PT97247B - PREPARATION PROCESS OF ANTIGEN COMBINATIONS OF HEPAPITE C VIRUS (HCV) FOR USE IN IMMUNOSENSES FOR ANTI-HCV ANTIBODIES - Google Patents

Abstract

This invention relates to combinations of HCV antigens having a wider range of immunological reactivity than any HCV antigen isolated. The combinations consist of an antigen from the C domain of the HCV polyprotein, and at least one additional HCV antigen, either from the NS3 domain, or from the NS4 domain, or from the S domain, or from the NS5 domain, the same combinations being in the form of a fusion protein, of a simple physical mixture, or of the individual antigens bound to a common solid matrix.

Description

Description of the invention

Applicants have performed additional serological studies with HCV antigens, which confirm that none of the HCV polypeptides that have been identified to date is, when isolated, immunologically reactive with all sera. The lack of an isolated polypeptide that is universally reactive with all sera from individuals with HCV may be due, among other factors, to inter-strain variation in HCV epitopes, to inter-individual variation in humoral response,

and / or the serological variation depending on the stage of the disease.

These additional studies have also enabled applicants to identify combinations of HCV antigens that provide more efficient detection of anti-HCV antibodies than would be allowed by any isolated HCV polypeptide.

Thus, an aspect of this invention is the production of a combination of HCV antigens, comprising:

(a) a first HCV antigen from the domain

Ç; and (b) at least one additional HCV antigen, selected from the group consisting of (i) an HCV antigen from the domain

NS3;

NS4;

S? and (ii) an HCV antigen from domain (iii) an HCV antigen from domain (iv) an HCV antigen from domain

NS5.

In one embodiment, the HCV antigen combination is in the form of a fusion protein, composed of the antigens. In an alternative embodiment, the antigen combination is in the form of the individual antigens attached to a common solid matrix. In yet another embodiment, the combination of antigens is in the form of a mixture of the individual antigens.

Another aspect of the invention is a method for detecting anti-HCV antibodies in a component of a

a mammal suspected of containing said antibodies, said method comprising establishing contact between said component of the organism and the combination of HCV antigens, described above, under conditions that allow the antibody-antigen reaction, and detecting the presence of immune complexes in said antibodies and said antigens.

Another aspect of the invention is a method for detecting anti-HCV antibodies in a component of a mammalian organism that is suspected to contain said antibodies, said method comprising establishing contact between said component of the organism and a set of HCV antigens, simultaneously or sequentially, comprising:

(a) a first HCV antigen from the domain

Ç; and (b) at least one HCV antigen selected from the group consisting of:

(i) an additional HCV antigen from the domain

NS3;

(ii) an HCV antigen from the domain

NS4;

(iii) an HCV antigen from the domain

S; and (iv) an HCV antigen from the domain

NS5, under conditions that allow the antibody-antigen reaction, and the detection of the presence of immunocomplexes of said antibodies and said antigens.

Another aspect of the invention concerns a kit for the

carrying out an assay for detecting anti-HCV antibodies on a component of a mammalian organism suspected of containing said antibodies, characterized by the fact that it comprises, in packaged combination:

(a) said combination of HCV antigens;

(b) standardized control reagents; and (c) instructions for performing the test.

Brief Description of Drawings>

In the Drawings:

Fig. 1 represents the nucleotide sequence of the sense and antisense strands of the cDNA for the HCV polyprotein, and the amino acid sequence encoded by the sense strand.

Fig. 2 schematically represents the amino acid sequence of Fig. 1, showing the supposed domains of the HCV polyprotein.

Embodiments of the invention

Definitions The term HCV antigen means a polypeptide of at least 5 amino acids, more commonly at least 8 to 10 amino acids, which defines an epitope found in an HCV isolate. Preferably, the epitope is exclusive to HCV. When an antigen is designated by an alphanumeric code, the epitope is in the HCV domain that is specified by the alphanumeric term.

synthetic term, as used to characterize an HCV antigen, means that the HCV antigen was obtained by isolation from native sources or by recombinant or chemical synthesis.

domains term means the segments of the HCV polyprotein, shown in Fig. 2, which generally correspond to the supposed HCV structural and non-structural proteins. Domain designations generally follow the convention used to designate Flavivirus proteins. Domain locations, shown in Fig. 2, are approximate only. NS designations denote non-structural domains, while S indicates the domain of the envelope, and C indicates the domain of the nucleocapsid or nucleus (core).

The term fusion polypeptide means a polypeptide in which the HCV antigen (s) is (are) part of a single continuous chain of amino acids, the said chain not being naturally occurring. HCV antigens can be linked together directly, by peptide bonds, or be separated by intervening amino acid sequences. Fusion polypeptides can also contain amino acid sequences exogenous to HCV.

The term common solid matrix means to indicate a solid body to which the individual HCV antigens, or the fusion polypeptide composed of the HCV antigens, are linked, either by covalent bonds or by non-covalent means, such as hydrophobic adsorption.

The term component of a mammalian organism means a fluid or tissue from a mammalian being (e.g., a human being) that commonly contains antibodies produced by that mammal. Such components are well known in the art and include, without limitation, blood, plasma, serum, spinal fluid, lymphatic fluid, secretions from the respiratory, intestinal or genito-urinary tracts, tears, saliva, milk, white blood cells, and myelin.

immunologically reactive term means that the antigen in question will specifically react with an anti-HCV antibody commonly present in a significant proportion of sera from individuals infected with HCV.

immunocomplex term means the combination, or aggregate, formed when an antibody binds to an epitope present on an antigen.

HCV Antigen Combinations

Fig. 2 shows the supposed domains of the HCV polyprotein. The domains from which the antigens used in the combinations are derived are: C, S (or E), NS3, NS4, and NS5. Domain C is assumed to define the HCV nucleocapsid protein. This domain extends from the N end of the polyprotein to approximately amino acid 120 of Fig. 1. The S domain is believed to define the virion envelope protein, and possibly the matrix protein (Μ), and it is assumed to extend from approximately amino acid 120 to amino acid 400 of Fig. 1. The NS3 domain extends from approximately amino acid 1050 to amino acid 1640, and is assumed to constitute the viral protease. The NS4 domain extends from the terminal part of NS3 to approximately amino acid 2000. The function of the NS4 protein is currently unknown. Finally, the NS5 domain extends from about amino acid 2000 to the end of the polyprotein, and is supposed to define viral polymerase.

The sequence shown in Fig. 1 is the sequence of the HCV1 isolate. It is to be expected that the sequences of other strains of blood-borne HCV may differ from the sequence of Fig. 1, particularly in the envelope (S) and nucleocapsid (C) domains. The use of HCV antigens having such sequence diversity is intended to be within the scope of the present invention, provided, however, that this variation does not significantly degrade the immunological reactivity of the antigen towards sera from individuals infected with HCV.

In general, HCV antigens will comprise whole or truncated domains, the fragments of the domains being analyzed for antigenicity by those skilled in the art. The individual HCV antigens used in the combination will preferably comprise the immunodominant portion (i.e., the portion primarily responsible for the immunological reactivity of the polypeptide) of the specified domain. In the case of domain C, it is preferable that the antigen of domain C comprises a major part of the complete sequence of the domain. The antigen called C22 (see Example 4, infra) is particularly preferred. The S domain antigen preferably includes the hydrophobic subdomain located at the N end of the domain. This hydrophobic subdomain extends from approximately amino acid 199 to amino acid 328 of Fig. 1. The HCV antigen called S2 (see Example 3, infra) is particularly preferred. The downstream sequence of the hydrophobic subdomain can

be included in the S domain antigen, if desired.

An antigen of the NS3 domain presently preferred is an antigen called C33c. That antigen includes amino acids from 1192 to 1457, in Fig. 1. A preferred NS4 antigen is C100, which comprises from amino acid 1569 to 1931, in Fig. 1. A preferred NS5 antigen comprises from amino acid 2054 to 2464 , in Fig. 1.

HCV antigen may be in the form of a polypeptide composed entirely of an HCV amino acid sequence, or it may contain an exogenous sequence to HCV (ie, it may be in the form of a fusion protein containing an exogenous sequence ). In the case of an HCV antigen produced by recombination, the production of the antigen in the form of a fusion protein, using SOD, alpha factor or ubiquitin (see US patents with serial numbers 4.751.180, 4,870,008, and the patent application with serial number 390,599, of August 7, 1989 - the exhibition of which is incorporated herein - describing the expression of the SOD, alpha factor and ubiquitin fusion proteins), can increase the degree of expression and / or increase the solubility of the antigen in water. Fusion proteins, such as alpha factor or ubiquitin fusion protein, are processed by the expression host to remove the heterologous sequence. The alpha factor fusion protein is, however, an excretion system, while the ubiquitin fusion proteins remain in the cytoplasm.

In addition, the combination of antigens can be produced in the form of a fusion protein. For example, a continuous fragment of DNA encoding C22 and C33c can be constructed by cloning this fragment into an expression vector, which is used to express a C22 and C33c fusion protein. Similarly, C22 and C100 fusion proteins can be prepared; C22 and S2; C22 and an NS5 antigen; C22, C33c, and S2; C22, C100 and S2; and C22, C33c, C100 and S2. Alternative fragments of the exemplified domain can be used.

Preparation of HCV Antigens

The HCV antigens of the invention are preferably produced by recombination or by the known solid phase chemical synthesis. However, they can also be isolated from dissociated HCV, or from HCV particles, using affinity chromatography techniques, employing antibodies to the antigens.

When produced by recombination techniques, standard procedures can be employed to construct the DNA encoding the antigen, clone that DNA into expression vectors, transform host cells such as bacteria, yeast, insect, or mammalian cells, and express that DNA in order to produce the antigen. As previously indicated, it may be desirable to express the antigen as a fusion protein to increase the degree of expression, to facilitate purification, or to improve solubility. Examples of specific procedures for producing representative HCV antigens are described in the Examples, infra, and in the patent application with serial number 456,637.

Formulation of Antigens for Use in

Immunoassays

HCV antigens can be combined by producing a fusion protein composed of two or more antigens, by immobilizing the individual antigens on a common solid matrix, or by physically mixing them. Antigen fusion proteins can also be immobilized on (attached to) the solid matrix. Methods and means for covalently or non-covalently attaching proteins to solid matrices are well known in the art. The nature of the solid surface will vary depending on the type of test. For tests performed on microtiter domes, the solid surface will be the dome or bowl wall. For tests using spheres, the solid surface will be the surface of the sphere. In tests using rods (i.e., a solid body formed of a porous or fibrous material, such as fabric or paper), the surface will be the surface of the material from which the rod is made. In agglutination tests, the solid surface can be the surface of particles of latex or gelatin. When the individual antigens are attached to the matrix, they can be distributed homogeneously on the surface or be distributed according to a given pattern, such as bands, so that an antigen binding pattern can be discerned.

Simple mixtures of antigens comprise the same antigens in any suitable solvent or dispersant medium.

Types of Assays Using Antigen Combinations

HCV antigens can actually be used in

any type of assay that uses a known antigen for the detection of antibodies. A common feature in all of these assays is the fact that the antigen is brought into contact with the component of the organism suspected of containing anti-HCV antibodies, under conditions that allow the antigen to bind to any such antibody present in the component. Such conditions will typically be physiological temperature, pH and ionic strength, using an excess of antigen. Incubation of the antigen with the sample is followed by the detection of immunocomplexes containing the antigen.

heterogeneous competitive,

The design of immunoassays is subject to many variations, and the technique includes a wide variety of assay types. The protocols may, for example, use solid supports, or immunoprecipitation. Most assays involve the use of labeled antibody, or polypeptide; the markers can be, for example, enzymatic, fluorescent, chemiluminescent, radioactive, or dye molecules. Assays that amplify signals from the immune complex are also known; examples of the latter are assays using biotin or avidin, and enzyme-mediated, labeled immunoassays, such as ELISA assays.

immunoassay can be, without limitation, character or homogeneous, and of a standardized type or In a heterogeneous assay, the polypeptide is typically attached to a matrix or solid support, in order to facilitate the separation between the sample and the polypeptide, after incubation. Examples of solid supports that can be used include nitrocellulose (e.g., in the form of a membrane or

microtiter dome), polyvinyl chloride (eg, in microtiter sheets or domes), polystyrene latex (eg, in microtiter beads or plates), polyvinylidine fluoride (known as Immulon®, diazotized paper, nylon membranes, spheres activated, and Protein A beads. For example, Dynatech InunulonO ^ 1 or Immulon ^) 2 microtiter plates, or 0.25 inch polystyrene beads (Precision Plastic Bali) can be used in heterogeneous format. The solid support containing the antigenic polypeptides is typically washed after separation from the test sample, and prior to detection of bound antibodies. Standardized and competitive tests are already known in the art.

In a homogeneous assay, the test sample is incubated with the combination of antigens in solution. For example, the assay can run under conditions that will cause any antibody-antigen complexes formed to precipitate. Standardized and competitive types for these tests are also known in the art.

In a standardized assay, the amount of HCV antibodies that form the antibody-antigen complex is directly monitored. This can be achieved by determining the amount of labeled anti-xenogenic (e.g., anti-human) antibodies, which recognize an epitope present in the anti-HCV antibodies, which will bind as a result of complex formation. In a competitive assay, the amount of anti-HCV antibodies present in a sample is deduced by monitoring the competitive effect, relative to binding, of a known amount of labeled antibody (or other competitive ligand) present in the complex.

The complexes formed, comprising the anti-HCV antibody (or, in the case of competitive assays, the given amount of competitive antibody), are detected by any of a variety of known techniques, depending on the type of assay. For example, unlabeled anti-HCV antibodies present in the complex can be detected using a conjugate formed by an antixenogeneic Ig complexed with a marker (e.g., an enzyme marker).

In an immunoprecipitation or agglutination assay, the reaction between HCV antigens and the antibody forms a network, which precipitates out of the solution or suspension, and forms a visible precipitate layer or film. If no anti-HCV antibody is present in a test specimen, no visible precipitate is formed.

HCV antigens will typically be packaged as a kit for use in these immunoassays. The kit will normally contain in separate deposits the combination of antigens (either already bound to a solid matrix, or separated, with reagents to bind them to the matrix), antibody control formulations (positive and / or negative), antibodies labeled when the type assay requires them, and signal generating reagents (eg, enzymatic substrate) if the marker does not generate the signal directly. Instructions for carrying out the test will generally be included (e.g., written, recorded, on video cassette, on CD-ROM, etc.).

The following examples are intended to illustrate the invention and are not intended to limit it in any way.

Example 1: Synthesis of HCV C33c Antigen HCV C33c antigen contains a sequence from the NS3 domain. Specifically, the antigen includes amino acids 1192-1457 of Figure 1. This antigen was produced in bacteria as a fusion protein with human superoxide dismutase (SOD), as follows. The pSODcfl vector (Steiner et al. (1986), J. Virol. 58: 9) was fully digested by EcoRI and BAMHI, and the resulting EcoRI, BamHI digest fragment was linked to the following linker to form pcflEF:

GATC CTG GAA TTC TGA TAA

GAC CTT AAG ACT ATT TTA A

A cDNA clone coding for amino acids 1192-1457 was introduced in pcflEF, and having ends digested by EcoRI, in order to form pcflEF / C33c. This expression construct was transformed using E. coli D1210 cells.

The transformants were used to express a fusion protein, composed of SOD at the N-terminus and HCV C33c antigen, inside the chain at the C-terminus. Expression was made possible by inoculating 1500 ml of Luria broth containing ampicillin (100 μg / ml ) with 15 ml of a 12-hour culture of the transformants. The cells were grown to a

OF. 0.3, IPTG was added to produce a final concentration of 2 mM, and the culture continued until the cells reached a density of 1 OD, at which point they were collected by centrifugation at 3,000 xg at 4 ° C for 20 minutes. Stacked cells can be stored at -80 ° for several months.

In order to purify the SOD-C33c polypeptide, the bacterial cells in which the polypeptide was expressed were subjected to osmotic shock and mechanical disruption, the fraction being insoluble, containing SOD-C33c, isolated and subjected to differential extraction with an alkaline solution of NaCl, and the fusion polypeptide present in the purified extract, by chromatography on sepharose S and sepharose Q columns.

crude extract, resulting from osmotic shock and mechanical rupture, was prepared by the following procedure. 1g of the stacked cells were suspended in 10 ml of a solution containing 0.02 M Tris-HCl, pH 7.5, 10 mM EDTA, 20% sucrose, and incubated for 10 minutes on ice. A cell pellet was then obtained by centrifugation at 4,000 x g, for 15 min., At 4 ° C. After removing the supernatant, the cell pellets were resuspended in 10 ml of Al Buffer (0.01 M Tris-HCl, pH 7.5, 1 mM EDTA, 14 mM beta-mercaptoethanol [BME]), and incubated on ice for 10 minutes. The cells were again centrifuged to form a pellet at 4,000 x g for 15 minutes at 4 ° C. After removing the cleared supernatant (periplasmic fraction I), the cell pellets were resuspended in Buffer Al, incubated on ice for 10 minutes, and again centrifuged at 4,000 x g for 15 minutes at 4 ° C. The cleared supernatant (periplasmic fraction II) was removed, and the cell pellet resuspended in 5 ml of Buffer A2 (0.02 M Tris-HCl, pH 7.5, 14 mM BME, 1 mM EDTA, 1 mM PMSF). To obtain cell rupture, the suspension (5 ml) and 7.5 ml of lead-free Dyno-mill glass beads, washed with acid (0.10-0.15 mm in diameter; obtained through Glen-Mills , Inc.) were placed

in a Falcon tube and vortexed at maximum speed for two minutes, followed by cooling on ice of at least 2 minutes; the vortexing-cooling procedure was repeated four more times. After vortexing, the fine particle suspension was filtered through a glass funnel using low suction; the glass beads were washed twice with Buffer A2, and the filtrate and washes were combined.

The insoluble fraction of the crude extract was collected by centrifugation at 20,000 x g, for 15 minutes, at 4 ° C, washed twice with 10 ml of Buffer A2, and resuspended in 5 ml of MILLI-Q water.

A fraction containing SOD-C33c was isolated from the insoluble material, adding to the suspension NaOH (2 M) and NaCl (2 Μ), to obtain a final concentration of 20 mM of each, vortexing the mixture for 1 minute, centrifuging -a at 20,000 xg for 20 minutes at 4 ° C, and retaining the supernatant.

To purify SOD-C33c in sepharose S, the supernatant fraction was adjusted to a final concentration of 6 M urea, 0.05 M Tris-HCl, pH 7.5, 14 mM BME, 1 mM EDTA. This fraction was then applied to a S-Sepharose Fast Flow column (1.5 x 10 cm) that had been equilibrated with Buffer B (0.05 M Tris-HCl, pH 7.5, 14 mM BME, 1 mM EDTA ). After application, the column was washed with two volumes, equivalent to the column volume, of Buffer B. The elution and wash fractions were collected. The flow rate of application and washing was 1 ml / min; and the collected fractions were 1 ml. In order to identify the fractions containing SOD-C33c, aliquots of the fractions were analyzed by electrophoresis on 10% polyacrylamide gels containing SDS, followed by staining with Coomassie blue. Fractions are also analyzed by the Western Blot technique, using an antibody directed against SOD. Fractions containing

SOD-C33c were collected.

A more complete purification of SOD-C33c was carried out in a column of Q sepharose (1.5 x 5 cm) that had been equilibrated with Buffer B. Fractions collected containing SOD-C33c, obtained by chromatography in sepharose S, were applied on the column. The column was then washed with Buffer B, and eluted with 60 ml of a 0.0 to 0.4 M NaCl gradient in Buffer Β. The flow rate for application, washing, and elution was 1 ml / min; the fractions collected were 1 ml. All fractions from the sepharose Q column were analyzed as described for the sepharose S column. The peak of SOD-C33c corresponded to the fraction eluted from the column, the NaCl concentration of about 0.2 M.

SOD-C33c, obtained from the Q sepharose column, had a purity level greater than about 90%, as determined by analysis on polyacrylamide-SDS gels and by immuno-blot using an immuno-blot. monoclonal antibody directed against human SOD.

Example 2; Synthesis of HCV C100 Antigen The HCV C100 antigen contains sequences from the NS3 and NS4 domains. Specifically, it includes amino acids 1569-1931 in Figure 1. This antigen was produced in yeast. A 1270 bp cDNA fragment was prepared, coding for the above-mentioned amino acids and having ends digested by EcoRI.

The construction of a yeast expression vector, in which this fragment was directly fused to the S. cerevisiae ADH2 / GAP promoter, was achieved through a protocol that included amplification of the C100 sequence using a PCR method, followed by binding of the amplified sequence to a cloning vector. After cloning, the C100 sequence was removed, and, together with a sequence containing the ADH2 / GAP promoter, ligated to a large fragment of a yeast vector in order to obtain a yeast expression vector.

PCR amplification of the C100 was achieved using the pS3-56ci00m / 9 ^u vector as a template ^and had been linearized by digestion by Sall. The plasmid pS3-56, which is a derivative of pBR322, contains an expression cassette that is composed of the hybrid yeast ADH2 / GAPDH promoter, upstream of the human superoxide dismutase gene, and an alpha factor transcription terminator, a downstream.

The oligonucleotide primers used for amplification were designed to facilitate cloning into the expression vector, and to introduce a translation termination codon. Specifically, new HindIII (at -5 ') and Sall (at -3') restriction sites were generated with the PCR oligonucleotides. The oligonucleotide containing the Sall restriction site also codes for the stop codons, TAA and TGA. The oligonucleotide containing the HindIII restriction site also contains an untranslated leader sequence derived from the pgap63 gene, located immediately upstream of the AUG codon. The pEco63GAPDH gene has been described by Holland and Holland (1980) and by Kniskern et al. (1986). At

PCR primer sequences, used for direct expression of the

ClOOm were:

5 '

GAG

ACT

TGC

TTC

TCA AGC TAT CCC

TTC AAA ACA AAA TGG CTC AGA CAA AGC AGA GT 3 '

5 'GAG TGC TCG TCG ACT CAT TAG GGG GAA ACA TGG TTC CCC CGG GAG GCG AA 3'.

PCR amplification, using primers and mold, was performed with a Cetus-Perkin-Elmer PCR kit, and according to the manufacturer's instructions. The conditions of the PCR technique were 29 cycles of 94 ° C for 1 minute, 37 ° C for 2 minutes, 72 ° C for 3 minutes, and the final incubation at 72 ° C for 10 minutes. DNA can be stored at 4 ° C or -20 ° C for 12 hours.

After amplification, the PCR products were digested by HindIII and Sall. The main 1.1 kb product was purified by gel electrophoresis, and the eluted purified product was attached to a large restriction fragment by HindIII of pBR322. In order to isolate correct recombinants, competent HB101 cells were transformed with the recombinant vectors, and, after cloning, the desired recombinants were identified based on the predicted size of the HindIII-SalI restriction fragments that were removed from the clones. One of the clones that contained a HindIII-SalI restriction fragment of the correct size was designated as pBR322 / C100 ^- d. Confirmation that this clone contained the amplified C100 was made by direct sequential analysis of the HindIII-SalI restriction fragment.

expression vector containing C100 was constructed by ligating the HindIII-SalI restriction fragment from pBR322 / C100 ~ from a 13.1 kb BamHI-SalI restriction fragment from pBS24.1 to a restriction fragment from BamHI-HindiII, with 1369 bp, containing the ADH2 / GAP promoter (the latter fragment is described in EPO 164,556). The pBS24.1 vector is described in U.S. patent serial number 382,805, dated July 19, 1989. The fragment containing the ADH2 / GAP promoter was obtained by digestion of the pPGAP / AG / HindIII vector by HindiII and BamHI , followed by gel purification of the 1369 bp fragment.

Competent HB101 cells were transformed with the recombinant vectors; and the correct recombinants were identified by the generation of a fragment with 2464 bp and another fragment with 13.1 kb, created by digestion, by BamHI and Sall, of the cloned vectors. One of the cloned recombinant vectors considered correct was designated as pC100 ^- d # 3.

For the expression of C100, competent cells of Saccharomyces cerevisiae, strain AB122 (MATa leu2 ura3-53 prb

1-1122 pep4-3 prcl-407 [cir-0]) were transformed with the expression vector pC100 ~ d # 3. The transformed cells were seeded in plates with URA-sorbitol medium, and the individual transformants were streaked in Leu plates.

The individual clones were grown in Leu ^- medium, with 2% glucose, at 30 ° C, for 24-36 hours. One liter of medium with yeast extract and peptonate (YEP) containing 2% glucose was inoculated with 10 ml of the 12 hour culture, and the resulting culture was grown at 30 ° C, at a stirring rate of 400 rpm and at an aeration rate of 1 liter of air per 1 liter of medium per minute (ie, 1 vvm), for 48 hours. The pH of the medium was not controlled. The culture was grown in a fermenter

BioFlo II, manufactured by New Brunswick Science Corp .. After fermentation, the cells were isolated and analyzed for C100 expression.

The analysis of the expression of the C100 polypeptide by the transformed cells was performed on lysates of total cells and crude extracts, prepared from colonies isolated from yeast obtained from the Leu plates. Crude cell lysates and extracts were analyzed by electrophoresis on SDS polyacrylamide gels, and by Western Blot techniques. Western Blot techniques used rabbit polyclonal antibodies directed against the SOD-CIOO polypeptide expressed by yeasts as probes. The expected size of the C100 polypeptide is 364 amino acids. By gel analysis, the expressed polypeptide has a MW _r of 39.9K.

Both analytical methods demonstrated that the expressed C100 polypeptide was present in total cell lysates, but that it was absent from the crude extracts. These results suggest that the expressed C100 polypeptide may be insoluble.

Example 3: Expression of HCV S2 Antigen

The HCV S2 antigen contains a sequence from the hydrophobic N-terminus of the S domain. It includes amino acids 199-328 of Fig. 1.

protocol for the construction of the expression vector

encoding the S2 polypeptide and its expression in yeast was analogous to that used for the expression of the C100 polypeptide, described in Example 2.

template for the PCR reaction was the vector pBR322 / Pil4a, which had been linearized by digestion by HindIII. Pil4a is a cDNA clone that codes for amino acids 199-328.

The oligonucleotides used as primers for PCR amplification of the S2 coding sequence were as follows.

For the 5 'region of the S2 sequence:

5 'GAG TGC TCA AGC TTC AAA ACA AAA TGG GGC TCT ACC ACG TCA CCA ATG ATT GCC CTA AC 3';

and for the 3 'region of the S2 sequence:

5 'GAG TGC TCG TCG ACT CAT TAA GGG GAC CAG TTC ATC ATC ATA TCC CAT GCC AT 3'.

primer for the 5 'region introduces a restriction site for HindIII and an ATG initiation codon in the amplified product. The primer for the 3 'region introduces translation termination codons and a restriction site for Sal in the amplified product.

The conditions for PCR were 29 cycles of 94 ° C for one minute, 37 ° C for 2 minutes, 72 ° C for 3 minutes, and the final incubation was at 72 ° C for 10 minutes.

The main product of the PCR reaction was a 413 bp fragment, which was gel purified. The purified fragment was linked to the large fragment obtained from pBR322 digested by

HindIII and Sall, obtaining plasmid pBR322 / S2d.

The binding of the S2 fragment, obtained by restriction by HindIII-SalI, with the restriction fragment by BamHI-HindiII, with 1.36 kb, containing the ADH2 / GAP promoter, and with the large restriction fragment, by BamHI-SalI, of yeast vector pBS24.1, produced recombinant vectors, which were cloned. The correct recombinant vectors were identified by the presence of a 1.77 kb fragment, after digestion by BamHI and Sal. An expression vector, constructed from the amplified sequence, and containing the S2-encoding sequence, directly fused to the ADH2 / GAP promoter, is identified as pS2d # 9.

Example 4: Synthesis of HCV Antigen C Antigen HCV C22 antigen belongs to domain C. It comprises amino acids 1-222 of Figure 1.

The protocol for the construction of the expression vector, encoding for the C polypeptide and for its expression in yeast, was analogous to that used for the expression of the C100 polypeptide, described above, except in the following aspects.

template for the PCR reaction was pBR322 / Ag30a, which had been linearized by HindIII. Ag30 is a cDNA clone that codes for amino acids 1-222. The oligonucleotides used as primers for PCR amplification of the coding sequence for polypeptide C were as follows.

For region 5 'of sequence C:

5 'GAG TGC AGC TTC AAA ACA AAA TGA GCA CGA ATC CTA AAC CTC AAA AAA AAA AC 3', and

for region 3 'of sequence C:

5 'GAG TGC TCG TCG ACT CAT TAA CCC AAA TTG CGC GAC CTA CGC CGG GGG TCT GT 3'.

primer for the 5 'region introduces a restriction site for Hindlll in the amplified product, and the primer for the 3' region introduces translation termination codons and a restriction site for Sal. The PCR reaction was run in 29 cycles of 94 ° C for 1 minute, 37 ° C for 2 minutes, 72 ° C for 3 minutes, and the final incubation was at 72 ° C for 10 minutes.

The main product resulting from PCR amplification is a 381 bp polynucleotide. The binding of this fragment to the large restriction fragment, by SalI-HindIII, of pBR322, produced plasmid pBR322 / C2.

The binding of the restriction fragment, by HindIII-SalI, encoding polypeptide C and 381 bp, which was removed from pBR322 / C2, to the restriction fragment, by BamHI / HindIII, with 1.36 kb and containing the ADH2 promoter / GAP, and the large restriction fragment, by BamHl / SalI, from the yeast vector pBS24.1, produced recombinant vectors, which were cloned. The correct recombinant vectors were identified by the presence of a 1.74 kb fragment, after digestion by BamHI and Sal. An expression vector, constructed from the amplified sequence, and containing the sequence encoding C directly fused to the ADH2 / GAp promoter, is identified as pC22.

The analysis of the expression of polypeptide C by transformed cells was performed on lysates of total cells and crude extracts, prepared from colonies of simple yeasts obtained from these Leu ^- plates. Cell lysates and

'''χίί crude extracts were analyzed by electrophoresis on SDS polyacrylamide gels _and d. Polypeptide C is expected to have 122 amino acids, and, by gel analysis, the expressed polypeptide has a MW _r of approximately 13.6 Kd.

Example 5: Synthesis of the NS5 polypeptide

This polypeptide contains a sequence from the N-end of the NS5 domain. Specifically, it includes the amino acids 2054-2464 of Fig. 1. The protocol for the construction of the expression vector encoding the NS5 polypeptide, and for its expression, was analogous to that used for the expression of C33c (see Example 1).

Example 6: Radioimmunoassay (RIA) for anti-HCV antibodies

The HCV antigens of Examples 1-5 were tested in an RIA assay for their ability to detect anti-HCV antibodies in the serum of individuals clinically diagnosed with HCV (Non-A, Non-B) and in the serum drawn from blood of paid donors.

RIA was based on the technique of Tsu and Herzenberg (1980), in SELECTED METHODS IN CELLULAR IMMUNOLOGY (W.H. Freeman & Co.) pages. 373-391, In general, immunotiter plates (Immulon 2, Removawell strips) are coated with purified HCV antigen. The coated plates are incubated with the serum samples or the appropriate controls. During incubation, the antibody, if present, binds immunologically to the solid phase antigen. After removing the unbound material and washing the microtiter plates, human antibody-NANBV antigen complexes are detected by incubation with 125 sheep anti-human immunoglobulin, labeled with I. The unbound labeled antibody is removed by aspiration, and the plates are washed. Radioactivity in individual domes is determined; the amount of human anti-HCV antibody bound is proportional to the radioactivity in the dome.

Specifically, 100 μΐ aliquots containing 0.1 to 0.5 gg of HCV antigen in 0.125 M sodium borate buffer, pH 8.3,

0.075 M NaCl (BBS) was added to each dome of a microtiter plate (Dynatech immulon 2 Removawell Strips). The plate was incubated at 4 ° C overnight in a humid chamber, after which the antigen solution was removed and the domes were washed three times with BBS containing 0.02% Triton X-100 (BBST). To avoid non-specific binding, the domes were coated with bovine serum albumin (BSA), by adding 100 μΐ of a solution of 5mg / ml BSA in BBS, followed by incubation at room temperature for 1 hour; after this incubation, the BSA solution was removed. The reaction between the antigens present in the coated domes and the serum was promoted by adding 100 μΐ of serum samples, diluted 1: 100 in 0.01 M sodium phosphate buffer, pH 7.2, 0.15 M NaCl (PBS) containing 10 mg / ml BSA, and incubating the domes containing serum for 1 hour at 37 ° C. After incubation, serum samples were removed by aspiration, and the domes were washed 5 times with BBST. The amount of antibody bound to the antigen was determined by the binding of sheep IgG F '(ab) 2, labeled with I, to the coated domes. Have been added to each

dome aliquots of 100 μΐ of the labeled probe (specific activity 5-20 μθχ / μ9), and the plates were incubated at 37 ° C for 1 hour, followed by removal of the excess probe by aspiration, and 5 washes with BBST. The amount of bound radioactivity present in each dome was determined by counting on a counter that detects gamma radiation.

Table 1 below shows the results of the tests

carried out with the serum coming from individuals diagnosed as being carriers HCV. Table 1 INDIVIDUAL ANTIGEN S2 C22 C100 C33c NS5 CVH IVDA P P P (+ + +) P P CVH IVDA P P P (+) P P CVH IVDA P P P (+) P P CVH WE. P P P P P AVH NOS HS N N N N N AVH NOS HS P N N N N AVH NOS HS P N N N N AVH NOS HS P / N N N N N AVH PTVH N N N P / N N AVH NOS HS N N N N N AVH WE N N N N P AVH PTVH N N 'N N N AVH IVDA N P N P P AVH PTVH P P / N N N P AVH WE N P N N N AVH IVDA • N P N P P AVH NOS HS P / N N N N N AVH PTVH N N N N N CVH IVDA P P P P P CVH IVDA P P P P P AVH NOS HS N N N N N CVH PTVH P P N N N AVH PTVH P N P (+) P (+++) N CVH PTVH N P P P P CVH NOS HS P P P P N CVH WE N P P / N P P

INDIVIDUAL

ANTIGEN

S2 C22 C100 C33c NS5 CVH IVDA N N N P N AVH IVDA P P P P P AVH IVDA P P P P P CVH IVDA P P P P P AVH IVDA P / N F N P P AVH IVDA N P P P N CVH PTVH P P / N N N N CVH NOS N N N N N CVH NOS N N N N N CVH IVDA P P P P P AVH IVDA P P P P P CVH PTVH P P P P P AVH PTVH? N P P P P AVH IVDA N P N P N AVH NOS N N N N N AVH NOS N N N N N CVH NOS N P N N P CVH NOS P P N N N CVH NOS HS P P P P P CVH PTVH P P N P P AVH (nurse) P P N N N AVH IVDA P P P P N AVH IVDA N P P (4 ·) P (+++) N & VH (nurse) P / N P N N N AVH PTVH P / N P P N P AVH NOS N P / N N N P AVH NOS N P N P N AVH PTVH P P / N N N N AVH PTVH N ' P N P P AVH PTVH P P P P P AVH PTVH N P N N P CVH PTVH P / N P P (+) P (+++) N AVH PTVH N P / N P (+) P (+++) P

INDIVIDUAL

ANTIGEN

S2 C22 C100 C33c NS5 AVH PTVH P <? ) P N P CVH PTVH N P N P P CVH PTVH N P P P P CVH PTVH N N N N N AVH NOS N N N N N AVH nurse P P N N N CVH PTVH N P N N P AVH IVDA N P N P / N N CVH PTVH P P P (+) P (+++) P AVH NOS P P N N N AVH NOS P / N P N N P AVH PTVH P / N P P P P AVH NOS N P P P P / N AVH IVDA N P N N P AVH NOS N P / N N N N AVH NOS P P N N P AVH PTVH N P P P P crypto P P P P P CVH NOS N P P P P CVH NOS N N N N N AVH PTVH N P P (+) P (++) N AVH PTVH N P / N P (+) P (++) P AVH PTVH N P / N P (+) P (++) P CVH IVDA P P P P P CVH IVDA P P P P P CVH IVDA P P P P P CVH IVDA P P P P P AVH NOS N P N N N CVH IVDA P P P P P / N AVH IVDA P P P P N AVH NOS P P N N N AVH NOS P P N N N CVH PTVH P P N N P / N

INDIVIDUAL

ANTIGEN

S2 £ 22 C100 C33c NS5 AVH PTVH N P N P P AVH NOS N N N N N AVH NOS N P N N N AVH NOS P N N N N CVH NOS N N N N N AVH NOS N P / N N N N AVH IVDA N P P. P P crypto N P N N P / N crypto P P P / N P P AVH IVDA N N P P N AVH IVDA N P P P N AVH NOS N N N N N AVH NOS N N N N N CVH IVDA P F P P P CVH PTVH N N · N N N CVH PTVH P P P (+) P (+++) P CVH PTVH P P P (+) P (+++) P CVH NOS P / N N N N N CVH NOS N N N N N CVH PTVH P P P P P CVH PTVH P P P P P CVH PTVH P P P P P AVH IVDA N P P P P CVH NOS N N N N N CVH NOS N N N N N CVH PTVH P P P P P AVH NOS P P N N P / N AVH NOS N Ρ / N N N N CVH PTVH P P N N P CVH NOS N P N N N N AVH NOS N r N N N AVH NOS N P N N N CVH PTVH N P N N N

ANTIGEN

INDIVIDUAL

S2 C2 2 C100 C33c NS5 AVH IVDA N P N P P AVH NOS P N N N N CVH NOS N N N N N CVH NOS N N N N N CVH IVDA P P P P P CVH IVDA P / N P P P P CVH IVDA P P P P P CVH IVDA N P P P P AVH NOS N P N N N CVH IVDA N P N N P CVH IVDA N P N N P AVH PTVH P P N P P AVH PTVH P P N P P CVH NOS N P / N N N P / N CVH NOS N P N N N CVH NOS N N N N N CVH PTVH P P P P P CVH PTVH P P P P P CVH PTVH P P P P P AVH IVDA N P N N P AVH IVDA N P P (++) P (+) P CVH PTVH P P P P P AVH PTVH N P P P P CVH PTVH? N P P P P CVH PTVH? P / N P P P P CVH NOS HS • P P N N N CVH IVDA P P P P N CVH PTVH N P P P P CVH PTVH P P P P P / N CVH NOS P P P P P CVH IVDA P P P P P CVH PTVH P P P P N CVH PTVH P P P P P

individual

ANTIGEN

S2 C22 C100 C33c NS5 CVH NOS N N N N P / N CVH NOS N P / N N N P / N CVH PTVH P P P P P CVH NOS N P N N N CVH NOS N N N N N CVH NOS P P N N P / N CVH NOS N N N N N CVH NOS HS P P P P P CVH NOS HS P P P P P CVH PTVH N N N N N AVH PTVH N P P P P AVH NOS - - CVH PTVH N P P / N P (+++) N crypto P P P P P crypto P P P P P crypto N P N N N crypto N P P P P CVH PTVH P P P P P crypto N N N N N crypto N P N N P / N crypto N P N N P crypto P P P P P crypto N P N P N crypto - crypto - - CVH NOS - - AVH-IVDA N P N P (+) P

INDIVIDUAL _____ ANTIGEN

S2 C2 2 C100 C33c AVH-IVDA N P / N N P (++ HAV = hepatitis acute viral ÇVH = hepatitis chronic viral

PTVH = post-transfusion viral hepatitis

IVDA = intravenous drug addict

Crypto = cryptogenic hepatitis

NOS = source not evident

P = positive

N = negative

According to these results, no single antigen reacted with all sera. The C22 and C33c antigens were the most reactive, and S2 reacted with some sera from some putative cases of acute HCV infection with which no other antigen reacted. Based on these results, the combination of two antigens, which would provide the greatest detection range, is that of C22 and C33c. If the detection of a maximum of acute infections was desired, S2 would be included in the combination.

Table 2, below, shows the results of tests performed on paid blood donors.

Table 2 Antigens

Donor

C100

N

N

P

N

N

N

N

N

Ç33c

N

N

P

N

N

N

N

N czz

N

N

P

N

N

N

N

N

S2

N

N

P

N

N

N

N

N

NS5

N

N

P

N

N

N

N

N

Donor

21 22 ’27

C100 ANTIGENS C33c C22 N N N N N N N N N N N N N N N N N N N N N N N N N N N P P P P P N P P N N N N N P P P P P N N N N N N N N N N N N N N N N N N N N N P P P N N N N N N N · N N N N P N N N N N N N N N N N N N N N N N N N N N

S2

N

N

N

N

N

N

N

N

N

P

P

P

N

N

P

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

NS5

N

N

N. N N N N N N P P P N P P N N Ν 'N N N N P P N N P P N N N N N P P

Τ ’

ί * · *

ANTIGENS

Donor C100 C33c C22 S2 NS5 43 N N N N N 44 N N N N N 45 N N N N N 46 N N N N N 47 P P N N P 48 N N N N N 49 N N N N N 50 N N N N N 51 N P P N P 52 N N N N N 53 N P P N P 54 P P P P N 55 N N N N N 56 N N N N N 57 N N N N N 58 N N N N N '59 N N N N N 60 N N N N • N '61 N N N N N 62 N N N N N 63 N N N N N 64 N N N N N 65 N N N N N 66 N N N N N 67 N N N N N 68 N · N N N N 69 N N N N N 70 P P P P P 71 N N N N N 72 N N N N N 73 P P P P N 74 N N N N N 75 N N N N N 76 N N N N P

ANTIGENS

C100

N

N

N

N

N

N

P

N

N

P

N

N

P

P

N

P

N

N

N

N

N

N

P

N

P

NN

P

N

N

N

P

P

C33c

N

N

N

N

N

N

P

N

N

P

N

N

P

P

N

P

N

N

N

N

N

P

P

N

P

N

N

N

P

N

N

N

P

P

C22

N

N

N

N

N

N

N

P

N

P

N

N

P

P

N

P

N

N

N

N

N

P

P

N

P

N

N

N

P

N

N

N

P

P

S2

N

N

N

N

N

N

N

N

N

P

N

N

P

P

N

N

N

N

N

N

N

P

P

N

P

N

N

N

P

N

N

N

P

N

NS5

N

N

N

N

N

N

N

N

N

N

N

N

P

N

P

N

N

N

N

N

N

P

P

N

P

N

N

N

N

N

N

N

P

P

Donor

111

112

113

114

115

116

117

118

119

120 121 122

123

124

125 1-26

127

128 129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

C100 Antigens C33c C22 S2. NS5 P P P N P N N N N N P P P * P P N N N N N N N N N N P P P P P N N N N N N N N N N N N N N N P P P P P N N N N N N P P N P N N N N N N N N N N N N N N N P N N N N N N N N N N N N N N N N N N N P P P P N N N N N P N N N N N N N N N N N N N N N N N N N N N- N N N N N N N N N N N N N N N N N N N P N N N N P N P P P N N N N N N N N N N N N - N N N

Antigens

Donor

145

146

147

148

149

150

151

152

153

154

155

156

157

158

159

160 161 162

163

164

165

166

167

168

169

170

171

172

173

174

175

176

177

178

C100

N

N

N

N

N

N

N

N

N

P

N

N

N

N

N

N

P

N

N

P

N

P

N

N

N

NN

N

N

N

N

N

N

N

C 3 3 c N N N N N N N N N P N N N N N N P P N N P N P N N N N N N N N N N N N N

C22

N

N

N

N

N

N

N

N

N

P

N

N

N

N

N

N

P

N

N

P

N

P

N

N

N

N

N

N

N

N

N

N

N

N

S2

N

N

N

N

N

N

N

N

N

P

N

N

N

N

N

N

P

N

N

N

N

N

N

N

N

N

N

N

N

N

N

NS5

N

N

N

N

N

N

N

N

N

P

N

N

N

N

N

N

P

N

N

P

N

P

N

N

N

N

N

N

N

N

N

Donor

C100 antigens

179

180 181 182

183

184

185

186 187 | 188 * 189

190

191

192

193

194

195

196 '197

198

199

200

N

N

N

N

P

N

N

N

N

N

N

N

N

N

N

N

N

N

N

P

N

P

C33c

N

N

N

N

P

N

N

N

N

P

N

N

N

N

N

N

N

C22

N

N

N

N

P

N

N

N

N

P

N

N

N

S2

N

N

N

N

P

N

N

N

N

N

N

N

N

NS5

N

N

N

N

P

N

N

N

N

N

N

N

N

N

N

N

N

N

P

N

P

N

P

N

P

The results obtained with paid donors generally confirm the results obtained from the sera of infected individuals.

Example 7: ELISA Determinations of Anti-HCV Antibodies Using a Combination of HCV Antigens Plates coated with a combination of the C22 and C33c antigens are prepared as follows. A solution containing a

coating buffer (50 mM sodium borate, pH 9.0), 21 ml / plate, BSA (25 (ig / ml), C22 and C33c (2.5 μg / ml each), is prepared immediately before adding to the plates Removeawell

Immulon I (Dynatech Corp.). After mixing for 5 minutes, 0.2 ml of the solution is added to the plates per dome, covering and incubating the plates for 2 hours at 37 ° C, after which the solution is removed by aspiration. The domes are washed once with 400 μΐ of washing buffer (100 mM sodium phosphate, pH 7.4, 140 mM sodium chloride, 0.1% (w / v) casein, 1% (w / v)

Triton x-100, 0.01% (w / v) Thimerosal). After removing the washing solution, 200 μΐ of Postcoat solution is added per dome (10 mM sodium phosphate, pH 7.2, 150 mM sodium chloride, 0.1% (w / v) casein, 3% sucrose and phenylmethylsulfonylfluoride (PMSF), 2 mM), covering the plates in a non-watertight manner to prevent evaporation, and allowing them to remain at room temperature for 30 minutes. The domes are then aspirated to remove the solution, and lyophilized until dry, overnight, without heating the support. The prepared plates can be stored at 2-8 ° C in sealed aluminum TM bags containing a desiccant (3 g packs of Sorb-it).

For ELISA determination, 20 μΐ of serum sample or control sample is added to a dome containing 200 μΐ of sample diluent (100 mM sodium phosphate, pH 7.4, 500 mM sodium chloride, 1 mM EDTA , 0.1% (w / v) casein, 0.01% (w / v) Thimerosal, 1% (w / v) Triton X-100, 100 μ9 / ιη1 yeast extract). The plates are sealed, and incubated at 37 ° C for two hours, after which the solution is removed by aspiration, and the domes are washed three times with 400 μΐ of wash buffer (phosphate buffered saline (PBS) containing 0.05% Tween 20). The washed domes are treated with 200 μΐ of horseradish horseradish IgG conjugate (HRP), contained in an Ortho conjugate diluent solution (10 mM sodium phosphate, pH 7.2, 150 mM sodium chloride, 50% (v / v) fetal bovine serum, 1% (v / v) equine serum treated by heat, K3Fe (CN) g 1 mM, 0.05% (w / v) Tween 20, 0.02% ( p / v) of Thimerosal). The treatment is carried out for 1 hour at 37 ° C, the solution is removed by aspiration, and the domes are washed three times with 400 ml of washing buffer, which is also removed by aspiration. To determine the amount of bound enzyme conjugate, 200 μΐ of the substrate solution (10 mg O-phenylenediamine dihydrochloride per 5 ml of developer solution) is added. The developer solution contains 50 mM sodium citrate, adjusted to pH 5.1 with phosphoric acid, and 0.6 μΐ / ml of 30% H2O2. The plates containing the substrate solution are incubated in the dark for 30 minutes, at room temperature, the reactions being stopped by adding 50 μΐ / ml of 4N sulfuric acid, and determining the

From.

Similarly, ELISA techniques can be performed using fusion proteins of C22 and C33c, and C22, C33c and S2 and combinations of C22 and C100, C22 and S2, C22 and an NS5, C22, C33c and S2 antigen, and C22, C100 and S2.

Modifications to the embodiments of the invention, described above, which are obvious to those skilled in the fields of molecular biology, immunology, and related fields are intended to be within the scope of the following claims.

Claims (16)

1. A process for the manufacture of a composition useful in an immunoassay for the detection of hepatitis C, which is characterized by the combination of: (a) a first ACV antigen from the domain Ç; and (b) at least one additional HCV antigen, selected from the group consisting of (I) an HCV antigen from the domain NS3; (II) an HCV antigen from the domain NS4; (III) an HCV antigen from the domain S; and (IV) an HCV antigen from the domain NS5. 2. A process for preparing a combination of synthetic hepatitis C virus (HCV) antigens, characterized by the fact that it comprises: (a) a first HCV antigen consisting essentially of the C domain; and (b) a second HCV antigen from the domain NS3. A process as claimed in claim 2, characterized in that the first HCV antigen is C22, and the second HCV antigen is C33c. A process as claimed in claim 2, characterized in that it includes (c) a third HCV antigen from the domain S. A process as claimed in claim 3, characterized in that it includes (c) the HCV S2 antigen. 6. A process for the manufacture of a composition useful in an immunoassay for the detection of hepatitis C, characterized by the fact that it comprises the combination of: (a) a first HCV antigen consisting essentially of domain C; and (b) a second HCV antigen from the domain NS4. A process as claimed in claim 6, characterized in that the first HCV antigen is C22, and the second HCV antigen is C100. A process as claimed in claim 6, characterized in that it includes (c) a third HCV antigen from the domain S. A process as claimed in claim 7, characterized in that it includes (c) the HCV S2 antigen. 10. A process as claimed in claims 1, 2, 3, 4, 5, 6, 7, 8 or 9, characterized in that the combination is in the form of a fusion polypeptide. A process as claimed in claims 1, 2, 3, 4, 5, 6, 7, 8 or 9, characterized in that the combination has said first HCV antigen and said additional antigens, individually linked to a common solid matrix. A process as claimed in claim 11, characterized in that the solid matrix is the surface of a microtiter plate dome, sphere, or rod. A process as claimed in claims 1, 2, 3, 4, 5, 6, 7, 8 or 9, characterized in that the combination is in the form of a mixture of said first HCV antigen and said (s) additional HCV antigen (s). 14. A method for the detection of antibodies against hepatitis C virus (HCV) in a component of a mammalian organism suspected of containing said antibodies, this method being characterized by the fact that it comprises the establishment of contact between said component of the organism with the HCV synthetic antigen combination of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13, under conditions that allow the antibody-antigen reaction, and the detecting the presence of immunocomplexes of said antibodies and said antigens. 15. A method for the detection of antibodies against the hepatitis C virus (HCV) in a component of a mammalian organism suspected of containing said antibodies, this method being characterized by the fact that it comprises the establishment of ¢ ,,. · Β »Contact between said component of the organism with a set of synthetic HCV antigens, comprising: (a) a first HCV antigen from the domain Ç; and (b) at least one additional HCV antigen, selected from the group consisting of (I) an HCV antigen from the domain NS3; NS4; S; and (II) an HCV antigen from domain (III) an HCV antigen from domain (IV) an HCV antigen from domain NS5, under conditions that allow the antibody-antigen reaction, and the detection of the presence of immunocomplexes of said antibodies and said antigens. 16. A kit for carrying out an assay for the detection of antibodies against hepatitis C antigen (HCV) on a component of a mammalian organism suspected of containing said antibodies, characterized by the fact that it comprises, in packaged combination: (a) the combination of the synthetic HCV antigens of claim 1; (b) standardized control reagents; and (c) instructions for performing the test.