AU657497B2

AU657497B2 - HCV-specific peptides, agents therefor and the use thereof

Info

Publication number: AU657497B2
Application number: AU87002/91A
Authority: AU
Inventors: Stefan Brust; Manfred Gerken; Udo Krupka; Werner Stuber
Original assignee: Behringwerke AG
Current assignee: Siemens Healthcare Diagnostics GmbH Germany
Priority date: 1990-11-03
Filing date: 1991-11-01
Publication date: 1995-03-16
Anticipated expiration: 2011-11-01
Also published as: JP2003192697A; EP0484787A3; DK0770679T3; EP0484787A2; EP0484787B1; JP4097162B2; JP4097191B2; DK0484787T3; EP0770679B2; ATE318309T1; DE59109221D1; ES2255068T5; EP0770679A2; ATE206717T1; DK0770679T4; AU8700291A; EP0770679B1; JPH04330098A; ES2255068T3; CA2054798A1

Abstract

New peptides (I) which react specifically with antibodies to hepatitis C virus (HCV) have amino acid sequences corresp. to portions of the HCV C-100-3 construct (see WO8904669). DNA sequences coding for (I), and antibodies with specific affinity for (I), are also new.

Description

P/00/01o 215l Regulation 3.2(2)

AUSTRALIA

Patents Act 1990 74 SD V

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT I I Application Number, Lodged: Invention Title: HCV-SPECIFIC PEPTIDES, AGENTS TH-EREFOR AND THE USE THEREOF "the following statement Is a full description of this Invention, Including the best method of performing it known to u BEHRINGWERKE AKTIENGESELLSCHAFT HOE 90/B 032K Ma 871 Dr. Pfe/Zi Ma 897 Ma 910 Ma 917 Description HCV-specific peptides. agents therefor and the use thereof 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.

S*

s Go.. The invention also relates to an immunochemical method for the simultaneous detection and/or for the simulo 15 taneous determination of a plurality of different anti- *0 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- OO6* 0 associated and distinguishable from other virus-induced clinical pictures which are caused by different hepatitis 0 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 investigations to define, in accordance with the transmission route, at least two types of non A/non B hepatitis (NANBH): epidemic hepatitis virus (enterically transmitted NAMBV), which is transmitted by water and foodstuffs, and post-transfusion hepatitis virus (blood transmitted NANBV), which is transmitted by blood, needle sticks or similar routes. Besides these routes of infection there are also known to be transmissions which, as -2- S- 2 sporadically occurring NANBV ("community acquired NANBV"), have no evident association with the two types mentioned. Although the exact number of agents or viruses causing NANBH is unknown, so-called hepatitis C virus (HCV) has recently been identified as a causative pathogen of this disease (WO 89/04 669).

Until recently, clinical diagnosis was based mainly on serological determination of antigens and/or on antibodies 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 abovementioned determinations were negative.

oo* 0** Besides this, so-called surrogate markers have also been e .15 used, such as, for example, GPT (glutamic-pyruvic transaminase, 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 20 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 of cases of post-transfusion 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.

3 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 the meaning 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 immunoassay (ELISA or EIA), radioimmunoassay (RIA), immunofluorescence assay (IFA), radioimmunoprecipitation (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 detecting HCV-specific antibodies (anti-HCV). The construct C- 100-3, which is expressed by genetic engineering in yeast :20 clls, from the NSP 3/4 region comprises 363 amino acids, evse 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 I 4 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 statements 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 falsenegative way. The false-positive samples may comprise up to 40% in a group of healthy blood donors (WEINER et al. Lancet 1990, Vol. 336, p. 695). The lack of a speci- 15 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-positive are still not reliably identified.

S. 0 0 0* o 0 2 To eliminate these disadvantages, other groups have to* selected particular sequences of the ORF region of C-100- 3 (from WO 89/04 669, Fig. 1) and employed them in an 2 5 immunoassay (Peptides sp42, 117, 67 and 65, Fig. On o 25 investigation of NANB-infected chimpanzees it was not o. 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 et al. Int. Symp. on Viral Hepatitis and Liver Disease 1990 Houston, USA).

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, et al. Int. Congress of Virology 1990, Berlin, FRG).

An improvement is said to have been achieved with synthetically prepared core peptide (OKAMOTO 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 alid 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.

S S 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 ,40 confirmed by a comparative investigation using the a polymerase chain reaction (PCR) to detect HCV RNA.

of a However, it is evident from the 26 anti-core HCV-positive samples found in an investigation on 606 blood donors precisely that only 10 cases could be 5 confirmed by PCR. Conversely, 16 donors 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.

1 I -6- It has now been found, surprisingly, that certain polypeptides 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 concentration, and thus a high epitope density, is achieved in an immunochemical detection method with the peptides t* 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 :25 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 AA.

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 following sequences: 121 175 SGKPAI IPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLJQTASRQA 1 337 363 HVGPGEGAVQWM2=LAFASRGNHVS P 11 Bevorzugte Polypeptide sind: Preferred polypeptides are: 121 175 S GIPAI IPDRBVLYEFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQA 6lie.

*o &a 121 160 SGKPAIIPDREVLYEFDEMEECSQHLPYIEQG'9MLAEQF 4 073: 128 160 PDREVLYREFDEMEECS QHLPYIEQGMLAEQF 136 160

EFDEMEECSQHLPYIEQGMMLAEQF

~.ve e.d a 6 a.

S

4 071 144 160

SQHLPYIEQGMMLAEQF

161 175 X9,ALGLL.,ITASRQA 144 175

SQHLPYIEQGMMLAEQFKQKALGLLQTASR",,

135 164 REFDEMECSQHLPYIEQGMMLAEQF1(QKA I 11 8- 121 135 S GKPAII PDPXVLYR 129 143

DR.EVLYREFDMEC

3 1, a 37 151 FPDMECsQHLpyjE 145 159 QHLPYIEQGM@1LAEQ 402-o.- 153 16

GMXLAEQFKQKALGL

0 0410000 O 0 0000 00 00 7 4091 *L 345 359

VQWNRLIAFASR.-

Sequences from the carboxyl- and amino-terminal region of formula 1 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: 4 060: 121 139

SGKPAIIPDREVLYREFDE

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 S S I is relevant for early identification of anti-HCV.

•goS Preferred in this connection are the abovementioned polypeptides 4071 and 4072, and peptides 4054, 4053 and S. 4052.

10 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 (Fl and F2).

Examples of preferred peptides have one of the following S amino-acid sequences: S U 15 AA1-DREVLYR-BA,

(S)

o*ooo« o •.AAa 2 -QHLPYIE-BAb 2 (Fl) AA,3-KQKALGL-BAb 3 (F2) where AA and BA are any desired amino acid, and al a3 and bl 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.

I i II 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: MSTNPKPQRKTWNTNRRPQDVKPGGGQIVGVY (III) Preferred peptides are: 1 MSTNPKPQRKTKRNTNRRQDVKFPGGGQI SP 8 26 QRXKTGRiNRRPQDVKFPG SP 23 0 so10 26 S0KTKRNTNRRPQDVFPG SP 12 26 KRNTNPRPQDVKFPG SP 30 D 0 14 26 e NTNRRPQDVKFPG SP 30 C 16 26 NRRPQDVKFPG SP 30 B 0005SS a S 18 26 RPQDVKFPG SP 30 A 8 24 QRKTKRNTNRRPQDVKF SP 31 8 22 QRITKRNTNPRRPQDV SP 32 The peptides SP 10 and SP 23 are particularly Preferred.

I I S I 11 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 reaction.

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 5 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 00* G. HCV-specific IgG and/or IgM antibodies, and it is genErally possible to discriminate between positive and negative samples with extremely high accuracy. In addition, there is generally no interferene by the on:* 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 S* plasma of human origin, and inactivation of the samples at about 56"C for about 60 minutes generally gives rise S* 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 HCVspecific antibodies from convalescent or chronically infected patients and/or HCV antibodies from acute phases of infection are described, and polypeptides and mixtures 12 of polypeptides which are suitable as Ibasis 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 further ,ore relates to antibodies which have a biospecific affinity for ,t 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 antigens 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.

a*f. 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 invention 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 onle 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 f.r raising antibodies in mammals, especially in humans.

The invention also relates to the use of the antibodies described above forx 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.

4 4 S, 13 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 small 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 '0 peptides may have better diagnostic 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 peptidos 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 particles. Thus, particularly suitable as carrier or bridge are, for example, human serum albumin and/or polylysine. It is likewise possible to modify the peptides by an extension with 1 to 40, preferably 1 to 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.

Thu), for example, the invention also relates to peptides of the following formula AAn-QRKTKRNTNRRPQDVK-BAm 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 It is often advantageous for peptides to be derivatized in a variety of ways, such as, for example, by aminoterminal 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 methylamine. Modifications of this type may alter the net charge on the polypeptide and improve the physicochemical S 15 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 epitopes 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.

.•gO 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 immunological properties of the peptides. Thus, for example, methionine is prone to spontaneous oxidation, which can be prevented by replacement by norleucin without essentially changing the antigenic properties of the polypeptide.

The person skilled in the art is aware that given amino- .acid sequences can be subjected to a wide variety of o* 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, Gln; 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 hydrophobic amino acids, such as, for example, Phe Ala Phe Ala Phe.

1i 16 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 complementary 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, radiometric, visual or by the aggregation, 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 antibodies 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 SI Il S, 17 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 formation 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 2- or multistep 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 S" *.15 specific so-called capture antibodies (for example anti- IgM) or affinity reagents (for example protein A).

S

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, polyamide 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- S 18 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 pg/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 allow the use of tiny amounts of, for example, 0.01 pg/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 6 6**.15 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 nmarkers are enzymes, especially alkaline phosphatase and/or horseradish peroxidase or chemoluminogens such as, for example, acridinium esters.

S I 19 The labeling is carried out by methods which are described for the said markers in the state of the art.

Where the antibodies are labeled with peroxicase, 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 peptidespecific antibodies, such as, for example, precipitations, aggregation or light scattering. It is known that S 15 the peptides can also be employed not derivatized for inhibition of these measurement principles like also the 6* methods mentioned before.

It is possible to use for detection of the antigens immunodiagnostic methods which make use of polyclonal or S 20 monoclonal antibodies which are prepared with the aid of the peptides or derivatives thereof according to the invention. The embodiments suitable for the detection a method are known to the person 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 principle 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 2or 3-step methods. Thus, multistep methods can be carried out with unlabeled detecting antibodies which are f I .I I 20 e e *54 ire e0Se Se c 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 impr-ved, as is possible, for example, by coupling to serum albumin or keyhole limpet hemocyanin Schaffhausen in Hybridoma Technologie in the Biosciences and Medicine, ed. T.A. Springer, Plenum Press Nl, 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 15 phase or in the detection reagent or in both, and an antibody determination, an antigen detection or combinations with other analytes is carried out, An unexpected advantage of the novel peptides of the present invention is that they allow reliable determination 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 invention, 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, a

OS.,

a Ii 0rre 5555 Jrr

S

S I I 21 available since 1989, permit simultaneous non-differentiating detection of anti-HIV 1 and/or anti-HIV 2 K8rner 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 combination tests is also based on a cross-reactivity of anti-HIV 1 with HIV 2 antigens Busch et al., Transfusion 30/2, 184 187, 1990), as also conversely the anti-HIV 2 detection is enhainced 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 straightforward 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 determination according to the present invention. Despite the 20 improvement, associated therewith, in the performance 9* 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 commercial 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 determining 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 affiliations are present as in the case of HCV (flavivirus) and HIV (retrovirus). Within the meaning of the invention, different antibody specificities mean antibodies which S22 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 properties 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 immunochemically in a single test when different epitopes of different pathogens in each case are immobilized on a carrier.

i5 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 features 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 25 of an anti-HIV/anti-HCV test according to the invention e the specificity obtained was higher than would be given by the total of the two single tests, with the consequence 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 immobilized on a carrier and the detection and/or the 23 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 nondifferentiating 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 invention by, for example, in an immunometric test, the antigens which are used having different virus-specific 1 5 labels, such as, for example, HIV 1 antigens with peroxidase, HIV 2 antigens with alkaline phosphatase and HCV *el antigens with p-galactosidase. Successive or simultaneous determination of the simultaneously bound antibody specificities is then possible with different enzyme .20 substrates.

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

0 The invention thus further relates to an immunochemical 25 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 differentialting or non-differentiating, preferably nondifferentiating, manner.

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

S-24 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 concentration 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 re'iable determination 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- S. positive results.

S*o. Preferably used are single epitopes and/or combinations thereof, especially of polypeptides of HIV 1 and/or HI.V 2 20 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.

*sees* The following polypeptides are particularly preferred: 1. HIV 1 (numbering 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 p -25 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 AA 121-AA 175 XI non-structural protein 3 (NSP .AA 1-AA 265 XII structural protein (core): AA 1-AA Especially preferred are mixtures of the said polypeptides, especially for a non-differentiating anti-HIV/ anti-UCV' screening test, some being described by way of example without confining the conceivable possibilities thereto: XIII gp 41 HIV 1 al gp 36 HIy 2 .L5 NSP 3 HCV XIV gp 41 HIV 1 :2 gp 36 HIV 2 NSP 4 H-CV core HCV XV gp 41HIVlI gp 36 HIV 2 NSP 3 flCV NSP 4 HCV core HCV 625 XVI gp 41HIV I p 24 HIV 1 0 gp 36 HIV 2 NSP 3 HCV core HCV or XVII gp, 41 HIV 1 p 24 HIV 1 V I I, I 26 gp 36 HIV 2 p 24 HIV 2 NSP 3 HCV NSP 4 HCV core In cieneral, better immunological properties for diagnostic use are achieved with the said p~eptide mixtures.

The following peptides of HIV 1, HIV 2 and HCV have proven particularly suitable: XVIII SPH 9 (HIV 10 gp 41): 586 620 RI LAVERYLXDQQLLGIWGCSGKLI CTTAVPWNAS eve. XIX SPH 20 (l11ly 2, gp 36): *GSo* 578 613

RVTAIEKYLQDQARLNSWGCAFRQVC-HTTVPWVNDS

XX SP 4083 (HCV, NSP 4): 121 175 S GKPAI IPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQA *10 or parts thereof, for .'.aiple XXI a mixture of SP 4060 and SP 4082 (HCV, NSP 4): o 1 1 19SP46 *144 175 SQHLPYIEQGMMILAEQFKQKALGLLQTASRQA SP 4082 XXII SP 10 (HCV, core): 1 MS TNPKPQRXTKRNTNRRPQDVKFPGGGQI and/or I I Ii S- 27 XXIII SP 31 (HCV, core): 8 24

QRKTKRNTNRRPQDVKF-NH

2 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. Conversely, it may also be advantageous, for example for epidemiological problems, to exclude a specific antibody in the non-differentiating detection by omitting corresponding peptides in order, for example, to determine with a mixture of HIV 1 and HCV peptides only anti-HIV 1/anti- HCV simultaneously and not, for example, anti-HBV/antij0 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.

i A considerable advantage of the method according to the invention is that a plurality of antibodies against different 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.

20 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.

28 Example 1 Preparation of peptide solutions and coating of microtiter 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 distilled 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 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 1 or 1 4, in order to obtain by dilution in 0.1 M sodium bicarbonate the overall final concentrations which :15 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 l1 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 0 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 ~p 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 Example 2 Preparation of a peroxidase-labeled antibody against human immunoglobulin of IgG class (h-IgG) and TMB substrate for detection Antibodies against h-IgG were raised by the method of 29 KOEHLER and MILSTEIN for preparing monoclonal antibodies (Nature 256, 495, 1975), different monoclonal antibodies with the same antigen specificity being identified by the method described by STXHLI et al. of Immunological Methods 32, 297 304, 1980). After purification by gel chromatography and dialysis against phosphate-buffered saline (PBS, pH 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. Immunol. Meth. 62, 123 131, 1983).

2-Iminothiolane hydrochloride (supplied by Sigma, Cat.

No. I 6256) was reacted wich horseradish peroxidase 15 (POD), obtained from Boehringer Mannheim, Cat. No.

S 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 pS//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/l 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% RTween 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-(hydroxymethyl)-1,3-'propanediol (tris), 0.1 M sodium chloride (NaC1) and 0.1% RTween pH 8.4. Rabbit polyclonal antibodies 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 I I I t S- 30 substrate system or a substrate preparation containing hydrogen peroxide and tetramethylbenzidine (TMB), 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 mmol/l, in double-distilled water and adjusted to pH 1.5 with 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 H 2 0 2 as urea/ hydrogen peroxide adduct were added to 900 ml of doubledistilled water. After dissolution was complete, the mixture was made up to 1 1 with double-distilled water.

15 TMB substrate preparation: One part by volume of stock solution 1 and 10 parts by volume of stock solution 2 t 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 incubation of anti-human IgG/POD conjugate and the enzyme substrate system with o-phenylenediamine (OPD) as substrate, photometric measurement at 492 nm and establishment 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 31 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 chimp'nzee serum or plasma with the two commercial products was such that a polyclonal antibody raised in rabbits against human IgG was used for detection. 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 concentration 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.

20 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).

SI II 32 Example 4 Determination of human antibodies of immunoglobulin class G against HCV in the ELIBA with the peptides according to the invention 50 pl of serum or plasma were added to 50 Jl of sample buffer containing 0.3 M tris, 0.3 M NaCl, 20% boviserine and 0.1% RTween 20 in wells of microtiter plates which were coated as described vwth peptides or peptide mixtures. 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/l RTween 20 in PBS. Then 100 ji of conjugate in the final dilution were added to the wells, preferably using a preliminary dilution of 1 3000 in tris, 0.5% Tween ,56 and a final dilution of 1 26 in conjugate buffer. After a incubation at 37°C for 30 min, the content of the wells was removed by aspiration and again washed five times.

i* Subsequently 100 pl of TMB substrate preparation were added to each well, incubated at 20 22*C for 30 min, O0 and incubation was stopped by addition of 100 Al of 1 normal sulfuric acid. The extinction of the colored solution was measured at a wavelength of 450 nm (E 450 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.

a Tab. 1 summarizes results obtained from the determination, 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).

0 0 4 33 Tab. 1 Results elf the determination of anti-HCV with an ELISA containing the peptide 4083 and with a mixture of smaller peptides on the solid phase with htman samples Positive with peptide ELISA (2 pg 4083/mi) (2 ,.Lg 4060/mi and 2 4ig 4082/mi) retest positive initial positive retest pos itivc

I.

.1 big 'I 0 0444 dOd b 20 *j.

4* 4 4,

I.

"ObO 0 4 LA S~d ad anti-HCV positive samples from France n=15 15 15 from Austria n=17 17 17 17 from Germany n=20 20 20 from USA n=49 49 49 49 total n=101 1011) illl 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 2) 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.

I I 1 4 I 34 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 from Austria n 26 26 26 from Germany n 29 29 29 from USA n 53 53 53 -15 total 143 143" 143") 0 i 0C *'S -lobes :*9 0 00 0 a'.

C

20 Paired sera/plasmas (see distribution plot of healthy blood donors in Figt 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 25 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 (HP1) 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 pepttde ELISAs is also noticeable. At the samle 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 extreiely 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 human samples with ELISAs based on the use of smaller sequences (15 amino acids) of the formula 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 0* "5 surface of microtiter plates according to Example 1; the results are reported as extinctions at 450 nm (E 4 5 0 negative finding below 0.10 E).

S

*4056 4055 4053 4052 4081 4091 Mixture 0'"0 c4056 S Proben- 4055 4053 Nr. 4052 4081 4091 000. Ug/ml 2 2 2 2 2 20.5 .9 0 BC90-89 >2.50 0.20 1,40 252 0.20 >2,50 2,10 0.80 1.60 2,00 268 >2,50 0120 >2.50 >2.50 >2.50 9 84 1.20 1,60 1,70 >2.50 >2,50 .25 >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 >2 50 >2,50 >2.50 242 0,50 1,20 1 40 >2.50 137 0,40 0 3 5 0,70 286 0.60 0,15 0,45 0.15 0,90 192 0.20 >2,50 100 W >2,50 235 0.15 0.40 0,20 0,30 0,75 0.40 I 1 S- 36 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/ml each), where mixtures of smaller peptides comprising about 15 AA proved thoroughly suitable: 4056/4055 and 4052 (0.5 pg/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.

Example *0 *Optimization of the ELISA determination The modifiable parameters of the ELISA determinati. n 0 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 25 pr)limina-y dilution was varied at fixed coating concen- *0 t'tins. 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.

I I 37 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 ratlos are reported as quotients of the specific signals to the limit, and values greater than 1 indicate a positive result and values smaller than 1 indicate a negative result.

Peptide ELISA Commercial Serum (4083) test (HP1) Dilution 2 pg/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 1: 128 2.5 0.8 nea.

1: 256 1.5 1: 512 1.1 1:1024 0.6 ne. 0.2 HC 90-90 1: 1 25 6

S

00 *o*o 0 000e e g.

C

S..

.0* C S

OC.

1 2 1 4 1 8 1 16 1 32 1: 64 1: 128 1: 256 1: 512 1:1024 1:2048 25 25 25 25 11,8 5.9 3.3 1.8 1.08 0.4 ne, 0.4 6 6 6 6 5.4 3.

1.4 9ljj ne&.

0.4 0.3 0.2 0 0 0 0e C

C

@000gg

S

0 S.e.C.

HC 90-252 1 2 4 8 16 32 64 128 256 25 25 25 20 12.2 6.5 4,2 2,4 2.4 mar) 97 rnarg.

6 6 3,2 2.7 1.2 0- 8 nl 1) marg. marginal I I 38 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 1: 2 1: 4 1: 8 1 16 1: 32 1: 64 1:128 1:256 1 1 1: 10 1: 100 25 25 20 8,5 3,7 2,9 1.4 flct 0.3 6 6 6 2.9 D_,2 flegf 0.4 0.2 6 2.1 0A .4 ec HC 90-83 25 20 6.5 OS 0 0 S

S.

S

S..

S5 S 0 0~

S

.5S @0

*SSS

S S S. 0 HC 90-240 1: 1 25 6 1: 10 8 1.5 1: 100 1.4 D_.

2 n c HC 90-239 1:41 25 6 1: j.0 7.8 1: 100 1.4 ne-i HC 90-89 1: 1 25 6 1: 10 25 6 1: 100 9 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 on the 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, dil'.fted samples which already gave repeated negative reactions in the 39 commercial test (HP1) were still measured as significantly 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 pg/ml each) 4074/4082 (0.5 pg/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- S* ing to the invention is specific for HCV and is not subject to any known interferences such as, for example, o cross-reactions of other antibodies, interference by heat 20 in&ativation or the like. This also applies correspondo** ingly to other stated novel peptides or peptide mixtures, S"with which uniformly a high serum concentration (dilution 1 2 in sample buffer) was used in order to increase the sensitivity, which was possible owing to the purity of 25 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 likewise 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 (HPI).

3 0 SO @8 8 8 8 8 8 6 6 S 8 6 @6 6 *8 8 S S 0 S 6S @6 @6 @8 6 C S 5 6 3 a 0 Tab. Comparison of the analytical sensitivity of an ELISA based on a core peptide (SplO, 2 pg/mi) with the state of the art. For this, serial dilutions of anti-HCV-positive human sera in negative htuman serum were prepared and the results of the tests with an ELISA based on a peptide according to the invention (SPlO) were compared with the commercial HCV ELISA (HP1) and with a peptide described as immunorelevant according to Fig. 2 in the literature (OKAMOTO et All the data represent absorbances (E 4 50 the results to be regarded as reactive being underlined.

Serum No. Peptide SPlO according Peptide disclosed in the Commercial to the invention literature (OKAM4OTO) anti-HCV ELISA Dilution AA 1 30 AA 39 74 (HPl) 2 pg/mi 0.25 pg/mi 2 pg/mi 0.25 yg/mi HC 90-85 1: 1 1 :32 1 :64 1 128 1 :256 1 :512 1 :1024 1 :2048 2,500 2,500 1,586 1,032 O0 601 0,328 0,188 0,080 2,500 2,500 0,742 0,783 0,519 0,232 0,122 0,034 2,500 1.008 0.548 0,250 0,101 0,060 2,500 0,878 0.477 0.183 0,098 0,051 2,500 0,904 0,342 0.153 HC 90-225 1:1 1 16 2,500 2,500 2,500 2,500 1> 2,500 2,500 2,500 29366 2, 500 2050n S 0 *0 5.

0 0 0 0 000 *.a 0 0 0 0 *0 a -g C *0 *a, 00 0 0 0 a 0% 00 t S 6* CC Continuation of Tab. 1: 1: 1: 1: 1: 1: 32 64 128 256 512 1024 2,500 1.669 0,971 0,561 0,486 0,295 2,065 1,520 0.790 0,374 0,191 0,141 2.020 0,655 0.4 14 0.281 0,146 0,080 1.619 0,883 0,544 0,215 0,115 2, 500 1,759 0.909 0.380 0,122 HC 90-270 1:1 2.500 2.500 2,500 2,500 2,500 1 8 2,500 2.500 2.500 2,222 1,902 1. 16 2,500 2,500 1,478 1,016 0,980 1 32 1.512 1.408 0,743 0,4780,1 1 64 0,898 0.802 0,322 0,216 0,259 1 128 0.536 0.483 0,121 0,096 0,179 1 256 0,312 0,275 0.050 0,041 HC 90-354 1: 1 1 :32 1 64 1 :128 1 :256 1 512 1 1024 1 2048 2,500 2,500 2,500 1,703 0,918 0.582 0.326 0,159 2.500 2,500 1,833 0.873 0.410 0.310 0.178 0.084 2,500 1.917 10282 0.685 0,367 0,145 0.072 20500 00786 0,527 0,249 0,082 2, 500 0,352 0.200 +1- 42 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 equivalent to SP 10 with respect to sensitivity, and both peptides are superior to the peptide described in the literature.

Although the literature-analogous peptide SP 12 (AA 47does not correspond exactly to the peptide described by OKAMOTO et al. (AA 39-74), the results on the intermediate 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 attributable to the carboxyl-terminal region of SP 10 contained therein (overlapping region AA 24-30 shown in Fig. 3).

S

Se..

s

S

B.

5 a.

0 i I V I I 43 Tab. 6: Comparison of the immunoreactivities 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. All data represent extinctions (E4 5 0 .0

•S.

OSg 45 5* SP 23 SP 10 SP i Samples AA 8-26 AA 1-30 AA 24-53 2 ug/ml 2 g/ml 2 ug/ml pos. contr. 2.500 2.500 0.405 neg. contr. 0.051 0.049 0.030 Dilutions HC-90-225 1 16 2.203 2.500 0.428 1 64 1.329 1.367 1 256 0.454 0.386 0.024 1 1024 0,195 Q.142 HC-90-354 1 16 2.500 2.500 1 64 1.413 2.156 0.059 1 256 0.418 0.496 1 1024 0.1§2 Anti-HCV-positive samples (commercial test HP 1) 15

S

HC 90-371 HC 90-336 HC 90-453 HC 90-570 22500Q 2,5002 2,086Q.

2. 500 >2,500 0,224 0.058 0.088 This surprising advantage in sensitivity becomes particularly clear on testing undiluted samples. Thus, all 11

I

44 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 literature finds only 6 samples positive, and in no case was there a positive reaction with the commercial test (HP1).

With respect to the samples 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.

ee *4

I

00 *4 S a. 3 V S. 3 S S S

S*~

9 9 S 53 *L S 5* a a.

*65 5 S Ce~g~ u S. S C 5 5*s.

3 Tab. 7 Comparison of the sensitivity of a peptide ELISA (SP10 2 pg/ml) with the state of the art.

For this, 11 selected native human anti-HCV-positive samples were tested with an ELISA based on a peptide according to the invention, comparing with a commercial test (HP1) and with a peptide described as immunorelevant according to Fig. 3 in the literature (OKAMOTO et al.).

All the data represent extinctions The anti-HCV-positive sample HC 90-495 was also tested as control.

Serum No. Peptide accordina Peptide described in the Cnmmnr-ial WrC r.Tc to the invention 2 pg/ml cut off: 0.100 E literature (OKAMOTO) SP12 2 pg/ml cut off: 0.100 E (HP1) cut off 0.454 E 90-224 90-284 90-289 90-300 90-323 90-493 90-509 90-512 90-531 90-536 90-494 1,952 2,417 2,500 2,500 2,500 2,500 2,500 2,500 2,208 2.500 1,279 2,005 0,451 1,220 0,288 2,500 0,051 0,081 0,049 0,257 0,234 n.d.

marg.

Neg.

marg.

0,114 0,095 0,278 0,208 0,313 0,088 0,083 0,110 0,081 0,069 0,114

I

Neg.

L

Neg.

Neg. Neg.

Neg.

HC 90-495 2,500 2.500 2,500 Pos.

S1 I S 46 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 sensitivity 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- 6 HCV-positive samples (material and comparative test w results marketed by Boston Biomedica, USA). All ELISA data are ratio values, which describes the ratio of 20 sample extinction to cut-off. Values 1.0 are regarded as negative and 1.0 are regarded as positive.

4* 9 S C C. *g 0 S S S

C

COS See .4 C S.

0 eS~ C

C

0~ *Ce 9 0 S 5 See 008 e.g C -S 5 0 0 SO S SO* 0 p Tab. 8 Hember

I.D.

Number Comparative HCV ETA S/Co results HCV ELISA ELISA with peptide according to the invention (SP1OI2 pg/mi) S/co S/Co RESULT PHVlOI-01 PHIV101-02 PHVOI-03 PHVI01-04 PHV1O1--05 PHVIOI-06 PRVIOI-07 pIHv101-08 PRV1O1-09 PftV1O1-1O pliviol-li PHVIOI1-12 PHVIOI-13 PHVIOI-14 3.717 0.167 1.880 1.757 3.*4 &4 1.640 2.*281 3.009 1.855 3.*649 0.662 3.018 2.805 1.912 3.686 0.964 0.268 1.323 3.076 2.169 1.642 1.624 1.588 1.119 3.098 1.820 2.205 2.770 0.*846 3.240

POS

N

POS

18 0.25 0.90 C C S. S g S C g e .55 g;g

C

g egg *Ogeg Ce...

CCC,.

CCC

C r Continuation of Tab. 8

MDIBER

I.D.

Ntnber CONFItIlofY DATA RIBA 5-1-1 C-100-3 C33C C22-C RESUtrV ALT" a ullti-IBcO IIlsAg antJLIIV* antI-IFLV* PIIVIO-I01 PtIV101-02 PHVIOI-03 PIV101-04 PIVO-06 PIVO-07 PRlV10-08 PIVI11-09 PIVOL-11 PIVIO1-12 PHVIOI-13 PHVOL-14

PIVIOI-I

4 4 4 4.

1 +1- 4/- 2 POS

N

+1 N 4 4 POs

N

1 4 [lOS 1 4 [os 1 4 PM5 1 4 [OS 2 4 [OS 4 1 POQ 4 [l~S

N

[OS

N

[Os

N

[OS

POS

11OS 1

I

+1-

-I-

-1

I-

+1- P

POS

[VS

*POS Positive, N Negative, NA Not applicable Less than 2 x upper limit of normal, G Greater than 2 x upper limit of normal.

ALT results are -those read following sterile filtration and vialing and may not reflect the ALT status of the donor at the time of donatidn.

0 1 q I 49 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 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 20 genetic engineering). According to this test, sample 03 ought in fact to be classified as negative.

The tests on the second panel summarized in Tab. 9 led to similar observations: 19 of the total of 22 anti-HCVpositive samples are found, surprisingly, to be extremely *25 strongly positive with extinctions greater than (corresponds to ratio 25). The three anti-HCV-negative sera contained in the panel are correctly classified as negative (ratios 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 core-specific antibodies according to the confirmatory test.

Tab. 9 shows the comparison of the sensitivity of a peptide ELISA (SP 10, 2 pg/ml) with the state of the art for native anti-HCV-positive samples in the panel 4. 1 1 so PHV 201, comprising well -characterized anti-HCV-positive samples with different anti-HCV titers. (Material and comparative test results markceted by Boston Biomedical 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 resuilt and values 1.0 represent a positive result.

V O 0 0** 00 *6

S

*5* *5 0 0 0S 0 0.

0 0 *0.

0 0 Tab. 9

MEMBER

I.D.

NUMBER

Comparative results HCV ETA HCV EIISA ELISA with peptide according to the invention (SP 10/ 2 pg/ml) RESULT* S/Co t'BV2O1-01 I'I1V201-03 I'IIV21-03 ITIV201-06 11IV201-06 I11V201-09 IIIVZMi-00 111V201-11 IIIv l-12 l11V201-I 3 PlIV201-12 I'lV201-13 IIV201-16 PlIV201-17 1'r4201-10 llIV201-19 IV201-21 N1V201-22 lllIV201-23 lIV201-24 IlIV201-25 1-69 3.660 3.79r 0.271 3.411 3.711 0.260 1.515 2.530 2.114 2.003 3.641 4.115 2.69 4.15 4.1.15 4.115 3.530 0.490 2.611 -1.430 1.721 4.115 2.500 4.115 1.4715 5. OF 3.946 0.17G 2.591 4.005 0.108 1-244 1-.214 2.145 2.169 4.769 5.662 4.301) 561) 5.662 5.662 3.943 1.520 2.371 2.666 1.690 5.662 2.631 5.660 i"vs IN'M IIVS

IVSS

POS

"os

['OS

I'OS

fVS

IN)S

"OS

1'03

OS

r 0. 1 0.25 0.25 0.25 06 Continuation-f H DIEDE C fI 0 0 D00.

0~~ 0 0 6 00 #6 0 6 0 06 066 *So 00 6 ONFIRIK17MY DATA on 2.0 ELISA w1th peptide accordii, to the invention (SP 10/ 2 pg/ml) Ltu-IIIV* aItI-Ir V* S/Co tun___ 5-1-1 C-1o-3 C33C C22-C RESljt'Pr,* A1 .111A 4 is asnl PlIV201-01 I'IV201 -02 PP1201-03 I11V201-04 PlIV201-05 PlV201-06 'I1J20 1 -00 i'11V2(JI-DO 'lIV201-09 PlIV201-10 111201-11 1-12 PIM1201-13 NIV2OI-14 IlIV201-156 P11V201-16 l'llV201-10 PlIV201-19 'lV201-20 PlIV201-21 PlIV201-2 IPJ201-23 lIV201-24 PlIV201-25 2 2 2 1 2 2 2 2 4 2 2 4 1 i 0/- 4 2 4 2

-I

1/- 2 4 4 4 2 +1- 1 3 4

I

'los

POS

'los

POS

I

POS

46s pos PO5

POSS

MlS

H

P05

POS

'lOS

POS

tIOS

N

POS

N

1"SS PuS 11

N

M4

N

I'MS

1W

N

N 14

POS

raS 1

N

N 11

N

14

N

POS

H

N

14 14

H

N

N4

N

0.25 0.75 0.25 0,25 '25- *POS Positive, N Negative, I Indeterminant, NA Not applicable Less than 2 x upper limit of normal, G Greater than 2 x uppfer limit ALT results are those read following sterile filtration and vialing and may the ALT status of the donor at the time of donation.

of normal.

not reflect f I I I S- 53 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 infectious 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 determination 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 20 (ratios).

S

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

:25 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 1ISP 4 region. The data for the first 3 time values are extinc- 30 tions which serve to fix the limits, from which the subsequent ratios of specific signal and limit have been formed.

9 0O 06

S

a 4 4 4 0 44 4 4* 4 S a *5 *.4 Ow.

as a a 4 4 S S 0 0 4 0 006 a aS.. 4 ~0 54 5 S *ea Tab. 10 A Animal No. 123* ALT Commercial 4083 4083 4060 test (HPi) 4081 Time (weeks) 2 pg/mi 0.5 pg/mi 2 pg/ml each 0 inoculation 2 4 Extinctions (E) define the limits: 0,500 0,600 0.450 0,7 E

E

ratios 29 140 170 23 23 0,6 0,4 0,7 0,4 0.5 0,6 0.7 0.6 0.5 0,6 0.6 0,032E45 0.027 E 0.013 E 0.10 E 04U01 0.02 0.01 0,02 0.01 0.03 5,3 pos.

7,4 pos.

5.7 Pos.

2,7 pos.

1,5 Dos.

0,041 E45 0,024E 0.025 E 0,10 E 0.03 0,03 0,02 0,03 0.03 0,03 1. 5 Dos, 1. 8 ]Dos, 1.1 R2O.z.

0.7 0,4 0,026 0,014E 0.019 E 0.10 E 0,03 0,03 0,02 0,03 0,03 0.03 0,6 0.7 0,3 O 3 confirmed as NANB-infected by cnfimedas ANBinfcte byelectron microscopic investigation of liver bose biopsies a a a. a.

a a a 4 a a 0 aS a a a. a a a *a a..

*5O ft a a a a.

a a. S S S Tab. 10 B Animal No. 048 ALT Commercial 4083 4083 4060 4073 4090 test (HPI) 4081 Time (weeks) 2 pg/ml 0.5 pg/ml 2pg/ml each 2 pg/ml 2 pg/ml Sinoculation 20 0,5 E 492 0,018 E 450 0,027 E 450 0,008 E 0,036 E 45 0,028 -inoculation 0 0 492 450 450 450 450 450 2 25 0,5 E 0.041 E 0,070 E 0,007 E 0.041 E 0.050 E 4 23 0,5 E 0,026 E 0.050 E 0.009 E 0.039 E 0,037 E Extinctions (E) define the limits:he 0,70 E 0.10 E 0.10 E 0.10 E 0,10 E 0.10 E ratios (r) 32 r 0,6 r 0,3 1 r 0,5 r 0,2 r 0,6 r 0,6 12 100 r 0,4 r 1,0 marg. r 1,0marg- r 0,1 r 0,95 marg- r 2,0 pos.

14 112 r 0,7 r1l.7 pos. r 4.9 pos. r 0,1 r 6.9 pos. r16,2 pos.

16 30 r 1,0 r 7,4 pos. r 3,8 pos. r 0,2 r12,2 Pos r23,2 pos.

18 25 r 0,7 r 7,2 pos. r 2,8 pos. r 0,2 r 7,8 pos. r18,9 pos.

30 r 0,8 r 8,6 pos. r 4,7 pos. r 0,1 r12,8 pos. r16,4 pos.

22 25 r 0,8 r 8,4 pos. r 5,4 pos. r 0,1 rll,7 pos r22.3 pos, 24 23 r 0,8 r 9,8 pos. r 7,2 pos. r 0,2 r15,5 pos. r20,0 pos.

26 20 r 0,8 r10,8 pos. r 6,0 pos. r 0,2 r13,0 nos r17,4 pos.

28 20 r 0,8 )r 9,8 Pos. r 5,4 pos. r 0,2 rll,7 pos. r 8,9 pos.

20 r 1,0 margr 7,6 pos. r 6,3 pos. r 0,2 rll,5 Dos. r12.9 pos.

32 25 r 1.3 pos. rl6,2 pos. r17,4 pos. r 1.3 pos. r24,5 pos. r23,3 pos.

34 25 r 2,0 Dos. r18,4 pos. r22,5 pos. r 2.0 os, r >25 pos. r >25 pos..

36 28 r 2,4 pos. r16,1 pos, r22,0 pos. r 2.0 Pos. r >25 pos. r >25 Pos.

38 28 r 2,1 Pos. r17,9 pos. r >25 pos. r 2,1 pos. r >25 pos r >25 pos.

26 r 2,9 pOS. r >25 pos. r >25 pos. r 2,5 pos. r >25 pos. r >25 pos.

1) marg. marginally positive 0 9 a. 9 9 9

S

a a 0 9

S

~S *SE

S..

S

9* 0 5 SO SR S S S 0.

*5s Tab. A0 C Animal No. 147 ALIT Comimercial 4083 4083 4060 4074 test (HP1) 4081 4081 Time (weeks) 2 pg/mi 0.5 pug/mi 2 Ag/mi each 2 pg/mi each 0 inoculation 2 4 Extinctions (E) define the limits: 0,60 0,50 0.45

E

0 068E45 0:043 E 0,066 E 0 042 E 450 0:029 E 0,033 E 0 *008 E45 0:012 E 0,015 E 0 027 E45 0:032 E 4 0,023 E 0,70 E 0,10 E 0,10 E 0. 10 E 0010 E ratios 52 110 52 42 48 48 0.5 0.5 008 0,6 0,6 0,4 0,5 0,6 0,7 O's O'8 Id2 IdL r 0,2 r JA r 6,5 rll,8 r 7,6 r12, 5 rlO, 9 r 8,9 rll, 3 r13, 3 r19,*9 tDos. r20, 8 nos. r >25 130S,

DOSE

gos.

Dos, P08 Pos 9 Dos V Dos I

DOS,

Dos, 12Os, r 0,2 r 0.3 r 5,9 r 3,7 r 6,0 r 4,9 r 6,2 r 6,9 r14 .1 r19,7 r2 4.7 r >25 Dos.

DOS.

Pos.

Dos.I pos.

p05.

DOS.

Dos.I Dos..

DOS I Pos.

0.1 0.1 0.2 0,2 0.1 0,1 0,2 0,4 0.9 1.6 2.8 2,5 3,4 r 0,3 r 0,2 r 0,3 r 005 r 1,0 r jLZ r 2.1 r 3,5 r 3,4 r 7,6 r 9,5 rll,0 r15,0 marg.) DgB,,

POSI

DROS 0

DPOS.

Dos, 120s,

IDOS,-

Dos.

DPOS 0 DPOS Dos, Dos I 1) marg. marginally positive 57 The resylts 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 central amino-acid sequence 2,.20 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-terminal sequences) and 4090 which comprises the central structure.

Similar results of an early identification of HCV antibodies, almost simultaneously with ALT, were produced by the investigations on animal No. 147 in which a reliable HCV antibody detection is possible approximately simul- 30 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 chimpanzees infected with H-CV. The results with the commercial test (liPI) are presented in comparison with a synthetiprepared core peptide according to the invention (SP 10, 2 pg/ml), the results being presented as ratios of specific signal and limit.

6 off# too**.

tI I I I 59 'Animal No. 048 ALT Commercial HCV ELISA (HP1) SP 2 pg/mi Time (weeks) 0 =inoculation 065 E 4 9 2

E

0,09 E 450 0,21 E 0413 E Extinctions define the limits: 0,70 E 0,10 E ratios (r) 34 32 100 1.12 30 00 4 0@ 00 0 0000 040

I

*000 0* 0 004 0e 0 0 @0 *00*00 0 0004 04 @0 4 0,4 0,6 0,4 r 007 100 0,7 r 0,8S 0,8 0,8 r 0,8 r 0,8 r 1,0 r 103 r 2,0 r 2,4 r 2,1 ,r 2,9 0'.50 r >2060 P.22-L r >20o, 0 2, >20,0 Ros >20,0 Dos >20,0 Dos 19,0 Ros 2000 Dos,.

r 18,5 11g.

1004 Dg.L r 14,2 99A.

1706 I~2 1504 Dos >20,0 ROS#~ >20,0 Dos,~- >20p0 Dos,.

1) Targ.

'DOS

PQL.

22L 12OU f 1) marg. marginally positive I I 1 4 60 Animal No. 147 Time (weeks) ALT Commercial HCV ELISA (HP1) SP 2 pqg/ml 0 inoculation 0.60 E492 0.50 E 0,45 E 0.011 E450 0,032.

0,029 Extinctions define the limits: 0,70 E 0.10 E ratios (r) 52 110 52 42 48 48 r 0,5 r 0,5 r 0,8 r 0,6 r 0,6 r 0,4 r 0,5 r 0,6 r 0,7 r 0,8 r 0,8 r Li r l/ r 0,2 r 0,1 r 0,2 r 0,4 r 0,3 r 0.2 r 0,3 r 0,2 r 0.4 r >20,0 os.

not tested not tested r >20,0 pos, 1) marg. marginally positive 61 The results presented in Tab. 11 A and B with a novel core peptide also underline the advantages of the peptides according to the invention, especially by comparison 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).

0* Overall, the peptides according to the invention and the use thereof in immunochemical detection methods prove to 20 be considerably more sensitive than all hitherto described methods based on proteins prepared by genetic engineering and othnr 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 O considerably less sensitive to non-specific bindings, 4".30 which is expressed not least by a drastic reduction in the background compared with the commercial test (HP1) (max. 0.10 E 5 o, compared with max. 0.4 E4, 2 with the commercial test both for samples of animal and of human origin so that a considerably sharper negative/ positive discrimination is possible. Finally, advantageous aspects to be mentioned are the shorter overall f t 1 I S 62 I duration and the greater 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 mixture 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 :I order to obtain by dilution in 0.1 M sodium bicarbonate the overall final concentrations which are indicated 0 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 pl 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 200C 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/l 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 A concentration of 1 pg of 4083/ml and 1 pg of SP has proven suitable for coating the peptide mixture, this 63 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 pg/ml each *«0i PHVI01-01 1 16 1 2 I* PHV101-02 negative negative PRV101-03 1 4 1 1 PHV101-04 1 512 1 64 PHVI01-05 1 2 1 1 PHV101-06 1 128 1 32 S..PHV101-07 1 128 1 128 PHVI01-08 1 64 1 16 PHV101-09 1: 64 1 16 PHVI01-10 1 256 1 128 PHV101-11 1 32 1 16 P 5HV101-12 1 512 1 :128 PHV101-13 1 16 1 64 PHV101-14 1: 64 1 32 PHVI01-15 1 128 1 64 64 Tab. 13: BBI Mixed-titer anti-HCV PANEL PHV 201

MEMBER

I.D.

NUMBER

ELISA according the invention SP 4083/SP 10 1 g/iml each to Commercial anti- HCV test (HP2) 4660 4 I N a 41 64 a ebg a a,* .a« o 'a PHV201-01 PHV201-02 PHV201-03 PHV201-04 PHV201-05 PHV201-06 PHV201-07 PHV201-08 PHV201-09 PHV201-10 PHV201-11 PHV201-12 PHV201-13 PHV201-14 PHV201-15 PHV201-16 PHV201-17 PHV201-18 PHV201-19 PHV201-20 ,PHV201-21 PHV201-22 PHV201-23 PHV201-24 PHV201-25 1 1000 1 1000 1 128 negative 1 64 1 64 negative 1: 4 1 1000 1 4 1 512 1 32 1 256 1 64 1 512 1 512 1 128 1 16 negative 1: 4 1 500 1 16 1 256 1 256 1 1000 1 128 1 500 1 32 negative 1 64 1 256 negative 1 16 1 512 1 16 1 128 1 16 1 256 1 64 1 1000 1 512 1 256 1 128 negative I 4 1 256 1 256 1 512 1 512 1 512 S. 65 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) Ojos acetic acid.

b '.20 These 4 stock solutions were mixed in various volumebased 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 pl of each of the diluted solutions were k« 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/1 bovine serm albumin in phosphate-buffered physiological saline (PBS, pH and the test plates were then dried over silica gel at Exaxnile 9 Optimization of the concentration of HlIV 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. for the four stock solutions described in Example 8, all four'modifiable 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 pg/ml): Peptide Peptid e Peptide Peptide XVIII XIX XXXXII 2 2 2 2 1 2 2 2 2 2 2 se 0, 02; 2 2 2 to,0,1 2 2 2 0,05 2 2 2 Peptide Peptide Peptide Peptide XVITI XIX XX XXII 2 1 2 2 2 0,5 2 2 2 092 2 2 .2 04f 2 2 2 0,05 2 2 2 2 1 2 etc. to 0.05 0.5 005 04,05 04.05 4 t 67 These mixtures were immobilized as described in Example 8 on micvotiter plates and evaluated in the ELISA as desc, "bed 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-negative samples were likewise tested in order to be able to define the background reaction 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-HIVand anti-HCV-positive samples with, at the same time, a low background in the tests on anti-HIV- and anti-HCVnegative samples.

Coating mixtures which were usually favorable were found on the basis of these criteria, from which the following mixture was selected: 00e5 I *too XXI SPH 9 0.500 pg/ml SSPH 20 0.250 pg/ml SP 4083 0.500 Pg/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 establishment of limits).

These establishments were applied uniformly and unchanged to following Examples 10 to Example Determination of human antibodies of the immunoglobulin G class against HIV 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 Commercially available 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 :20 according to the invention.

oo 0 9.

0 *o o*o oo* o Tab. Determination of anti-HIV 1 with the method according to the invention compared with an anti-EIV 1/2 combination test (Enzygr stR anti HIV 1/2, HP 3) Strongly reactive means extinctions 2.5 in 'the photometric evaluation; all 76 anti-HIV 1-positive samples are H-CV-negative 0 Method Carmercial anti-IIIV 1/2 Number of Origin of according combination samples samples to the test invent ion n 12 Europe na= 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 reatitive coinfections) n =76 n =76 n =76 positive positive

~S

9 0 9009ee

S

it I 70 Tab. 16 Determination of anti-HIV 2 with the method according to the invention compared with an anti-HIV 1/2 combination test (EnzygnostR anti HIV 1/2, HP 3) Strongly reactive means extinctions 2.5 in the photometric 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 coinfections) n 28 n =28 n =28 positive positive

S

556VS

S

tI I I 71 I

I

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 of f 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 of of the according to Commercial anti- HCV ELISAs HP 1 HP 2 samples samples the invention n =61 Anti HCV Positiv~e HC 90-346 -351 -516 -570 Europe n 57 n 57 n 57 Europe strongly reactive n 4 2, LaA 102.+ strongly strongly reactive reactive ZAZ negative negative IAI (H 2.9 JLQ Ig.Q

C*O

C

CCC C

C

Ce..

C.

S

C**

C*

el.

Cr S AC CC

CO

CC C

C

050CC

C

n =53 USA n- 51 n -51 n -51 Anti HCV strongly strongly strongly positive reactive reactive reactive USA n 2 NC 90-511 ij+ 12 (3)99 -566 A negative 5L00 n 114 n- 114 n -11l n 114 Positive positive Positive -72 ExamPTLeg 11: Determination of the analytical sensitivity of the ELISA according to the invention Zor anti-fly and anti-HCV compared with tl,,,e state of the art As a model for the assessment of the analytical sensitivity of the ELISA according to the invention, titrations of positive samples were prepared and tested in the ELISA described in Example 9. In total, diLh)tions 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 l.iiting sensitivity defined as the last preliminary dilut'ion still reactive in the particular test system using the new limit (mean of the negative controls plus 0.250 threshold).

The results of these limitiig sensitivity tests are compiled in Tab. 18 (anti-HIV Tab. 19 (anti-Hly 2) and .and Tab. 20 '(anti-HCV)' as ex~tinctio~ns.

toot*: 0 0 Tab. 18 Determinption of the limiting sensitivity of the ELISA accordin. .,to the invention for anti-HXV 1 com Fared with the anti-HIV 1/2 combination test (Enzygrnost 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 E 5 ).All anti-HIV-positive samples had a negative reaction in HP 2.

S amxpl e Preliminary dilution Extinctions in the ELISA according to the invention cut off=0.282 0.D.

Extinctions in the anti- HIV 1/2 combination test (HP 3) cut off=0.268

O.D.

I S 64 0 to 669 Of 8803/26 undiluted 2s500 20500 1 :128 2s500 2,500 Anti 256 2,334 2,185 HIV 1- 512 10408 1,412 positive 1024 00825 0,837 2048 0,4672 0,5 4096 0,240 -0,216 8000 0@137 -0,135 8611/144 undiluted 2,500 2,500 1. 128 2,162 2.014 Anti 256 1,366 1,028 HIV 1- 512 00806 0,648 positive 1024 0JA.29 0,5 2048 0,280 0,258 4096 00153 0,136 8000 00088 0.076 8803/24 Anti 1 400 1,2 ,8 HIV Ipositive 22281.

Anti HCV- 1 .6 2 5; 2LZ2192 0,014 positive. (negative) 66*t C I 9* 6

I

6 6**6t~

I

74 Tab. 19 Determination of the limiting sensitivity of the ELISA according to the invention for anti-HIV 2 compared 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 (E 450 All anti-HIV-positive samples had a negative reaction in HP 2.

Sample Preliminary dilution Extinctions in the ELISA according to the invention cut off=0.282

O.D.

Extinctions in the anti- HIV 1/2 combination test (HP 3) cut off=0.268

O.D.

'.S

U*

V S 4 0@*S *449 4 4* is 4 4 4 *1 a ft 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 0.365 0 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 0,656 0,667 2048 9.320 0.302 4000 0.166 0,172 8804/116 Anti 1 400 1.Q45 1.020 HIV 2pos-'tive 22281 Anti HVpositive 1 256 a.2.12 0.014 (negative) 0 1k 1 4 ,t 75 Tab. 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-HIVand anti-HCV-negative sera, and these dilutions were tested as in Example 3. All the data are extinctions

(E

45 nm) Sample Preliminary dilution Extinctions in the ELISA according to the invention cut off=0.270 Commercial anti-HCV ELISA (HP 2) cut off=0.481 HC 90-85 undiluted 2,500 20500 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 10045 1,707 1 512 09648 00871 1 1024 .Q.3 2 2 0,492 1 2048 04207 0,255 HC 90-225 undiluted 2,500 2.500 1 16 2,500 2,500 Anti 1 32 20214 2o500 HCV- 1 64 1,816 2,500 positive 1 128 1,488 2,008 1 256 0,689 1,120 1 512 0.580 0,,501 1 1024 0,206 0,375 1 2048 0,148 0,111 Anti.HCVpositive HC 90-270 1 256 0299 1,54A HC 90-354 1 128 Q.271 0,76 re*i

V..

6 S Anti HIV 1positive 8803/26 8811/144 8803/24 undiluted undiluted undiluted 2.90 2599 2..0.Q 0,110 0,098 0,068 (negative) 9 9 Anti HIV 2positive 8804/103 8804/117 8804/116 undiluted undiluted undiluted 2,.Q 2500 2. SQ 0075 0,081 0.099 (negative) v *I I 76 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 combination 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 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 according 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.

*4 It is furthermore evident from the data in Tab. 18 to that the strong reactivity of the anti-HIV- and anti-HCVpositive samples in the novel ELISA is particularly advantageous because the anti-HIV samples reacted nega- ,.:t30 tively in the specific anti-HCV test and the anti-HCVpositive samples reacted negatively in the specific anti- 94 HIV test.

0 a a 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 invention compared with an anti-HIV 1/2 combination test have been reproduced in Tab. 21.

Tab. 21: All data are extinctions (E 450 and values above the particular limit are to be regarded as positive.

*4 9 *4 **4 e o4 4 4 9. 78 Tab. 2 1 Sample code

ELISA

according to the invention Commercial anti- HIV 1/2 ELISA Commercial anti-HCV ELISA (HP2) I A 0,023 0,066 negative 2 A 0.020 0,055 3 A 0.055 0,193 4 A 1,163 2,500 A 2,500 2,500 6 A 2,500 2,500 7 A 2,500 20500 8A> 2,500 2,500 9 A 2,500 2,500 1 C 2 C 3 C 4 C

C

6 C 7 C 9 C

C

11 C 12 C 13 C 14 C 15 C 16 C 17 C 18 C 0,.014 0.015 0.054 0.500 0,630 0,889 0,942 1,010 1,081 1,564 2.500 11 It It of 0,022 0,054 0,145 1o138 1.707 2,500 of 11 It It if of negative of

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it it of of be S S .5 5*e 0e*e be..

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a 0S** 0* C. S

S

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C

G BBI 80 81 82 83 84 86 87 88 89 0.012 0,013 0,018 0,478 0,733 0,612 0,763 16296 1,309 1.544 06056 0.053 00059 1,112 2,500 2,500 2,500 2,500 2,500 2,500 negative If cut off 0.281 0,285 0,481 79 The results in Tab. 21 make it clear that the ELISA according to the invention detects low-titer anti.HIV 1positive samples just as reliably as the anti-HIV 1/2 combination test (Enzygnost R anti-HIV 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 (Enzygnost anti-HIV 1/2).

The sample panel supplied by Boston Bicmedica; 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.

0*S 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.

II

r\ e I I I so Tab. 22 ID No. ELISA according Commercial anti- Commercial to the invention, HIV 1/2 anti-HCV ELISA HCIV ELISA BO 1-01 02 03 04 06 07 08 09 11 12 13 14 cut of f 2,500 1.979 2,056 1,751 1,781 1,428 1,183 0,641 0.984 09682 1,302 2,032 1,017 1. 079 2t500 0.279 14872 1,575 2,280 1,960 2,044 1,72~8 1,564 1#152 1,499 00940 1,650 0,540 1,854 10484 2,090 0,2 2s500 2.023 negative

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S~ ~35 0 81 The results in Tab. 22 reveal that, compared with the anti-HIV 1/2 combinrtion 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). Additional 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 anti-HCV ELISA (HP 2).

00 0 The data on the ELISA according to the invention and commercial represent so-called end point titers with "25 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.

*0 *oeo

S

82 TAb. 23: EBI low-titer anti-HCV PANEL P1W 101

MEMBER

I. D.

NUMBER

ELISA according to the invention Commxercial anti- HCV test (HP 2) PHV1 01-01 PHV101-02 PHV101-03 PHV101-04 PHV1 01-05 PHV1 01-06 PHV101-07 PHV1O1-08 PHV1O1-09 PHV101-10 PHV1 01-11 PHV1 01-12 PHV101-13 PHV101-14 PHV101-15 I 16 negative 1: 4 1 :512 1: 2 1 :128 1 :128 1 64 1 64 1 256 1. 32 1 512 1 :16 1 :64 1 :128 I1: 2 negative 1: 64 1 32 1 :128 1 :16 1 :16 1 :128 1 :16 1: 64 1 32 1: 64

S*

beS Ope 0 0 $S060 S( i 83 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-negative 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 detection limits for anti-HCV in the PHV 101 panel than the state of the art.

Example 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 ion-specific reactions, which lead to false positive results in the ELISA, a total of n 512 healthy blood donors was used. Serum and 0 plasma were obtained simultaneously from these donors in order to be able also to investigate a possible interfering 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 S* and anti-HIV 1/2 HP 3) (so-called initial tests) were 30 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.

Sa a 84 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 classified 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.

9 i 9** a.

be s e 9 e t

S

O

a 3. 09 90 0 9 0 0 9 9 9 On. 6.* p 9 6 33 9. p SW 0 0 @9 ~&q boo ooUe Go 009 0 *O boo Tab. 24 Method according to the invention Number ratios reactive sera/ sera/ plasmas plasmnas Anti-J{IV 1/2 Anti-HCv Number reactive sera/ plasmas ratios sera/ plasmas Number reactive sera/ Dlasxuas (Hp 2) ratios sera/ plasmas Sample Code 3 /2 0/ 0 0 /0 9104 -PE-1 2 3 2.1 /1.3 1,0 /1.2 1,1 /0,9 nieg.

neg.

0 /0 1/ I 9104-HTJV-1 neg. 1.8 1.6 neg.

0 /0 0 /0 5 /4 0 104 -HCV-1 2 3 4 neg.

neg.

"I'eg neg.

neg.

0 2,5 1,4 /1.2 1,3 0,8 -/101 Tab. 0 *0 *7 Results of the specificity after repeat tests on the 9 reactive sera from Tab. 24 n 512 sera and n 512 paired plasmas from a total of 512 healthy blood donors novel peptide ELISA serum plasma anti-H'IV 1/2 (HP 3) serum plasma aniti-HCV (HP 2) serum plasma Number of falsepositive results 3 2 1 1 5 4 in initial tests initial specificity 99.41 I99.60 99.80 /99.80 99.02 /99.22 Nuimber of falsepositive results 2 2 1 1 4 4 after repeat retest specificity ()99.60 /99.60 99.80 /99.80 99.22 /99.22 I I I 87 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 t* the invention, 1 serum (and one corresponding 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 ELIOA were not identical.

After repeat tests had been carried out on these nine reactive sera, the picture which emerged was that depicted 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 sera 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.

fee*** In summary, it can be said that the novel peptide ELISA for the simultaneous non-differentiating detection of anti-.-V 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 t t

I

88 two single tests anti-HIV 1/2 on the one hand and anti- FCV on the other hand: the data from the tests on panels, i.e. native samples containing anti-HIV I 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 determination of anti-HIV 1, anti-HIV 2 and anti-HCV.

o g. It was additionally found, completely surprisingly, when 20 determining the susceptibility of this novel test, to interference that, by reason of the very good specificity, 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- HIV 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 formulae 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 S. 89 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 nonspecific, 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.

.e.

a* O@ee a.

Se *005S a

Claims

1. A mixture of peptides composed of at least one of the peptides of group A which group consists of the peptides: MSTNPKPQRKTKRNTNRRPQDVKFPGGGQI 8 26 QRIKTK1TNRRPQDVKFPG 2 XTKRNTN'R PQDVKFPG 12 26 KRNTNRRPQDVKFPG 14 26 NTNRRPQDVKFPG 16 26 NP.RPQDVKFPG 18 26 RPQDVKFPG 8 24 QRKTKRNTNRRPQDVKF 8 22 SP SP 23 SP SP 30 D SP 30 C SP 30 B SP 30 A 4 00.0 6 6 0 0 SP 31 QRKTKRNTNRRPQDV SP 32 and at least one of the peptides of group B (n~on- structural protein of HCV),, which group consistM of the pept ides: ,408l3:- 121 SGXPAX IPDREVLiYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQA 91 4074: 121 160 S GKPAI IPDREVLYREFDEMEECSQHLPYIEQGMMLAEQF 4073: 128 160 PDREVI 1 YREF'DEMEECS QULPY IEQGMMLAEQF 4072: 136 160 EFDEMEECSQHLPYIEQGMMLAEQF .4071: 144 160 SQHLPYIEQGMMLAEQF 4081: 161 175 KQKALGLLQTAS1AQA .4082: 144 175 SQHLPYIEQG4NLAEQFKQKALGLLQTASRQA 4090: 135 164 REFDEMEECSQHLPYIEQGM4LAEQIFKQKA 4056:

121. 135 SGKPAIIPDREVLYR 129 143 DflEVLYREFDEMEEC sot ft.. 0*666 f.00

4054. 137 151 FDEMEECSQHLPYIE 145 159 QHLPYIEQGMMLAEQ 153 167 GMMLAEQFKQKALGL 121 139 SGk,'PAIIPDREVLYREFDE 2. The mixture of claim 1 consisting of MSTNPKPQRKTKRNTNRRPQDVKFPGGGQI :09:and .2.:121 175 SGKPAI IPDREVLYREFDEMEEC2QH LPYIEQGMMLAEQFKQKALGL LOT ASRQA. 3. Mixture of claims 1 and 2 characterized in that the amino acid sequence of the peptides has been modified in comparison to the natural sequences by replacement or deletion of one or more amino-acids in such a way that the immunomedical reactivity is maintained or Improved. 4. The mixture as claimed in at least one of claims I to 3, wherein the peptides are linked to one another directly or via carrier. The mixture of at least one of claims 1 to 4, wherein the peptides are immobilized to a solid support. 93 6. The mixture of claim 1, wherein the peptides have been synthesized by peptide chemistry. 7. The mixture of at least claims 1 to 6 characterized in that it contains additional HIV peptides, which may be recombinant or synthetici and may be combined with e- ee~binnt and synthetic .peptides according to claim 6. DNA 8. AAsequence, which codes for the peptides of a mixture according to at least one of the claims 1 to 9. Antibody having a specific affinity for a mixture of peptides according to a least one of the claims 1 to 6. Immunochemical method for the detection and/or determina- tion of antibodies against HCV, wherein a mixture of peptides according to at least one of claims 1 to 7 is used. 11. Analytical method for the detection and determination of HCV eaz ricd intha e-spef-i p -sitep4s a hybrid- S"o ization reaction wherein a probe is used which in it's spe. i-e-prt is complementary to a DNA sequence according to claim 8. oo 12. The method of claim 10, wherein the mixture of peptides is immobilized on a carrier. 13. The method of claim 12, wherein the carrier is selected from the group comprising polystyrene, polyvinyl chloride, polyamide and other sy^S:hetic polymers, natural polymers such as cellulose and derivatized natural polymers such as b: cellulose acetate and nitrocellulose, as well as glass, especially as glass fibers. 14. The method of claim 13, wherein the carrier is poly- Sstyrene. 4Lr' 4.A de-oi anJ./or de. 4 4-mmahonl 6f 14ICV' aiif 4 (i'te The method a-z-elme e&4.-_-e4m--24 wherein the detecticni and/or the determination of ar epitope-bound antibody is carried out with the aid of enzyme-labeled, fluorescence- labeled, chemilumiriescence-labeled, biotin-labeled or radioactively labele'd antibodies against the epitope-bound antibodies. 16. Method of claim 15, wherein the enzymes alkaline phospha- tase and/or horseradish peroxidase are used as enzyme labeles for the antibodies. 17. The method of claim 12, characterized in that the "Immuno- Dot" method is used. 18. The method of using mixtures of peptides as claimed in at least one of olaims 1 to 7 for raising antibodies in mammals, especia'lly in humans. 19. The use of the antibodies as claimed in claim 9 for diagnostic and therapeutic purposes. a. 0 20. The use of antibodies as claimed in claim 19, wherein the diagnostic method is a heterogeneous immunoassay. Inm4,,n1 -e V1-f@ n *3 4 at~~~_a47 determination of HCV antibodies hre-t-Hi__ test kit contains one 2 jor-ec rriers on which peptide mixtures as cL 4 i~i1:n~it1 at one-of--e.-alms-I--te-a e-mmobl-i-z ed. DATED this 9th day of November, 1993. E A KTI EN G.E.ELLS.QHj&E ve&. $*too: WATERMARK PATENT 'TRADEMARK ATTORNEYS THE ATRIUM 290 BURWOOD ROAD HAWTHORN VICTORIA 3122 "'RA4,,AUSTRALIA Sequence listing:HO 90B02 HOE 90/B 032K Sequence No. 1 HCV amino-acid sequence 121 175 SGKPAI IPDREVLYREFDEMEECSQHLPYI EQGMMLAEQFKQKAL-GLLQTASRQA Sequence No. 2 HCV amino-acid sequence 121 160 SGKPAI IPDREVLYREFDEMEECSQHLPYIEQGMMLAEQF *9 a B. B *0OS B 0Be B a. S 0Oe S. B a. Sequence No. 3 HCV amino-acid sequence 128 16 PDREVLYREFDEMEECS QHLPY IEQGMMLAEQF Sequence No. 4 HCV amino-acid sequence 136 16 EFDEMEECSQHLPYIEQGMMLAEQF 0 0 Sequence No. HCV amino-acid sequence B BOSS,, a S .0.55, S 144 160 SQHLPYIEQGMMLAEQF Sequence No. 6 HCV amino-acid sequence 161 175 KQKAILGLILQTASRQA Sequence No. 7 HCV amino-acid sequence 144 175 SQHLPYIEQGMIAEQFKQKALGLLQTASRQA Sequence No. 8 HCV amino-acid sequence 135 164 REFDEMEECSQHLPYIEQGMMLAEQFKQKA Sequence No. 9 HCV amino-acid sequence :121 135 *****SGKPAIIPDREVLYR a Sequence No. HCV amino-acid sequence 129 143 DREVLYREFDEMEEC a,...:Sequence No. 11 HCV amino-acid sequence 137 151 FDEMEECSQHLPYIE Sequence No. 12 HCV amino-acid sequence 145 159 QHLPYIEQGMMLAEQ Sequence No. 13 HCV amino-acid sequence 153 167 GMLAEQFKQKAJGL Sequence No. 14 HCV amino-acid sequence 345 359 VQWMNRLIAFASRGN Sequence No. HCV amino-acid sequence 121 139 SGKPAIIPDREVLYREFDE :Sequence No, 16 HCV amino-acid sequence. a a to** SeuneN.1 6 Sequence No. 17 HWV amino-acid sequence 8 26 QRKTKRNTN.'MPQDVKFPG S SP 2.3 Sequence No, HCV amino-acid sequence 26 KTMTNRPQD'VKFPG SP Sequence No. 21 HCV amino-acid sequence 12 26 IWN~iPQDVKFPG SP 30 D Sequence No. 22 HCV amino-acid sequence 14 26 a.:NTNMRPQDVKFPG SP 30 C Wood Sequence No. 23 H-Cy amino-acid sequence B..16 26 NRRPQDVKFPG SP 30 B Sequence No. 24 IiCV amino-acid sequence 18 26 SRPQDVKFPG SP 30 A Sequence No. HCV amino-acid sequence 8 24 QRIK'KRNTNRP.PQDVXF P 1 SP 31 Sequence No. 26 HCV amino-acid sequence 9e me *9 0.0 C .me C a. w mm. 1 0 ii m 0990 ~I 0 mm.' 9 00 C 4 C C QRIKTKRNTNRRPQDV Sequence No. 27 HCV amino-acid sequence -DVE'VLYR- Sequence No. 28 HCV amino-acid sequence -QHLPYIE- Sequence No. 29 HCV amino-acid sequence -KQKALGL- Sequence No. NCV amino-acid sequence -0,RKTIRNTNRRPQDVK- Sequence No. 31 HIV amino-acid sequence SP 32 586 620 RI LAVERYLKDQQLLGIWGCSGKLI CTTAVPWNAS Sequence No. 32 HIV amino-acid sequence 578 613 RVTAIEKYLQDQARLNSWGCAFRQVCHTTVPWVNDS