DE19549390C2 - Spliced peptide compositions for the diagnosis and detection of hepatitis C virus (HCV) infections - Google Patents

Description

The invention relates to new spliced peptide compositions for blocking the non- specific reactivity of certain NS-3 conformation epitopes from hepatitis C virus (HCV). The invention further relates to the use of these peptide compositions in Immunoassay kits for the detection of hepatitis C virus (HCV) infections.

HCV-induced non-A, non-B hepatitis (NANBH) is the most common form of post- Transfusion hepatitis. Therefore, there is an urgent need for sensitive and specific diagnostic detection methods to be among potential blood donors and others Identify people carrying the virus who could transmit the disease. It Therefore, accurate detection procedures are required to identify infected blood and blood products To be able to separate donor blood with a high degree of certainty.

The etiological agent HCV was cloned and identified by several groups; see. Houghton et al., EP-B 0 318 216, Okamoto et al. (1990) JPn. J. Exp. Med. 60: 167, Houghton et al., EP-A 0 388 232, and Kato et al. (1990) Proc. Natl. Acad. Sci. USA 87: 9524; Arima et al. (1989a) Gastroenterologia Japonica 24: 540; Reyes et al. (1990) Science 247: 1335; Arima et al. (1989b) Gastroenterologia Japonica 24: 545; Maeno et al. (1990) Nucleic Acids Res. 18: 2685.

The HCV genome is approximately 10 kilobases (kb) long and encodes a single one Polyprotein, which is processed into structural proteins and non-structural proteins. Outgoing from the amino terminus, the polyprotein closes the capsid and envelope proteins of the structural region and the NS-1 to NS-5 proteins of the non-structural region.

Many of the antigenic areas of HCV have been identified. Peptides and recombinant  Proteins from these areas show different degrees of sensitivity and Selectivity in the detection and diagnosis of HCV carriers. It was about antigens Areas reported in every major HCV protein. An overview of many identified The German gives antigenic areas and some peptides suitable for the detection of HCV Patent application (09.01.1995) DE 195 00 394.2-41. Recently about additional peptides and / or antigenic sites in the core protein have been reported; Core residues 1-84 and 9-177; U.S. PS 5,350,671 and in NS-3 protein (PCT / US94 / 07088; WO93 / 09253; EP-A 0 388 232). Hosein et al. identified an NS-3 conformational epitope at residues 1378-1459; see. PCT / US94 / 07088. Note: The amino acid numbering system used here corresponds to that numbering system used for the HCV-1 polyprotein.

For mapping antigenic determinants (epitopes) within certain antigenic areas of HCV serological analyzes were performed; see. Wang (1992), U.S. Patent 5,106,726; PCT / US93 / 08638, PCT / US94 / 07088 and German patent application DE 195 00 394.2-41. At These mapping studies have used synthetic peptides to do well to examine characterized HCV serum samples and they allowed the identification of particularly immunoreactive antigenic areas of HCV as well as the identification of some Mixtures of peptides that are suitable for the detection of HCV antibodies. However, the Tests using these peptides are not always known HCV antibody-containing sera after. In addition, such tests occasionally give false positive results for the HCV Antibody detection (does not require donor blood to be removed from blood banks).

The present invention overcomes many of these disadvantages by providing spliced ones Peptide compositions that associate non-specific immunoreactivity with certain Block NS-3 conformation epitopes.

The invention relates to a peptide composition comprising a linear "spliced" peptide, its conjugates and its polymers, which determine the non-specific reactivity of certain NS Block 3 conformation epitopes in HCV immunoassays, whereby the C-terminal amino acid of Peptide contains a COOH or CONH₂ group, and the spliced peptide one Amino acid sequence comprises selected from the group consisting of SEQ ID NOS: 48-50 or an analog of the spliced peptide is with an amino acid sequence of a strain / isolate of HCV from the corresponding NS-3 regions of the spliced peptide (Table 8). Just like that the spliced peptides can be modified to substitute, delete and degenerate  Contain positions and include the preferred peptides of SEQ ID NO: 51 and Formula X a. The invention further relates to a spliced peptide composition in which the polymerized spliced peptide by the general formula

(Peptide) ₂X
(Peptide) ₄X₂X
(Peptide) ₈X₄X₂X
(Peptide) ₁₆X₈X₄X₂X

is shown in which X is an amino acid or an amino acid analogue with two Is amino groups and a carboxyl group, each group capable of a peptide bond form.

The invention further relates to the use of a kit for the detection of HCV antibodies or for the diagnosis of HCV infections, comprising a first peptide composition, comprising at least one HCV peptide or HCV protein with an NS-3 conformation epitope and an effective amount of a second spliced peptide composition as above described. The kit is preferably an ELISA assay kit, a sandwich assay or a PHA Assay kit. In a particular embodiment, the HCV peptide, its conjugates and its polymers, selected from the group consisting of: (i) a peptide 4 (SEQ ID NO: 4), (ii) a peptide analog having an amino acid sequence of a strain / isolate of HCV in one Area corresponding to peptide 4, (iii) a linear peptide, the carboxy terminus of Peptide is a -COOH or -CONH₂ group, the peptide being specifically immunoreactive with Hepatitis C virus (HCV) antibodies, and the 81 amino acid peptide Prototype sequence of SEQ ID NO: 4 corresponds, is immunoreactive with HCV-NS-3 antibodies and contains the following substituted or degenerate positions: position 3: norvaline, Position 7: norvaline or Val: Ala: Thr: Phe: Tyr: Nva in a ratio of 3: 3: 1: 1: 1: 1, position 8: valine or norvaline, position 16: norvaline, position 20: arginine, position 26: norleucine, position 33: Norleucine, position 39: serine, position 46: norvaline, position 52: alanine, position 60: serine, Position 63: norleucine, position 68: serine and position 74: norvaline, and (iv) one of the peptides 5-21 (SEQ ID NOS: 5-21). The invention further relates to the use of the HCV peptide in which the polymerized HCV peptide by the general formula

(Peptide) ₂X
(Peptide) ₄X₂X
(Peptide) ₈X₄X₂X
(Peptide) ₁₆X₈X₄X₂X

is shown in which X is an amino acid or an amino acid analogue with two Is amino groups and a carboxyl group, each group capable of a peptide bond form.

In a preferred embodiment, the first peptide composition is mixture A, B, C, D or E. Furthermore, the invention relates to the use of an ELISA test kit for Detection of HCV antibodies or to diagnose HCV infection with a solid phase, coated with a peptide composition comprising mixture D or E and one such Spliced peptide compositions described above in soluble form in one Sample diluent. In a further embodiment, the kit also contains (c) one negative control, (d) a positive control, and (e) antibodies to human IgG antibodies, these antibodies being labeled with a reporter molecule. In preferred embodiments, the spliced peptide composition comprises the spliced Peptide SEQ ID NO: 48 or 51.

Fig. 1 shows the immunoreactivity of a dilution series of human monoclonal antibody (B12.F8; Cerino (1993), J. Immunol 151: 7005-7015.), Which is specific for the HCV core protein, ((▲) peptide mixture A Example 5), (⚫) peptide mixture E (Example 5) and (○) a commercially available HCV antibody detection kit (Ortho HCV 3.0; Ortho Diagnostic Systems, Inc.).

FIG. 2 shows the immunoreactivity shows a dilution series of human monoclonal antibody (60H9 [9] D10E6; Cerino (1991), J. Immunol. 147: 2692- 2696), the specific NS-4 HCV protein for the (▲) Peptide mixture A (example 5), (⚫) peptide mixture E (example 5) and (○) a commercially available HCV antibody detection kit (Ortho HCV 3.0).

Figure 3 shows the immunoreactivity of a dilution series of a human monoclonal antibody (CM3.B6; Mondelli (1994), J. Virol. 68: 4829-4836),  which is specific for the HCV-NS-3 protein, with (▲) peptide mixture A (example 5), (⚫) peptide mixture E (Example 5) and (○) and a commercially available HCV antibody detection kit (Ortho HCV 3.0).

According to the invention, intensive serological analyzes have one refinement and one advanced definition of immunoreactive peptides, which are used for the detection of HCV Antibodies and for the diagnosis of HCV infections. Surprisingly found that synthetic peptides that are multiple in a prototype HCV sequence Contain substitutions with natural (usual) and non-natural (unusual) amino acids, are extremely effective for the detection and diagnosis of HCV infections. This multiple times Substituted peptides can also contain degenerate positions with sequence variability to provide high selectivity and sensitivity for a variety of HCV strains.

A position in a prototype sequence in which an amino acid residue is replaced by another residue is substituted is referred to as a "substituted position". Similarly, a position in a prototype sequence in which there is no amino acid residue as a "deleted position" designated. Finally, a position in a prototype sequence, in the course of Peptide synthesis is a single amino acid residue through a mixture of two or more Amino acid residues were replaced (as in the case of the peptide libraries below) as "degenerate position".

The spliced peptides of the invention can be linear or branched. The invention closes Polymers, conjugates and mixtures of the peptides.

The invention relates to a peptide composition comprising a spliced peptide which non-specific immunoreactivity of certain NS-3 conformation epitopes in HCV Immunoassays blocked. Generally the spliced peptides are in aqueous buffer solutions soluble.

As a linear peptide, the spliced peptide consists of an amino acid sequence selected from the group SEQ ID NOS: 48-50 (Table 8) or an amino acid sequence of a strain / isolate of HCV from the corresponding NS-3 regions of one of these peptides. This group of Peptides are called "spliced" peptides because each of these peptides has a large internal one  Exhibits deletion in the NS-3 region. Therefore, the peptides have a deletion of the amino acids 33 to 57 of SEQ ID NO: 4. Since residues 31 to 32 and 58 to 59 are the same (val- Ala), these residues occur only once at the splice point. A preferred spliced peptide of the invention is the peptide SEQ ID NO: 48.

Serological analyzes of this region have shown that the spliced peptide is multiple Substitutions of natural and unnatural amino acids in the prototype HCV sequence can have and yet the effectiveness of blocking the non-specific reactivity of the NS-3 conformation epitope remains high. Therefore, the spliced peptides can be a specific one Pattern of substitutions, deletions or degenerate positions relative to the following Have prototype sequence:

Thus the spliced peptide can have the formula X. Peptides of Formula X are those peptides that block the non-specific reactivity of NS-3 conformation epitopes in HCV immunoassays that essentially maintain the reading frame of the 35 amino acid prototype sequence of SEQ ID NO: 48 and that contain the following substituted positions :
Position 7: norleucine, position 17: norleucine, position 24: glutamic acid and position 28: norvaline.

In a preferred embodiment, the spliced peptide is the peptide SEQ ID NO: 51.

The prototype, its analogs, or modified spliced peptides can be used with a carrier are connected or polymerized as described below. If the peptide is in polymerized in a branched form, it can be represented by the following formulas will.

(Peptide) ₂X
(Peptide) ₄X₂X
(Peptide) ₈X₄X₂X
(Peptide) ₁₆X₈X₄X₂X

in which X is the residue of an amino acid or an amino acid analogue with two amino groups and a carboxyl group, each group capable of forming a peptide bond. The linear part of the spliced peptides (modified or unmodified) has a length in that Range as described below for the other peptides according to the invention is. Preferably the spliced peptides are from about 30 to about 50 in length Amino acid residues. Similarly as described above, amino acids can be attached to both termini the spliced peptides are added (e.g. KKK to the amino terminus or the rest M) the carboxy terminus). Furthermore, strain dependent variations can be as follows described be balanced.

The spliced peptides (including both the prototype and its analogues or a modified peptide) are used in a method for the detection of HCV antibodies or for Diagnosis of HCV infections used in a patient as tracked: (i) provision a first peptide composition comprising one or more peptides or proteins an NS-3 conformation epitope, (ii) contacting an effective amount of these first Peptide composition with a serum, tissue, tissue extract or body fluid the patient in the presence or after pretreatment with an effective amount of spliced peptide composition of the invention in an immunoassay method for a sufficient time to complex between the first peptide composition and a Form antibodies in the serum, tissue, tissue extract or liquid, (iii) and determining the complex with a detection device. Preferably that is Immunoassay method an ELISA assay, a sandwich assay or a PHA assay.

The spliced peptide composition can conveniently be in the diluent to be provided. It is preferred in a concentration range of 5-100 µg / ml of about 50 µg / ml. The concentration in the spliced peptide, suitable for a certain assay format, can be easily determined by titration of the amount of spliced peptide in the Sample diluents are determined by determining the amount of peptide that is required to remove the non-specific absorption, d. H. the amount of peptide which no further increase in inhibition is achieved. The sufficient time for the reaction of the first peptide compositions with an HCV antibody in the sample is known or can  can be easily obtained by determining the time required for the reaction to complete becomes. The presence of the spliced peptide in the sample diluent or that Pretreatment of the sample with a spliced peptide composition changes the Response time is not essential.

For use according to the invention, the first peptide composition comprises in one preferred embodiment a peptide with an NS-3 conformation epitope selected from: (i) peptide 4 (SEQ ID NO: 4), (ii) a peptide analog having an amino acid sequence of one HCV strain / isolate in a region corresponding to peptide 4, (iii) a linear peptide, in which the carboxyl group at the carboxy terminus of the peptide is a COOH or CONH₂ Group is that is peptide-specific immunoreactive with hepatitis C virus (HCV) antibodies and the peptide is essentially the reading frame of the 81 amino acid prototype sequence of SEQ ID NO: 4 maintains, is immunoreactive with HCV NS-3 antibodies and the following substituted or degenerate positions contains:

Position 3: Norvalin, position 7: Norvalin or Val: Ala: Thr: Phe: Tyr: Nva in the ratio 3: 3: 1: 1: 1: 1, position 8: valine or norvaline, position 16: norvaline, position 20: arginine, Position 26: norleucine, position 33: norleucine, position 39: serine, position 46: norvaline, Position 52: alanine, position 60: serine, position 63: norleucine, position 68: serine and Position 74: norvaline and

(iv) any of peptides 5-21 (SEQ ID NOS: 5-21) shown in Table 13. Each of these Peptides can be conjugated or polymerized to a carrier. When the peptide polymerizes it is represented by the following formulas:

(Peptide) ₂X
(Peptide) ₄X₂X
(Peptide) ₈X₄X₂X
(Peptide) ₁₆X₈X₄X₂X

in which X is the residue of an amino acid or an amino acid analogue with two amino groups and a carboxyl group, each group capable of forming a peptide bond. Furthermore, the first peptide composition mixture A, mixture B, mixture C, mixture D or mixture E. The composition of mixtures A and B are shown in Example 5.  The composition of mixture C contains peptides 19, 25, 28 and 33.

Each of these peptides of the first peptide composition has a special pattern of Substitutions, deletions or degenerate positions in its peptide residue relative to one of the six prototype sequences given below.

Furthermore, some of the peptides contain degenerate positions, i.e. H. make these peptides "Libraries" of peptides; see. WO95 / 11998. ("Peptide libraries" are also called "Structured synthetic antigen libraries" or "SSAL".) At peptide libraries degenerate amino acid positions those positions of known sequence variability as they due to strain-dependent differences in different HCV isolates. At the degenerate positions are thus either of two or more Amino acid residues. The residues which are possible for a given position are known on the basis of Sequence variations that occur at this position within the sequence of the peptide are determined. The ratio of the amino acids used in the synthesis for a particular position can are represented either by the frequency with which the given residues in the known Variants occur, or by a simple equimolar distribution of the possible amino acids at this position. Not every need for the present use according to the invention Sequence position that can be subject to a virus strain-dependent variation is taken into account will. A linear peptide used in the present invention can be from about 30 to about 100 Contain amino acid residues, preferably about 40 to about 85 amino acid residues.

The peptides used according to the invention can also have some further amino acids, including non-natural (unusual) amino acids, which are bound to the terminal amino acids. For example, the sequence KKK (lysine-lysine-lysine) can be bound to the amino terminus of each of the peptides used according to the invention. In the case of branched peptides, a residue M (methionine) can be bound to the carboxy terminus of the peptide residue, ie between the peptide residue and the branch. Likewise, the peptides can carry a cysteine residue at the carboxy terminus to utilize the thiol group of the cysteine to form a covalent bond to an electrophilic group, e.g. B. to an N ^α- chloroacetyl-modified amino acid or a maleimide-derivatized α or ε-amino group of a lysine residue which is bound to the amino terminus of another peptide.

As is known, HCV is subject to frequent mutations. There are several variants of Strains / isolates known, e.g. B. PT, J, J1 and J4; see. Houghton (1989), Okamoto (1990), Houghton (1990) and Kato (1990), supra. It is expected that more Virus strain variants exist; see. Bukh et al., Proc. Natl. Acad. Sci. USA 90: 8234-8238 (1993). Adaptations of the prescribed sequences to those resulting from stem variations  conservative substitutions can be made provided the pattern of the substituted, deleted or degenerate positions are maintained in a given peptide. To this In this way, the peptides used according to the invention can be modified by changing their antigenicity do not affect, strain-dependent variations that balance within different HCV isolates exist.

Thus, the immunoreactivity of the invention is due to the feasible changes Peptides used with HCV antibodies retained the pattern of the substituted, deleted or degenerate position. The changes can be made in Substitutions, insertions, deletions or degenerations at about 2 to about 5 positions or alternatively exist in up to about 10% of the positions in a peptide of Tables 1, 3, 5 and 8. In addition, the feasible changes are derived from known HCV strains.

The peptides used according to the invention are synthesized and against a selection of HCV sera (HCV serum panel) tested to determine how Determine immunoreactivity of the peptides.

The peptides used according to the invention can also be used to form conjugates become, d. H. the peptides can be directly or indirectly in a manner known per se Carrier proteins such as bovine serum albumin (BSA), human serum albumin (HSA) or an Erythrocytes or latex particles are bound.

The term "natural" or "common" amino acids denotes the 20 amino acids that usually found in proteins, namely alanine, aspartic acid, asparagine, arginine, Cysteine, glycine, glutamine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, Phenylalanine, proline, serine, threonine, tyrosine, tryptophan and valine. The expression natural Amino acids include both the D and L forms.

The term "non-natural" or "unusual" amino acids includes both the D and the L-form of any other amino acid, whether it is within a protein or otherwise occurs in nature, or whether it is produced synthetically. Examples of non- natural amino acids include β-alanine, ornithine, norleucine, norvaline, Hydroxyproline, thyroxine, gamma-aminobutyric acid, homoserine and citrulline.  

The linear peptides used according to the invention have the general formula

[Peptide] -Y

in which Y represents a COOH or CONH₂ group. The linear peptides include mixtures, Conjugates and polymers. The peptides include at least one antigenic determinant that is specifically immunoreactive with antibodies to HCV.

The branched peptides used according to the invention have one of the following general formulas:

(Peptide) ₂X
(Peptide) ₄X₂X
(Peptide) ₈X₄X₂x
(Peptide) ₁₆X₈X₄X₂X

in which X is the residue of an amino acid or an amino acid analogue with two amino groups and a carboxyl group, each group capable of forming a peptide bond. Preferably X is a lysine residue or the residue of a lysine analog such as ornithine. The Amino acid analogue can be an α-amino acid, a β-amino acid or any other natural one or non-natural amino acid with two amino groups and one carboxyl group, which are used for Formation of peptide bonds are available. Preferred branched peptides of the invention are Dimers, tetramers and octamers, in particular those peptides that form the core structure of the Branching have a lysine, d. H. in which the residue X is a lysine residue. Branched dimers and octamers are particularly preferred.

The length of the peptide residue of the linear or branched peptides can be about 30 vary to about 100 amino acid residues. The preferred peptides used in the invention contain about 30 to about 60 amino acid residues.

The peptide compositions used according to the invention also relate to one or more the peptides of the formulas V-IX.  

Peptides of the formula V are those which essentially fall within the framework of the 52 amino acids Preserve prototype sequence of residues 1 to 52 of SEQ ID NO: 3, which with HCV core Antibodies are immunoreactive and contain the following substituted positions:

Position 8: ornithine or lysine, position 16: ornithine or lysine, position 24: Hydroxyproline, position 29: norvaline, position 37: hydroxyproline, position 43: norleucine and position 45: norvaline or leucine.

The peptides of formula V can also be one or more of the following substituted or degenerate positions include:

Position 4: Pro: Gly in the ratio 9: 1, position 10: Thr: Asn in the ratio 7: 3, position 21: norvaline, position 30: norvaline, position 36: valine or norleucine, position 48: Thr: Pro in the ratio 9: 1 and position 52: threonine or Thr: Ala: Glu: Lys in the ratio 1: 1: 1: 1.

The peptides of the formula VI are those peptides which essentially fall within the scope of 47 Protect amino acid prototype sequence from SEQ ID NO: 2 with HCV NS-4 antibodies are immunoreactive and contain the following substituted positions:

Position 1: asparagine, position 2: glutamine, position 3: arginine, position 4: Hydroxyproline, position 5: serine or threonine, position 12: norvaline or isoleucine, Position 15: ornithine, position 22: aspartic acid, position 27: norvaline, position 31: Aspartic acid, position 39: asparagine and position 45: norvaline or valine.

The peptides of formula VI can also be one or more of the following substituted or degenerate positions include:

Position 6: Ile: Val in a ratio of 6: 4, position 7: Ile: Val: Ala in a ratio of 6: 2: 2, position 10: Arg: Lys in the ratio 8: 2, position 16: Glu: Ala in the ratio 8: 2, position 19: Aspartic acid, position 24: Ser: Ala in the ratio 4: 6, position 25: Gln: Ser in the ratio 4: 6, position 26: His: Lys: Arg in the ratio 8: 1: 1, position 28: Pro: Ala in the ratio 8: 2, Position 29: Tyr: Leu in a ratio of 8: 2, Position 30: Norleucin or Norvalin, Position 32: Gln: Glu in the ratio 8: 2, position 34: Met: Gln in the ratio 8: 2, position 35:  Met: Gln: Arg in the ratio 4: 4: 2, position 36: Leu: Met: Ile in the ratio 8: 1: 1, position 40: Phe: Leu in the ratio 8: 2, position 42: Gln: Ser in the ratio 8: 2 and position 44: Ala: Ile in the ratio 8: 2.

The peptides of formula VII are those peptides which essentially fall within the scope of 44 Protect amino acid prototype sequence from SEQ ID NO: 1 with HCV NS-5 antibodies are immunoreactive and contain the following substituted positions:

Position 2: ornithine, position 10: norvaline, position 17: glutamic acid, position 23: Norvaline, position 31: hydroxyproline and position 37: hydroxyproline.

The peptides of formula VII can also degenerate one or more of the following Items include:

Position 8: Pro: Leu: Val in the ratio 8: 1: 1, Position 12: Thr: Ser: Pro in the ratio 5: 4: 1, Position 15: Lys: Asp: Arg in the ratio 4: 4: 2, position 19: Glu: Val: Gln in the ratio 5: 4: 1, position 21: Pro: Ala in a ratio of 9: 1, position 22: Val: Thr in a ratio of 8: 2, Position 24: His: Leu: Ala in the ratio 8: 1: 1, Position 27: Pro: Ala in the ratio 8: 2, Position 30: Pro: Ser in a ratio of 9: 1, Position 32: Lys: Pro: Arg in a ratio of 8: 1: 1, Position 33: Ser: Ala: Lys: Gln in the ratio 4: 4: 1: 1, position 34: Pro: Thr in the ratio 8: 2, position 36: Val: Ile: Thr in the ratio 5: 4: 1, position 41: Lys: Arg in the ratio 4: 6, Position 42: Lys: Arg in the ratio 7: 3 and position 44: Thr: Ala in the ratio 9: 1.

The peptides of the formula VIII are those peptides which essentially fall within the scope of the 40th Protect amino acid prototype sequence from SEQ ID NOS: 35, those with HCV-NS-4- Antibodies are immunoreactive and contain the following substituted positions:

Position 8: norleucine, position 13: ornithine, position 20: hydroxyproline, position 28: Arginine and position 35: ornithine.

Peptides of the formula IX are those which essentially fall within the framework of the 55 amino acids Prototype sequence of SEQ ID NO: 36 maintained with HCV NS-5 antibodies are immunoreactive and contain the following substituted positions:

Position 8: ornithine, position 16: norvaline, position 24: asparagine, position 32: ornithine, Position 40: norvaline and position 48: norleucine.

The peptides of the formulas V-IX can be linear or branched and those according to the invention Use includes polymers, conjugates and mixtures of these peptides according to the here described embodiments.

The term "HCV NS-3 antibody" includes an antibody, or a serum or one Plasma sample containing antibodies that are immunoreactive with peptide 4 (SEQ ID NO: 4) or bind to it. The term "HCV core protein antibody" includes an antibody or Serum or a plasma sample containing antibodies that are linked to peptide 3, e.g. B. VIIIE (SEQ ID NO: 3) are immunoreactive or bind to it. The term "HCV NS-4 antibody" includes an antibody, or a serum, or a plasma sample containing antibodies associated with Peptide 2, i.e. H. IIH (SEQ ID NO: 2) are immunoreactive or bind to it. The expression "HCV- NS-5 antibody "includes an antibody or a serum or plasma sample that Contains antibodies that are immunoreactive with or bind to peptide 1 (SEQ ID NO: 1).

The peptide compositions used according to the invention can consist of one or more Peptides. These compositions preferably contain 1 to 10 Peptides and particularly preferably 1 to 4 peptides and particularly preferably 1 to 6 peptides.

In addition, the peptide compositions used according to the invention contain mixtures of these peptides. The effective one for the diagnosis of HCV or for the detection of HCV antibodies Ratio of peptides in peptide compositions, the mixtures of the invention Containing peptides can be easily determined. In general, these relationships are in the Range from about 1 to about 50 by weight of peptide. Preferred Peptide compositions for the diagnosis and detection of HCV infections are the Mixtures A to E. Mixture A contains peptides 25, 29, 33 and 19, mixture B contains peptides 25, 27-octamer, 33 and 19, mixture C the peptides 19, 25, 28 and 33, mixture D the peptides 19, 33, 38, 39, 46 and 47 and mixture E the peptides 19, 37, 43, 45, 46 and 47. A preferred one Embodiment provides the mixture E with a weight ratio of 1.5: 1: 1: 0.5: 1: 2 for the Peptides 43, 46, 37, 45, 47 and 19.

The peptide compositions, peptides and mixtures described above are for  Detection of HCV antibodies in body fluids and for the diagnosis of HCV infections suitable. To determine the effectiveness of the peptides according to the invention Detection of HCV antibodies will test the peptides for their immunoreactivity with samples previously tested by screening thousands of patient and normal sera Immunoreactivity with HCV have been selected. Such HCV-specific serum panels are commercially available and examples of such serum panels and methods are available suitable panels are given here in the examples.

The strategy for serological confirmation of effectiveness depends on the expected Properties of the antigenic determinants to be examined. For example, that Screening in universal immunodominant areas, such as the gp41 transmembrane peptide of HIV-1, by means of a single representative serum sample from a patient who was treated with the Virus is infected. In antigenic areas, not in all infected patients must be recognized, or for which antibodies are formed late or only transiently, must be large Serum panels can be used for screening. Both screening methods can are used according to the invention to carry out the immunological analysis of HCV by means of the Peptides according to the invention to improve significantly. However, the latter method is with Examination for superior selectivity and sensitivity of the peptides according to the invention particularly useful.

The identification of antigenic determinants also depends on the serum panels used. Each more precisely, the panel represents a population that is most likely related to one Determinant is seropositive, the greater the likelihood that the antigen Determinant can be identified and carefully mapped. To expand the Reactivity spectrum of an assay that already identified antigenic determinants or Epitopes should therefore be used to screen a large number of samples from patients which may be infected but are seronegative to known ones Are antigens or epitopes.

The process of "serological confirmation" is particularly difficult if that too identifying antigenic determinant only in a subpopulation of an infected one Patient group triggers antibodies. If such antigenic areas aim for detection must be paid special attention to synthetic peptides, the very weak reactivity demonstrate.  

In this regard, the low background absorption of synthetic peptides allows especially peptides with non-natural amino acids, the exact detection weaker Reactivity. In some cases, absorptions of 50 mA (mA = milliabsorption) are opposed Background reading is sufficiently significant and can be gradually improved Amino acid sequence of a peptide lead to the identification of important antigenic determinants. With a sufficient laboratory routine, consistent and reliable results can also be achieved obtained when working in the range of absorptions below 200-300 mA.

The peptides can be prepared in a manner known per se, e.g. B. by the Merrifield method; see. J. Am. Chem. Soc. 85 (1963), 2149-2154 and the variety of improvements available this technique, cf. Synthetic Peptides: A User’s Guide, Grant, Ed. (1992), W.H. Freeman & Co., New York; Jones (1994) The Chemical Synthesis of Peptides, Clarendon Press, Oxford.

Another difficulty that arises when using the Peptides in Compared to recombinantly expressed proteins can be kept to a minimum, is high number of false positive results due to the presence of antigenic material which is produced together with the recombinant proteins derived from HCV is cleaned. For example, certain normal patients have antibodies against Escherichia coli or yeast proteins with antigenic material of the expression systems cross-react as used for diagnostic tests based on recombinant proteins will. Sera from such normal patients can have a false positive response in such Show immunoassays. Such false reactions are in the inventive Immunoassays excluded.

In addition, the peptide compositions according to the invention become synthetic manufactured so that the product quality can be easily controlled. The is accordingly Reproducibility of the test results ensured. Furthermore, for each test are only very small amounts of peptide are required and the cost of making the peptides is relative low. As a result, the cost of screening body fluids for antibodies against HCV and for the diagnosis of HCV infections relatively low. Another advantage is Using peptide libraries the broader reactivity spectrum with respect to a larger one To name the number of HCV strains. Degenerate positions of the peptide libraries set in "Open" diagnostic tool that shows the strain-specific sequence variations (and the  resulting variation in antibody specificity), which results from the large number of HCV Tribes gives, balances. Thus peptide libraries increase the ability of the antigenic peptides for reactivity with antibodies specific for a wider range of HCV variants are.

The peptides and peptide compositions according to the invention can in a kit for Detection of HCV antibodies and for the diagnosis of HCV infections in body fluids from mammals, e.g. B. serum, tissue extracts, tissue fluids, in vitro cell culture supernatants and cell lysates can be used as test reagents in an immunoassay. Anyone can do it suitable immunoassay can be used, especially an enzyme-bound Immunoabsorption assay (enzyme-linked immunoadsorbent assay; ELISA), an enzyme Immunodotassay, a passive hemagglutination assay (e.g. a PHA test), an antibody Peptide-Antibody-Sandwich-Assay, a Peptide-Antibody-Peptide-Sandwich-Assay or others known. Such methods are known and described in numerous standard works, e.g. B. Harlow et al., (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. In a preferred embodiment, the spliced peptides and using the peptide compositions in an ELISA test kit comprising (a) a solid one Phase coated with mixture D or E and (b) a sample diluent comprising one of the spliced peptide compositions according to the invention. In another In one embodiment, the kit also contains (c) a negative control sample, (d) a positive Control sample and (e) antibodies against human IgG antibodies, these antibodies with are labeled with a reporter molecule. In a preferred embodiment comprises the first peptide composition is mixture E and the spliced peptide composition is spliced peptide 48 or 51.

The immunoassays are used to screen body fluids and tissues for the Presence of HCV-reactive antibodies used. They make the diagnosis easier for the doctor of HCV infections. Examples of body fluids that can be examined include Blood and blood fractions, such as plasma and serum, saliva or any other liquid that May contain antibodies to HCV.

The test kit is used, for example, as follows. A sample to be examined, e.g. Legs A mammalian body fluid, optionally diluted with sample diluent for a period of time and under such conditions with the peptide in the container  Brought into contact, which are sufficient so that possibly existing antibodies can bind. After separation of unbound material, e.g. B. by washing with sterile phosphate buffered saline, the secondary complex with labeled antibodies against human IgG antibodies contacted. These antibodies bind to the secondary Complex to form a tertiary complex. Since the second antibody with a Reporter molecule are marked, the tertiary complex can be added when detection agent is added to prove. The reporter molecule can be an enzyme, a radioisotope, a fluorophore bioluminescent molecule, a chemiluminescent molecule, bioton, avidin, streptavidin or the like. For the ELISA test, the reporter molecule is preferably an enzyme.

The following examples serve to illustrate the invention and are not intended to be limiting understand.

example 1 ELISA test procedure

The wells of a microtiter plate (96-well plate) were separated for 1 hour at 37 ° C with 5 µg / ml peptide using 100 µl per well in 10 mM NaHCO₃ buffer, pH 9.5 coated unless otherwise stated. The peptide-coated cells were covered with Incubated 250 µl of a 3% by weight gelatin solution in PBS for 1 hour at 37 ° C block nonspecific protein binding sites. Then three times with PBS washed, which contains 0.05% by volume of TWEEN 20, and dried. The test samples, the HCV Antibody-positive patient sera were 1:20 in volume ratio with PBS diluted, the 20 vol .-% normal goat serum, 1 wt .-% gelatin and 0.05 vol .-% TWEEN 20 contains. 100 ul of the diluted samples were placed in each of the wells and 30 minutes incubated at 37 ° C.

The cells were then washed six times with 0.05% by volume TWEEN 20 solution in PBS washed to separate unbound antibodies. Horseradish-peroxidase-conjugated Goat anti-human IgG antibodies were used as a secondary antibody tracer to the binds the HCV-antibody-peptide-antigen complex in the positive wells. 100 µl of  Peroxidase-labeled goat anti Human IgG antibody in a dilution of 1: 1800 in 1% by volume normal Goat serum, 0.05 vol% TWEEN 20 solution in PBS was added to each Wells were added and incubated for a further 15 minutes at 37 ° C.

The wells were then six times with 0.05% by volume TWEEN 20 solution washed in PBS to remove unbound antibody, and then washed with Treated 100 ul of the substrate mixture, the 0.04 wt .-% o-phenylenediamine (OPD) and 0.12 vol.% Hydrogen peroxide in a sodium citrate buffer, pH 5.0, contains. This mixture of substrates was used for the detection of Peroxidase labeling used, whereby a colored product forms. The Reactions were stopped by adding 100 µl of 1.0 M H₂SO₄ and the absorption at 492 nm (A₄₉₂nm) measured.

Example 2 Immunoreactivity of nuclear protein-reactive peptides

The immunoreactivity of peptides 26 and 37 (Table 1) was compared with one group known HCV serum samples from HCV panels 2, 3 and a serological one Conversion sample (serol 1-5) determines the antibodies against the core protein according to the ELISA test according to Example 1 using a Contains peptide coating at 1 µg / ml. The results in Table 2 show that  peptide 37 reacts much more strongly with HCV core protein antibodies than that Peptide 26.

Example 3 Immunoreactivity of NS-4 reactive peptides

U.S. Patent 5,105,726 (Table 5) describes a rare serum sample (NAB-2-2) that preferably with the five amino-terminal residues of peptide 2 (SGKPA) responded. Although this sequence is part of an epitope that is important for Detection of samples with NS-4 reactivity, peptide 39 (Table 3) showed that the sequence contains SGKPT, non-specific immunoreactivity. This led to Identification of five false positive samples under 1000 randomly selected blood donor samples (Table 4).

The sequence SGKPT was found to be homologous to highly conserved A region in helicases (Gorbalenya et al. (1989), Nucleic Acid Res. 17: 4713-4730). The HCV helicase (in the NS-3 range) has the sequence SGKST, whereby the amino acids G and K are not variable among the HCV strains are.

Accordingly, several peptides were synthesized (peptides 42-44, Table 3) and for immunoreactivity with actual HCV positive samples analyzed as well as for the lack of non-specific immunoreactivity the false positive samples identified with peptide 39. The complete deletion of these five residues (peptide 44) eliminated the non-specific cross reaction in the false positive samples, but also decreased the reactivity with the known HCV-positive sample (Table 4). The modification of these five residues (Peptides 42 and 43, each having an amino-terminal sequence from NQRpS have resulted in peptides not using false positive samples are immunoreactive, but the strong immunoreactivity with the known HCV showed positive sample NAB-2-2 (Table 4).

Example 4 Immunoreactivity of NS-5 reactive peptides

The immunoreactivity of an NS-5 reactive peptide (peptide 45, Table 5) was determined with a panel of known HCV sera from HCV panel 3 according to the ELISA test Example 1 determined. The peptide coating was 2 µg / ml. The results show that peptide 45 has a comparable immunoreactivity as peptide 33 (Table 6).

Example 5 Detection of HCV antibodies by peptide mixtures

The peptide mixture A with the peptides 25, 29, 33 and 19, wherein the the latter peptide conjugated to BSA was in a weight ratio of 2: 2: 0.5: 8 (µg / ml) used for coating. This mixture was in an ELISA test according to Example 1 with peptide mixture B, which the Peptides 25, 27-octamer, 33 and 19 contained and in a weight ratio of 2: 2: 0.5: 8 (µg / ml) was used for coating, the the latter peptide 19 was conjugated to BSA.

Peptide 19 was synthesized using m-maleimidobenzoyl-N- hydroxysuccinimide ester (MBS) according to the manufacturer (Pierce Chemical Co.) or according to Kitagawa et al. (1976) J. Biochem. 79: 233-236 with Conjugated BSA.

To investigate the sensitivity and specificity of mixtures A and B the mixtures were tested with a specially selected serum panel, the Sera containing specific immunoreactivity to nuclear protein, NS 3, NS-4 or NS-5 areas of HCV showed. This panel was created by Screening of sera against peptide VIIIE (SEQ ID NO: 3) Identify core protein-specific sera against peptide IIH (SEQ ID NO: 2), to identify NS-4 specific sera against Pep11 (Wang, 1992, supra), to identify NS-5 specific sera and against peptide 4 (SEQ ID NO: 4), to identify NS-3 specific sera. The identified in this way Sera were then diluted with normal human sera to make one showed weak to moderate reactivity to these same peptides (i.e., to have an absorption at 492 nm in the range 0.3 to 2.0 had). The diluted samples then provided the special panel Investigation of detection sensitivity (Table 7), which is used to test the  Mixture A and B was used. Mixtures A and B had comparable ones Sensitivities (Table 7, top panel).

Mixture D contained peptides 19, 33, 38, 39, 46 and 47, the Peptide 19 was conjugated to BSA. Peptides 39, 46, 38, 33 and 47 were in a weight ratio of 1.5: 1: 1: 0.5: 1 (µg / ml) for 16 hours Room temperature, as described in Example 1, applied to ELISA plates. The peptide coating solution was aspirated from the well and through 100 µl of the peptide 19-BSA conjugate at a concentration of 2.0 µg / ml in Phosphate buffered saline, pH 7.2, replaced for 1 hour at 37 °. The Mix D tests were carried out according to Example 1.

Mix E contained peptides 19, 37, 43, 45, 46 and 47. Mix E was in applied to ELISA plates in the same way as mixture D using of peptides 43, 46, 37, 45 and 47 at a weight ratio of 1.5: 1: 1: 0.5: 1 (µg / ml). The Mix E tests were performed with 25 µg / ml peptide 51 im Sample diluent performed.

Mixtures D and E were on the same panel as Mixtures A and B tested. The results in the lower part of Table 7 show that the mixtures D and E as a whole had a sensitivity comparable to that of Mixtures A and B. In two samples, core protein 2 and core protein 3, showed mixtures D and E are more sensitive to these antibodies than Mixtures A and B.

Example 6 Mixtures of peptides

Mixture A (Example 5) and Mixture E (Example 5) were tested for detection sensitivity with a dilution series of human monoclonal antibodies which were specific for a) the core protein of HCV, B12.F8 (Cerino (1993), J. Immunol. 151 : 7005-7015) b) the NS-4 protein from HCV, 60H9 [9] D10E6 (Cerino (1991), J. Immunol. 147: 2692-2696) and c) the NS-3 protein conformation epitope, CM3 .B6 (Mondelli (1994) J. Virol. 68: 4829-4836). The same dilution series was tested in parallel with a known anti-HCV test kit (Ortho HCV 3.0), which is composed of a mixture of recombinant proteins from the region of the core protein and the NS-3, NS-4 and NS-5 proteins . The analytical sensitivity in the detection of the antibodies directed against the three areas mentioned above against all three areas was two to eight times higher when using peptide mixtures A and E than when mixing the recombinant proteins of the known kit, cf. Fig. 1-3. In addition, mixture E showed higher sensitivity to the detection of HCV antibodies than mixture A.

Example 7 Quantification of test efficiency in patient and donor populations

A total of 1034 samples used in the PCR analysis (Wang et al., 1992, Gastroenterology 103: 609) or according to the Chiron RIBA® HCV 2.0 Strip Immunoblot assay (Ortho Diagnostic Systems, Inc., Raritan, NJ) HCV positive with regard to mixture A in the ELISA according to Examples 1 and 5 tested. These samples were from NANBH patients with chronic or acute hepatitis, hemophiliacs, patients who have received transfusions several times, HCV-infected patients who showed seroconversion and blood donors who considered infected with HCV. All 1034 as positive confirmed samples were positive with mixture A at one in this test average signal-background ratio of 10.

A collection of 3154 randomly selected blood donor samples was collected in tested in the same way in the ELISA with the peptide mixture A. The specificity of the Tests in the blood donor population was greater than 99.5%. The middle signal Background ratio of the samples was 0.3. So they delivered as positive Samples confirmed a medium signal to background ratio that was 33 times higher was when that was obtained with the randomly selected blood donor samples Relationship.

A collection of 1395 randomly selected blood donor samples was included the mixture E, as described in Example 5, tested. The initial Response rate was 0.4%, which corresponds to a test specificity of 99.6%. The initially reactive samples were re-tested and all were determined to be negative (except marginally for 3 out of 4 samples). The result of 100% test Specificity shows the high specificity of this test format in one Donor population.  

Example 8 Immunoreactivity of another NS-4 reactive peptide

The immunoreactivity of peptides 35 (aka V, SEQ ID NO: 35) and 46 (Table 8) was determined with a panel of known HCV sera from HCV panel 3, showed the antibodies against the NS-4 protein in the ELISA test according to Example 1. The results are shown in Table 9. Peptide 46 showed reactivity comparable to that of the prototype HCV-1 strain peptide 35.

Example 9 Spliced peptides

Peptide 4, a control peptide with an NS-3 conformation epitope and spliced Peptides 48, 49 and 50 were applied to plates for ELISA tests as in Example 1 described. Peptides 48-50 showed little or moderate reactivity with the NS-3 conformational antibodies present in the HCV panel 3 Serum samples, shown by the significant decrease in absorption for the Most of the samples in Table 10. Peptide 48 remained the most immunoreactive to the linear epitope in this NS-3 region. About that in addition, the three peptides reacted with sera that were known to be false positive To give results with peptide 4 in HCV tests, as by the NYBC Samples were shown in Table 10. This showed its usefulness as a reagent to selectively remove the non-specific immunoreactivity that is associated is with the NS-3 conformation epitope of peptide 4.

Peptides 48 and 49 were used at a concentration of 50 µg / ml Given sample dilution and with known HCV-positive sera and known HCV false positive sera as described in Example 1 below Tested using ELISA plates coated with peptide 4. Table 11 shows that the immunoreactivity of the true positive samples (HCV- Panel 3 samples) was inhibited over a range of -26% to 23%. The Most of the samples showed a maximum of 15% inhibition and several Samples showed increased absorption (which increased Detection sensitivity for really positive sera allowed). On the other Page 11 further shows that the immunoreactivity of the false positive  Samples (NYBC samples) differ significantly due to the presence of the spliced peptide in which sample diluent was inhibited. In the tested At concentrations, inhibition with peptide 48 was greater than 85% in 4 of 6 Rehearse. The inhibition was titratable for that in a dose-dependent manner Majority of samples.

Peptide 51 was used at a concentration of 25 µg / ml for sample dilution given and tested in an ELISA, with the mixture E on microtiter plates were applied by coating as described in Example 5. At a Quadruple test on two false positive samples (980421 and 820905) was done reduced non-specific immunoreactivity to 90 and 94%, respectively with a known HCV-NS-3 reactive sample the immunoreactivity was not was changed.

Similar results were seen with a number of false positive serum samples obtained, which were tested with mixture D in the ELISA test.

Example 10

Mixtures A, D and E were, as described in Example 5, with three HCV positive serum samples tested (by PCR and / or RIBA). mixture A gave false negative signals with these three samples, whereas both Mixture D and mixture E gave strongly positive signals, as in Table 17 shown.

Sample T14916 was selected as a rare sample that was identified in the PCR test HCV positive and which shows that it contains antibodies that with NS-3 and NS-5 proteins, but not with the core protein or NS-4- Proteins reacted. The improvement in implementation for the sample T14916 is on the addition of peptide 47 (an NS-5 reactive peptide) to the Mixtures D and E attributed. If the peptide 47 and its prototype Peptide 36 (also known as pep12 in EP-A 0 468 527) on the sample alone T14916 were tested, the absorption was at 1.146 and 1.089 at 492 nm.

Table 2

Immunoreactivity of the core reactive peptides

Table 4

Elimination of false positives with NS-4 reactive peptides

Table 6

Immunoreactivity of the NS-5 reactive peptides

Table 7

Detection sensitivity and specificity of peptide mixtures

Table 9

Immunoreactivity of the NS-4 reactive peptides based on peptide 35 (V)

Table 10

Specificity of the spliced peptides

Table 11

NS-3 inhibition test

Table 12

Mixture

For the following sequence listing are not for the abbreviations following natural (unusual) amino acids used:

Norvalin: Nva
Norleucine: Nle
Ornithine: Orn
Hydroxyproline: Hyp.

SEQUENCE LOG

Claims (22)

1. A spliced peptide composition comprising a linear spliced peptide in which the C-terminus of the peptide is a -COOH or -CONH₂ group, the spliced peptide is a Amino acid sequence selected from SEQ ID NO: 48-50, where SEQ ID NO: 48-50 components of this claim and an amino acid sequence of a strain / isolate of HCV from the corresponding NS-3 region of the spliced peptide, and wherein the spliced Peptide the non-specific reactivity of the NS-3 conformation epitopes in HCV immunoassays blocked. 2. A spliced peptide composition comprising a linear spliced peptide in which the Carboxy terminus of the peptide is a -COOH or -CONH₂ group, the spliced Peptide the non-specific reactivity of the NS-3 conformation epitopes in HCV immunoassays blocked, and the spliced peptide of the 35 amino acid prototype sequence of SEQ ID NO: 48 corresponds to and contains the following substituted positions: Position 7: norleucine, position 17: norleucine, position 24: glutamic acid and position 28: norvaline, the 35 amino acid prototype sequence of SEQ ID NO: 48 forming part of this claim is. 3. A spliced peptide composition according to claim 2, wherein the spliced peptide is Peptide is SEQ ID NO: 51, with SEQ ID NO: 51 being part of this claim. 4. A spliced peptide composition according to any one of claims 1 to 3, wherein the spliced peptide is conjugated to a carrier. 5. A spliced peptide composition according to any one of claims 1 to 3, wherein the spliced peptide is polymerized to a polymerized peptide.   6. A spliced peptide composition according to claim 5, in which the polymerized peptide is represented by the general formula (peptide) ₂X
(Peptide) ₄X₂X
(Peptide) ₈X₄X₂X
(Peptide) ₁₆X₈X₄X₂X in which X is an amino acid or an amino acid analogue having two amino groups and one carboxyl group, each group being able to form a peptide bond. 7. Using a kit comprising

• (i) a first peptide composition comprising at least one HCV peptide or HCV protein with an NS-3 conformation epitope; and
• (ii) an effective amount of a second spliced peptide composition according to any one of claims 1 to 6

in an immunoassay for the detection of HCV antibodies or for the diagnosis of HCV Infections in serum, tissue, tissue extract or body fluid. 8. Use according to claim 7, wherein the immunoassay is an ELISA assay, a sandwich Assay or a PHA assay. 9. Use according to claim 7, wherein the first peptide composition is an HCV peptide with an NS-3 conformation epitope. 10. Use according to claim 9, wherein the HCV peptide is selected from the group consisting of:

• (i) a peptide 4 (SEQ ID NO: 4),
• (ii) a peptide analog with an amino acid sequence of a strain / isolate of HCV in a region corresponding to peptide 4,
• (iii) a linear peptide, the carboxy terminus of the peptide being a -COOH or -CONH₂ group, the peptide being specifically immunoreactive with hepatitis C virus (HCV) antibodies, and the peptide of the 81 amino acid prototype sequence of SEQ ID NO: 4, is immunoreactive with HCV NS-3 antibodies and contains the following substituted or degenerate positions:
  • Position 3: norvaline, position 7: norvaline or Val: Ala: Thr: Phe: Tyr: Nva in a ratio of 3: 3: 1: 1: 1: 1, position 8: valine or norvaline, position 16: norvaline, position 20: Arginine, position 26: norleucine, position 33: norleucine, position 39: serine, position 46: norvaline, position 52: alanine, position 60: serine, position 63: norleucine, position 68: serine and position 74: norvaline, and
• (iv) one of peptides 5-21 (SEQ ID NOS: 5-21),

wherein SEQ ID NOS: 4-21 form part of this claim. 11. Use according to claim 10, wherein the HCV peptide is conjugated to a carrier. 12. Use of the peptide according to claim 10, wherein the HCV peptide to a peptide is polymerized. 13. Use of the peptide according to claim 12, wherein the polymerized peptide is represented by the general formula (peptide) ₂X
(Peptide) ₄X₂X
(Peptide) ₈X₄X₂X
(Peptide) ₁₆X₈X₄X₂X in which X is an amino acid or an amino acid analogue having two amino groups and one carboxyl group, each group being able to form a peptide bond. 14. Use according to claim 7, wherein the first peptide composition mixture A, the which contains the peptides (SEQ ID NO) 25, 29, 33 and 19, mixture B which contains the peptides (SEQ ID NO)  25, 27, 33 and 19, mixture C which contains the peptides (SEQ ID NO) 19, 25, 28 and 33, Mixture D, which contains the peptides (SEQ ID NO) 19, 33, 38, 39, 46 and 47, or mixture E comprising the peptides (SEQ ID NO) 19, 37, 43, 45, 46 and 47, the SEQ ID NO: 19, 25, 27, 28, 29, 33, 37, 38, 39, 43, 45, 46 and 47 form part of this claim.   15. Use according to claim 14, wherein the immunoassay is an ELISA assay Sandwich assay or a PHA assay. 16. Use according to any one of claims 7 to 15, comprising the spliced A peptide composition according to any one of claims 1 to 6 in a sample diluent. 17. Use an ELISA kit

• (a) a solid phase coated with a peptide composition comprising mixture D containing the peptides (SEQ ID NO) 19, 33, 38, 39, 46 and 47 or mixture E containing the peptides (SEQ ID NO) 19 , 37, 43, 45, 46 and 47, wherein SEQ ID NO: 19, 33, 37, 38, 39, 43, 45, 46, and 47 form part of this claim, and
• (b) a spliced peptide composition according to any one of claims 1 to 6 in soluble form in a sample diluent,

for the detection of HCV antibodies or for the diagnosis of an HCV infection. 18. Use according to claim 17, further comprising

• (c) a negative control sample,
• (d) a positive control sample, and
• (e) Antibodies against human IgG antibodies, these antibodies being labeled with a reporter molecule.

19. Use according to claim 17 or 18, wherein the peptide composition the mixture E and the spliced peptide composition spliced peptide 48 or 51 (SEQ ID NOS: 48 or 51), with SEQ ID NOS: 48 and 51 forming part of this claim.