B13	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;
B14	nt	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;
B15	&Circlesolid;	&Circlesolid;		◯	◯	&Circlesolid;	&Circlesolid;
B16	&Circlesolid;	&Circlesolid;	◯	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;
B17	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;
B18	nt	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;
B19	nt	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;
B20	◯	◯		&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;
01-421533	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;
S23	&Circlesolid;	&Circlesolid;	◯	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;

	TABLE IV
	Peptide antigens
				2a, 2b, 2c, 4, 6,
Serum	2b, 4, 6	2b, 2c, 4, 6	2a, 2b, 2c, 4, 6	9a, 9b,
S1	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;
S2	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;
S3	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;
S4 (*)
S5	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;
S6	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;
S7 (*)
S8	◯
S9	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;
S10			◯	◯
S11	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;
S12	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;
S13	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;
S14	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;
S15	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;
S16	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;
S17	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;
S18	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;
S19 (*)
S20			◯	&Circlesolid;
S21	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;
S22	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;
S23	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;
S24	&Circlesolid;	&Circlesolid;	&Circlesolid;	&Circlesolid;
(*) Negative sera

	TABLE V
	Peptide
		2a, 2b,	2d, 2f,	8a, 11
	Serum	4, 6, 9a	4a, 6c, 9c	12
	1	+	+	−
	2	−	−	−
	3	+	+	+
	4	+	+	+
	5	−	−	+
	6	−	−	−
	7	+	+	+
	8	−	−	−
	9	+	+	−
	10	+	+	−
	11	+	+	−
	12	+	+	+
	13	+	+	−
	14	+	+	−
	15	+	+	−
	16	+	+	−
	17	−	−	−
	18	−	−	+
	19	+	+	+
	20	+	+	−
	21	+	+	−
	22	+	+	+
	23	+	+	+
	24	+	+	−
	25	+	+	−
	26	+	+	+
	27	+	+	−
	28	+	+	−
	29	−	−	−
	30	−	−	−
	31	−	−	−
	32	+	+	−
	33	−	−	−
	34	+	+	+
	35	+	+	+

	TABLE VI
	Peptide
	Serum	2d	2f	4a	6b	6c, 6d	9c	11
	1′	+	−	+	+	+	−	−
	2	+	−	+	−	−	−	−
	3′	−	−	−	+	+	+	−
	4′	+	−	+	+	+	−	−
	5′	−	+	+	+	+	−	−
	6′	−	−	−	−	−	−	−
	7′	−	−	−	−	−	−	−
	8′	−	−	−	−	−	−	−
	9′	+	+	+	+	+	+	+
	10′	−	−	−	−	−	−	−
	11′	+	+	+	+	+	−	−
	12′	−	−	−	+	+	+	+

	TABLE VII
	Peptide
Serum	2h	4b	4c	6a
1″	+	−	−	+
2″	−	−	−	+
3″	−	+	+	−

32 18 amino acids amino acids linear peptide 1Ser Gly Lys Pro Ala Ile Ile Pro Asp Arg Gly Val Leu Tyr Arg Glu 5 10 15Phe Asp 33 amino acids amino acids linear peptide 2Glu Cys Ser Gln His Leu Pro Tyr Ile Glu Gln Gly Met Met Leu Ala 5 10 15Glu Gln Phe Lys Gln Lys Ala Leu Gly Leu Leu Gln Thr Ala Ser Arg 20 25 30Gln 15 amino acids amino acids linear peptide 3Glu Cys Ser Gln His Leu Pro Tyr Ile Glu Gln Gly Met Met Leu 5 10 15 18 amino acids amino acids linear peptide 4Met Met Leu Ala Glu Gln Phe Lys Gln Lys Ala Leu Gly Leu Leu Gln 5 10 15Thr Ala 21 amino acids amino acids linear peptide 5Met Met Leu Ala Glu Gln Phe Lys Gln Lys Ala Leu Gly Leu Leu Gln 5 10 15Thr Ala Ser Arg Gln 20 6 amino acids amino acid linear peptide 6His Leu Pro Tyr Ile Glu 5 9 amino acids amino acid linear peptide 7Ser Gln His Leu Pro Tyr Ile Glu Gln 5 7 amino acids amino acid linear peptide 8Lys Ala Leu Gly Leu Leu Gln 5 9 amino acids amino acid linear peptide 9Gln Lys Ala Leu Gly Leu Leu Gln Thr 5 10 amino acids amino acid linear peptide 10Gln Lys Ala Leu Gly Leu Leu Gln Thr Ala 5 10 19 amino acids amino acid linear peptide 11Ala Val Gln Thr Asn Trp Gln Lys Leu Glu Thr Phe Trp Ala Lys His 5 10 15Met Trp Asn 15 amino acids amino acid linear peptide 12Asn Pro Lys Pro Gln Lys Lys Asn Lys Arg Asn Thr Asn Arg Arg 5 10 15 9 amino acids amino acid linear peptide 13Lys Asn Lys Arg Asn Thr Asn Arg Arg 5 10 amino acids amino acid linear peptide 14Gln Lys Lys Asn Lys Arg Asn Thr Asn Arg 5 10 15 amino acids amino acid linear peptide 15Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg 5 10 15 16 amino acids amino acid linear peptide 16Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly Gly Val 5 10 15 12 amino acids amino acid linear peptide 17Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile 5 10 9 amino acids amino acid linear peptide 18Lys Phe Pro Gly Gly Gly Gln Ile Phe 5 12 amino acids amino acid linear peptide 19Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val 5 10 9 amino acids amino acid linear peptide 20Lys Phe Pro Gly Gly Gly Gln Ile Val 5 9 amino acids amino acid linear peptide 21Gln Asp Val Lys Phe Pro Gly Gly Gly 5 15 amino acids amino acid linear peptide 22Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro Arg Arg 5 10 15 47 amino acids amino acid linear peptide 23Gln Leu Phe Thr Phe Ser Pro Arg Arg His Trp Thr Thr Gln Gly Cys 5 10 15Asn Cys Ser Ile Tyr Pro Gly His Ile Thr Gly His Arg Met Ala Trp 20 25 30Asp Met Met Met Asn Trp Ser Pro Thr Thr Ala Leu Val Met Ala 35 40 45 18 amino acids amino acid linear peptide 24Met Ala Trp Asp Met Met Met Asn Trp Ser Pro Thr Thr Ala Leu Val 5 10 15Met Ala 45 amino acids amino acid linear peptide 25Arg Gly Pro Arg Leu Gly Val Arg Ala Thr Arg Lys Thr Ser Glu Arg 5 10 15Ser Gln Pro Arg Gly Arg Arg Gln Pro Ile Pro Lys Ala Arg Arg Pro 20 25 30Glu Gly Arg Thr Trp Ala Gln Pro Gly Tyr Pro Trp Pro 35 40 45 21 amino acids amino acid linear peptide 26Arg Gly Pro Arg Leu Gly Val Arg Ala Thr Arg Lys Thr Ser Glu Arg 5 10 15Ser Gln Pro Arg Gly 20 20 amino acids amino acid linear peptide 27Ser Gln Pro Arg Gly Arg Arg Gln Pro Ile Pro Lys Ala Arg Arg Pro 5 10 15Glu Gly Arg Thr 20 15 amino acids amino acid linear peptide 28Lys Ala Arg Arg Pro Glu Gly Arg Thr Trp Ala Gln Pro Gly Tyr 5 10 15 11 amino acids amino acid linear peptide 29Gln Lys Lys Ala Ala Arg Asn Thr Asn Arg Arg 5 10 15 amino acids amino acid linear peptide 30His Trp Thr Thr Gln Gly Ser Asn Ser Ser Ile Tyr Pro Gly His 5 10 15 18 amino acids amino acid linear peptide 31Ser Ser Ile Tyr Pro Gly His Ile Thr Gly His Arg Met Ala Trp Asp 5 10 15Met Met 16 amino acids amino acid linear peptide 32Pro Glu Gly Arg Thr Trp Ala Gln Pro Gly Tyr Pro Trp Pro Leu Tyr 5 10 15

Claims

1. An HCV peptide antigen consisting of SEQ ID NO: 1 and optionally bound directly or indirectly to a label or solid support.

2. A method for determining the presence of HCV specific antibody in a sample, comprising the steps of:incubating the sample with an HCV peptide antigen according to claim 1, under conditions favoring the binding of any HCV antibody in said sample to said HCV peptide antigen, and determining said binding in the incubated sample as an indication of the presence of HCV specific antibody.

3. A protein complex comprising an HCV peptide antigen according to claim 1, coupled to a non-HCV peptide.

4. The protein complex according to claim 3, wherein said protein complex is biotinylated.