CA2054798C - Hcv-specific peptides, agents therefor and the use thereof - Google Patents

Abstract

The invention relates to polypeptides for the immunochemical determination of HCV-specific antibodies and HCV antigens, and to agents suitable for this method and the use thereof. The invention also relates to an immunochemical method for the simultaneous detection and/or for the simultaneous determination of a plurality of, different antibody specificities against pathogens which are different in each case in a single test.

Description

BEHRINGWERKE AKTIENGESELLSCHAFT HOE 90/B 032K - Ma 871 Dr. Pfe/Zi Ma 897 Ma 910 Ma 917 Description HCV-specific peptides acients therefor and the aase thereof The invention relates to polypeptides for the immuno-chemical determination of HCV-specific antibodies and HCV
antigens, and to agents suitable for this method and the use thereof.

The invention also relates to an immunochemical method for the simultaneous detection and/or for the simul-taneous determination of a plurality of different anti-body specificities against pathogens which are different in each case in a single test.

Non A/non B hepatitis (NANBH) is, as transmissable disease or group of clinical pictures, regarded as virus-associated and distinguishable from other virus-induced clinical pictures which are cau:sed by different hepatitis viruses such as hepatitis A virus (HAV), hepatitis B
virus (HBV), hepatitis D virus (HDV) and hepatitis E
(HEV). Finally, it is also possible to diagnose hepatitis caused by cytomegalovirus (CMV) or Epstein-Barr virus (EBV).

It is possible on the basis of epidemiological investiga-tions to define, in accordance with the transmission route, at least two types of non A/non B hepatitis (NANBH): epidemic hepatitis virus (enterically trans-mitted NANBV), which is transmitted by water and food-stuffs, and post-transfusion hepatitis virus (blood transmitted NANBV), which is transmitted by blood, needle sticks or similar routes. Besides these routes of infec-tion there are also known to be transmissions which, as - 2 -= 2054798 sporadically occurring NANBV ("community acquired NANBV"), have no evident association with the two types mentioned. Although the exact number of agen=ts or viruses causing NANBH is unknown, so-called hepatitis C virus (HCV) has recently been identified as a causative patho-gen of this disease (WO 89/04 669).

Until recently, clinical diagnosis was based mainly on serological determination of antigens and/or on anti-bodies directed against them, the tests being specific for parameters from the group of hepatitis pathogens HAV, HBV, HDV, HEV, CMV or EBV. In this so-called exclusion method, NANBH was diagnosed only when all the abovemen-tioned determinations were negative.

Besides this, so-called surrogate markers have also been used, such as, for example, GPT (glutamic-pyruvic trans-aminase, also called ALT - alanine aminotransferase) or anti HBc (hepatitis B core-specific antibodies). However, these aids are neither sensitive nor specific enough to be regarded as reliable. It is therefore possible to avoid only a small part of the cases of post-transfusion hepatitis, which occur in about 10% of transfused patients, by investigating blood donors. The urgent need for introduction of a specific test is underlined by the fact that NANBV is regarded as responsible for about 90%
of cases of post-transfusic?n hepatitis. The main problem with this disease is the fact that between 25 and 55% of infected people suffer chronic liver damage.

The discovery of HCV has provided the basis for a specific detection of HCV or HCV-specific antibodies. The isolation and characterization of HCV and corresponding cDNA replicates of parts of the HCV genome is the subject of WO 89/04 669, where HCV is assigned to the family of flavi-like viruses. There are also descriptions therein of the use of HCV antigen for detecting HCV-specific antibodies, and of the raising of antibodies for the diagnostic determination of antigen in patient's blood.

2054710#8 However, use is made of genetically engineered proteins, especially so-called non-structural proteins (NSP) from the open reading frame region (ORF), which are used as reactants in immunochemical detection methods.

An immunochemical detection comprises within theimeaning of this invention all processes which, as homogeneous (in solution) or heterogeneous (with solid phase) in vitro methods, permit the determination of antigens and/or antibodies of immunoglobulin classes A, D, E, G or M
(IgA, IgD, IgE, IgG or IgM) in body fluids such as serum, plasma, saliva, cerebrospinal fluid or urine. Examples of these methods, which are also called immunoassay, are enzyme imxnunoassay (ELISA or EIA), radioimmunoassay (RIA), immunofluorescence assay (IFA), radioimmunopre-cipitation (RIPA), agar gel diffusion etc.

For example, WO 89/04 669 makes use of the antigen C-100-3, mentioned therein, in the ELISA procedure for detect-ing HCV-specific antibodies (anti-HCV). The construct C-100-3, which is expressed by genetic engineering in yeast cells, from the NSP 3/4 region comprises 363 amino acids, whose sequence is depicted in Fig. 1, where the numbering system corresponds to that in the abovementioned patent.
However, the maximum sensitivity which can be achieved with the ELISA method for determining anti-HCV in samples of human origin is assumed to be about 80% in the case of chronic NANB patients and about 30% in the case of acute NANB patients. These findings on blood from human patients are supported by corresponding investigations on chimpanzees infected with NANBV.

In them, positive anti-HCV detection on the basis of the C-100-3 construct does not succeed until about 6 - 18 weeks after the ALT increase has taken place, which in turn can be observed as sign of disease 3 - 10 weeks after inoculation of NANBV. Thus, it emerges that 9 - 26 weeks after infection of a chimpanzee is the total time - 4 - 2~~4798 before HCV-specific antibodies can be detected with the immunoassay according to the state of the art using the C-100-3 construct.

WO 90/11 089 describes further HCV amino acid sequences which can be used to detect anti-HCV. However, no state-ments are made about the diagnostic relevance of particular protein sections, nor are there examples of immunodominant epitopes.

The fundamental problem in the detection of anti-HCV with the methods disclosed in the literature is that there are still samples which react in a false-positive and false-negative way. The false-positive samples may comprise up to 40% in a group of healthy blood donors (WEINER A., et al. Lancet 1990, Vol. 336, p. 695). The lack of a speci-fic and sensitive HCV test therefore means that a not inconsiderable proportion of samples which react falsely are wrongly discarded in blood donation centers and, at the same time, patients who are actually anti-HCV-posi-tive are still not reliably identified.
To eliminate these disadvantages, other groups have selected particular sequences of the ORF region of C-100-3 (from WO 89/04 669, Fig. 1) and employed them in an immunoassay (Peptides sp42, 117, 67 and 65, Fig. 2). On investigation of NANB-infected chimpanzees it was not possible to achieve an improvement in the diagnostic value. On the contrary, the observations made therein led to the conclusion that none of the described peptides is suitable to serve as basis for a reliable IgG or IgM
determination, because the sensitivity is inadequate and the diagnostic gap of at least 7 to 17 weeks until antibodies are detected means that there is no reduction in time compared with conventional detection methods.
Furthermore, it was found that sp42 in particular, with a diagnostic gap lasting 20 to 40 weeks, is unsuitable for a diagnostic test (SAFFORD J., et al. Int. Symp. on Viral Hepatitis and Liver Disease 1990 Houston, USA).

- 5 - 205479$

However, the polypeptide called sp67 is said to be immunodominant although only 86% identification of anti-HCV-positive samples was possible with this peptide (DAWSON, G.J., et al. Int. Congress of Virology 1990, Berlin, FRG).

An improvement is said to have been achieved with synthe-tically prepared core peptide (OKILMOTO H., et al. Japan.
J. Exp. Med. 1990, Vol 60, No. 4, p. 223 - 233). This entailed selection of a sequence from the total of 3 potential antibody binding sites of the core amino-acid sequence 1-120 (OKAMOTO, H. et al., Japan. J. Exp. Med.
1990, Vol. 60, No. 3, p. 167 - 177) with the aid of physicochemical predictive criteria specified by HOPP and WOOD (Proc. Nat. Acad. Sci. 1981, Vol. 78, p. 3824 -3828). This peptide is depicted in Fig. 3 and comprises 36 amino acids numbered from No. 39 to No. 74.

In contrast to the anti-C-100 'test, it was possible to detect HCV antibodies in some anti-HCV-positive sera with an ELISA based on this core peptide. This finding was confirmed by a comparative investigation using the polymerase chain reaction (PCR) to detect HCV RNA.
However, it is evident from the 26 anti-core HCV-positive samples found in an investigation on 606 blood donors (4.3%) precisely that only 10 (1.65%) cases could be confirmed by PCR. Conversely, 16 donors (2.65%) gave false positive reactions with this peptide. Thus, although the said core peptide is said to be more suitable in some cases for an anti-HCV detection method than the anti-C-100 test, the core peptide of the state of the art is generally associated with considerable susceptibility to interference.

The present invention was thus based on the object of developing a test able to detect HCV infections as early as possible and with high specificity.

It has now been found, surprisingly, that certain poly-peptides from the area of the ORF region of C-100-3 and/or the amino-terminal region of the HCV core region are particularly suitable for detecting HCV-specific antibodies, and a distinct increase in sensitivity compared with peptides of the state of the art with, at the same time, significantly reduced susceptibility to interference from unwanted false positive reactions can be achieved.

It has furthermore been found that a high antigen concen-tration, and thus a high epitope density, is achieved in an immunochemical detection method with the peptides according to the invention. This is possible particularly when, owing to =the absence of interfering contaminations, the investigation of highly concentrated patients' samples is possible.

It was also completely surprising that the peptides according to the invention carry immunodominant epitopes for early and late HCV antibodies and thus can be employed particularly advantageously for longitudinal investigations.

Hence the invention relates to peptides or peptide mixtures which react specifically with antibodies against an HCV infection, the amino-acid sequences thereof containing parts of the ORF region of C-100-3 and/or of the amino-terminal HCV core region.

The terms peptides and polypeptides are used within the scope of the invention as equivalent to peptides with up to about 80 AF,.

The peptides according to the invention preferably comprise amino-acid sequences from AA 121 to AA 175 (formula I) and from AA 337 to AA 363 (formula II) of the HCV genome section described as C-100-3, with the follow-ing sequences:

SGKPAI IPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQA I

HVGPGEGAVQWMNRLIAFASRGNHVSP II
Preferred polypeptides are:

4083:

SGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQA
4074:

SGKPAI I PDREVLYREFDEMEECSQHLPYIEQGMMLAE,QF
4073:

PDREVLYREFDEMEECSQHLPYIEQGMMLAEQF
4072:

EFDEMEECSQHLPYIEQGMLAEQF
4071:

SQHLPYIEQGMMLAEQF
4081:

KQKALGLLQTASRQA
4082:

SQHLPYIEQGMMLAEQFKQKALGLLQTASRQA
4090:

R,EFDEMEECSQHLPYIEQGMMLAEQFKQKA

- 8 - 2 0~ ~~x 4056:

SGKPAIIpDREV'LaR
-49-5 -5.*

DREVLYREFDEMEEC
4054t FDEMEECSQHLPYIE

QHLPYXEQGMAiLA.EQ
40~

GMMLAEQFKQKALGL
4Q91:

VQ[+TMNRLIAFASRGN
Sequences from the carboxyl- and amino-terminal region of formula I in particular have proven beneficial for detecting late antibodies of an HCV infection, such as, for example, the peptides 4081, 4056, 4055, and 4060 hereinafter:

- 9 - ~~~~~~~
AQ -O-L

S G KPA I I P DRE<7 LYRE F DE

Surprisingly, the carboxyl-terminal region of the ORF
region of C-100-3 has proven particularly suitable for detecting late antibodies, containing amino acids of the formula II or parts thereof, for example 4091.

It has also been found that the central region of formula I is relevant for early identification of anti-HCV.
Preferred in this connection are the abovementioned polypeptides 4071 and 4072, and peptides 4054, 4053 and 4052.

Furthermore 3 epitopes have been located on the peptide of formula I, one of which recognises a late antibody (S) and the others recognise early antibodies (F1 and F2).
Examples of preferred peptides have one of the following amino-acid sequences:

AAsl-DREVLYR-BAbi ( S ) AABa-QHLPYIE-BAb2 ( F1) AAB3-RQICALGL-BAb3, (F2 ) where AA and BA are any desired amino acid, and al - a3 and bi - b3 are each, independently of one another, integers greater than or equal to zero.

It is advantageous to select the amino-terminal sequences together with the carboxyl-terminal amino-acid sequences of the formula I and the sequence of the formula II. The central amino-acid sequence of the formula I from about 130 to 160 can be used separate therefrom.

-a0-The peptides according to the invention furthermore comprise parts of the amino-terminal HCV core region, preferably that amino-acid sequence from AA 1 to AA 35 of the HCV genome section described as core, with the following sequence:

MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVY (III) Preferred peptides are:

P'iSTNPKPQRKTKRNTNRRPQDVKFPGGGQI SP 10 g 26 K'TKRNTNRRPQDVKFPG SP 30 KRNTNRRpQDVITpG SP 30 D

The peptides SP 10 and SP 23 are particularly preferred.

It is possible with the aid of the peptides according to the invention of the core region to identify both early antibodies from acute phases of infection and late antibodies, which considerably improves the sensitivity of the detections based on these peptides compared with the state of the art. Another advantage is the high specificity of the peptides, which generally results in a minimal number of samples with a false positive reac-tion.

It is generally advantageous to use peptides, linked peptides or peptide mixtures which are specific for early and late HCV antibodies, because it is,possible with both types of binding site to identify both samples from early and samples from late phases of infection. It is also possible reliably to identify seroconversions caused by HCV-specific IgG and/or IgM antibodies, and it is gener-ally possible to discriminate between positive and negative samples with extremely high accuracy. In addition, there is generally no interference by the expression system required for protein preparation by genetic engineering, or other host cell contaminations which are unavoidable for growing viruses. Reliable determination of HCV-specific antibodies is also generally ensured in sera, citrated, heparinized or EDTA
plasma of human origin, and inactivation of the samples at about 55 C for about 60 minutes generally gives rise to no false positive results. Finally, these peptides according to the invention can be used to prepare specific antibodies with whose aid, owing to the immunochemical determination of corresponding antigens in cell-free patient's blood, the diagnostic gap can be further closed.

According to the subject-matter of the invention, a series of novel peptides which preferably identify HCV-specific antibodies from convalescent or chronically infected patients and/or HCV antibodies from acute phases of infection are described, and polypeptides and mixtures - 12 - 2054'98 of polypeptides which are suitable as basis for screening tests for the non-differentiating detection, which is, however, highly sensitive and little susceptible to interference, of anti-HCV antibodies are described.

The invention furthermore relates to antibodies which have a biospecific affinity for at least one of the peptides, described above, of the ORF region of C-100-3 or of the core region.

The immunochemical determination of corresponding anti-gens with the antibodies according to the invention in cell-free patient's blood makes it possible to establish the presence of HCV even before endogenous antibodies appear.

The invention furthermore relates to an immunochemical method for the detection and/or for the determination of HCV antibodies with the peptides according to the inven-tion as antigen.

The invention also relates to a method for the purpose of differential diagnosis between an early and late phase of infection, where in each case one or more peptides which react specifically to early antibodies and one or more peptides which react specifically to late antibodies are reacted with the sample in separate mixtures.

The invention furthermore relates to the use of the peptides described above for raising antibodies in mammals, especially in humans.

The invention also relates to the use of the antibodies described above for diagnostic and therapeutic purposes.
The invention therefore further relates to agents which contain at least one of the antibodies or peptides according to the invention alone or in combination with other peptides or antibodies.

- 13 - 2 05 4 79 $

The said immunoreactive peptides can be prepared by synthesis or genetic engineering, preferably by synthesis by the methods known to the person skilled in the art.
The chemical synthesis of the peptides can be carried out, for example, as described by BARANI, G. and MERRIFIELD, R.B. in "The Peptides, Analysis, Synthesis and Biology", Vol. 2, Academic Press 1980, ed.
Erhard Gross, Johannes Meyenhofer, especially as a polypeptide or as mixture of several sma11 peptides with overlapping or non-overlapping amino-acid sequence.

The polypeptides prepared by genetic engineering include fusion proteins whose fusion portion has subsequently been eliminated. Also included are polypeptides which have been modified where appropriate, for example by glycosylation, acetylation or phosphorylation.

These amino-acid sequences can be synthesized both as a polypeptide and as mixture of several small peptides with overlapping or non-overlapping amino-acid sequence.

It has also been found that mixtures of individual peptides may have better diagr-ostic properties for an immunochemical anti-HCV detection than single peptides of the said structures. It is particularly advantageous to use mixtures of peptides of the ORF region of C-100-3 and of the core region.

The invention therefore further relates to mixtures of peptides containing one or more of the peptides according to the invention.

In another embodiment, 2 or more of the said peptides, preferably 2 to 10, especially 2 to 4 peptides are linked with or without bridge. The length of the peptides is preferably 6 to 15 amino acids. It is even possible for polymeric forms of two or more peptides to be prepared by methods known to the person skilled in the art and bound to a carrier such as, for example, protein or latex - 14 - ~0 5 particles. Thus, particularly suitable as carrier or bridge are, for example, human serum albumin and/or polylysine< It is likewise possible to modify the pep-tides by an extension with 1 to 40, preferably 1 to 20, in particular 1 to 10 amino acids. The additional regions and structures may have a beneficial effect=on the physicochemical behavior of the complete peptide, but the immunoreactivity of the peptides or parts thereof ought to be retained.

The invention therefore also relates to peptides which are linked to one another with or without bridge, or can also be bound to a carrier.

Modifications of this type generally alter the passive adsorption or covalent bonding property to the solid phase in a.beneficial manner, have an advantageous effect on the coupling method or act more strongly as antigen when raising polyclonal or monoclonal antibodies directed against the peptides.

Thus, for example, the invention also relates to peptides of the following formula AAõ- QRKTKRNTNRRP QI7VK-BAs, where AA and BA are any desired amino acid, and n and m are each, independently of one another, integers from 0 to about 60, in particular from 1 to 40.

It is often advantageous for peptides to be derivatized in a variety of ways, such as, for example, by amino-terminal or carboxyl-terminal attachment of one or more amino acids, preferably cystein, in order, for example, to achieve the linkage of peptides with one another or to a carrier, thioglycolic acid amidation, carboxyl-terminal amidation such as, for example, with ammonium or methyl-amine. Modifications of this type may alter the net charge on the polypeptide and improve the physicochemical a~~~7H

properties of the peptide or facilitate covalent bonding of the peptide to a solid carrier, to carrier proteins or to another peptide. The person skilled in the art is also aware that the peptides according to the invention can also be prepared among themselves or with themselves by genetic engineering or synthesis in such a way' that a plurality of immunorelevant epi=topes is located on one peptide.

Thus the invention furthermore relates to peptides with an amino-acid sequence according to the invention which has been modified by replacement, addition or deletion of one or more amino acids.

In general, modifications of this type do not result in direct alterations of the immunoreactivity of a peptide, but it is perfectly possible to achieve improved immuno-logical properties of the peptides. Thus, for example, methionine is prone to spontaneous oxidation, which can be prevented by replacement by norleucin without essen-tially changing the antigenic properties of the polypep-tide.

The person skilled in the art is aware that given amino-acid sequences can be subjected to a wide variety of alterations such as, for example, deletions, insertions or substitutions, which may be associated with various advantages. Modifications of this type relate, for example, to combinations such as Gly, Ala; Val, Ile, Leu;
Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; Phe, Tyr; Ala, Ser; Ala, Thr; Ala, Val; Ala, Pro; Ala, Glu; Leu, G3.n;
Gly, Phe; Ile, Ser and Ile, Met.

it may likewise be advantageous to improve the adsorption properties of the polypeptide in the form of addition of a hydrophobic sequence comprising about 2 to 20 hydro-phobic amino acids, such as, for example, Phe Ala Phe Ala Phe.

The invention furthermore relates to DNA sequences which code for at least one of the peptides according to the invention.

The invention also relates to an analytical method for the detection and/or for the determination of HCV, employing as specific step a hybridization reaction in which at least one nucleic acid probe which is complemen-tary in its specific part with at least one of the DNA
sequences according to the invention is used.
An immunochemical detection generally comprises processes which, as homogeneous (in solution) or heterogeneous (with solid phase) methods, permit the determination of antigens and/or antibodies. Examples of these, which are also called immunoassays, are enzyme immunoassay (ELISA
or EIA), radioimmunoassay (RIA), immunofluorescence assay (IFA), radioimmunoprecipitation assay (RIPA) or agar gel diffusion assay etc.

These numerous, very diverse methods differ in specific embodiments in the marker used for detection or the measurement principle (for example photometric, radio-metric, visual or by the aggregaition, scattered light or precipitation behavior) and in the solid phases. The person skilled in the art is aware that separation of bound and free sample antibodie:s or antigen is, although widely used, not absolutely necessary, such as, for example, in so-called homogeneous assays. Heterogeneous immunoassays are preferred, especially heterogeneous ELISA methods.

The person skilled in the art is likewise aware that the term "confirmatory test" describes only the use of an immunoassay, similar to the way that the tests called dot methods only describe by name the way in which the antigen is immobilized.

In the antibody detection, it is a prerequisite for an immunochemical detection method that there is contact of the sample with the described peptide sequences during the course, in order to form an antigen-antibody complex in a particular step of the particular method or, in competition and inhibition tests, to prevent the forma-tion thereof by adding suitable labeled reagents.

In the direct method it is possible for the antibodies to be contacted with peptides bound to solid phases or with labeled peptides or with both, it being immaterial whether the fundamental method is, as 1-, 2- or multi-step method, based on the principle of the 2nd antibody test or the immunometric test design (double antigen sandwich) either with identical or different peptides (or peptide mixtures) on the solid phase and as liquid reagent for the detection and in combination with specific so-called capture antibodies (for example anti-IgM) or affinity reagents (for example protein A).

The peptides can be bound to the solid phase covalently, by adsorption or using specific antibodies or similar affinity methods, for example via the biotin/avidin complex, but preferably by adsorption.

Suitable as carrier material for the solid phase are plastics such as polystyrene, polyvinyl chloride, poly-amide and other synthetic polymers, natural polymers such as cellulose and derivatized natural polymers such as cellulose acetate and nitrocellulose, as well as glass, especially as glass fibers. Polystyrene is preferred as carrier material.

The carriers can be in the form of beads, rods, tubes and microtiter plates or in the form of suspensions such as, for example, latex particles. Sheet-like structures such as strips of paper, small plates and membranes are likewise suitable. The surface of the carriers can be both permeable and impermeable for aqueous solutions.
Preferred carriers are beads, tubes, wells, micro-particles, strips of paper and membranes. Particularly preferred carriers are microtiter plates, latex particles, polystyrene beads or particles amenable to magnetic attraction.

The peptide concentration for coating the carrier is generally about 0.01 - 20 jug/ml, preferably 0.01 -pg/ml, particularly preferably 2 - 10 pg/ml. It is particularly advantageous to use synthetically prepared polypeptides whose high purity and strong antigenicity 10 allow the use of tiny amounts of, for example, 0.01 -2.0 mg/ml, preferably 0.1 - 0.5 pg/ml. The binding capacity of the carrier, in particular when polystyrene is used, is generally not saturated so that it is normally possible to coat with a plurality of different polypeptides, in particular with 2 - 5, especially with 3 - 4 different polypeptides, which is a particular advantage.

When the peptides are used as labeled derivatives for -the detection, suitable coupling techniques are all those known to the person skilled in the art. It is also possible to arrange multistage methods such as, for example, preformed peptide-antibody complexes in which the antibody carries the label, or high affinity systems such as, for example, biotin/avidin with labeling of one of these reactants.

Examples of markers which can be used are radioactive isotopes, fluorescent or chemiluminescent dyes. It is also possible to use enzymes which are detected, for example, by chromogenic, luminogenic or fluorogenic substrate systems, or by subsequent amplification systems with a second enzyme which is activated by the first, as markers.

Preferably used as markers are enzymes, especially alkaline phosphatase and/or horseradish peroxidase or chemoluminogens such as, for example, acridinium esters.

- 19 -. 2054798 The labeling is carried out by methods which are des-cribed for the said markers in the state of the art.
Where the antibodies are labeled with peroxidase, the periodate technique of NAKANE et al., 1974, J. Histochem.
Cytochem. 22, 1084 - 1090, can be used, or a method of ISHIKAWA et al., 1983, J. Immunoassay 4, 209 - 327, in which the partners are linked by a heterobifunctional reagent.

Besides these methods, the peptides can also be used for sensitization of suitable surfaces such as, for example, latex or erythrocytes in order to measure, automatically or visually, physicochemical changes induced by peptide-specific antibodies, such as, for example, precipita-tions, aggregation or light scattering. It is known that the peptides can also be employed not derivatized for inhibition of these measurement principles like also the methods mentioned before.

It is possible to use for detection of the antigens immunodiagnostic methods which make use of polyclonal or monoclonal antibodies which are prepared with the aid of the peptides or derivatives thereof according to the invention. The embodimen=ts suitable for the detection method are known to the persoxi skilled in the art and comprise forming antibody-antigen complexes in a particular step or inhibiting the complex formation in a competition method by adding a labeled antigen.

Suitable as solid phases, markers or measurement prin-ciple for establishing an antigen test are all the possibilities described for the corresponding antibody determination, with the competition principle and the double antibody sandwich technique being particularly preferred as immunochemical method. It is immaterial in this context whether the methods are designed as 1-, 2-or 3-step methods. Thus, multistep methods can be carried out with unlabeled detecting antibodies which are - 20 .- 2034798 determined with the aid of another antibody which is directed against them and is appropriately labeled. It is advantageous for the raising of the antibodies to modify the peptides in such a way that their immunogenic property is improved, as is possible, for example, by coupling to serum albumin or keyhole limpet hemocyanin (B.S. Schaffhausen in Hybridoma Technologie in the Biosciences and Medicine, ed. T.A. Springer, Plenum Press NY, London, 1985).

Finally, the present invention can also be applied when using an immunodiagnostic element which contains the solid phase and, in dry form, a part or even all the reagents required, where in this case too the novel peptides are contained either on the side of the solid phase or in the detection reagent or in both, and an antibody determination, an antigen detection or combina-tions with other analytes is carried out.

An unexpected advantage of the novel peptides of the present invention is that they allow reliable determina-tion of HCV antibodies. Furthermore, early antibodies from acute phases of infection are also identified with the aid of these peptides, which considerably improves the sensitivity of the detections based on these peptides compared with the state of the art and, moreover, allows differentiation between acute phases on the one hand and chronic or convalescent stages on the other hand when the peptides with the appropriate binding sites for these antibody types (early or late antibodies) are used separately from one another in two different test methods. Finally, another advantage which emerges is the high specificity of the peptides of the present inven-tion, which leads to a minimization of the number of samples with a false positive reaction.

In view of the virus safety in blood donation centers on the one hand and for reasons of cost on the other hand, so-called combination tests have been developed and, available since 1989, permit simultaneous non-differen-tiating detection of anti-HIV 1 and/or anti-HIV 2 (K. Korner et al., Lab.Med. 14, 159 - 161, 1990). This development was possible owing to the great similarity which the two HIV sub-types display towards one another and, moreover, they belong to the same virus class. Thus the anti-HIV 1 determination in such anti-HIV 1/2 com-bination tests is also based on a cross-reactivity of anti-HIV 1 with HIV 2 antigens (M. Busch et al., Trans-fusion 30/2, 184 - 187, 1990), as also conversely the anti-HIV 2 detection is enhanced by the reactions which take place between anti-HIV 2 and HIV 1 antigens. On this basis the establishment of a combined detection of two antibody specificities proves to be relatively straight-forward when antigens which are related or structurally analogous are used. The detection of a pathogen of non A
non B hepatitis, the so-called hepatitis C virus (HCV), was the prerequisite for establishing an anti-HCV deter-mination according to the present invention. Despite the improvement, associated therewith, in the performance features of screening tests, the problem again arises also with modern anti-HCV tests that the investigation of individual donors with anti-HIV combination tests on the one hand and with anti-HCV single tests on the other hand entails considerable effort and considerable additional costs.

All commerci,al embodiments to date of the anti-HCV
determination and the majority of anti-HIV combination tests are based on genetically engineered HCV or HIV
polypeptides. However, there has also been no success to date in deterniining a plurality of different antibody specificities in only a single test mixture containing a plurality of different antigens simultaneously in one immunochemical detection when it is not possible to use similar antigens, since different virus class affilia-tions are present as in the case of HCV (flavivirus) and HIV (retrovirus). Within the meaning of the invention, different antibody specificities mean antibodies which - 22 .- ~0547rnlS

have very low or zero cross-reactivity with one another.
It was assumed that a test of this type for detecting a total of three antibody specificities against a plurality of different viral antigens tends to be worse in terms of the sensitivity and in terms of the susceptibility to interference (specificity) as a consequence of mutual interfering interactions than the corresponding proper-ties of the particular single test.

Surprisingly, it has now also been found that a plurality of different antibodies or antibody specificities against different pathogens in each case can be detected immuno-chemically in a single test when different epitopes of different pathogens in each case are immobilized on a carrier.

Furthermore, the sensitivities found with the method according to the invention at least correspond to the sensitivities of single tests. Thus, for example, the sensitivity feature.s determined for anti-HIV 1/2 and anti-HCV for the method according to the invention at least correspond to those of single tests.

It was therefore also completely surprising that the susceptibility to interference by unwanted false positive reactions was reduced with the method according to the invention. Thus, for example, on testing the specificity of an anti-HIV/anti-HCV test according to the invention the specificity obtained was higher than would be given by the total of the two single tests, with the conse-quence that overall fewer blood donations may be wrongly discarded.

The invention therefore also relates to an immunochemical method for the detection and/or for the determination of a plurality of different antibody specificities against different pathogens in each case, which comprises one or more epitopes of the particular pathogens being immobil-ized on a carrier and the detection and/or the - 23 - ~7 9 19 determination of the said pathogens being carried out in a single test.

For certain problems such as, for example, longitudinal investigations, it may be desirable to undertake non-differentiating simultaneous determination of different pathogens. This means that no distinction is made between the various antibodies but only a yes response or no response (negative for all antibody specificities) is obtained.
Also desirable where appropriate is differentiating determination of the simultaneous antibody detection.
This is directly possible within the scope of the inven-tion by, for example, in an immunometric test, the antigens which are used having different virus-specific labels, such as, for example, HIV 1 antigens with peroxi-dase, HIV 2 antigens with alkaline phosphatase and HCV
antigens with p-galactosidase. Successive or simultaneous determination of the simultaneously bound antibody specificities is then possible with different enzyme substrates.

An alternative form of differentiation is the inhibition of one antibody specificity by the specific addition of the corresponding antigen to the sample.

The invention thus further relates to an immunochemical method for the simultaneous detection and/or simultaneous determination of different antibody specificities, where the detection and/or the determination is carried out in a differentiating or non-differentiating, preferably non-differentiating, manner.

Different pathogens according to the invention mean pathogens against which antibodies of different specificity and, in general, very low or zero cross-reactivity are directed. Examples of these are HIV 1 + 2, HCV, HTLV I + II, HBV or Treponema pallidum, preferably HIV and HCV.

- 24 _. 2074798 It is particularly advantageous to immobilize on a carrier antigens of the said pathogens, especially of HIV
1, HIV 2 and HCV, which have a high density or concentra-tion of binding sites (epitopes) for the corresponding antibodies. It is possible in this way, for example, to identify seroconversions, a discrimination of positive and negative samples is possible with an accuracy which is generally high, interference by the expression system which is necessary for preparation of proteins by genetic engineering cannot generally occur, other host cell contaminations which are unavoidable in growing viruses are not generally present, in general reliable determina-tion of HIV- and HCV-specific antibodies with different specificity is ensured in sera, citrated, heparinized and EDTA plasmas of human origin, and inactivation of the samples at 56 C for 60 min normally results in no false-positive results.

Preferably used are single epitopes and/or combinations thereof, especially of polypeptides of HIV 1 and/or HIV 2 and HCV, which are suitable for a highly sensitive anti-HIV/anti-HCV detection, and, furthermore, suitable polypeptide mixtures as basis for a screening test for a combined antibody detection.

The invention thus also relates to polypeptide mixtures of HIV 1 and/or HIV 2 and HCV.

The following polypeptides are particularly preferred:
1. HIV 1(numberinq system of Ratner et al., Nature 1985, 313, 277 - 284):
IV transmembrane protein (gp 41): AA 580 - AA 630 V envelope protein (gp 120): AA 490 - AA 540 VI core protein (p 24): AA 240 - AA 390 2. HIV 2(numbering system of Gyader et al., Nature 1987, 326, 662 - 669):
VII transmembrane protein (gp 36): AA 570 - AA 620 - 25 .- 2054798 VIII envelope protein (gp 110): AA 480 -.AA 530 IX core protein (p 26): AA 230 -.AA 380 3. HCV (numbering system of WO 89/04669 and WO 90/11089):
X non-structural protein 4 (NSP 4): AA 121-AA 175 XI non-structural protein 3 (NSP 3): .AF, 1-AA. 265 XII structural protein (core): AA 1-AA 80 Especially preferred are mixtures of the said polypep-tides, especially for a non-differentiating anti-HIV/
anti-HCV screening test, some being described by way of example without confining the conceivable possibilities thereto:
XIII gp 41 HIV 1 gp 36 HIV 2 XIv gp 41 HIV 1 gp 36 HIV 2 core HCV

Xv gp 41 HIV l gp 36 HIV 2 core HCV

XVI gp 41 HIV 1 p 24 HIV 1 gp 36 HIV 2 core HCV
or XVII gp 41 HIV 1 p 24 HIV 1 _ 26 gp 36 HIV 2 p 24 HIV 2 core In general, better immunological properties for diagnos-tic use are achieved with the said peptide mixtures.

The following peptides of HIV 1, HIV 2 and HCV have proven particularly suitable:

XVIII SPH 9 (HIV 1, gp 41):

RILAVERYLICDQQLLGIWGCSGKLICTTAVPWNAS
XIX SPH 20 (HIV 2, gp 36):

RVTAIEKYLQDQARLNSWGCAFRQVCHTTVPWVNDS
XX SP 4083 (HCV, NSP 4):

SGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQA
or parts thereof, for example XXI a mixture of SP 4060 and SP 4082 (HCV, NSP 4):
127, 139 SGI{PAIIPDREVLYREFDE SP 4060 SQHLPYIEQGPgiLF,EQFKQEALGLLQTASFiQA SP 4082 XXII SP 10 (HCV, core):

MS TNPKPQRIC'!'KRNTNRRPQDVKFPGGGQI
and/or - 27 .. 2054798 XXIII SP 31 (HCV, core):
$ 24 It is likewise possible to integrate further peptides from different protein regions of HIV 1, HIV 2 or HCV, as long as these polypeptides are immunorelevant. Con-versely, it may also be advantageous, for example for epidemiological problems, to exclude a specific antibody in the non-differentiating detection by omitting corres-ponding peptides in order, for example, to determine with a mixture of HIV I and HCV peptides only anti-HIV 1/anti-HCV simultaneously and not, for example, anti-HBV/anti-HIV 1/anti-HCV. It is also possible for the peptides which are not identical to HCV, such as, for example, precisely HIV, to be derivatized and modified in a manner analogous to that described above for the HCV peptides.
A considerable advantage of the method according to the invention is that a plurality of antibodies against dif-ferent pathogens can be detected in a single test and, moreover, a specificity or sensitivity which is still as high as that which can be achieved with single tests is achieved.

Fig. 1 Primary sequence of the amino acids of the ORF
region of C-100-3 (from WO/89/04669) Fig. 2 Comparison of the sequences, according to the invention, of the formula I and II with known peptides from the ORF region of C-100-3 Fig. 3 Comparison of the sequences, according to the invention, of the HCV core protein with known peptides The examples described hereinafter represent embodiments of the invention without, however, confining it to them.

Example 1 Preparation of peptide solutions and coating of micro-titer plates with these peptides or peptide mixtures Serial 2-fold dilutions in 0.10 M sodium bicarbonate pH 9.6 were made up from stock solutions of the peptides according to the invention in 50% acetic acid in dis-tilled water containing 1 to 10 mg of peptide/ml, that is to say a series with the concentrations 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, 0.2, 0.1, 0.05 and 0.01 g i0 of peptide/ml was obtained. The procedure was analogous for mixtures of single peptides, the stock solutions additionally being mixed in various ratios, for example : 1 or 1: 4, in order to obtain by dilution in 0.1 M
sodium bicarbonate the overall final concentrations which are indicated above but which in the case of mixtures of several peptides contain the latter in equal concentrations (in the case of 1. 1 mixture) or in various ratios to one another.

In each case, 100 ;.1 of each dilution were placed in 16 TM
wells of microtiter plates, type B supplied by Nunc, Roskilde, Denmark. The test plates charged with the di2. utions were left at 20 C for 18 hours and then the solutions in the wells were removed by aspiration and the wells were washed 3 - 4 times with 300 p1 of a solution of 10 g/l bovine serum albumin in phosphate-buffered physi_ological saline (PBS, pH 7.4) by filling and removal by aspiration, and the test plates were subsequently dried over silica gel at 20 C.

Example 2 Preparation of a peroxidase-labeled antibody against human immunoglobulin of IgG class (h-IgG) and T'!B
substrate for detection Antibodies against h-IgG were raised by the method of KOEHLER and MILSTEIN for preparing monoclonal antibodies (Nature 256, 495, 1975), different anonoclonal antibodies with the same antigen specificity being identified by the method described by STkLI et al. (J. of Immunological Methods 32, 297 - 304, 1980). After purification by gel chromatography and dialysis against phosphate-buffered saline (PBS, pH 7.4), the pool containing the monoclonal antibody fraction (4 mg of protein/ml) was subsequently reacted with N-gamma-maleimidobutyryloxysuccinimide (GMBS), obtained from Behring Diagnostics, as described by TANAMORI et al. (J. Immunol. Meth. 62, 123 - 131, 1983).

2-Iminothiolane hydrochloride (supplied by Sigma, Cat.
No. I 6256) was reacted with horseradish peroxidase (POD), obtained from Boehringer Mannheisn, Cat. No.
413470, as described by KING et al. (Biochem. 17, 1499 -1506, 1978). An IgG-POD conjugate was prepared from the GMBS-IgG conjugate and the iminothiolane-POD conjugate as described by TANAMORI et al. (supra).

The resulting solution of -the IgG-POD conjugate had a protein content of 360 g/ml. The ratio of POD to IgG was determined as 2.8. The solution was subsequently diluted to 500 ng/ml IgG-POD with a solution of 50 ml/1 fetal calf serum (FCS), 5 g/1 polyoxyethylene(20)sorbitan monolaurate (RTween 20) in PBS and was called anti-IgG/
POD conjugate. For use in the ELISA, the subsequent dilution in tris buffer pH 7.4, containing 0.5% "~Y'ween 20, was varied (between 1 : 100 and 1 : 20,000 dilution) in order to prepare uniformly a 1 : 26 final dilution in conjugate buffer, containing 0.1 M 2-amino-2-(hydroxy-methyl)-1,3-propanediol (tris), 0.1 M sodium chloride (NaCl) and 0.1% RTween pH 8.4. Rabbit polyclonal anti-bodies prepared according to the state of the art were adjusted so that a dilution of 1+ 25 was likewise achieved for use.

Used for the detection of anti-IgG/POD conjugate was a - 30 _ 2054798 substrate system or a substrate preparation containing hydrogen peroxide and tetramethylbenzidine (TMF3), which was prepared from two stock solutions.

Stock solution 1: TMB dihydrochloride was dissolved while stirring in a concentration of 5 g/l, i.e. of 16 mxnol/l, in double-distilled water and adjusted to pH 1.5 with 5 normal hydrochloric acid. Penicillin G was added to this solution while stirring in a final concentration of 200 mg/l, i.e. of 0.56 mmol/l.

Stock solution 2: 1.4 ml of glacial acetic acid, 1.5 ml of 1 normal NaOH and 250 mg, i.e. 3 mmol, of H202 as urea/
hydrogen peroxide adduct were added to 900 ml of double-distilled water. After dissolution was complete, the mixture was made up to 1 1 with double-distilled water.

TMB substrate preparation: One part by volume of stock solution 1 and 10 parts by volume of stock solution 2 were mixed together. =
Example 3 State of the art Employed as example was a commercially available ELISA
test kit (HP1) in which the C-100-3 construct which is described in WO 89/04669 and is prepared in yeast by genetic engineering is used as antigen. The procedure for investigating human sera and plasmas was that indicated in the manufacturer's pack insert, for example 1 : 11 sample dilution and 1 h incubation of serum, 1 h incuba-tion of anti-human IgG/POD conjugate and the enzyme substrate system with o-phenylenediamine (OPD) as sub-strate, photometric measurement at 492 nm and establish-ment of limits (a threshold of 0.40 E is added to the mean of the negative controls).

An entirely analogous procedure was used with another commercially available ELISA (HP 2) which, besides the C-100 construct, contains additional epitopes from the core region and from the NSP 3 region (c33c), that is to say the original test kit was used, observing all the statements about procedure stated by the manufacturer -as described above, for investigating human samples.

By contrast, the procedure for determining HCV-specific antibodies in chimpanzee serum or plasma with the two commercial products was such that a polyclonal antibody raised in rabbits against human IgG was used for detec-tion. For this purpose, the IgG fraction of rabbit antiserum was, as described in Example 2, purified, dialyzed and labeled with peroxidase (POD). The final concentration was adjusted to about 4 times the concen-tration of the monoclonal anti-IgG/POD conjugate in order reliably to fashion over the cross-reaction, validated in preliminary tests, of the antibodies against human IgG
for chimpanzee IgG.

It was necessary to modify the establishment of limits as described in Tab. 10 A - C because the initial values of all the chimpanzees already exhibited elevated values (see Tab. 10 A - C).

The comparative anti-HIV 1+ 2 combination test for the non-differentiating determination of HIV 1 and HIV 2 antibodies is a commercially available product (HP 3) which is based on synthetically prepared peptides of HIV
1 and HIV 2. The procedure for this test was also that of the manufacturer's pack insert, for example sample dilution 1: 2, 30 min incubation of serum, 30 min incubation of conjugate (anti-human IgG/POD) and the enzyme substrate system with tetramethylbenzidine (TMB) as substrate, photometric measurement at 450 nm and establishment of limits (a threshold of 0.250 was added to the mean of the negative controls).

Example 4 Determination of human antibodies of immunoglobulin class G against HCV in the ELISA with the peptides according to the invention 50 l of serum or plasma were added to 50 l of sample buffer containing 0.3 M tris, 0.3 M NaC1, 20% boviserine and 0.1% RTween 20 in wells of microtiter plates which were coated as described with peptides or peptide mix-tures. After incubation at 37 C for 30 min, the content of the wells was removed by aspiration, and the wells were washed five times with washing buffer containing 1 g/1 RTween 20 in PBS. Then 100 l of conjugate in the final dilution were added to the wells, preferably using a preliminary dilution of 1: 3000 in tris, 0.5% Tween 20 and a final dilution of 1: 26 in conjugate buffer. After incubation at 37 C for 30 min, the content of the wells was removed by aspiration and again washed five times.
Subsequently 100 l of TMB substrate preparation were added to each well, incubated at 20 - 22 C for 30 min, and incubation was stopped by addition of 100 l of 1 normal sulfuric acid. The extinction of the colored solution was measured at a wavelength of 450 nm (E450) with a PBS blank as reference.

Samples classified as anti-HCV positive were those which produced an E450 greater than 0.10, samples classified as anti-HCV marginal were those whose E450 was in the range from 0.05 to 0.10, and samples classified as anti-HCV
negative were those which produced an E450 below 0.05.

Tab. 1 summarizes results obtained from the determina-tion, described in Example 1, 2 and 4, of human samples with the peptide 4083 according to the invention and with a mixture of smaller sequences of the formula I. The data in Tab. 2 were obtained analogously with the peptide SP
10 according to the invention, the results of both ELISAs being compared with those of HP 1 (Example 3).

Tab. 1 Results of the deternnination of anti-HCV with an ELISA
containing the peptide 4083 and with a mixture of smaller peptides on the solid phase with human samples Positive with peptide ELISA
(2 pg 4083/m1) (2 Ug 4060/mi and 2 ug 4082/ml) initial retest retest positive positive positive anti-HCV
positive samples from France n=15 15 15 15 from Austria n=17 17 17 17 from Germany n=20 20 20 20 from USA n=49 49 49 49 total n=101 1011, 1011) 1012) paired sera/plasma from healthy blood donors n = 259 sera 2 1 n.d.
n = 259 plasmas 1 0 n.d.
n = 97 samples showed extinctions >> 2.5 E which resulted in ratios > 25 at a limit of 0.10 E
n = 94 samples showed extinctions > 2.5 E.

Only 4 and 7 samples, respectively, reacted more weakly, but with E450 values between 0.8 and 2.5 and a limit of 0.10 E still rather strongly.

Tab. 2 Results of the determination of anti-HCV with an ELISA
containing the novel peptide SP 10 on the solid phase with human samples Positive with peptide ELISA (2 pg SP 10 /ml) Anti-HCV positive initial retest samples positive positive from France n = 35 35 35 from Austria n = 26 26 26 from Germany n = 29 29 29 from USA n = 53 53 53 total 143 1431) 143"

Paired sera/plasmas (see distribution plot of healthy blood donors in Fig. 2) n= 500 sera 0 0 n = 500 plasmas 0 0 1) n = 142 samples showed extinctions > 2.5 E, which results in ratios >> 25 at a limit of 0.1 E; only one sample reacted more weakly with a value of 1.2 E, but still considerably more strongly than with the commercial ELISA (HP 1).

All of the tested human samples which were classified as anti-HCV positive with a commercial test (HPI) were likewise found to be positive with all ELISAs based on the peptides according to the invention. Besides the very good agreement with the results of the commercial test (HP1), the very strong signal formation in the peptide ELISAs is also noticeable. At the same time, the results of the longitudinal investigation of healthy blood donors makes it clear that the susceptibility of the test to interference is extremely low. Thus, on testing of healthy donors of serum and plasma there was found to be only extremely minimal non-specific binding to the peptides according to the invention, i.e. only a few false positive results were obtained.

Tab. 3 presents further results obtained on testing of humar- samples with ELISAs based on the use of smaller sequences (15 amino acids) of the formula (I) according to Examples 1,2 and 4. It is clear that the peptides are suitable for epitope definition of HCV antibodies and as mixture of several peptides, preferably containing 3 to 6 peptides, for the determination of anti-HCV.

Tab. 3 Reactivities of human anti-HCV-positive samples in ELISAs with smaller peptides comprising 15 amino acids on the surface of microtiter plates according to Example 1; the results are reported as extinctions at 450 nm (E,so) {- = negative finding below 0.10 E).

4056 4055 4053 4052 4081 4091 Mixture Sample 4055 No. 4052 jug/m1 2 2 2 2 2 2 0.5 each BC90-89 >2.50 0,20 - - - - 1,40 252 - - 0.20 - >2.50 - 2.10 90 0.80 - - - 1.60 - 2.00 268 >2,50 - 0.20 - >2.50 >2,50 >2.50 84 - 1.20 1.60 - 1,70 >2.50 >2,50 225 >2.50 >2.50 - - >2,50 >2,50 >2.50 270 2.00 1.70 - - 2,20 >2.50 >2.50 229 0.20 - - 1.10 1,00 - 1.50 288 >2.50 >2.50 - 0.50 >2,50 >2.50 >2,50 290 >2.50 >2.50 - 0.20 >2050 >2.50 >2.50 242 - 0.50 - - 1,20 1.40 >2.50 137 - - - - 0.40 0,35 0,70 286 - - 0.60 0.15 0,45 0.15 0.90 192 0.20 >2.50 - 1100 - - >2,50 235 0.15 - 0.40 - 0,20 - 0.30 85 - - - 0,75 - - 0,40 The reactivities obtained with a mixture of peptides according to the invention are shown. It is evident that, owing to the very strong signal formation of the peptide ELISA, a reliable discrimination between positive and negative results is achieved. Analogous results were also achieved with the following peptides or peptide mixtures according to the invention.
4074/4081 (2 pg/ml each) 4074/4082 (0.5 and 0.125 pg/ml) 4060/4071/4081 (2 pg/m1 each), where mixtures of smaller peptides comprising about 15 AA proved thoroughly suit-able:
4056/4055 and 4052 (0.5 g/ml each).
No dependence of the reactivity of the samples on their geographical origin was detectable in any case on testing with all the peptides according to the invention.
Exaniple 5 Optimization of the ELIS.A.determination The modifiable parameters of the ELISA determination which were varied were mainly the peptide concentration used for the coating, or in the case of mixtures of peptides their total concentration and ratio to one another, at a constant conjugate concentration of 1 : 3000 and 1 : 26 dilution. In addition, the conjugate preliminary dilution was varied at fixed coating concen-trations. Both modifiable variables were validated with regard to specificity by testing blood donors' sera and plasmas and with regard to sensitivity by determination of anti-HCV in positive groups. Furthermore, the limiting sensitivity was measured in the form of the analytical sensitivity by serial dilution of anti-HCV-positive samples (1 : 2, 1 : 4 etc. in anti-HCV-negative sera) and compared with the data of a commercial test in just the same way as with the results achieved using the peptide described in the literature.

The results obtained with human samples are compiled in Tab. 4 by way of example.

Tab. 4 Comparative titration of anti-HCV-positive sera and plasmas in the peptide ELISA (4083) and a commercially available test. The ratios are reported as quotients of the specific signals to the limit, and values greater than I indicate a positive result and values smaller than 1 indicate a negative result.

Peptide ELISA Commercial Serum (4083) test (HP1) Dilution 2 g/ml 4083 in neg. serum limit 0.10 E limit 0.454 E
HC 90-84 1: 1 > 25 > 6 1: 2 > 25 > 6 1: 4 > 25 > 6 1: 8 > 25 > 6 1: 16 24 > 6 1: 32 10.7 4,1 1: 64 6.1 2.0 1: 128 2.5 9-9 Imgz 1: 256 1.5 0.5 1: 512 1.1 0.5 1:1024 D_z,¾ nea, 0.2 HC 90-90 1: 1 > 25 > 6 1: 2 > 25 > 6 1: 4 > 25 > 6 1: 8 > 25 > 6 1: 16 > 25 > 6 1: 32 11.8 5,4 1: 64 5.9 3,7 1: 128 3.3 1.4 1: 256 1.8 ,Q.2 1: 512 1.08 0.4 1:1024 A.--I flea. 0.3 1:2048 0.4 0.2 HC 90-252 1: 1 > 25 > 6 1: 2 > 25 > 6 1: 4 > 25 4.5 1: 8 20 3.2 1: 16 12.2 2,7 1: 32 6.5 1.2 1: 64 4,2 P-1 npg 1: 128 2,4 1) 0.5 1: 256 9.17, marg.
1~ marg. = marginal Continuation of Tab. 4 Peptide ELISA Commercial Serum (4083) test (HP1) Dilution 2 pg/ml 4083 in neg. plasma limit 0.10 E limit 0.454 E
HC 90-296 1: 1 > 25 > 6 1: 2 > 25 > 6 1: 4 20 > 6 1: 8 8.5 5.0 1: 16 3.7 2.9 1: 32 2,9 Q..ZnS-cj-.
1: 64 1..4 0.5 1:128 Q,,,.$ necz= 0.4 1:256 0.3 0.2 HC 90-83 1: 1 > 25 > 6 1: 10 20 2.1 1: 10 0 6.5 Q-A n2i HC 90-240 1: 1 > 25 > 6 1: 10 8 1.5 1:100 1.4 1. 1 nea_ HC 90-239 1: 1 > 25 > 6 1: 10 7.8 2.-0 1:100 1.4 " ngg, HC 90-89 1: 1 > 25 > 6 1: 10 > 25 > 6 1:100 9 3=5 HC 90-137 1: 1 > 25 > 6 1: 1 21 4..2 1:100 4 1.4 The results in Tab. 4 make it clear that the sensitivity of the ELISA based onthe peptides according to the invention, measured by the limiting sensitivity of serum titrations, is at least as good as the commercial test (HP1) which was tested comparatively with identical serum dilutions. In fact, in many cases, diluted samples which already gave repeated negative reactions in the - 39 -= 2054798 commercial test (HP1) were still measured as signifi-cantly positive with the peptide ELISA so that the overall result was a better detection limit of HCV
antibodies with the peptides according to the invention.
Analogous results were likewise achieved with other peptide concentrations, peptide sequences or mixtures of the novel peptides; such as, for example, 4083 (0.25 pg/ml) 4074/4081 (0.25 g/ml each) 4074/4082 (0.5 g/ml each) 4074/4082 (0.5 and 0.125 pg/ml) 4060/4082 (2 pg/ml each) Beyond the determination of the limiting sensitivity, in the case of optimized systems the specificity was also examined by means of non-HCV-specific antibodies and other potential interfering factors, and it became clear that the anti-HCV determination with the peptides accord-ing to the invention is specific for HCV and is not subject to any known interferences such as, for example, cross-reactions of other antibodies, interference by heat inactivation or the like. This also applies correspond-ingly to other stated novel peptides or peptide mixtures, with which uniformly a high seruin concentration (dilution 1 : 2 in sample buffer) was used in order to increase the sensitivity, which was possible owing to the purity of the peptides and the high antigen density on the solid phase.

The comparisons summarized in Tab. 5 between another core peptide according to the invention, SP10, and HP1 like-wise make it clear that the peptide according to the invention has, compared with the state of the art, advantages which in turn comprise improved limiting sensitivities, i.e. increased analytical sensitivity.
Thus, the 4 titrations in Tab. 5 were determined a factor of 2 to 4 more sensitively than with the published peptide, and the factor was even 8 to 32 compared with a commercial test (HP1).

_ 40 - ~~54798 rn~ ~s x b ~ ="~ v Q~
N R! 04 ~
s~ cna~b a a ov~i~wa~~~ a 049 ctl 0 r w o N m O
tA ctl A U N =~ > u9 c~wf r~d Itf ORi ~x+, Q U U i a s=
b H~r-1 Q a~i o O cv N
=H rl yd 4J ~=-I A n n (U+4 9 N ~ p 0 b x 04,N O c: rl 1 , O rl N
'JI =v U N p =~ rA
U N ,~ q - Cr 0 S1 ~,1õ~ =rl H ~ O e0 6s ~0 rl o 'O
O d-) N 4 ~ ~ ~ N 119 ~ ~ ~G Ln r) O4.) 41 0-5 N O O O O ~V N
U) w.[ 3 b~ ~ U O 8 A A
R9 O +) 'p 0 w N
A O=rl ~ = Q) d+
- O 47~ N
V2 O 040 U N d, i ~-i O ~p qp O r~ O O O
H=~-I !!] F U U 'L~ O O e!' l~l O 6D O O
+J ~ o Uz = S-I \ it1 o t11 N ri O tf1 Ll1 UrC d-~ N M C) = = e = o = o, fCS TJ N S-1 0 O a ~ N N rl O C1 Q C~ N N
A A A
p cvbm orocvNro ~ ,~ .-= ~
~a~i~~~~
-I cn Tt N O N .-1 => N tn R

04 b ~
N A Q 0~
O O O N e"1 ' N N v O O
N f 1=~-I 1=d 0 tdl d O ad' CO rl t'1 N M O O
N CC 4-3 tA U N 1n LL9 1`= 1- If1 N r1 0 Y1 lty tA N Gd 0 cfi 1tl U 0 = = o = = = = = =
O Q O ~ ~,~ N N O O o O O O N N
f 10 ~. A A A A
b~r' S~r >~' ~
U = . I A *-a 4) +~ f-I ~ U) =1-I .G N caa O
>4 CO 3~ vVi~ ~ r r + i ~ =~ M ~1 O o~o N~o ca o0 0 0 0 O O dJ r"1 o N t6 O O
ro.N 5 +) .0 O -P 4J H ~ In cn Ln O ko eaf O sn tn = e. = = = A
~4 a) V a' = e ~ N N r+ r+ O O O a N N
~ u D i~~--~ w ~ c~ n n A A
N 0'O 04 U+
44 4-3 oro.'d ~i~p,`C=r~+
o~~~c0a~ i~
cn N (D 4 o a) 0 rI 1* o ~~=~b ~io=.> ~ 0 RJ 4-3 =d ;3 -P 4-J N ~' tOV ~ ~ ~ 0 sn , a,.s~ ro s.~ a~ U = ~ In (n oc ri N sea ra N N.=a 1 es so 0o s= oe == ee 1 =e ~ v3~a,ba~ z ~

Ch H !`d i 4 P'd -1 7 4 9 I $~1 r~
rri E (~ =s~i x - -' e e e ~ a e o ~1 ~1 O N O N O ~e ~ O N O
o IL1 O O N o O co Lt9 h O tt1 o itY !'% E"1 r9 !n 01 01 N M 6n e"8 N
= s = 0 b s te y m N Pi O O O N r-0 O O O N O O
A A A
@% M V {C1 tL1 O N 4O QO %D rl O L4 N
m O e1 rl rl O N rd t- Oi eP O 0c N 0D
~ CO -n N rl In N O ~' O O It9 S'~ tt9 O
o o 6 o s o =
rl O O= O N o o+ o o a A A
O Ln sr r l t0 O O O 60 e'1 N ri O .O A N Ln P LR9 N
N Ln rl CD V C0 O O i~ v N N LL"f O ri t0 6:0 10 ep t~
O 1O ep N r1 O tf1 t~ ~tN C~ t~1 a ~ O tt1 1 N t0 M rl O
o = e = o - s O o = = = = e e N O O O O O N N rl O O O O N ri e^ii~ O O O O
A A A

u1 O O e1 e-1 e-1 O O O W N P9 U1 O O P1 t='1 O O O v w N m t~ m CN O O O O O CO t~ O O P1 t~ rl e-I 11 CO
O ln P t"1 ~-I e-I 1f1 lf1 tf1 eP OD eM tn tC1 CO CO ~' M r~ d = s = = s s = v + s e a = a N+ i O O O O N N N~ I O O O N N rd O O O O O
A A A A

d 9~ rl rl !D un O O O N O !D O O O t"9 0 tV 1D h O tG !v ~D O ~ O O O ri 0- e'1 O O O O a~l s0 N 6K1 1C1 l0 B- ~ et N U1 6tl lf1 t~1 ~f 6t1 1~ Il1 BC1 ia C1 6A C"9 ri = ~ p ! s s o s e =
N ei O O O O N N N a-1 O O O N N N ~i O O d O
A A A A A A A
Ln ro ~

w p V RV d CO !O N N O %0 O 1D N N ei' 4; t~7 ¾p N 1f1 r1 O 0 %D N v N tCf qr N I* N Ln e-1 O d U C1 !D e-i N Lf1 r4 [- W rl F9 %0 ri N 1!1 i1 i kO rd N L41 r I N
=ri N ( 1 .P .. .. ,. .. .. .. e .. .. .. .. .. .. e .. .. .. .. .. .. ..
~ d o 0 ri rl W-4 a-t a-1 r-1 C, w! ~4 am1 ~i rd r4 M s-0 ri eA wl yd r4 r=I

r~:

U

Evaluation of another core peptide according to the invention (SP 23) gave similar results. The comparative results presented in Tab. 6 reveal that SP 23 is equiva-lent to SP 10 with respect to sensitivity, and both peptides are superior to the peptide described in the ]. iterature .

Although the literature-analogous peptide SP 12 (AA 47-75) does not correspond exactly to the peptide described by OKAMOTO et al. (AA 39-74), the results on the inter-mediate sequence (SP 11, AA 24-53, Tab. 6) make it clear that no immunorelevant epitopes are present in the following sequence region (AA 39 - 47), and the very weak reactivity of SP 11 which is still detectable is attribu-table to the carboxyl-terminal region of SP 10 contained therein (overlapping region AA 24-30 shown in Fig. 3).

Tab. 6:
Comparison of the i.mmunoreactivities in the ELISA between 2 peptides according to the invention and an amino-acid sequence which is located between the published sequence (AA 39-74, OKAMOTO et al.) and the novel structures (SP 11, AA 24 - 53, see also Fig. 3). All data represent extinctions (Epso ~~=

Samples AA 8-26 AA 1-30 AA 24-53 2Ag/ml 2}cg/ml 2 gJml pos. contr. > 2.500 > 2.500 0.405 neg. contr. 0.051 0.049 0.030 Dilutions 1 16 2.203 > 2.500 0.428 1 64 1.329 1.367 Q.õ16,4_ 1 256 0.454 0.386 0.024 1 : 1024 0i195 0.142 1 16 > 2.500 > 2.500 0.164 1: 64 1.413 2.156 0.059 1 : 256 0.418 0.496 1 1024 Q.124 , Q,.-M
Anti-HCV-positive samples (commercial test HP 1) HC 90-371 ~ 2s-Q85 0,224 HC 90-336 > Z.L' OQ > 2s= Q2235 HC 90-453 > ,2.500 > 2.sL00 0.058 HC 90-570 1.827 2.222 0.088 This surprising advantage in sensitivity becomes parti-cularly clear on testing undiluted samples. Thus, all 11 - 44 - 20547"M
of the 11 anti-HCV-positive samples shown in Tab. 7 were found to be reactive with the peptide according to the invention, whereas the peptide described in the litera-ture finds only 6 samples positive, and in no case was there a positive reaction with the commercial test (HP1), With respect to the sampl s which agree in reacting positively with both peptides, it is noticeable that the peptide according to the invention reacts significantly more strongly under identical test conditions, which provides considerable advantages especially for positive/
negative discrimination.

N
re4-J ro~ a 0t!]=~+1 Ga L, ~
0, o a ow 1-, =
`~~I -A - ; t;t;~t;~, 0 Gl ~1 ~ W ~ 91 Q! ~ 41 a! 0 ~ .., v Q z z z z z z z z z +J 43 0 N la 0 o x 0 v 0 eo 6 r, eo N1 o ri m ~' O
N j ' ~'' ~ U r+ 0 c- o ~1 ao cn r1 co ~o 1-4 O
N ro a O N N t"1 O O rl O t~ w=1 Il~
r-{ = o s = s = o a o 0 ~ o O O O O O O O O N
=~ 60d SUd '=~d 0 A
.=e 0 0 tT 0 O
r-i 4R!=~ a .~

9 4 M >
O tn ,, ~ 0 a a) =~ tr+.,',~ H
-H tr1 44 -i~ ~ O Csa ~ ~ 4) Q) ww Rl vi'nN~ ro ~0 o x z x r ~
W 0' H ai m1-1 o in r-+ ca ea o r+ ~ a~ r~ as o N U U'~ 0 ~ ~ 0 l!1 N W O tn Co tl' U1 f' f = O
.. 0 d' N N tC1 O O O N N G U 1 bd N 4-) 4.1 =
U ~ = -i= ON a m Oa O o a Oa O
H 0 -11 ~ N O N N
~~p~k ~ ~ .N-i .N A A
aro a ~ u~ ~
i ci co 0 00 w 4+it9>o o =H a) =H
r-i 4J
oou 4 ~0~~ ~ -N o =~ ~~~ 0 U N ~~ N t~ O O O O O O e0 O~+ O
V1 0+=~1 }~ 0 ="+ e 111 ri o o O o O Fe o 0 t~ f., rci r. tn si' 66! 111 1~1 !`9 l~1 tl9 N f~ N 1t!
Q1 Ql =r{ (p ~ =H :% .. = a s = o a a U1 41 'O td W (L) W ai N N N N N N N N N ri N 10 .

0~ 010 6d ~i =~I .[ N 0 A A A A A A A
'ri 4-! ~(d la ~ q W c~I2 U
O~NUN0 r+viNt) ~ i =rl Q~ ttl ~ a (a =PI =rl Q) ~0 ~= ~' O~ O M r1 ~ N aei t0 +~= 119 1-I ~. O.!'C~ 4 "~ N O Co O N 1 O rl @'9 t"1 9% 0 RI ¾J =ei dJ 0 N N N M C 1 el' tn tn u1 N1 tl' tl' C1i td 4.) 41 1 1 I I 1 ! I 1 1 I I 1 w C4 "i tA 0 O O O O O O O O O
t- U W 0 04 i ~ z 0 J1 ~Y1 ~tt 4J1 ~t 01 0- tA - 9- ~1 tU 0 E-i t!a These findings (Tab. 7) derived from the titrations (Tab. 5) and preselected native human sera with the core peptides are confirmed by the results of the tests on commercially available serum panel (LOT # PHV 101 and LOT # PHV 201, Boston Biomedica, USA).

Specifically, the results of the so-called low-titer anti-HCV panel are summarized in Tab. 8 and the results of the mixed-titer anti-HCV panel are summarized in Tab. 9. The results of the data obtained with the core peptide according to the invention are compared to the data obtained with commercial tests as state of the art.
Tab..8 Tab. 8 shows the comparison of the sensi=tivity of a peptide ELISA (SP 10, 2 pg/ml) with the state of the art for native anti-HCV-positive samples in the panel PHV 101, comprising 15 low-titer well-characterized anti-HCV-positive samples (material and comparative test results marketed by Boston Biomedica, USA). All ELISA
data are ratio values, which describes the ratio of sample extinction to cut-off. Values < 1.0 are regarded as negative and > 1.0 are regardled as positive.

~
_ 47 .'., I.a .

~
~
~
=~
04 .~
~t F-4 =~ .~
N
.jJ ~ PJ $' O O 0 k-l ~ I c0 tt1 c d1 tfl tff U1 t1f u1 t1Y u1 ~!1 U1 W yJ v~ ri N N N N N N N N N N N
A A A A A A A A A A A

U~ In Np Co N t~ Upl 0 tpn V1 tpIJ L'I tPJ 0 A~ G~ QQi 6~ G~ W LL~ Gl~ R~+ PI P~ W
va H RV ao M N aP eo Oh oo O Ln O % 0 ^la ~-l O e0 N P~0 V N c0 rl B1 N O P a Rr ~ ~ C~7 C9 9i N M O ai ~C t0 ti f rl O OD N P ~ N
= = = = = o = s s = = = m r, U O O ri M N a-1 r 4 1-9 rl M r I N N O M
~4 Iy =,~
i-I H P P O P 6A' O r+l 61 tP1 1 N OD Ln N 60 fti Ds7 O r! W C LL'f O O O tS1 ~= e0 v 1 O r4 W
~ C~ P H O P ~ 1o N O OD tp e0 O m S1 60 = o o m m m = = = m = m m U'~' ~ M e-d ri M ~=i N t`l yd P9 O M tV a-1 ~"9 e=i N M V M %D P O M a4 N M iL9 O O O O O '~S O O P"~ 1 11 P' ~ ~ C'~ @ 1~
1 1 1 1 1 1 1 1 1 1 i 1 1 1 1 ri rd W-1 r^0 r4 ri rd e-i v-d aml w=1 r4 rd r4 r4 co f~ N ~ ~ ~ ~ ~ ~ ~ A -I t-4 P4 -1 '.4 ri 0-4 = ~ ~ ~ ~ ~ ~ ~ ~ '~ ~ ~ ~ ~
~r Q4 &4 iSa 8m R~ 6~
~ H 13e 6a 04 !S+ N aa N 04 ~

2 0 5 4 7 49 $

Z Z Z Z Z Z.Z Z Z Z Z Z Z Z Z ~I 0) Id r N
Of-1 H O
O
Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z =N

s-I 'CJ

=ri UI
L~ N =~ .
t! Z Z Z Z
rj 41 rU
1=I O
* I tU =rl a a.a a a a'-a c~ a a a a a a a ~b~
(1) N 3 O
rl id =W =rl t A C7 ~-1 Zz U 11 44 o a.=H w 0 r-IHO
mu~
U N =s=~ N
I O tA
rV av I - e =~ =cr e ~, r~ r~ .r Z 0 =~-I

E- .II 44 =ri O
r-Ui 1 084 V ~1 1 .* I -4 -4 ~ w tV -a v ~= ~ rn Z +J r-~I
4 =Pi rl 4 O 1~ O b ev e^^~ 'y =.1 44 co =~ ~ o =rl H b=I
-~j= 1 ~ I I I ~_. I + 1 1 ~ b ~ =I ~
+ I + +
H ~ ~0 O N O
o-a O +~1 .w 1 ~ I \ 1 \ I =w -m N z m ~ 'P =
+ + + =~ + + N 4J W

=,j .=-1 N ry ~P tn 60 P % O a=i N N9 w U1 4J O O O O 0 0 0 O O W ~I nt vi 1 'J R9 =J (A
I I 1 1 I I I 1 1 1 1 1 I I I --14 tu 0 ".9 y 3a .=i =N =~i ~=1 vi o.d <W ..d .=~ .~I m-4 .==1 .=1 .=i .-1 d, =N =I=-'f .' Ca7 O O O O O O O O O O O O O 0 =11 t!I td .=r ~ ..a -4 .-i -4 -4 -4 ~ --4 -4 tfa tA =N =IJ

0 ato H
tn il S=I
0 a E-i Q) a* ~

It is evident from the data in Tab. 8 that, with one exception (PHV 01-03), all anti-HCV-positive samples give a correctly positive reaction with the novel peptide. It is noticeable, by comparison with the commercial test, that the signal strength, expressed by the ratio sample signal: cut off, was found to be significantly stronger with the peptide according to the invention than with the assays tested for comparison, which expresses a distinctly improved reliability of the novel peptide ELISA.

According to these data (ratio > 25, which corresponds to extinctions > 2.5), the samples are low-titer only according to the definition of the state of the art and are found to be, surprisingly, strongly reactive with the novel peptide.

The one exception (sample 03) is explained by the data of the comparative results on the confirmatory test which demonstrates absence of core-specific antibodies in this sample (C22c is the core-protein prepared in E. coli by genetic engineering). According to this test, sample 03 ought in fact to be classified as negative.

The tests on the second panel surnmarized in Tab. 9 led to similar observations: 19 of the total of 22 anti-HCV-positive samples are found, surprisingly, to be extremely strongly positive with extinctions greater than 2.5 (corresponds to ratio > 25). The three anti-HCV-negative sera contained in the panel are correctly classified as negative (ratios < 1.0). Finally, samples Nos. PHV 201-08, -10 and -20 are also found to be correct within the scope of the problem because these samples exhibit no (-08 and -10) or few (-20) core-specific antibodies according to the confirmatory test.

Tab. 9 shows the comparison of the sensitivity of a peptide ELISA (SP 10, 2 g/m1) with the state of the art for native anti-HCV-positive samples in the panel ~~~4798 PHV 201, comprising well-characterized anti-HCV-positive samples with different anti-HCV titers. (Material and comparative test results marketed by Boston Biomedica, USA). All the ELISA data are given as ratios which represent the quotients of sample extinction and cut-off value. Values < 1.0 represent a negative result and values > 1.0 represent a positive result.

_ ~ 51 .,j -P
a~ =~

w ~
Q+ O M

~rl f: N =ac N Ta N N
3~o C op o C
LP1 Lf1 LP1 7l1 L'1 LP9 t!'1 LP1 L=1 Ln Ln 6P1 0 0 {19 U1 119 61"f 3J9 LP1 O O N N N N N N N N N N N N N N Pl N N N N N
F-+UPaU nn n nn n nn n ns~nnn I~nnnnn U tA, W R1~V) a N U) dJ H
a .
6f~ L^1 .^.. ~.^. L'~ =^~ Lr. 'C' 61 CS f9~ N^,,. N N P1 O! ~ L9 o N dl c` 4 Z. %A `:. "` .-> %Z C %3 P'1 A9 . ~-I U C a C~ .~ 1t1 Q ~ N N!d 1 T+ V M !6~ O= tf1 f~1 s9 tZ 4~ !:~ %. x ~ .
= . . = . . . = . . . . . . . . . .
~+ M P`1 G> N'~ J ==~ ~1 N N T L'~ =C 1f9 N'f tr1 P9 ~a N N.~ 6P1 N tP1 ~

it P"5 .~l !=. ar ~-1 H tT :J LI1 'V L"1 O% L'f 6f1 tf1 C C> =a 0 -W tf1 o tf1 o`? C~ !== .. ... y"f w P1 M1 -.l:. - - ==w r1 C9~ .r r'1 N.a ^~ av a W !e~ "V P"o N g 1~ N 6n Yf "~ C.7 V" ='~ 'd' =~ w~ wM !11 r a"a T!*= f 6P'f .~{
s s s =
rz > =~r1r~ ~ e~e ~~~aNNNf~.4- N9 =eQtn Nr=^w Nr O U

.w N r'S ~ 1d1 s~ 1~ C~ ~~==a N P! ^~ 6H ~D P~ 9= C~ oma e Q e=y ~= !A . ..
d~ a ~ C ~ O O ~ ~ ~ ~ e~ +', .~c N ~ ~ ~ .~a .;v eV t ~d Pi ~ t r~ N
W W ..a =ar .w ~ IIIII I ~a .w .w .a .ot ~a . a .n .a .a ..+ .r w .ti ..=+ =~
.y .~t a W At~ C~ o0 oCaoo C?oC ~:a~ OC c~o~ ~:~
õC~ ~," ='~ N N N N N N N N N N N N N N N N N N N N N N N N N
E-4 z m w.=. a e '~. a. .. a.e m~. Ls m m. w ao Ge .m w. ~ ~ 'w a'n E

2054y~98 -m 'ti a4J ~
o~ , 4j 01 N 1 1 , t 1 ~=~p a e~V ee e~+, n, . V
~i' W r-1 a~ = o o ' ~^i 1~
ti) O 0 tl9 td1 iP1 N1 L^1 6A 1l1 m L 1 L 1 L'1 Lf1 L!1 6,'1 Ln Lrl 6,=1 LPi {d9 tm ~y,i 1~ /~ \ N N N N h N N N N N N N N Caj N N N N N O
( ya~-tn nn r~ nn n nns~r.nnnn Annnnn Ow a 4-) 44 ~

ZZ: ZZioZ~Z8~~ZZ ZZZZZ ~2Z. ~^ 3~ o~t =~1 ta R

eI O $d (0 TI`I zzzZZZZZZZZZZ~zazzzzZzzZz .0 t~ =~1 >sf ~~b ra, =~
i S]~ND
yI zzzzz r_zzxzzzzzmzzazxzzz~z 04 m z4.) di u No U O .r=l z~zzzzz;;~z~.z xx~~o . $, .N -r ~ =.~ ~s ~ ' ~M_ =rM _^ _ a aru44 a r ~q qoy = M P1 == , M ~ N Yd PA IW I 0 le~ r = bM b! il W 44 m= =1 1~ ~ V
M1I ~ Y.^ w 0, r-I f=! A
e N 0 4J=0 `n tn mEn0 -1 o=~
1~ 1-1 O f=t - , NN *a ~ vg 1 '~ = I I QY 1 e 1 M 4T ~ '~' a~ 7^, 1 ~ ~` ~` ~ s .~=) ri O tC
;444 ~ i N a I t. r 1 r~~Nr~N~~e~r ~=~ 1~ew a r~~ 4J ib1 rd~
O U ~= 0 04$4 z 04 ~ wN 1 1 I 1 I 1 I t ii O tOAoOG
ro L=r O 1 N -+ 1 \ ~=1 1 "s 1 \ \ N ~ \ "~ ~ '~ N w o~ \ I P9 ~
E. e. ..a .r =~ 4. v Z
N i, 44 o C N!V L 1 a N N PV Nv w t0-4 a.d \qv N@ to Irl ~ 0 * y ) ~ ~ 4~, co ..N~~ ~neAw~e~o ~Nt~1~at1,At~0e~o.rN~q~=u~
N N N =~ aj4J
0 0'6~ C> o o b o o ..a .~ +a! s~l e~ aw sv ~ W t~l N N

7 ~ i t 1 I t 1 1 1 1 1 1 I 1 I 1 1 I 1 I 1 I 1 9 I Oto 'I to C ,q + as w - - - ..s - s.a mi P1 0) :3 ~~=~~ OP~ O C~'ea 0 C OOOO G? OOOcoOOO Fa tAr;-4 2: 2t 2: 2: N N N N N N N i=V N N N N N N N N N N N N N N
rr u ~
Z ~
U~"",~ C+e.~ ce G~o ~: E:cobA E-+01 a* ~.0 Example 6 Determination of anti-HCV in chimpanzee sera with the peptides according to the invention in the ELISA
Various peptides and peptide mixtures adsorbed in wells of microtiter plates were investigated with sera from chimpanzees which had been infected with various infec-tious doses of NANBV. The sequential samples taken every 2 weeks after inoculation underwent measurement of not only GOT but also GPT with commercial tests, and all samples were determined in parallel and in the ELISA with peptides according to the invention. Similar to the commercial test (HP1) in Example 3, in the peptide ELISA
the polyclonal rabbit antibodies, labeled with POD, were used concentrated 4-fold for the detection, using the enzyme substrate TMB with photometric measurement at 450 nm. The test procedure corresponded to the determina-tion of human anti-HCV described above, with 0.1 E as fixed limit and presentation of the results of Tab. 10 as ratio of the specific extinctions to this cut-off value (ratios).

The results listed in Tab. 10A to 10C and 11A and 11B with peptides from the NSP 4 region re:present so-called ratios with which the quotient of specific signal cut off is defined.

Tab. 10 A- C
Anti-HCV determination in chimpanzee samples with a commercial test (HP1) and ELISAs with peptides or peptide mixtures according to the invention from the 1NSP 4 region. The data for the first 3 time values are extinc-tions which serve to fix the limits, from which the subsequent ratios of specific signal and limit have been formed.

_ 54 -u 00 a~ www O e~v~~ 0 melNmrlch ~
::r 000 .-1 OO OOO% hcnMM
C~ ~ 9 = = a a = = 9.

0 to N LI $1 $.i M 14 D4 W W W k 1d 0 =11 O
O a) W w[s'I W 6~ d f16 O OOOOOOtflo a-h44' ~
m ~ = e Q O uOMO= Oi =rl sri e =09 O Q O OOOrl0 s1~ O 3d bd ~1 ba ~J 74 $d }~l 7d kd ~1 0 b O
LO rl Nhm G1iddWO
~ MNrI 0 ~-iNa-INr~M r' \
000 H 000000 t=9qw hh1S1 M Ol M A 9 9 9= a= O= f = w ~) ~ oao 0 00o00ott1h6i9Nr1 -4 cr N }d Xd Sd LI fd ~1 ~1 $d id 1.1 7d 04 U
rn N V
~ =ri ~1i a w~ww 00o w u~ o oo~ s~
~ ![1 ~D m h o0 a9 t` C ln l4 h OD L19 !D %D
N . o s s = o e eOa==OO s a=Os V
0 ~ O O O O O O O O O O O
U+) ~4 34 b.13rkIS.1!=1W 34 kM
N
H tclt~t7 Q-OO~t10000N1t~90 N N N t'9tl1U1td1~h ~ e'NNC9 4-) U
A QI

=~
;t.
re Sd M 0 W "
N =i -N ra N
~ 0 ~ () õH d.) .f.~
tC =r1 rl 3 0 Ci N tn 4-)4~a ~ 4V wONqw t OON:fi%0 U
N~rw0 Pa~~rtr4 N N N N
o Q
un W W W

cnot-N Ilf f"1 O
q 0) 000 H %D O NNqie?l70d 0190M1116L16t11L9 p 0 = 07 07 Oo Oe ~ = N1 O 'D N
00 I~' C M=NANANA AN
N
.d4 C 1Nr4 s4 NNv-i riN
La W Sa !"d 6+ W 14 W W ld $4 14 W k+ 11 fa O
!!1 ry .
~ W W W Ed W O O 0 O d O d O O O O O O
~ ~
M 0) M%w M O LO
C- Z. O O O rd t0 0 01 N W W t` td1 P tl9 t8'f tt1 111 tt1 tSY
q m= m m a o m s a s e ao s s eN N N N
N 000 O OO~NhNrItt1M~-iri~= A A A A
rl ri r-1 ri r1 H ri N
~+ ~d ~d ~d ~i 7d 5.1 ~=1 S.1 ~A ~1 k b.i kl ~=I 3a U

Op U) q~\ W W W W ~~~ O O
co r rn q 000 0 000 ri Nrle-INNrIriNNNNMOOr-!ln zv (N
= m= m a e a= e e= e= s m 00 0 000 00000000r-dN NN
0 b i Sa LI Sr 44 LI 3d bd ba !d &d !=1 Ll !+
rI ~
~ W W W ool N V1 U! I!I dOd0 U1 N N N 1- O O ~~11 ~O O
r') N l, lC) 0 00 U1 O O O a-1 lf1 0 1 oD CO 1~ ~t N C 1L1 O 1S1 lC1 q . o = s = = + = = = = = m o = 0 e N
N
cM O 000 0 Or1~ MN~IL1l~1o LC1lDhNN A A
O id !4 ~=1 $d $4 ~1 ~1 L1 i=1 $1 ~d ~4 $4 Ifl OH ko W O O O 0 0 0 0 O O O O 0 O 0 00 Z3. 000 rl MOl~Q!=N~O=7'C9COOO~ON~=elOtttl 0 == q e a m : e m m==== s= a N
d~ N 000 O Or Ir1P1~ODO0Q~OQ1P~tAt01~ A
r-I -= {=1 ~1 ~=I {=1 ~ I ~1 f=1 1 1d ~d ~=I ~=1 ~1 ~=I =1 00 tCS r-1 n =~x ~ W ~s~ ~~ ton N W ~1 W W d alftl9ul h w v POc9co ao toao 00v rllT
O O m==
C) 4-3 O O Oo O= Om e eo 00 00 00 07 0P r1m NN = s Ne ~

~a !d !.1 S.1 !4 ~=1 Sd 1.i bd 5d S.1 S=1 Sd 5i ~t =PI
E O
O Ic1 4=1 N O N O lL1 6~ t[1 M b O O It! tL1 OD em D ~~y NNN t90~~MNMNNNNNNNNNN ,.i e-I

00 O W ~ b~
to f0 t!) W 0 O O 04 ~
z O U =A =~
CD 0 P ~= ~, fC! v =rl ~i =a 4-) (a =H ',4 A ='i ~ II k' Q)=~
~ GlN~'mCDONaO'~00N=~ ~OSnO ,., H ~ H qN~ WTJr-1 v-+rI.~~a-1NNNNNMMMMM+T ~{

- 56 _ 2054798 U O ~
N 'y DO
ro oo~ o ~ ~N~ d c94\ N M N O
d CT 000 e-9 Me=11[90I rIt[fsr'o ld100 h ~ = = e = o 0 o a = = .
O 000 0 0000~1 MMt~~leollt'1 ei N rl e~
$d ~d ~i ~1 ~J bi $1 ~d ~d ~d ~d bd f=i U O
ro u I
cor-i www W ~0 ~ad o rg eoNLn O r=1 r-1 0 O tT 000 H rirlNNHriNIW 1ICttil-0 ~ t . o . O. O. a O= O. OO a . O . O .=
o O O O O O N N M
`f' N
$4 w 3~ $4 $4 !=+ Fa $a Sa }a ba Sa #a O
Ln ~ Q1M w W d 0 01 1 0 1ad 6.Y 6.sf WW
C) ~ N M O
:a. 000 r^d NM W~01~OC-N 1r1l- t~t~1 = a s . = a~y! O a= o= o a= q (y ~ ~ 000 O 00 lf1 Mt0 V=t9~0ep01~ A
Co r1 r1 N
xr $d f-1 $4 51 k 34 $d $1 ~i ~=1 O
Ln ~~~ W ol1sI~~~~~p~~~
M m OOO H Ned~tff&Q10tl1~9101MS"0-C U1 ~ o s s . =rp~ o a~ = e o s=. o aN
Q 000 O O 10r=dt~NOODrIMC1 O/~
et N r9 "^I rl rl yd r=i N
k 14 $d W 14 14 bd M $d N

m WwW ~0 v 0 0 1C9 O
~p tt~ ^s4' t~ t~1 tt1 SO m~0 d' i[1 ~D ?~ W O
O O O o= O= s Oq O= Q= Oo O= O. Oa O= Oa Oo =~
4-) O O ~~ 4-) 3=1 ~d ;d S=1 bd rbl ~d ~1 ~=1 ~=1 ~=I S d =1 `rl CO

H Oa Ocno Nu1o ~nO~nNN~~~n~
MNM lnY`r4~=!~~ltlln~'a0"~'Mad' ~
.o r.. ~=
=,-I
r ~
t~ 0 -i co Pa ~
m (tt tA O
~ II
t~ 0 0) 0 0 4 z 4) U ==-I 4-) 0 0 ~~~
r-{ '--I -0 =='{ Ci 0 +J R1 rE4 ~ II d~ 4-4 =
rtS i~ rl ~C ~==I tOOON~ 1DOON~'60oi1O
H rC H N=W w'd~ 9-4 p+NNNNNMMMMMV

- 5' 2054r198 The results presented in Tab. 10 A - C underline the advantages of the peptides according to the invention, especially on comparison with the corresponding results from the commercial test (HP1) and the ALT course.

Thus, for example, animal No. 123 is not found to be antibody-positive at all by commercial test (HP1) although NANBH was detected by electron microscopy of liver biopsies. By contrast, a reliable HCV antibody detection is possible with the ELISA based on 4083 almost at the same time as the ALT increase.

In particular, the chronological congruence of the peptide ELISA results with the increased ALT levels is evident for animal No. 048 where with all the described peptides or peptide mixtures there is a very reliable, in the sense of a sharp positive/negative discrimination, anti-HCV detection almost at the same time as the ALT
increase and on average 18 weeks before the commercial test (HP1). The comparisons among the peptides make it particularly obvious that the cer-tral amino-acid sequence makes a considerable contribution to the recognition of the early HCV antibodies by the complete peptide of the formula I(4083), as is made clear by the comparison of the mixture of 4060 and 4081 (amino- and carboxyl-ter-minal sequences) and 4090 which comprises the central structure.

Similar results of an early identification of HCV anti-bodies, almost simultaneously with ALT, were produced by the investigations on animal No. 147 in which a reliable HCV antibody detection is possible approximately simul-taneously with the ALT increase, again about 16 - 18 weeks earlier than with the commercial test (HP1).

Tab. 11 A and B
Anti-HCV determination in serum samples from 2 chimpan-zees infected with HCV. The results with the commercial test (HP1) are presented in comparison with a syntheti-cally prepared core peptide according to the invention (SP 10, 2 g/ml), the results being presented as ratios of specific signal and limit.

- 59 -=
Animal No. 048 ALT Commercial SP 10-ELISA
Time (weeks) HCV ELISA (HP1) 2 g/ml 0 = inoculation 20 0,5 E492 0 09 E450 25 0 5 E 0,21 E
23 0,5 E 0,13 E
Extinctions (E) define the limits:
0,70 E 0,10 E

8 XA_tjos (r) 34 r 0,4 0,50 32 r 0,6 r >20,0 12os.
12 100 r 0,4 r >20,0 pos.
14 112 r 0,7 r>20p0 Ros.
16 30 r 1 0 r >20,0 gos.
18 25 r 0,7 r >20,0 Ros.
30 r 0,8 r >20,0 pps.
22 25 r 0,8 r 19,0 p-os.
24 23 r 0,8 r 20,0 pos, 26 20 r 0,8 r 18,5 nos.
28 20 r 0,8 r 10,4 oos=
20 r 1,0 _ drgl) r 14,2 pps.
32 25 r 1,3 Izos. r 17,6 uos.
34 25 r 2g0 pos. r 15,4 R s.
36 28 r 2,4 320s. r >20,0 pos.
38 28 r 2,1 ngg- r>20,0 PQS.
: 26 r 2,9 pos. r >20,0 R2L&.
1) marg. = marginally positive 60 - 2 0 5) 4 7 9 8 Animal No. 147 ALT Commercial SP 10-ELISA
Time (weeks) HCV ELISA (HP1) 2 pg/m1 0 = inoculation 30 0,60 E492 0'013, E450 25 0,50 E 0,032 30 0,45 E 0,029 Extinctions (E) define the limits:
0,70 E 0,10 E
16 ratios (r) 52 r 0,5 r 0,2 18 75 r 0,5 r 0,1 20 110 r 0,8 r 0,2 22 40 r 0,6 r 0,4 24 75 r 0,6 r 0,3 26 70 r 0,4 r 0,2 28 55 r 0,5 r 0,3 30 52 r 0,6 r 0s2 32 42 r 0,7 r 0.4 34 48 r 0,8 r >20,0 nos 36 48 r 0 8 not tested 38 35 r~. as, not tested 40 45 rpos. r>20.0 p s.

1) marg. = marginally positive The results presented in Tab. 11 A and B with a novel core peptide also underline the advantages of the pep-tides according to the invention, especially by com-parison with the corresponding results of the commercial ELISA (HP1) and the ALT course.

In particular, the chronological congruence of the core peptide ELISA results with the increased ALT levels is clear for animal No. 048 where there is a very reliable, in the sense of a sharp positive/negative discrimination, anti-HCV detection almost at the same time as the ALT
increase and on average 20 weeks before the commercial ELISA (HP1).

Similar results of an early identification, almost simultaneous with ALT, of HCV antibodies were produced by the investigations on animal No. 147 in which a reliable HCV antibody detection is in turn possible about 4 weeks earlier than with the commercial test (HP1).

Overall, the peptides according to the invention and the use thereof in immunochemical detection methods prove to be considerably more sensitive than all hitherto des-cribed methods based on prote:i.ns prepared by genetic engineering and other synthetic peptides of the state of the art. With improved sensitivity in later phases of infection, the novel peptides additionally provide considerable advantages in that reliable determination of early HCV antibodies is possible and thus the currently existing diagnostic gap is significantly reduced.
Furthermore, the peptides according to the invention are considerably less sensitive to non-specific bindings, which is expressed not least by a drastic reduction in the background compared with the commercial test (HP1) (max. 0.10 E45D compared with max. 0.4 E4az with the commercial test (HP1)), both for samples of animal and of human origin so that a considerably sharper negative/
positive discrimination is possible. Finally, advantage-ous aspects to be mentioned are the shorter overall duration and the grea=ter precision of determination owing to the sample volume of 50 pl which can be pipetted more accurately.

Example 7:

Preparation of peptide solutions for producing a ffiizture of NSP 4 peptide 4083 and core peptide SP 10, and coating of microtiter plates with this peptide mixture Serial 2-fold dilutions in 0.10 M sodium bicarbonate pH 9.6 were made up from stock solutions of the peptides SP 10 and 4083 in 50% acetic acid in distilled water each containing 6 mg of peptide/ml, that is to say a series with the concentrations 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, 0.2, 0.1, 0.05 and 0.01 pg of peptide/ml was obtained. The procedure was analogous for mixtures of single peptides, the stock solutions additionally being mixed in various ratios, for example 10 : 1 or 1 : 4, in order to obtain by dilution in 0.1 M sodium bicarbonate the overall final concentrations which are indicated above but which in the case of mixtures of several peptides contain the latter in equal concentrations (in the case of 1: 1 mixture) or in various ratios to one another.

In each case, 100 l of each dilution were placed in 16 wells of microtiter plates, type B supplied by Nunc, Roskilde, Denmark. The test plates charged with the dilutions were left at 20 C for 18 hours and then the solutions in the wells were removed by aspiration and the wells were washed 3 - 4 times with 300 pl of a solution of 10 g/1 bovine serum albumin in phosphate-buffered physiological saline (PBS, pH 7.4) by filling and removal by aspiration, and the test plates were subsequently dried over silica gel at 20 C.

A concentration of 1 pg of 4083/ml and 1mg of SP 10/ml has proven suitable for coating the peptide mixture, this concentration representing the basis for the results which are summarized in Tab. 12 to 14 and were obtained as described in Examples 4 and 5. In contrast to previous comparisons with the state of the art, all subsequent comparisons will relate to a commercial anti-HCV ELISA of the 2nd generation (HP 2) as described in Example 3.
Tab. 12 and 13:
The data on the ELISA according to the invention and commercial HP 2 represent so-called end point titers with which the highest preliminary dilution, which was found to be reproducibly positive in a given test, of a serum in anti-HCV-negative serum is defined.

Tab. 12: BBI Low-titer anti-HCV PANEL PHV 101 MEMBER ELISA according to Commercial anti-I.D. the invention HCV test NUMBER SP 4083/SP 10 (HP2) 1 g/ml each PHV101-01 1 16 1: 2 PHV101-02 negative negative PHV101-03 1: 4 1 : 1 PHV101-04 1 : 512 1 : 64 PHV101-05 1: 2 1: 1 PHV101-06 1 : 128 1 : 32 PHV101-07 1: 128 1 a 128 PHV101-08 1 : 64 1 : 16 PHV101-09 1 : 64 1 : 16 PHV101-10 1: 256 1 : 128 PHV101-11 1: 32 1: 16 PHV101-12 1 : 512 1 : 128 PHV101-13 1 : 16 1 : 64 PHV101-14 1: 64 1 : 32 PHV101-15 1 : 128 1 : 64 Tab. 13: BBI Mixed-titer anti-EICV PANEL PHV 201 MEMBER ELISA according to Commercial anti-I.D. the invention HCV test NUMBER SP 4083/SP 10 (HP2) 1 g/ml each PH'V201-01 1: 1000 1 : 128 PHV201-02 1: 1000 1 : 500 PHV201-03 1: 128 1 : 32 PHV201-04 negative negative PHV201-05 1: 64 1 : 64 PHV201-06 1: 64 1 : 256 PHV201-07 negative negative PHV201-08 1: 4 1 : 16 PHV201-09 1: 1000 1 : 512 PHt72 O 1-10 1: 4 1 : 16 PHV201-11 512 1 : 128 PHV201-12 1: 32 1 : 16 PHV201-13 1 : 256 1: 256 PHV201-14 1 : 64 1: 64 PHV201-15 1 : 512 1: 1000 PHV201-16 1 : 512 1: 512 PHZ7201-17 1 : 128 1: 256 PHV201-18 1 : 16 1 : 128 PHV201-19 negative negative PHV201-20 1: 4 1: 4 PHV201-21 1: 500 1: 256 PHV201-22 1 i 16 1 a 256 PHV201-23 1: 256 512 PHV201-24 1: 256 512 PHV201-25 1: 1000 1: 512 Tab. 14:
Results of the tests on 930 healthy blood donors from whom serum and plasma were obtained simultaneously.
Specificity Specificity in % after in % after initial tests repeats n= 930 sera 99.5 99.7 n = 930 plasmas 99.6 99.7 Example 8 Preparation of peptide solutions for producing mixtures according to formula XIV with peptides of the formulae XVIII, XIX, XX and XXII, and coating of microtiter plates with these peptide mixtures The polypeptides SPH 9 (formula XVIII), SPH 20 (formula XIX), 4083 (formula XX) and SP 10 (formula XXII) were dissolved at a concentration of 6 mg/ml in 50% (v/v) acetic acid.

These 4 stock solutions were mixed in various volume-based ratios as described in Example 4 and diluted in 0.10 M sodium bicarbonate (pH 9.6) so that the total concentration of the polypeptides was between 0.2 and 8 pg/ml.

In each case 100 l of each of the diluted solutions were placed in 16 wells of microtiter plates, type B supplied by Nunc, Roskilde, Denmark. The filled test plates were incubated at 20 C for 18 hours. The solutions were then removed by aspiration and the wells were washed 3 - 4 times with 300 pl of a solution of 10 g/l bovine serum albumin in phosphate-buffered physiological saline (PBS, pH 7.4), and the test plates were then dried over silica gel at 20 C.

Example 9 Optimization of the concentration of HIV 1, HIV 2 and HVC
peptides for the coating mixture, and optimization of the ELISA determination and evaluation criteria Starting with a ratio of 1: 1: 1: 1(V/V) for the four stock solutions described in Exairtple 8, all four modifi--able contents were varied independently of one another but keeping the three others constant in each case in order to obtain the following final concentrations of the individual peptides of HIV 1, HIV 2 and HCV in the coating solution (in g/m1):

Pepticle Peptid e Peptide Peptide xvTli xix xX xxll 0,5 2 2 2 0,2 2 2 2 0,1 2 2 2 0,05 2 2 2 Peptide Peptide Peptide Pept;ide XVIII XIX XX XXII

2 0,5 2 2 2 0,2 2 2 2 0,]. 2 2 2 0.05 2 2 etc. to 0.05 0 05 0,05 0 05 - 67 - 20e9 4r! .0-18 These mixtures were immobilized as described in Example 8 on microtiter plates and evaluated in the ELISA as described in Example 4 and 5, with the concentration of the peroxidase-labeled antibody likewise being optimized against human immunoglobulin G.

The samples used to evaluate the optimization were composed of low-titer anti-HIV 1, anti-HIV 2 and anti-HCV
samples which were prepared by serial dilutions of corresponding positive human sera in negative human serum. In addition, several anti-HIV and anti-HCV-nega-tive samples were likewise tested in order to be able to define the background reaction (i.e. the non-specific binding on a given coated microtiter plate).

Set up as selection criterion was a signal of maximum specificity, i.e. high limiting sensitivity in the determination of the titration series of human anti-HIV-and anti-HCV-positive samples with, at the same time, a low background in the tests on anti-HIV- and anti-HCV-negative samples.

Coating mixtures which were usually favorable were found on the basis of these criteria, from which the following mixture was selected:

XXI SPH 9 0.500 g/ml SPH 20 0.250 g/ml SP 4083 0.500 og/ml SP 10 0.125 pg/ml A conjugate concentration of Example 2 of 1 : 3000 with a uniform additional final dilution of 1: 26 was found to be favorable, the test being carried out as in Example 4. In contrast to the previous establishment of limits, under these conditions samples having an extinction at 450 nm which is greater than the mean of the negative controls plus an addition of 0.250 O.D. were classified as anti-HIV- and/or anti-HCV-positive (new - 68 - ~~~4798 establishment of limits).

These establishments were applied uniformly and unchanged to following Examples 10 to 15.

Example 10 Determination of human antibodies of the immunoglobulin G class against i3iV 1, HIV 2 and HCV in the ELISA

The samples shown in Tab. 15 - 17 were tested as described in Example 4 under optimized conditions of Example 9 with the peptide mixture of the formula XIV.
With regard to anti-HIV 1 or 2, the reactivities of the method according to the invention were compared with an anti-HIV 1/2 combination test (EnzygnostR anti-HIV 1/2, supplied by Behringwerke AG, HP 3). Commercially avail-able Western blots (anti-HIV 1 and anti-HIV 2) supplied by DuPont were used for control. HCV was detected with the aid of two different ELISA methods (HP 1 and HP 2).
As is made clear by the data in Tab. 15 to 17, anti-HIV 1 and anti-HIV 2, as well as anti-HCV, are detected safely and reliably in samples of human origin with the method according to the invention.

Tab. 15 2054798 Determination of a.nti-HIiT 1with the method according to the invention compared with an anti-HIV 1/2 combination test (EnzygnostB anti HIV 1/2, HP 3) Strongly reactive means extinctions > 2.5 in the photo-metric evaluation; all 76 anti-HIV 1-positive samples are HCV-negative Method Cce[anercial anti-F3IV 1/2 Number of Origin of according combination samples samples to the test invention n = 12 Europe n = 12 n = 12 (anti- strongly strongly HIV 1 reactive reactive positive) n= 57 West n= 57 n= 57 (anti- Africa strongly strongly HIV 1 reactive reactive pos.) n= 7 West n= 7 n= 7 (HIV 1/ Africa strongly strongly HIV 2 reactive reactive coinfec-tions) n= 76 n= 76 n= 76 positive positive Tab. 16 Determination of anti-HIV 2 with the method according to the invention compared with an anti-HIV 1/2 combination test (Enzygnose anti HIV 1/2, HP 3) Strongly reactive means extinctions > 2.5 in the photo-metric evaluation; all 28 anti-HIV 2-positive samples are HCV negative Method Commercial Number of Origin of according anti-HIV 1/2 samples samples to the combination invention test n= 21 West n= 21 n= 21 (anti- Africa strongly strongly HIV 2 reactive reactive positive) n= 7 West n= 7 n= 7 (HIV 1/ Africa strongly strongly HIV 2 reactive reactive coinfec-tions) n= 28 n= 28 n= 28 positive positive - 71 - ~~54793 Tab. 17 Detection of anti-HCV with the ELISA method according to the invention Samples were termed strongly reactive when they achieved extinctions > 2.500. So-called ratios are stated for the moderately strongly reactive samples, which means the quotient of extinction of the sample to cut o.ff of a given ELISA. Values below 1.0 are, by agreement, termed negative. Values above 1.0 as positive, the reactivity being stronger as the resulting ratio increases; all anti-HCV-positive samples are HIV-negative Number Origin Method Commercial anti-of of the according to HCV ELISAs samples samples the invention HP 1 HP 2 n = 61 Europe n= 57 n 57 n= 57 Anti HCV strongly strongly strongly positivLo reactive reactive reactive Europe n '- 4 HC 90-346 > 9.0 ++ 2j7 (+) > ,~i,.,_Q ++
- 3 51 ,A + ne ga t i v e ,;}õLa +
-516 g$ + negative J'Q +
-570 Aj + ILI (+) > 510 +

n= 53 USA n= 51 ng51 n- 51 Anti HCV strongly strongly strongly positive reactive reactive reactive USA n S 2 HC 90-511 $,,z + III (+) 3.99 +
-566 A,_1 + negative > ,5"9Q +
n = 114 n=114 n = 111 n- 124 Positive positive positive Example 11:
Determination of the analytical sensitivity of the ELISA
according to the invention for anti-HIV and anti-HCV
compared with the state of the art As a model for the assessment of the analytical sensitiv-ity of the ELISA according to the invention, titrations of positive samples were prepared and tested in the ELISA
described in Example 9. In total, dilutions of three each of anti-HIV 1- and anti-HIV 2-positive human sera in anti-HIV- and anti-HCV-negative serum were prepared and the limiting sensitivity defined as the last preliminary dilution still reactive in the particular test system using the new limit (mean of the negative controls plus 0.250 threshold).

The results of these limiting sensitivity tests are compiled in Tab. 18 (anti-HIV 1), Tab. 19 (anti-HIV 2) and .and Tab. 20 (anti-HCV)_as extinctions.

~ ~~ 5 3 ~~ ~~ z~ ~~n ~~
Tab. 18 Determination of the limiting sensitivity of the ELISA
according to the invention for anti-HIV 1 comvared with the anti-HIV 1/2 combination test (Enzygnost Anti-HIV
1/2).

Three confirmed anti-HIV 1-positive samples underwent preliminary serial two-fold dilution with human anti-HIV
1- and anti-HCV-negative sera, and these dilutions were tested as in Example 3 or in accordance with the pack insert of the manufacturer of the comparative test. All the data are extinctions (E450)= All anti-HIV-positive samples had a negative reaction in HP 2.

Sample Preliminary Extinctions in Extinctions dilution the ELISA in the anti-according to the HIV 1/2 com-invention bination test (HP 3) cut off=0.282 cut off=0.268 0.D. O.D.
8803/26 undiluted > 2,500 + > 2,500 +
1 : 128 > 2,500 + > 2,500 +
Anti 256 2,334 + 2,185 +
HIV 1- 512 1,408 + 1,412 +
positive 1024 0,825 + 0,837 +
2048 D,._4&7 + 0,457 +
4096 0,240 - 0,216 -8000 0,137 - 0,135 -8611/144 undiluted > 2õ500 + > 2.500 +
1 : 128 20162 + 2,014 +
Anti 256 1õ366 + 1,028 +
HIV 1- 512 0,806 + 0,648 +
positive 1024 Qc-42A + 0.454 +
2048 0,280 - 0 258 -4096 0.153 - 0,136 -8000 0,088 - 0,076 -Anti 1 : 400 ~ + 2458~ +

positive Anti HCV- 1 256 2. 219 + 0,014 positive (negative) Tab. 19 Determination of the limiting sensitivity of the ELISA
according to the invention for anti-SIV 2 comnvared with the anti-HIV 1/2 combination test (Enzygnost Anti-HIV
1/2).

Three confirmed anti-HIV 2-positive samples underwent preliminary serial two-fold dilution with human anti-HIV
1- and anti-HCV-negative sera, and these dilutions were tested as in Example 3 or in accordance with the pack insert of the manufacturer of the comparative test. All the data are extinctions (E450). All anti-HIV-positive samples had a negative reaction in HP 2.

Sample Preliminary Extinctions in Extinctions dilution the ELISA in the anti-according to the HIV 1/2 com-invention bination test (HP 3) cut off=0.282 cut off=0.268 O.D. O.D.
8804/103 undiluted > 2,500 + > 2,500 +
1 : 128 > 2,500 + 2,500 +
Anti 256 2,420 + 2,045 +
HIV 2- 512 1,488 + 1,339 +
positive 1024 0,737 + 0,705 +
2048 Q,aaa65 + Qj $5 +
4000 0,196 - 0,190 -8804/117 undiluted > 2,500 + > 2,500 +
1 : 128 > 2,500 + > 2,500 +
Anti 256 1,860 + 1,902 +
HIV 2- 512 1,233 + 1,100 +
positive 1024 0, Ei56 + 0,667 +
2048 P.O,;= + D_j 0 2 +
4000 0,166 - 0,172 -Anti 1 : 400 1,945 + +

positive Ant,3..FICST- 1 256 2 219 + 0.014 positive (negative) Tab. 20 Determination of the limiting sensitivity of the ELISA
according to the invention for anti-HCV.

Four confirmed anti-HCV-positive samples underwent preliminary serial two-fold dilution with human anti-HIV-and anti-HCV-negative sera, and these dilutions were tested as in Example 3. All the data are extinctions (E450 nM) =
Sample Preliminary Extinctions in Commercial dilution the ELISA anti-HCV
according to ELISA (HP 2) the invention cut off=0.481 cut off=0.270 HC 90-85 undiluted > 2 500 + > 2,500 +
1 : 16 > 2,500 + > 2,500 +
Anti 1 : 32 2,299 + > 2,500 +
HCV- 1 : 64 1,858 + > 2,500 +
positive 1 : 128 1,515 + > 2 500 +
1 : 256 1,045 + 1,707 +
1 : 512 0,648 + 0,871 +
1 : 1024 0 322 + 0.492 +
1 : 2048 0,207 - 0,255 -HC 90-225 undiluted > 2,500 + > 2,500 +
1 : 16 > 2,500 + > 2,500 +
Anti 1 : 32 2,214 + > 2,500 +
HCV- 1 : 64 1,816 + > 2,500 +
positive 1 : 128 1,488 + 2,008 +
1 : 256 0,689 + 1,120 +
1 : 512 0,580 + D'= +
1 : 1024 Q`Z86 + 0,375 -1 : 2048 0,148 - 0 111 -Anti,HCV-positive HC 90-270 1 : 256 0222 + 1.544 +
HC 90-354 1 : 128 &,221 + 28291 +
Anti positive 8803/26 undiluted > ~~500 + 0,110 -8811/144 undiluted > 2 00 + 0,098 -8803/24 undiluted > 2"500 0m068 Anti = (negative) positive 8804/103 undiluted > 2.500 + 0,075 -8804/117 undiluted > 2.500 + 0,081 -8804/116 undiluted > 2.500 + 0 099 -, (negative) ~ a r - 76 - 2 t~'e~ r~ ~t ~(3 The procedure was analogous for anti-HCV in that serial dilutions of four anti-HCV-positive samples were prepared as described in Tab. 20, tested and evaluated in analogy to anti-HIV and compared with the results with the commercial anti-HCV ELISA of the 2nd generation (HP 2).
In addition, the specificity of the reactivities of individual donations were also tested in the individual tests by also investigating the anti-HIV 1- and anti-HIV
2-positive samples undiluted in the anti-HCV test and, conversely, the anti-HCV-positive samples in anti-HIV 1/2 combination tests (EnzygnostR anti-HIV 1/2).

The results compiled in Tab. 18 to 20 make it clear that the limiting sensitivity of the ELISA according to the invention for both anti-HIV 1 and anti-HIV 2 corresponds to the limiting sensitivity of the anti-HIV 1/2 combina-tion test. The HCV limiting sensitivity of the ELISA
according to the invention also corresponded to the limiting sensitivity of a commercially available anti-HCV
ELISA (HP 2). However, while a total of at least two tests (anti-HIV 1/2 combination test and at least one anti-HCV test) are necessary to detect these three antibody specificities accordinci to the state of the art, this detection is possible with the method according to the invention just as reliably and with comparable sensitivity using only one test mixture.

It is furthermore evident from the data in Tab. 18 to 20 that the strong reactivity of the anti-HIV- and anti-HCV-positive samples in the novel ELISA is particularly advantageous because the anti-HIV samples reacted nega-tively in the specific anti-HCV test and the anti-HCV-positive samples reacted negatively in the specific anti-HIV test.

_ 77 -Example 12 Determination of anti-HIV 1-positive samples from early stages of infection (seroconversions) with the ELISA
according to the invention.

Tests were carried out on a total of 3 patients from whom sequential blood samples were repeatedly obtained at defined times during very early phases of an HIV 1 infection. These serum panels are commercially available (Boston Biomedica Inc., USA). The results which were obtained with the ELISA according to the ~nvention compared with an anti-HIV 1/2 combination test have been reproduced in Tab. 21.

Tab. 21:
All data are extinctions (E450 .) ~ and values above the particular limit are to be regarded as positive.

Tab. 21 Sample ELISA Commercial anti- Commercial code according to HIV 1/2 ELISA anti-HCV
the invention ELISA (HP2) 1 A 0,023 0,066 negative 2 A 0,020 0,055 A
3 A 0,055 0,193 4 A 1,163 + > 2,500 +
A > 2,500 + > 2,500 + n 6 A > 2,500 + > 2,500 + e1 7 A > 2,500 + > 2,500 + 91 8 A > 2,500 + > 2,500 + "

9 A > 2,500 + > 2,500 + 'e 1 C 0,014 0,022 negative 2 C 0õ015 0,054 "
3 C 0,054 0,145 t9 4 C 0,500 + 1,138 + 11 5 C 0,630 + 1,707 + "
6 C 0,889 + > 2,500 + "
7 C 0,942 + 10 8 C 1,010 + 11 ' 9 C 1,081 + 10 i: 1,564 + t1 i 11 C > 2,500 + n u 12 C n oe n 13 C u u C n ee 16 C u n n 17 C 1 ei 18 C n aa G BBI
80 0,012 0,056 negative 81 0,013 0,053 82 0,018 0,059 83 0,478 + 1,112 +
84 0,733 + > 2,500 +
85 0,612 + > 2,500 + n 86 0,763 + > 2,500 +
87 1,296 + > 2,500 +
88 1,309 + > 2,500 +
89 1,544 + > 2,500 +

cut off 0,281 0,285 0,481 The results in Tab. 21 make it clear that the ELISA
according to the invention detects low-titer anti-HIV 1-positive samples just as reliably as the anti-HIV 1/2 combination test (EnzygnostR anti-HIV 1/2). It is likewise evident from comparison with the data in Tab. 21 that the earliest time at which first detection of HIV 1-specific antibodies is possible with the novel peptide ELISA is also possible at least as early as with specific single anti-HIV 1 tests as state of the art.

Example 13 Determination of low-titer anti-HIV 1-positive samples with the ELISA according to the invention Tab. 22 summarizes the results which were obtained in the tests on a commercially available anti-HIV low-titer panel with the novel HIV/HCV peptide ELISA compared with an anti-HIV 1/2 combination test (EnzygnostR anti-HIV
1/2).

The sample panel supplied by Boston Bicanedica; Inc. , USA, comprises a total of 15 samples which were classified by means of anti-HIV 1 determination methods as low-titer anti-HIV 1-positive.

Tab. 22 Results of the tests on the low-titer anti-HIV 1 panel supplied by Boston Biomedica Inc., USA

All the data are extinctions at a wavelength of 450 nm.
Values above the stated limits are positive.

- 80 - 205d798 Tab. 22 ID No. ELISA according Commercial anti- Commercial to the invention HIV 1/2 anti-HCV
ELISA HCV ELISA

BO 1-01 > 2,500 1,872 > 2,500 +
02 1,979 1,575 2.023 +
03 2,056 2,280 negative 04 1,751 1,960 05 1,781 2,044 06 1,428 1a728 07 1,183 1,564 08 0,641 1,152 09 0,984 1,499 "
"
0,682 0,940 11 1,302 1,650 12 2,032 0,540 1,833 +

13 10017 1,854 negative 14 1,079 1,484 "
> 2,500 2,090 > 2p500 +

cut off 0,279 0,265 0,479 The results in Tab. 22 reveal that, compared with the anti-HIV 1/2 combination test (EnzygnostR anti-HIV 1/2), all the samples are found to be comparably strongly reactive with the HIV/HCV peptide ELISA according to the invention.

It is interesting that four samples in the panel are in fact found to be more strongly reactive with the novel peptide ELISA than with the anti-HIV 1/2 combination test (EnzygnostR anti-HIV 1/2) (No. 1, 2, 12 and 15). Addi-tional tests on these samples revealed the simultaneous presence of HCV antibodies, which again confirms the reliability of a non-differentiating detection of anti-HIV 1/-HIV 2 and -HCV by the method according to the invention.

Example 14 Determination of low-titer anti-HCV-positive samples with the ELISA according to the invention Tab. 23 contains the results which were obtained with-the novel HIV 1/HIV 2 and HCV peptide ELISA and the low-titer anti-HCV panel supplied by Boston Biomedica Inc., USA.
This Tab. 23 also contains data which were obtained on the same panel with a modern ant:i-HCV ELISA (HP 2).

The data on the ELISA according to the invention and commercial represent so-called end point titers with which the highest preliminary dilution, which was found to be reproducibly positive in a given test, of a serum in anti-HCV-negative serum is defined.

Tab. 23: BBI low-titer anti-HCV PANEL PHV 101 MEMBER ELISA according Commercial anti-I.D. to the invention HCV test (HP 2) NUMBER

PHV101-01 1: 16 1 2 PHV101-02 negative negative PHV101-03 1: 4 1: 1 PHV101-04 1: 512 1 64 PHV101-05 1: 2 1: 1 PHV101-06 1 : 128 1 32 PHV101-07 1: 128 1 128 PHV101-08 1 : 64 1 16 PHV101-09 1 : 64 1 16 PHV101-10 1 : 256 1 : 128 PHV101-11 1 : 32 1 : 16 PHV101-12 1: 512 1 : 128 PHV101-13 1 : 16 1 : 64 PHV101-14 1: 64 1 : 32 PHV101-15 1 : 128 1 : 64 - 83 - 2 0 O, 17 9 8 The results in Tab. 23 make it clear that all 14 positive samples were found reliably and unambiguously to be positive with the ELISA according to the invention. It is noteworthy in this connection that the signal strength is very high, which is attributable to high reactivity.
This high reactivity is also reflected in the limiting sensitivities found in Tab. 23. These detection limits were defined by subjecting the native human samples to preliminary serial two-fold dilutions in anti-HCV-nega-tive serum and then to testing as in Example 4, where the last preliminary dilution stage which is still found to be reactive represents the so-called final titer.

On this basis, the data in Tab. 23 reveal that the novel HIV 1/HIV 2/HCV peptide ELISA has in fact better detec-tion limits for anti-HCV in the PHV 101 panel than the state of the art.

Example 15 Determination of the rate of non-specific reactions of the novel peptide ELISA in a group of healthy blood donors To determine the frequency of non-specific reactions, which lead to false positive results in the ELISA, a total of n = 512 healthy blood donors was used. Serum and plasma were obtained simultaneously from these donors in '25 order to be able also to investigate a possible interfer-ing effect of the coagulation system on the method.
Samples which were reactive in one of these three tests (ELISA according to the invention, anti-HCV ELISA HP 2 and anti-HIV 1/2 HP 3) (so-called initial tests) were repeated in the same test (so-called retesting) and, where the reactivity was reproducible, investigated in the anti-HIV 1 Western blot and anti-HCV ELISA (HP 1) as comparative methods.

It was not possible to confirm as anti-HIV 1- or as anti-HCV-positive by means of this confirmatory method any of the samples which emerged in this screening as reactive in one of the three ELISAs. Thus, all the samples which were rejected in the screening were consistently classi-fied as false-positive in the particular methods.

Tab. 24:
Screening of n = 512 healthy blood donors from whom 512 sera and 512 so-called paired plasma were obtained simultaneously. The findings after the initial tests are indicated; see Tab. 25 for the retest results.

U9 N !~ ~
s s e~ ~
M M r1 r9 ~^I
(j) s = s \ !o \ \
O
=rl tti Ul a +.) x ro ro ~ . .
~ m 0+
w U

=rl U
> U) ~ 0~
ro ~4 ~ \ \
z ~ ~ a o ~n ~
.
U2 = = s = + =
~
' (d +) N Rf G ~ c0 C~~ 1C ~
pa N
ri ~"/' ~ \ \ \
2 SU-i tUn 04 o .-d O
U M N Oi = = o U) a I ri C
s = = =
~ o ro ~ \ \ \ B+ tT tT ~+ ~ ~+
a~ ~a ro r+ o .t (1) d+ ro a~ ~ . . m ~ M 04 ..~
'CS
O
U
(d 0 a) .4 p Lo 0 ~ N +J \ ~ N
~ P 9 ~ ~ ~ \ \
.~ Z S-I m LL M O

rd w~ t~l t"~ ~' BS1 M M H ~

a b ~
(0 ~ o rl sa cn U ~` ~' -~ 86 Itt N

N
N N
vi N
_ d' =
~ U N '-1 C75 01 o a O1 1 w =~I ~
N N
`n o = N
N ~ ia d`
:n v Ch m ~ rn v, ~
.,.,, ~ ~ M o ~ -4 U) co --ro .
> M ~ ~, O-cn ~ Q, rn rn ~ x P4 ~ ~ 9 o 0 ~ ~
rt] )`I =
o a~ ' N

N
Q) +,1 IC) F-1 A
04 W ~ O
Q) y ~c w ~ O ~ N = N =

O. o, o, a a w ~ ~I o LO
~ cn N
4-) O O ah arn .,q a to O, =~ 0 .,~
f) O

N
Q) N
~ ri W N
O cG d" ^
... I dP
~
tA 04 U1 1 to to 1 to a4-)-I O -P -i U ~ ) d N -i a1 'C3 ~ ~O to . ~ ~
N L11 O r-I 0 N O 4=) t!] (tl S 1 0 ed to 41 U CO U] ,F) -1 =
1~ N= ~ w ,1 44 rC O M r1 44 H ~ ) O -1 N U
N a~I O 0 44 N ~ =t) 0 N N ~ 44 =,I
~ ~ ~ ~ ~ ~ ~ ~ ~
u') Ln O A O4-) C: U) N d-) M Re ~ II II R9 .C ~=O C; (=-1U =O ~ d~
7r Pa =rl rl t[f tA
'-1 -0-) H ~C 4~4 .L; .~ f-I

- 87 - 2 () 5 d '7 8 The results in the first series of tests (initial results) are compiled in Tab. 24. Whereas three sera (and two corresponding plasmas) were reactive in the method according to the invention, 1 serum (and one correspond-ing plasma) was found to be reactive with an anti-HIV 1/2 ELISA and five sera (and four corresponding plasmas) were found to be reactive with the anti-HCV ELISA. The donors reactive in the particular ELISA were not identical.

After repeat tests had been carried out on these nine reactive sera, the picture which emerged was that depic-ted in Tab. 25 that two sera (two paired plasmas) were retest reactive in the peptide ELISA according to the invention, one serum (one paired plasma) was reactive in the anti-HIV 1/2 ELISA, and four sera (four paired plasmas) were reactive in the anti-HCV ELISA.

On the basis that it was not possible to confirm any of these samples as anti-HIV- and/or anti-HCV-positive, the specificity data presented in Tab. 25 were determined for each of the ELISAs investigated and for sera and plasmas by calculating the percentage of samples found correctly to be negative (in total 512 ser.a and 512 plasmas).
Based on the data in Tab. 25, according to the state of the art - obligatory tests for anti-HIV and anti-HCV on each donation, a total of five donors would have had to be excluded:
One donor repeatably false-positive in the anti-HIV 1/2 test and four donors repeatably false-positive in the anti-HCV test. By contrast, the number of donors possibly found with a false reaction is only two in the ELISA
according to the invention.

in summary, it can be said that the novel peptide ELISA
for the simultaneous non-differentiating detection of anti-HIV 1, anti-HIV 2 and anti-HCV at least corresponds in terms of the currently valid criteria of sensitivity to the in each case optimal performance features of the - 88 - ~~54793 two single tests anti-HIV 1/2 on the one hand and anti-HCV on the other hand: the data from the tests on panels, i.e. native samples containing anti-HIV 1 and/or anti-HIV
2 and/or anti-HCV indicate comparable efficiency just like the tests on the limiting sensitivities and time of earliest recognition of low-titer anti-HIV 1 or anti-HIV
2 or anti-HCV samples. Whereas detection of these three antibody specificities is possible according to the state of the art in this manner only with three different or, in view of the anti-HIV 1/2 combination tests, with two different tests, the ELISA according to the invention has the advantage that, while the efficiency is the same, only one test need be carried out. Particularly for blood banks which are obliged to carry out tests for anti-HIV
1, anti-HIV 2 and anti-HCV, the novel peptide ELISA means a considerable reduction in effort and costs with at least the same reliability and safety in the determina-tion of anti-HIV 1, anti-HIV 2 and anti-HCV.

It was additionally found, completely surprisingly, when determining the susceptibility of this novel test to interference that, by reason of the very good specific-ity, i.e. only low number of false-positive findings, fewer retests and fewer confirmatory tests are necessary and, moreover, fewer donations have to be rejected than with the conventional screening procedure using an anti-IHIV 1/2 test and, separate therefrom, an anti-HCV test.
This means that the method according to the invention has economic and chemical advantages which are also offered by other constellations of HIV and HCV peptides of the fornlulae IV to XII and XIII to XVII.

The method according to the invention is also extremely suitable for other problems in which maximal optimization of limiting sensitivity is of most importance. Thus, it is possible to show under other test conditions or, for example, by calculation in Examples 10 to 14 that it is perfectly possible to produce sensitivity features of the method according to the invention which are significantly better than those of the state of the art if somewhat less favorable specificity data are accepted, although these still correspond to the state of the art.

If, for example, the limit (Example 9) were to be reduced to 0.1 extinction, all the limiting sensitivities for anti-HIV 1, anti-HIV 2 and anti-HCV would be considerably improved by a factor of at least 2. A total of five donors would result as the rate of samples with a non-specific, i.e. false-positive, reaction in the screening of the healthy blood donors (Example 15), the donors having been found to be repeatedly reactive, which corresponds absolutely to the result of the two single tests, although the sensitivity of the antibody detection was considerably improved.

Claims (34)

1. A peptide which reacts specifically with antibodies against HCV and consisting of one of the following amino acid sequences:

2. A peptide which reacts specifically with antibodies against HCV and consists of at least one of the following amino-acid sequences:

3. A peptide which reacts specifically with antibodies against HCV, which consists of at least one of the following amino-acid sequences AA n-QRKTKRNTNRRPQDVK-BA m where AA and BA are any desired amino acid, and n and m are each, independently of one another, integers from 1 to 40.

4. A peptide as claimed in any one of claims 1-3, whose amino-acid sequence has been modified by replacement of one or more amino acids while retaining the ability to react specifically with antibodies against HCV;

wherein said amino acid replacements are limited to the following combinations listed between semicolons: Gly, Ala; Val, Ile, Leu; Asp, Glu;
Asn, Gln; Ser, Thr; Lys, Arg; Phe, Tyr; Ala, Ser; Ala, Thr; Ala, Val; Ala, Pro;
Ala, Glu; Leu, Gln; Gly, Phe; Ile, Ser and Ile, Met.

5. A peptide mixture which contains at least two peptides as claimed in any one of claims 1 to 4.

6. A peptide mixture comprising (a) at least one HIV polypeptide and (b) at least one HCV polypeptide, wherein (a) the HIV polypeptide is HIV 1 (numbering system of Ratner et al., Nature 1985, 313, 277-284), wherein the HIV 1 polypeptide is selected from the group consisting of:
IV transmembrane protein (gp 41):

AA 580 - AA 630, V envelope protein (gp 120):
AA 490 - AA 540 and VI core protein (p 24):
AA 240 - AA 390;
or is HIV 2 (numbering system of Gyader et al., Nature 1987, 326, 662-669), wherein the HIV 2 polypeptide is selected from the group consisting of:
VII transmembrane protein (gp 36):
AA 570 - AA 620, VIII envelope protein (gp 110):
AA 480 - AA 530 and IX core protein (p 26):
AA 230 - AA 380;
and wherein (b) the HCV polypeptide (numbering system of WO
89/04669 and WO 90/11089) is selected from the group consisting of:
X non-structural protein 4(NSP 4):
AA121-AA175, XI non-structural protein 3(NSP 3):
AA 1- AA 265 and XII structural protein (core):
AA1-AA80.

7. A peptide mixture as claimed in claim 6, containing at least one HIV
polypeptide and at least one HCV polypeptide, wherein the HIV polypeptide is: a HIV 1 polypeptide defined as XVIII SPH 9 (HIV 1, gp 41), and having an amino acid sequence of or the HIV polypeptide is a HIV 2 polypeptide defined as XIX SPH 20 (HIV 2, gp 36), and having an amino acid sequence of and the HCV polypeptide is selected from the group consisting of:
a HCV polypeptide defined as XX SP 4083 (HCV, NSP 4), and having an amino acid sequence of a HCV polypeptide defined as XXII SP 10 (HCV, core), and having an amino acid sequence of , and a HCV polypeptide defined as XXIII SP 31 (HCV, core), and having an amino acid sequence of

8. A peptide mixture as claimed in claims 6 or 7, wherein the peptides are linked to one another directly or via a carrier.

9. A peptide or peptide mixture as claimed in any one of claims 1-8, which has been synthesized by peptide chemistry.

10. A DNA sequence which codes for a peptide as claimed in any one of claims 1-4.

11. An antibody which has a biospecific affinity for a peptide as claimed in any one of claims 1-4.

12. An immunochemical method for the detection or for the determination of HCV antibodies, which comprises contacting a bodily fluid with the HCV
peptides as claimed in any one of claims 1-4 and determining whether the bodily fluid binds with any of the HCV peptides, wherein the presence of binding indicates that the bodily fluid comprises HCV antibodies.

13. An analytical method for the detection or for the determination of HCV, which comprises employing as specific step a hybridization reaction in which at least one nucleic acid probe which is complementary in its specific part to at least one of the DNA sequences as claimed in claim 10 is used.

14. The method as claimed in claim 12, wherein for the purpose of differential diagnosis between an early and late phase of infection in each case one or more peptides which react specifically for early antibodies and one or more peptides which react specifically for late antibodies are reacted with a sample in separate mixtures.

15. The method as claimed in at least one of claims 12-14, wherein two or more peptides are linked to one another directly or via a carrier.

16. The method as claimed in claim 15, wherein the carrier is human serum albumin or polylysine.

17. The method as claimed in claim 15, wherein the carrier is selected from the group consisting of polystyrene, polyvinyl chloride, polyamide, other synthetic polymers, natural polymers, derivatized natural polymers and nitrocellulose, and glass.

18. The method as claimed in claim 17, wherein the natural polymer is cellulose.

19. The method as claimed in claim 17, wherein the derivatized natural polymers is cellulose acetate.

20. The method as claimed in claim 17, wherein the carrier is glass fibers.

21. The method as claimed in claim 17, wherein the carrier is polystyrene.

22. The method as claimed in claim 12 or 14, wherein the detection or the determination of a peptide-bound antibody is carried out with the aid of enzyme-labeled, fluorescence-labeled, chemiluminescence-labeled, biotin-labeled or radio-actively labeled antibodies against the epitope-bound antibodies.

23. An immunological test kit for the simultaneous detection or determination of HCV antibodies, where the test kit contains one or more peptides or peptide mixtures as claimed in any one of claims 1-9 and one or more carriers on which said peptides or peptide mixtures are immobilized.

24. A test kit as claimed in claim 23, wherein the carrier is selected from the group consisting of polystyrene, polyvinyl chloride, polyamide, other synthetic polymers, natural polymers, derivatized natural polymers and nitrocellulose, and glass.

25. The test kit as claimed in claim 24, wherein the natural polymer is cellulose.

26. The test kit as claimed in claim 24, wherein the derivatized natural polymers is cellulose acetate.

27. The test kit as claimed in claim 24, wherein the carrier is glass fibers.

28. The test kit as claimed in claim 24, wherein the carrier is polystyrene.

29. A test kit as claimed in any one of claims 23-28, wherein the simultaneous detection or the simultaneous determination is carried out with the aid of enzyme-labeled, fluorescence-labeled, chemiluminescence-labeled, biotin-labeled or radio-actively labeled antibodies against the epitope-bound antibodies.

30. A test kit as claimed in claim 29, wherein the enzymes alkaline phosphatase or horseradish peroxidase are used as enzyme-labeled antibodies.

31. The use of peptides or mixtures of peptides as claimed in any one of claims 1-9 for preparing antibodies in mammals.

32. The use as claimed in claim 31, wherein the mammals are humans.

33. The use of the antibodies prepared according to claim 31 for a diagnostic method comprising a heterogeneous immunoassay.

34. An immunological test kit for the simultaneous detection or determination of HIV 1 or HIV 2 and HCV antibodies, where the test kit comprises one or more of the peptide mixtures as claimed in claims 6 or 7 and one or more carriers on which said peptide mixtures are immobilized.