IMMUNOASSAYS FOR ANTI-HCV ANTIBODIES

Abstract: HCV immunoassays comprising an NS3/4a conformational epitope and a multiple epitope fusion antigen are provided, as well as immunoassay solid supports for use with the immunoassays.

Description: IMMUNOASSAYS FOR ANTI-HCN ANTIBODIES Technical Field The present invention pertains generally to viral diagnostics. In particular, the invention relates to immunoassays using multiple HCV antigens, for accurately diagnosing hepatitis C virus infection. Background Of The Invention Hepatitis C Virus (HCV) is the principal cause of parenteral non-A, non-B hepatitis (NANBH) which is transmitted largely through body blood transfusion and body fluid exchange. The virus is present in 0.4 to 2.0% ofthe general population in the United States. Chronic hepatitis develops in about 50% of infections and of these, approximately 20% of infected individuals develop liver cirrhosis which sometimes leads to hepatocellular carcinoma. Accordingly, the study and control ofthe disease is of medical importance. HCV was first identified and characterized as a cause of NANBH by Houghten et al. The viral genomic sequence of HCV is known, as are methods for obtaining the sequence. See, e.g., International Publication Nos. WO 89/04669; WO 90/11089; and WO 90/14436. HCV has a 9.5 kb positive-sense, single-stranded RNA genome and is a member ofthe Flaviridae family of viruses. At least six distinct, but related genotypes of HCV, based on phylogenetic analyses, have been identified (Simmonds et al., J. Gen. Virol. (1993) 74:2391-2399). The virus encodes a single polyprotein having more than 3000 amino acid residues (Choo et al., Science (1989) 244:359-362; Choo et al., Proc. Natl. Acad. Sci. USA (1991) 88:2451-2455; Han et al., Proc. Natl. Acad. Sci. USA (1991) 88:1711-1715). The polyprotein is processed co- and post-translationally into both structural and non-structural (NS) proteins. In particular, as shown in Figure 1, several proteins are encoded by the HCV genome. The order and nomenclature ofthe cleavage products ofthe HCV polyprotein is as follows: NH 2 -C-El-E2-P7-NS2-NS3-NS4a-NS4b-NS5a-NS5b-COOH. Initial cleavage ofthe polyprotein is catalyzed by host proteases which liberate three structural proteins, the N-terminal nucleocapsid protein (termed "core") and two envelope glycoproteins, "El" (also known as E) and "E2" (also known as E2/NS1), as well as nonstructural (NS) proteins that contain the viral enzymes. The NS regions are termed ' NS2, NS3, NS4, NS4a, NS4b, NS5a and NS5b. NS2 is an integral membrane protein with proteolytic activity. NS2, either alone or in combination with NS3, cleaves the NS2- NS3 sissle bond which in turn generates the NS3 N-terminus and releases a large polyprotein that includes both serine protease and RNA helicase activities. The NS3 protease serves to process the remaining polyprotein. Completion of polyprotein maturation is initiated by autocatalytic cleavage at the NS3-NS4a junction, catalyzed by the NS3 serine protease. Subsequent NS3-mediated cleavages ofthe HCV polyprotein appear to involve recognition of polyprotein cleavage junctions by an NS3 molecule of another polypeptide. In these reactions, NS3 liberates an NS3 cofactor (NS4a), NS4b and NS5a (NS5A has a phosphorylation function), and an RNA-dependent RNA polymerase (NS5b). A number of general and specific polypeptides useful as immunological and diagnostic reagents for HCV, derived from the HCV polyprotein, have been described. See, e.g., Houghton et al., European Publication Nos. 318,216 and 388,232; Choo et al., Science (1989) 244:359--362; Kuo et al., Science (1989) 244:362-364; Houghton et al., Hepatology (1991) 14:381-388; Chien et al., Proc. Natl. Acad. Sci. USA (1992) 89:10011-10015; Chien et al., J. Gastroent. Hepatol. (1993) 8:S33-39; Chien et al., International Publication No. WO 93/00365; Chien, D.Y., International Publication No. WO 94/01778. These publications provide an extensive background on HCV generally, as well as on the manufacture and uses of HCV polypeptide immunological reagents. Sensitive, specific methods for screening and identifying carriers of HCN and HCN-contaminated blood or blood products would provide an important advance in medicine. Post-transfusion hepatitis (PTH) occurs in approximately 10% of transfused patients, and HCN has accounted for up to 90% of these cases. Patient care as well as the prevention and transmission of HCN by blood and blood products or by close personal contact require reliable diagnostic and prognostic tools. Accordingly, several assays have been developed for the serodiagnosis of HCN infection. See, e.g., Choo et al., Science (1989) 244:359-362; Kuo et al., Science (1989) 244:362-364; Choo et al., Br. Med. Bull. (1990) 46:423-441; Ebeling et al., Lancet (1990) 335:982-983; van der Poel et al., Lancet (1990) 335:558-560; van der Poel et al., Lancet (1991) 337:317-319; Chien, D.Y., International Publication No. WO 94/01778; Valenzuela et al., International Publication No. WO 97/44469; and Kashiwakuma et al, U.S. Patent No. 5,871,904. A significant problem encountered with some serum-based assays is that there is a significant gap between infection and detection ofthe virus, often exceeding 80 days. This assay gap may create great risk for blood transfusion recipients. To overcome this problem, nucleic acid-based tests (NAT) that detect viral RNA directly, and HCN core antigen tests that assay viral antigen instead of antibody response, have been developed. See, e.g., Kashiwakuma et al., U.S. Patent No. 5,871,904. However, there remains a need for sensitive, accurate diagnostic and prognostic tools in order to provide adequate patient care as well as to prevent transmission of HCV by blood and blood products or by close personal contact. Summary ofthe Invention The present invention is based in part, on the finding that the use of NS3/4a conformational epitopes, in combination with multiple epitope fusion antigens, provides a sensitive and reliable method for detecting early HCV seroconversion. The assays described herein can also detect HCV infection caused by any ofthe six known genotypes of HCV. The use of multiple epitope fusion proteins also has the added advantages of decreasing masking problems, improving sensitivity in detecting antibodies by allowing a greater number of epitopes on a unit area of substrate, and improving selectivity. Accordingly, in one embodiment, the subject invention is directed to an immunoassay solid support consisting essentially of at least one HCN ΝS3/4a conformational epitope and a multiple epitope fusion antigen, bound thereto, wherein said NS3/4a epitope and/or said multiple epitope fusion antigen react specifically with anti- HCN antibodies present in a biological sample from an HCN-infected individual. The ΝS3/4a epitope may comprise the amino acid sequence depicted in Figures 3 A-3D, or an amino acid sequence with at least 80% sequence identity thereto, or 90% sequence identity thereto, or at least 98% sequence identity thereto, or any integer in between, so long as the sequence has protease activity. In certain embodiments, the NS3/4a conformational epitope consists ofthe amino acid sequence depicted in Figures 3A-3D. In additional embodiments, the multiple epitope fusion antigen comprises the amino acid sequence depicted in Figures 5A-5F, or an amino acid sequence with at least 80% sequence identity thereto, or 90% sequence identity thereto, or at least 98% sequence identity thereto, or any integer in between, so long as the sequence reacts specifically with anti-HCV antibodies present in a biological sample from an HCN- infected individual. In certain embodiments, the multiple epitope fusion antigen consists ofthe amino acid sequence depicted in Figures 5A-5F. In yet another embodiment, the subject invention is directed to an immunoassay solid support consisting essentially of at least one HCN ΝS3/4a conformational epitope and a multiple epitope fusion antigen, bound thereto, wherein said NS3/4a conformational epitope comprises the amino acid sequence depicted in Figures 3A-3D, or an amino acid sequence with at least 80% sequence identity thereto which has protease activity, and said multiple epitope fusion antigen comprises the amino acid sequence depicted in Figures 5A-5F, or an amino acid sequence with at least 80% sequence identity thereto which reacts specifically with anti-HCV antibodies present in a biological sample from an HCN-infected individual. In certain embodiments, the ΝS3/4a conformational epitope and the multiple epitope fusion antigen have at least 90%, 98% (or any integer between) sequence identity to the amino acid sequences of Figures 3A-3D and Figures 5A-5F, respectively, so long as the NS3/4a sequence has protease activity, and the multiple epitope fusion antigen reacts specifically with anti-HCN antibodies present in a biological sample from an HCN-infected individual. In certain embodiments, the ΝS3/4a conformational epitope consists ofthe amino acid sequence depicted in Figures 3A-3D, and the multiple epitope fusion antigen consists ofthe amino acid sequence depicted in Figures 5A-5F. In another embodiment, the invention is directed to an immunoassay solid support consisting essentially of at least one HCV NS3/4a conformational epitope and a multiple epitope fusion antigen, bound thereto, wherein said NS3/4a conformational epitope consists ofthe amino acid sequence depicted in Figures 3A-3D, and said multiple epitope fusion antigen consists ofthe amino acid sequence depicted in Figures 5A-5F. In still a further embodiment, the invention is directed to a method of detecting hepatitis C virus (HCN) infection in a biological sample, said method comprising: (a) providing an immunoassay solid support as described above; (b) combining a biological sample with said solid support under conditions which allow HCN antibodies, when present in the biological sample, to bind to said ΝS3/4a epitope and/or said multiple epitope fusion antigen to form a first immune complex; (c) adding to the solid support from step (b) under complex forming conditions a detectably labeled antibody, wherein said labeled antibody is reactive with said immune complex; (d) detecting second immune complexes formed between the detectably labeled antibody and the first immune complex, if any, as an indication of HCV infection in the biological sample. In still a further embodiment, the invention is directed to a method of detecting hepatitis C virus (HCV) infection in a biological sample, said method comprising: (a) providing an immunoassay solid support consisting essentially of at least one HCV NS3/4a conformational epitope and a multiple epitope fusion antigen, bound thereto, wherein said NS3/4a conformational epitope consists ofthe amino acid sequence depicted in Figures 3A-3D, and said multiple epitope fusion antigen consists ofthe amino acid sequence depicted in Figures 5A-5F; (b) combining a biological sample with said solid support under conditions which allow HCN antibodies, when present in the biological sample, to bind to said ΝS3/4a epitope and/or said multiple epitope fusion antigen to form a first immune complex; (c) adding to the solid support from step (b) under complex forming conditions a detectably labeled antibody, wherein said labeled antibody is reactive with said immune complex; (d) detecting second immune complexes formed between the detectably labeled antibody and the first immune complex, if any, as an indication of HCV infection in the biological sample. In another embodiment, the invention is directed to an immunodiagnostic test kit comprising an immunoassay solid support as described above, and instructions for conducting the immunodiagnostic test. In another embodiment, the subject invention is directed to a method of producing an immunoassay solid support, comprising: (a) providing a solid support; and (b) binding to the solid support at least one HCV NS3/4a conformational epitope and a multiple epitope fusion antigen, wherein said NS3/4a epitope and/or said multiple epitope fusion antigen react specifically with anti-HCV antibodies present in a biological sample from an HCV-infected individual. In certain embodiments, the conformational epitope comprises the amino acid sequence depicted in Figures 3A-3D, or an amino acid sequence with at least 80% sequence identity thereto, or 90% sequence identity thereto, or at least 98% sequence identity thereto, or any integer in between, so long as the sequence has protease activity; and the multiple epitope fusion antigen comprises the amino acid sequence depicted in Figures 5A-5F, or an amino acid sequence with at least 80% sequence identity thereto, or 90% sequence identity thereto, or at least 98% sequence identity thereto, or any integer in between, so long as the sequence reacts specifically with anti-HCV antibodies present in a biological sample from an HCV-infected individual. In still further embodiments, the NS3/4a conformational epitope consists ofthe amino acid sequence depicted in Figures 3 A-3D and the multiple epitope fusion antigen consists ofthe amino acid sequence depicted in Figures 5A-5F. In another embodiment, the invention is directed to a method of producing an immunoassay solid support, comprising: (a) providing a solid support; and (b) binding to the solid support at least one HCV NS3/4a conformational epitope and a multiple epitope fusion antigen, wherein said NS3/4a conformational epitope consists ofthe amino acid sequence depicted in Figures 3A-3D, and said multiple epitope fusion antigen consists ofthe amino acid sequence depicted in Figures 5A-5F. In still a further embodiment, the subject invention is directed to a multiple epitope fusion antigen comprising the amino acid sequence depicted in Figures 5 A-5F, or an amino acid sequence with at least 80% sequence identity thereto, or 90% sequence identity thereto, or an amino acid sequence with at least 98% sequence identity thereto, or any integer in between, which sequence reacts specifically with anti-HCV antibodies present in a biological sample from an HCV-infected individual. In certain embodiments, the multiple epitope fusion antigen consists ofthe amino acid sequence depicted in Figures 5A-5F. In other embodiments, the invention is directed to a polynucleotide comprising a coding sequence for the multiple epitope fusion antigen, a recombinant vector comprising the polynucleotide and control elements operably linked to said polynucleotide whereby the coding sequence can be transcribed and translated in a host cell, a host cell transformed with the recombinant vector, and a method of producing a recombinant multiple epitope fusion antigen comprising providing a population of host cells as above and culturing said population of cells under conditions whereby the multiple epitope fusion antigen encoded by the coding sequence present in said recombinant vector is expressed. These and other aspects ofthe present invention will become evident upon reference to the following detailed description and attached drawings. Brief Description ofthe Drawings Figure 1 is a diagrammatic representation ofthe HCV genome, depicting the various regions ofthe polyprotein from which the present assay reagents (proteins and antibodies) are derived. Figure 2 is a schematic drawing of a representative immunoassay under the invention. Figures 3 A through 3D depict the DNA and corresponding amino acid sequence of a representative NS3/4a conformational antigen for use in the present assays. The amino acids at positions 403 and 404 of Figures 3 A through 3D represent substitutions of Pro for Thr, and He for Ser, ofthe native amino acid sequence of HCV- 1. Figure 4 is a diagrammatic representation of MEFA 7.1. Figures 5A-5F depict the DNA and corresponding amino acid sequence of MEFA 7.1. Figures 6A-6C show representative MEFAs for use with the subject immunoassays. Figure 6A is a diagrammatic representation of MEFA 3. Figure 6B is a diagrammatic representation of MEFA 5. Figure 6C is a diagrammatic representation of MEFA 6. Figures 7A-7D are diagrams ofthe construction of psMEFA7. Figure 8 is a diagram ofthe construction of psMEFA7.1. Figure 9 is a diagram ofthe construction of pd.HCVla.ns3ns4aPI. Detailed Description ofthe Invention The practice ofthe present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, recombinant DNA techniques and immunology, within the skill ofthe art. Such techniques are explained fully in the literature. See, e.g., Fundamental Virology, 2nd Edition, vol. I & II (B.N. Fields and D.M. Knipe, eds.); Handbook of Experimental Immunology, Vols. I-IV (D.M. Weir and C.C. Blackwell eds., Blackwell Scientific Publications); T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick andN. Kaplan eds., Academic Press, Inc.). It must be noted that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "an antigen" includes a mixture of two or more antigens, and the like. The following amino acid abbreviations are used throughout the text: Alanine: Ala (A) Arginine: Arg (R) Asparagine: Asn (N) Aspartic acid: Asp (D) Cysteine: Cys (C) Glutamine: Gin (Q) Glutamic acid: Glu (E) Glycine: Gly (G) Histidine: His (H) Isoleucine: He (I) Leucine: Leu (L) Lysine: Lys (K) Methionine: Met (M) Phenylalanine: Phe (F) Praline: Pro (P) Serine: Ser (S) Threonine: Thr (T) Tryptophan: Trp (W) Tyrosine: Tyr (Y) Valine: Val (V) I. Definitions In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below. The terms "polypeptide" and "protein" refer to a polymer of amino acid residues and are not limited to a minimum length ofthe product. Thus, peptides, oligopeptides, dimers, multimers, and the like, are included within the definition. Both full-length proteins and fragments thereof are encompassed by the definition. The terms also include postexpression modifications ofthe polypeptide, for example, glycosylation, acetylation, phosphorylation and the like. Furthermore, for purposes ofthe present invention, a "polypeptide" refers to a protein which includes modifications, such as deletions, additions and substitutions (generally conservative in nature), to the native sequence, so long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification. An HCV polypeptide is a polypeptide, as defined above, derived from the HCV polyprotein. The polypeptide need not be physically derived from HCV, but may be synthetically or recombinantly produced. Moreover, the polypeptide may be derived from any ofthe various HCV strains and isolates, such as, but not limited to, any ofthe isolates from strains 1, 2, 3, 4, 5 or 6 of HCV. A number of conserved and variable regions are known between these strains and, in general, the amino acid sequences of epitopes derived from these regions will have a high degree of sequence homology, e.g., amino acid sequence homology of more than 30%, preferably more than 40%, when the two sequences are aligned. Thus, for example, the term "NS3/4a" polypeptide refers to native NS3/4a from any ofthe various HCV strains, as well as NS3/4a analogs, muteins < and immunogenic fragments, as defined further below. The complete genotypes of many of these strains are known. See, e.g., U.S. Patent No. 6,150,087 and GenBank Accession Nos. AJ238800 and AJ238799. The terms "analog" and "mutein" refer to biologically active derivatives ofthe reference molecule, or fragments of such derivatives, that retain desired activity, such as immunoreactivity in the assays described herein. In general, the term "analog" refers to compounds having a native polypeptide sequence and structure with one or more amino acid additions, substitutions (generally conservative in nature) and/or deletions, relative to the native molecule, so long as the modifications do not destroy immunogenic activity. The term "mutein" refers to peptides having one or more peptide mimics ("peptoids"), such as those described in International Publication No. WO 91/04282. Preferably, the analog or mutein has at least the same immunoactivity as the native molecule. Methods for making polypeptide analogs and muteins are known in the art and are described further below. Particularly preferred analogs include substitutions that are conservative in nature, i.e., those substitutions that take place within a family of amino acids that are related in their side chains. Specifically, amino acids are generally divided into four families: (1) acidic — aspartate and glutamate; (2) basic — lysine, arginine, histidine; (3) non-polar « alanine, valine, leucine, isoleucine, praline, phenylalanine, methionine, tryptophan; and (4) uncharged polar ~ glycine, asparagine, glutamine, cysteine, serine threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. For example, it is reasonably predictable that an isolated replacement of leucine with isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar conservative replacement of an amino acid with a structurally related amino acid, will not have a major effect on the biological activity. For example, the polypeptide of interest may include up to about 5-10 conservative or non-conservative amino acid substitutions, or even up to about 15-25 conservative or non-conservative amino acid substitutions, or any integer between 5-25, so long as the desired function ofthe molecule remains intact. One of skill in the art may readily determine regions ofthe molecule of interest that can tolerate change by reference to Hopp/Woods and Kyte-Doolittle plots, well known in the art. By "fragment" is intended a polypeptide consisting of only a part of the intact full-length polypeptide sequence and structure. The fragment can include a C-terminal deletion and/or an N-terminal deletion of the native polypeptide. An "immunogenic fragment" of a particular HCV protein will generally include at least about 5-10 contiguous amino acid residues ofthe full-length molecule, preferably at least about 15- 25 contiguous amino acid residues ofthe full-length molecule, and most preferably at least about 20-50 or more contiguous amino acid residues ofthe full-length molecule, that define an epitope, or any integer between 5 amino acids and the full-length sequence, provided that the fragment in question retains immunoreactivity in the assays described herein. For example, preferred immunogenic fragments, include but are not limited to fragments of HCV core that comprise, e.g., amino acids 10-45, 10-53, 67-88, and 120- 130 ofthe polyprotein, epitope 5-1-1 (in the NS4a/NS4b region ofthe viral genome) as well as defined epitopes derived from any ofthe regions ofthe polyprotein shown in Figure 1, such as but not limited to the El, E2, NS3 (e.g., polypeptide c33c from the NS3 region), NS4 (e.g., polypeptide clOO from the NS3/NS4 regions), NS3/4a and NS5 regions ofthe HCV polyprotein, as well as any ofthe other various epitopes identified from the HCV polyprotein. See, e.g., Chien et al., Proc. Natl. Acad. Sci. USA (1992) 89:10011-10015; Chien et al., J. Gastroent. Hepatol. (1993) 8:S33-39; Chien et al., International Publication No. WO 93/00365; Chien, D.Y., International Publication No. WO 94/01778; U.S. Patent Nos. 6,150,087 and 6,121,020. The term "epitope" as used herein refers to a sequence of at least about 3 to 5, preferably about 5 to 10 or 15, and not more than about 1,000 amino acids (or any integer therebetween), which define a sequence that by itself or as part of a larger sequence, binds to an antibody generated in response to such sequence. There is no critical upper limit to the length ofthe fragment, which may comprise nearly the full-length ofthe protein sequence, or even a fusion protein comprising two or more epitopes from the HCV polyprotein. An epitope for use in the subject invention is not limited to a polypeptide having the exact sequence ofthe portion ofthe parent protein from which it is derived. Indeed, viral genomes are in a state of constant flux and contain several variable domains which exhibit relatively high degrees of variability between isolates. Thus the term "epitope" encompasses sequences identical to the native sequence, as well as modifications to the native sequence, such as deletions, additions and substitutions (generally conservative in nature). Regions of a given polypeptide that include an epitope can be identified using any number of epitope mapping techniques, well known in the art. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, New Jersey. For example, linear epitopes may be determined by e.g., concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions ofthe protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports. Such techniques are known in the art and described in, e.g., U.S. Patent No. 4,708,871 ; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81:3998-4002; Geysen et al. (1985) Proc. Natl. Acad. Sci. USA 82:178-182; Geysen et al. (1986) o/ec. Immunol. 23:709-715. Using such techniques, a number of epitopes of HCV have been identified. See, e.g., Chien et al., Viral Hepatitis and Liver Disease (1994) pp. 320-324, and further below. Similarly, conformational epitopes are readily identified by determining spatial conformation of amino acids such as by, e.g., x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, supra. Antigenic regions of proteins can also be identified using standard antigenicity and hydropathy plots, such as those calculated using, e.g., the Omiga version 1.0 software program available from the Oxford Molecular Group. This computer program employs the Hopp/Woods method, Hopp et al., Proc. Natl. Acad. Sci USA (1981) 78:3824-3828 for determining antigenicity profiles, and the Kyte-Doolittle technique, Kyte et al., J. Mol. Biol. (1982) 157:105-132 for hydropathy plots. As used herein, the term "conformational epitope" refers to a portion of a full- length protein, or an analog or mutein thereof, having structural features native to the amino acid sequence encoding the epitope within the full-length natural protein. Native structural features include, but are not limited to, glycosylation and three dimensional structure. The length ofthe epitope defining sequence can be subject to wide variations as these epitopes are believed to be formed by the three-dimensional shape ofthe antigen (e.g., folding). Thus, amino acids defining the epitope can be relatively few in number, but widely dispersed along the length ofthe molecule, being brought into correct epitope conformation via folding. The portions ofthe antigen between the residues defining the epitope may not be critical to the conformational structure ofthe epitope. For example, deletion or substitution of these intervening sequences may not affect the conformational epitope provided sequences critical to epitope conformation are maintained (e.g., cysteines involved in disulfide bonding, glycosylation sites, etc.). Conformational epitopes present in the NS3/4a region are readily identified using methods discussed above. Moreover, the presence or absence of a conformational epitope in a given polypeptide can be readily determined through screening the antigen of interest with an antibody (polyclonal serum or monoclonal to the conformational epitope) and comparing its reactivity to that of a denatured version ofthe antigen which retains only linear epitopes (if any). In such screening using polyclonal antibodies, it may be advantageous to absorb the polyclonal serum first with the denatured antigen and see if it retains antibodies to the antigen of interest. Additionally, in the case of NS3/4a, a molecule which preserves the native conformation will also have protease and, optionally, helicase enzymatic activities. Such activities can be detected using enzymatic assays, as described further below. Preferably, a conformational epitope is produced recombinantly and is expressed in a cell from which it is extractable under conditions which preserve its desired structural features, e.g. without denaturation ofthe epitope. Such cells include bacteria, yeast, insect, and mammalian cells. Expression and isolation of recombinant conformational epitopes from the HCV polyprotein are described in e.g., International Publication Nos. WO 96/04301, WO 94/01778, WO 95/33053, WO 92/08734. Alternatively, it is possible to express the antigens and further renature.the protein after recovery. It is also understood that chemical synthesis may also provide conformational antigen mimitopes that cross-react with the "native" antigen's conformational epitope. The term "multiple epitope fusion antigen" or "MEFA" as used herein intends a polypeptide in which multiple HCV antigens are part of a single, continuous chain of amino acids, which chain does not occur in nature. The HCV antigens may be connected directly to each other by peptide bonds or may be separated by intervening amino acid sequences. The fusion antigens may also contain sequences exogenous to the HCV polyprotein. Moreover, the HCV sequences present may be from multiple genotypes and/or isolates of HCV. Examples of particular MEFAs for use in the present immunoassays are detailed in, e.g., International Publication No. WO 97/44469, and are described further below. An "antibody" intends a molecule that, through chemical or physical means, specifically binds to a polypeptide of interest. Thus, for example, an HCV core antibody is a molecule that specifically binds to the HCV core protein. The term "antibody" as used herein includes antibodies obtained from both polyclonal and monoclonal preparations, as well as, the following: hybrid (chimeric) antibody molecules (see, for example, Winter et al. (1991) Nature 349:293-299; and U.S. Patent No. 4,816,567); F(ab')2 and F(ab) fragments; Fv molecules (non-covalent heterodimers, see, for example, Inbar et al. (1972) Proc Natl Acad Sci USA 69:2659-2662; and Ehrlich et al. (1980) Biochem 19:4091-4096); single-chain Fv molecules (sFv) (see, for example, Huston et al. (1988) Proc Natl Acad Sci USA 85:5879-5883); dimeric and trimeric antibody fragment constructs; minibodies (see, e.g., Pack et al. (1992) Biochem 31:1579-1584; Cumber et al. (1992) J Immunology 149B: 120-126); humanized antibody molecules (see, for example, Riechmann et al. (1988) Nature 332:323-327; Verhoeyan et al. (1988) Science 239:1534- 1536; and U.K. Patent Publication No. GB 2,276,169, published 21 September 1994); and, any functional fragments obtained from such molecules, wherein such fragments retain immunological binding properties ofthe parent antibody molecule. As used herein, the term "monoclonal antibody" refers to an antibody composition having a homogeneous antibody population. The term is not limited regarding the species or source ofthe antibody, nor is it intended to be limited by the manner in which it is made. Thus, the term encompasses antibodies obtained from murine hybridomas, as well as human monoclonal antibodies obtained using human rather than murine hybridomas. See, e.g., Cote, et al. Monclonal Antibodies and Cancer Therapy, Alan R. Liss, 1985, p. 77. By "isolated" is meant, when referring to a polypeptide, that the indicated molecule is separate and discrete from the whole organism with which the molecule is found in nature or is present in the substantial absence of other biological macro- molecules ofthe same type. The term "isolated" with respect to a polynucleotide is a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences in association therewith; or a molecule disassociated from the chromosome. By "equivalent antigenic determinant" is meant an antigenic determinant from different sub-species or strains of HCV, such as from strains 1, 2, or 3 of HCV. More specifically, epitopes are known, such as 5-1-1, and such epitopes vary between the strains 1, 2, and 3. Thus, the epitope 5-1-1 from the three different strains are equivalent antigenic determinants and thus are "copies" even though their sequences are not identical. In general the amino acid sequences of equivalent antigenic determinants will have a high degree of sequence homology, e.g., amino acid sequence homology of more than 30%, preferably more than 40%), when the two sequences are aligned. "Homology" refers to the percent similarity between two polynucleotide or two polypeptide moieties. Two DNA, or two polypeptide sequences are "substantially homologous" to each other when the sequences exhibit at least about 50% , preferably at least about 75%, more preferably at least about 80%-85%, preferably at least about 90%, and most preferably at least about 95%-98% sequence similarity over a defined length of the molecules. As used herein, substantially homologous also refers to sequences showing complete identity to the specified DNA or polypeptide sequence. In general, "identity" refers to an exact nucleotide-to-nucleotide or amino acid-to- amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Percent identity can be determined by a direct comparison ofthe sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length ofthe shorter sequence, and multiplying the result by 100. Readily available computer programs can be used to aid in the analysis of homology or identity, such as ALIGN, Dayhoff, M.O. in Atlas of Protein Sequence and Structure M.O. Dayhoff ed., 5 Suppl. 3:353-358, National biomedical Research Foundation, Washington, DC, which adapts the local homology algorithm of Smith and Waterman Advances in Appl. Math. 2:482-489, 1981 for peptide analysis. Programs for determimng nucleotide sequence homology are available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, WI) for example, the BESTFIT, FASTA and GAP programs, which also rely on the Smith and Waterman algorithm. These programs are readily utilized with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package referred to above. For example, percent homology of a particular nucleotide sequence to a reference sequence can be determined using the homology algorithm of Smith and Waterman with a default scoring table and a gap penalty of six nucleotide positions. Another method of establishing percent homology in the context ofthe present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, CA). From this suite of packages the Smith- Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the "Match" value reflects "sequence homology." Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + Swiss protein + Spupdate + PIR. Details of these programs can be found at the following internet address: http://www.ncbi.nlm.gov/cgi-bin/BLAST. Alternatively, homology can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination ofthe digested fragments. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill ofthe art. See, e.g., Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization, supra. A "coding sequence" or a sequence which "encodes" a selected polypeptide, is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries ofthe coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A transcription termination sequence may be located 3' to the coding sequence. "Operably linked" refers to an arrangement of elements wherein the components so described are configured so as to perform their desired function. Thus, a given promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence when the proper transcription factors, etc., are present. The promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence, as can transcribed introns, and the promoter sequence can still be considered "operably linked" to the coding sequence. "Recombinant" as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, viral, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation is not associated with all or a portion ofthe polynucleotide with which it is associated in nature. The term "recombinant" as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. In general, the gene of interest is cloned and then expressed in transformed organisms, as described further below. The host organism expresses the foreign gene to produce the protein under expression conditions. A "control element" refers to a polynucleotide sequence which aids in the expression of a coding sequence to which it is linked. The term includes promoters, transcription termination sequences, upstream regulatory domains, polyadenylation signals, untranslated regions, including 5'-UTRs and 3'-UTRs and when appropriate, leader sequences and enhancers, which collectively provide for the transcription and translation of a coding sequence in a host cell. A "promoter" as used herein is a DNA regulatory region capable of binding RNA polymerase in a host cell and initiating transcription of a downstream (3' direction) coding sequence operably linked thereto. For purposes ofthe present invention, a promoter sequence includes the minimum number of bases or elements necessary to initiate transcription of a gene of interest at levels detectable above background. Within the promoter sequence is a transcription initiation site, as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eucaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes. A control sequence "directs the transcription" of a coding sequence in a cell when RNA polymerase will bind the promoter sequence and transcribe the coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence. "Expression cassette" or "expression construct" refers to an assembly which is capable of directing the expression ofthe sequence(s) or gene(s) of interest. The expression cassette includes control elements, as described above, such as a promoter which is operably linked to (so as to direct transcription of) the sequence(s) or gene(s) of interest, and often includes a polyadenylation sequence as well. Within certain embodiments ofthe invention, the expression cassette described herein may be contained within a plasmid construct. In addition to the components ofthe expression cassette, the plasmid construct may also include, one or more selectable markers, a signal which allows the plasmid construct to exist as single-stranded DNA (e.g., a Ml 3 origin of replication), at least one multiple cloning site, and a "mammalian" origin of replication (e.g., a SV40 or adenovirus origin of replication). "Transformation," as used herein, refers to the insertion of an exogenous polynucleotide into a host cell, irrespective ofthe method used for insertion: for example, transformation by direct uptake, transfection, infection, and the like. For particular methods of transfection, see further below. The exogenous polynucleotide may be maintained as a nonintegrated vector, for example, an episome, or alternatively, may be integrated into the host genome. A "host cell" is a cell which has been transformed, or is capable of transformation, by an exogenous DNA sequence. As used herein, a "biological sample" refers to a sample of tissue or fluid isolated from a subject, that commonly includes antibodies produced by the subject. Typical samples that include such antibodies are known in the art and include but not limited to, blood, plasma, serum, fecal matter, urine, bone marrow, bile, spinal fluid, lymph fluid, samples ofthe skin, secretions ofthe skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, organs, biopsies and also samples of in vitro cell culture constituents including but not limited to conditioned media resulting from the growth of cells and tissues in culture medium, e.g., recombinant cells, and cell components. "Common solid support" intends a single solid matrix to which the HCV polypeptides used in the subject immunoassays are bound covalently or by noncovalent means such as hydrophobic adsorption. "hrrmunologically reactive" means that the antigen in question will react specifically with anti-HCV antibodies present in a biological sample from an HCV- infected individual. "Immune complex" intends the combination formed when an antibody binds to an epitope on an antigen. As used herein, the terms "label" and "detectable label" refer to a molecule capable of detection, including, but not limited to, radioactive isotopes, fluorescers, chemiluminescers, chromophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, ligands (e.g., biotin, avidin, strepavidin or haptens) and the like. The term "fluorescer" refers to a substance or a portion thereof which is capable of exhibiting fluorescence in the detectable range. Particular examples of labels which may be used under the invention include, but are not limited to, horse radish peroxidase (HRP), fluorescein, FITC, rhodamine, dansyl, umbelliferone, dimethyl acridinium ester (DMAE), Texas red, luminol, NADPH and α-β- galactosidase. II. Modes of Carrying out the Invention Before describing the present invention in detail, it is to be understood that this invention is not limited to particular formulations or process parameters as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments ofthe invention only, and is not intended to be limiting. Although a number of compositions and methods similar or equivalent to those described herein can be used in the practice ofthe present invention, the preferred materials and methods are described herein. As noted above, the present invention is based on the discovery of novel diagnostic methods for accurately detecting early HCV infection. The methods rely on the identification and use of highly immunogenic HCV antigens which are present during the early stages of HCV seroconversion, thereby increasing detection accuracy and reducing the incidence of false results. In particular, the immunoassays described herein utilize highly immunogenic conformational epitopes derived from the NS3/4a region of the HCV polyprotein, and multiple epitope fusion antigens comprising various HCV polypeptides, either from the same or different HCV genotypes and isolates, such as multiple immunodominant epitopes, for example, major linear epitopes of HCV core, El, E2, NS3, 5-1-1, cl00-3 and NS5 sequences. The methods can be conveniently practiced in a single assay, using any ofthe several assay formats described below, such as but not limited to, assay formats which utilize a solid support to which the HCV antigens are bound. The NS3/4a region ofthe HCV polyprotein has been described and the amino acid sequence and overall structure ofthe protein are disclosed in, e.g., Yao et al., Structure (November 1999) 7:1353-1363; Sali et al, Biochem. (1998) 37:3392-3401; and Bartenschlager, R., J. Viral Hepat. (1999) 6:165-181. See, also, Dasmahapatra et al., U.S. Patent No. 5,843,752. The subject immunoassays utilize at least one conformational epitope derived from the NS3/4a region that exists in the conformation as found in the naturally occurring HCV particle or its infective product, as evidenced by the preservation of protease and, optionally, helicase enzymatic activities normally displayed by the NS3/4a gene product and/or immunoreactivity ofthe antigen with antibodies in a biological sample from an HCV-infected subject, and a loss ofthe epitope's immunoreactivity upon denaturation ofthe antigen. For example, the conformational epitope can be disrupted by heating, changing the pH to extremely acid or basic, or by adding known organic denaturants, such as dithiothreitol (DTT) or an appropriate detergent. See, e.g., Protein Purification Methods, a practical approach (E.L.V. Harris and S. Angal eds., IRL Press) and the denatured product compared to the product which is not treated as above. Protease and helicase activity may be determined using standard enzyme assays well known in the art. For example, protease activity may be determined using the procedure described below in the examples, as well as using assays well known in the art. See, e.g., Takeshita et al., Anal. Biochem. (1997) 247:242-246; Kakiuchi et al., J. Biochem. (1997) 122:749-755; Sali et al., Biochemistry (1998) 37:3392-3401; Cho et al., J. Virol. Meth. (1998) 72:109-115; Cerretani et al., Anal. Biochem. (1999) 266:192-197; Zhang et al., Anal. Biochem. (1999) 270:268-275; Kakiuchi et al., J. Virol. Meth. (1999) 80:77-84; Fowler et al., J. Biomol. Screen. (2000) 5:153-158; and Kim et al, Anal. Biochem. (2000) 284:42-48. Similarly, helicase activity assays are well known in the art and helicase activity of an NS3/4a epitope may be determined using, for example, an ELISA assay, as described in, e.g., Hsu et al, Biochem. Biophys. Res. Commun. (1998) 253:594-599; a scintillation proximity assay system, as described in Kyono et al., Anal. Biochem. (1998) 257:120-126: high throughput screening assays as described in, e.g., Hicham et al., Antiviral Res. (2000) 46:181-193 and Kwong et al., Methods Mol. Med. (2000) 24:97-116; as well as by other assay methods known in the art. See, e.g., Khu et al, J. Virol. (2001) 75:205-214; Utama et al., Virology (2000) 273:316-324; Paolini et al., J. Gen. Virol. (2000) 81:1335-1345; Preugschat et al., Biochemistry (2000) 39:5174- 5183; Preugschat et al., Methods Mol. Med. (1998) 19:353-364; and Hesson et al., Biochemistry (2000) 39:2619-2625. The length ofthe antigen is sufficient to maintain an immunoreactive conformational epitope. Often, the polypeptide containing the antigen used will be almost full-length, however, the polypeptide may also be truncated to, for example, increase solubility or to improve secretion. Generally, the conformational epitope found in NS3/4a is expressed as a recombinant polypeptide in a cell and this polypeptide provides the epitope in a desired form, as described in detail below. A representative amino acid sequence for the NS3/4a polypeptide is shown in Figures 3 A through 3D. The amino acid sequence shown at positions 2-686 ofthe figure corresponds to amino acid positions 1027-1711 of HCV-1. An initiator codon (ATG) coding for Met, is shown as position 1. Additionally, the Thr normally occurring at position 1428 of HCV-1 (amino acid position 403 of Figure 3) is mutated to Pro, and the Ser normally occurring at position 1429 of HCV-1 (amino acid position 404 of Figure 3) is mutated to lie. However, either the native sequence, with or without an N-terminal Met, the depicted analog, with or without the N-terminal Met, or other analogs and fragments can be used in the subject assays, so long as the epitope is produced using a method that retains or reinstates its native conformation such that protease activity, and optionally, helicase activity is retained. Dasmahapatra et al., U.S. Patent No. 5,843,752 and Zhang et al., U.S. Patent No. 5,990,276, both describe analogs of NS3/4a. The NS3 protease of NS3/4a is found at about positions 1027-1207, numbered relative to HCV-1 (see, Choo et al., Proc. Natl. Acad. Sci. USA (1991) 88:2451-2455), positions 2-182 of Figure 3. The structure ofthe NS3 protease and active site are known. See, e.g., De Francesco et al., Antivir. Ther. (1998) 3:99-109; Koch et al., Biochemistry (2001) 40:631-640. Changes to the native sequence that will normally be tolerated will be those outside ofthe active site ofthe molecule. Particularly, it is desirable to maintain amino acids 1- or 2-155 of Figure 3, with little or only conservative substitutions. Amino acids occurring beyond 155 will tolerate greater changes. Additionally, if fragments of the NS3/4a sequence found in Figure 3 are used, these fragments will generally include at least amino acids 1- or 2-155, preferably amino acids 1- or 2-175, and most preferably amino acids 1- or 2-182, with or without the N-terminal Met. The helicase domain is found at about positions 1193-1657 of HCV-1 (positions 207-632 of Figure 3). Thus, if helicase activity is desired, this portion ofthe molecule will be maintained with little or only conservative changes. One of skill in the art can readily determine other regions that will tolerate change based on the known structure of NS3/4a. The immunoassays described herein also utilize multiple epitope fusion antigens (termed "MEFAs"), as described in International Publication No. WO 97/44469. Such MEFAs include multiple epitopes derived from two or more ofthe various viral regions ofthe HCV polyprotein shown in Figure 1 and Table 1. In particular, as shown in Figure 1 and Table 1, An HCV polyprotein, upon cleavage, produces at least ten distinct products, in the order ofNH 2 - Core-El-E2-p7-NS2-NS3-NS4a-NS4b-NS5a-NS5b- COOH. The core polypeptide occurs at positions 1-191, numbered relative to HCV-1 (see, Choo et al. (1991) Proc. Natl. Acad. Sci. USA 88:2451-2455, for the HCV-1 genome). This polypeptide is further processed to produce an HCV polypeptide with approximately amino acids 1-173. The envelope polypeptides, El and E2, occur at about positions 192-383 and 384-746, respectively. The P7 domain is found at about positions 747-809. NS2 is an integral membrane protein with proteolytic activity and is found at about positions 810-1026 ofthe polyprotein. NS2, either alone or in combination with NS3 (found at about positions 1027-1657), cleaves the NS2-NS3 sissle bond which in turn generates the NS3 N-terminus and releases a large polyprotein that includes both serine protease and RNA helicase activities. The NS3 protease, found at about positions 1027-1207, serves to process the remaining polyprotein. The helicase activity is found at about positions 1193-1657. Completion of polyprotein maturation is initiated by autocatalytic cleavage at the NS3-NS4a junction, catalyzed by the NS3 serine protease. Subsequent NS3-mediated cleavages ofthe HCV polyprotein appear to involve recognition of polyprotein cleavage junctions by anNS3 molecule of another polypeptide. In these reactions, NS3 liberates an NS3 cofactor (NS4a, found about positions 1658-1711), two proteins (NS4b found at about positions 1712-1972, andNS5a found at about positions 1973-2420), and an RNA-dependent RNA polymerase (NS5b found at about positions 2421-3011). *Numbered relative to HCV-1. See, Choo et al. (1991) Proc. Natl. Acad. Sci. USA 88:2451-2455. The multiple HCV antigens are part of a single, continuous chain of amino acids, which chain does not occur in nature. Thus, the linear order ofthe epitopes is different than their linear order in the genome in which they occur. The linear order ofthe sequences ofthe MEFAs for use herein is preferably arranged for optimum antigenicity. Preferably, the epitopes are from more than one HCV strain, thus providing the added ability to detect multiple strains of HCV in a single assay. Thus, the MEFAs for use herein may comprise various immunogenic regions derived from the polyprotein described above. Moreover, a protein resulting from a frameshift in the core region of the polyprotein, such as described in International Publication No. WO 99/63941, may be used in the MEFAs. If desired, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more of one or more epitopes derived from the HCV polyprotein may occur in the fusion protein. For example, epitopes derived from, e.g., the hypervariable region of E2, such as a region spanning amino acids 384-410 or 390-410, can be included in the MEFA antigen. A particularly effective E2 epitope is one which includes a consensus sequence derived from this region, such as the consensus sequence Gly-Ser-Ala-Ala-Arg-Thr-Thr- Ser-Gly-Phe-Val-Ser-Leu-Phe-Ala-Pro-Gly-Ala-Lys-Gln-Asn, which represents a consensus sequence for amino acids 390-410 ofthe HCV type 1 genome. A representative E2 epitope present in a MEFA ofthe invention can comprise a hybrid epitope sparining amino acids 390-444. Such a hybrid E2 epitope can include a consensus sequence representing amino acids 390-410 fused to the native amino acid sequence for amino acids 411-444 of HCV E2. Additionally, the antigens may be derived from various HCV strains. Multiple viral strains of HCV are known, and epitopes derived from any of these strains can be used in a fusion protein. It is well known that any given species of organism varies from one individual organism to another and further that a given organism such as a virus can have a number of different strains. For example, as explained above, HCV includes at least 6 genotypes. Each of these genotypes includes equivalent antigenic determinants. More specifically, each strain includes a number of antigenic determinants that are present on all strains ofthe virus but are slightly different from one viral strain to another. For example, HCV includes the antigenic determinant known as 5-1-1 (See, Figure 1). This particular antigenic determinant appears in three different forms on the three different viral strains of HCV. Accordingly, in a preferred embodiment ofthe invention all three forms of 5-1-1 appear on the multiple epitope fusion antigen used in the subject immunoassays. Similarly, equivalent antigenic determinants from the core region of different HCV strains may also be present. In general, equivalent antigenic determinants have a high degree of homology in terms of amino acid sequence which degree of homology is generally 30% or more, preferably 40% or more, when aligned. The multiple copy epitope ofthe present invention can also include multiple copies which are exact copies ofthe same epitope. Figures 4 and 6A-6C show representative MEFAs for use in the present invention which are derived from HCV. However, it is to be understood that other epitopes derived from the HCV genome will also find use with the present assays. The DNA sequence and corresponding amino acid sequence of a representative multiple epitope fusion antigen, MEFA 7.1, is shown in Figures 5 A through 5F. The general structural formula for MEFA 7.1 is shown in Figure 4 and is as follows: hSOD- El(type 1)-E2 HVR consensus(type la)-E2 HVR consensus(types 1 and 2)-helicase(type l)-5-l-l(type l)-5-l-l(type 3)-5-l-l(type 2)-cl00(type l)-NS5(type l)-NS5(type 1)- core(types l+2)-core(types 1+2). This multiple copy epitope includes the following amino acid sequence, numbered relative to HCV-1 (the numbering ofthe amino acids set forth below follows the numbering designation provided in Choo, et al. (1991) Proc. Natl. Acad. Sci. USA 88:2451-2455, in which amino acid #1 is the first methionine encoded by the coding sequence ofthe core region): amino acids 1-156 of superoxide dismutase (SOD, used to enhance recombinant expression ofthe protein); amino acids 303 to 320 ofthe polyprotein from the El region; amino acids 390 to 410 ofthe polyprotein, representing a consensus sequence for the hypervariable region of HCV- la E2; amino acids 384 to 414 ofthe polyprotein from region E2, representing a consensus sequence for the E2 hypervariable regions of HCV-1 and HCV-2; amino acids 1193- 1658 ofthe HCV-1 polyprotein which define the helicase; three copies of an epitope from 5-1-1, amino acids 1689-1735, one from HCV-1, one from HCV-3 and one from HCV-2, which copies are equivalent antigenic determinants from the three different viral strains of HCV; HCV polypeptide C100 of HCV-1, amino acids 1901-1936 ofthe polyprotein; two exact copies of an epitope from the NS5 region of HCV-1, each with amino acids 2278 to 2313 ofthe HCV polyprotein; and two copies of an epitope from the core region, one from HCV-1 and one from HCV-2, which copies are equivalent antigenic determinants represented by amino acids 9 to 32, 39-42 and 64-88 of HCV-1 and 67-84 of HCV-2. Table 2 shows the amino acid positions ofthe various epitopes with reference to Figures 5 A through 5F herein. In one embodiment ofthe invention, depicted in Figure 2, a rapid capture ligand immunoassay is performed using a conformational epitope from NS3/4a and one or more multiple epitope fusion antigens, such as MEFA 7.1. The sample is combined with the antigens, which may be present on a solid support, as described further below. If the sample is infected with HCN, HCN antibodies to those epitopes present on the solid support will bind to the solid support components. Detection is by the attachment of a detectable marker (such as horse radish peroxidase (HRP) as shown in Figure 2) to a member ofthe antigen/antibody complex. Attachment may be by covalent means or by subsequent binding of detectably labeled antibodies, such as in a standard sandwich assay, or by enzyme reaction, the product of which reaction is detectable. The detectable marker may include, but is not limited to, a chromophore, an antibody, an antigen, an enzyme, an enzyme reactive compound whose cleavage product is detectable, rhodamine or rhodamine derivative, biotin, avidin, strepavidin, a fluorescent compound, a chemiluminescent compound, such as dimethyl acridinium ester (DMAE, Ciba Corning Diagnostics Corp.), derivatives and/or combinations of these markers. A detectably labeled anti-human antibody, capable of detecting a human IgG molecule present, can be conveniently used. In order to further an understanding ofthe invention, a more detailed discussion is provided below regarding production of polypeptides for use in the immunoassays and methods of conducting the immunoassays. Production of Antigens for use in the HCN Immunoassays As explained above, the molecules ofthe present invention are generally produced recombinantly. Thus, polynucleotides encoding HCN antigens for use with the present invention can be made using standard techniques of molecular biology. For example, polynucleotide sequences coding for the above-described molecules can be obtained using recombinant methods, such as by screening cDΝA and genomic Hbraries from cells expressing the gene, or by deriving the gene from a vector known to include the same. Furthermore, the desired gene can be isolated directly from viral nucleic acid molecules, using techniques described in the art, such as in Houghton et al., U.S. Patent No. 5,350,671. The gene of interest can also be produced synthetically, rather than cloned. The molecules can be designed with appropriate codons for the particular sequence. The complete sequence is then assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge (1981) Nature 292:756; Νambair et al. (1984) Science 223:1299; and Jay et al. (1984) J. Biol. Chem. 259:6311. Thus, particular nucleotide sequences can be obtained from vectors harboring the desired sequences or synthesized completely or in part using various oligonucleotide synthesis techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR) techniques where appropriate. See, e.g., Sambrook, supra. J-n particular, one method of obtaining nucleotide sequences encoding the desired sequences is by annealing complementary sets of overlapping synthetic oligonucleotides produced in a conventional, automated polynucleotide synthesizer, followed by ligation with an appropriate DNA ligase and amplification ofthe ligated nucleotide sequence via P R. See, e.g., Jayaraman et al. (1991) Proc. Natl. Acad. Sci. USA 88:4084-4088. Additionally, oligonucleotide directed synthesis (Jones et al. (1986) Nature 54:75-82), oligonucleotide directed mutagenesis of pre-existing nucleotide regions (Riechmann et al. (1988) Nature 332:323-327 and Verhoeyen et al. (1988) Science 239:1534-1536), and enzymatic filling-in of gapped oligonucleotides using T 4 DNA polymerase (Queen et al. (1989) Proc. Natl. Acad. Sci. USA 86:10029-10033) can be used under the invention to provide molecules having altered or enhanced antigen-binding capabilities, and/or reduced immunogenicity. Once coding sequences have been prepared or isolated, such sequences can be cloned into any suitable vector or replicon. Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice. Suitable vectors include, but are not limited to, plasmids, phages, transposons, cosmids, chromosomes or viruses which are capable of replication when associated with the proper control elements. The coding sequence is then placed under the control of suitable control elements, depending on the system to be used for expression. Thus, the coding sequence can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator, so that the DNA sequence of interest is transcribed into RNA by a suitable transformant. The coding sequence may or may not contain a signal peptide or leader sequence which can later be removed by the host in post-translational processing. See, e.g., U.S. Patent Nos. 4,431,739; 4,425,437; 4,338,397. In addition to control sequences, it may be desirable to add regulatory sequences which allow for regulation ofthe expression ofthe sequences relative to the growth of the host cell. Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may also be present in the vector. For example, enhancer elements may be used herein to increase expression levels ofthe constructs. Examples include the SN40 early gene enhancer (Dijkema et al. (1985) EMBO J. 4:761), the enhancer/promoter derived from the long terminal repeat (LTR) ofthe Rous Sarcoma Nirus (Gorman et al. (1982) Proc. Natl. Acad. Sci. USA 79:6777) and elements derived from human CMN (Bosliart et al. (1985) Cell 41:521), such as elements included in the CMN intron A sequence (U.S. Patent No. 5,688,688). The expression cassette may further include an origin of replication for autonomous replication in a suitable host cell, one or more selectable markers, one or more restriction sites, a potential for high copy number and a strong promoter. An expression vector is constructed so that the particular coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation ofthe coding sequence with respect to the control sequences being such that the coding sequence is transcribed under the "control" ofthe control sequences (i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence). Modification ofthe sequences encoding the molecule of interest may be desirable to achieve this end. For example, in some cases it may be necessary to modify the sequence so that it can be attached to the control sequences in the appropriate orientation; i.e., to maintain the reading frame. The control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector. Alternatively, the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site. As explained above, it may also be desirable to produce mutants or analogs of the antigen of interest. This is particularly true with NS3/4a. Methods for doing so are described in, e.g., Dasmahapatra et al., U.S. Patent No. 5,843,752 and Zhang et al., U.S. Patent No. 5,990,276. Mutants or analogs of this and other HCV proteins for use in the subject assays may be prepared by the deletion of a portion ofthe sequence encoding the polypeptide of interest, by insertion of a sequence, and/or by substitution of one or more nucleotides within the sequence. Techniques for modifying nucleotide sequences, such as site-directed mutagenesis, and the like, are well known to those skilled in the art. See, e.g., Sambrook et al., supra; Kunkel, T.A. (1985) Proc. Natl. Acad. Sci. USA (1985) 82:448; Geisselsoder et al. (1987) BioTechniques 5:786; Zoller and Smith (1983) Methods Enzymol 100:468; Dalbie-McFarland et al. (1982) Proc. Natl. Acad. Sci USA 79:6409. The molecules can be expressed in a wide variety of systems, including insect, mammalian, bacterial, viral and yeast expression systems, all well known in the art. For example, insect cell expression systems, such as baculovirus systems, are known to those of skill in the art and described in, e.g., Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, z ' nter alia, Invitrogen, San Diego CA ("MaxBac" kit). Similarly, bacterial and mammalian cell expression systems are well known in the art and described in, e.g., Sambrook et al., supra. Yeast expression systems are also known in the art and described in, e.g., Yeast Genetic Engineering (Barr et al., eds., 1989) Butterworths, London. A number of appropriate host cells for use with the above systems are also known. For example, mammalian cell lines are known in the art and include immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human embryonic kidney cells, human hepatocellular carcinoma cells (e.g., Hep G2), Madin-Darby bovine kidney ("MDBK") cells, as well as others. Similarly, bacterial hosts such as E. coli, Bacillus subtilis, and Streptococcus spp. , will find use with the present expression constructs. Yeast hosts useful in the present invention include ter alia, Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica. Insect cells for use with baculovirus expression vectors include, inter alia, Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni. Nucleic acid molecules comprising nucleotide sequences of interest can be stably integrated into a host cell genome or maintained on a stable episomal element in a suitable host cell using various gene delivery techniques well known in the art. See, e.g., U.S. Patent No. 5,399,346. Depending on the expression system and host selected, the molecules are produced by growing host cells transformed by an expression vector described above under conditions whereby the protein is expressed. The expressed protein is then isolated from the host cells and purified. If the expression system secretes the protein into growth media, the product can be purified directly from the media. If it is not secreted, it can be isolated from cell lysates. The selection ofthe appropriate growth conditions and recovery methods are within the skill ofthe art. The production of various HCN antigens, including antigens used in the multiple epitope fusion proteins described above, has been described. See, e.g., Houghton et al., U.S. Patent Νos. 5,350,671 and 5,683,864; Chien et al., J. Gastroent. Hepatol. (1993) 8:S33-39; Chien et al., International Publication No. WO 93/00365; Chien, D.Y., International Publication No. WO 94/01778; Chien et al., Proc. Natl. Acad. Sci. USA (1992) 89:10011-10015; Chien, D.Y., International Publication No. WO 94/01778; and commonly owned, allowed U.S. Patent Application Serial Nos. 08/403,590 and 08/444,818. . Immunodiagnostic Assays Once produced, the HCV antigens may be used in virtually any assay format that employs a known antigen to detect antibodies. A common feature of all of these assays is that the antigen is contacted with the body component suspected of containing HCV antibodies under conditions that permit the antigen to bind to any such antibodies present in the component. Such conditions will typically be physiologic temperature, pH and ionic strength using an excess of antigen. The incubation ofthe antigen with the specimen is followed by detection of immune complexes comprised ofthe antigen. Design ofthe immunoassays is subject to a great deal of variation, and many formats are known in the art. Protocols may, for example, use solid supports, or immunoprecipitation. Most assays involve the use of labeled antibody or polypeptide; the labels may be, for example, enzymatic, fluorescent, chemiluminescent, radioactive, or dye molecules, as discussed in detail above. Assays which amplify the signals from the immune complex are also known; examples of which are assays which utilize biotin and avidin, and enzyme-labeled and mediated immunoassays, such as ELISA assays. The immunoassay may be, without limitation, a heterogenous or a homogeneous format, and of a standard or competitive type. In a heterogeneous format, the polypeptide is typically bound to a solid matrix or support to facilitate separation ofthe sample from the polypeptide after incubation. A solid support, for the purposes of this invention, can be any material that is an insoluble matrix and can have a rigid or semi- rigid surface. Exemplary solid supports include, but are not limited to, substrates such as nitrocellulose (e.g., in membrane or microtiter well form); polyvinylchloride (e.g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like. Particular supports include plates, pellets, disks, capillaries, hollow fibers, needles, pins, solid fibers, cellulose beads, pore-glass beads, silica gels, polystyrene beads optionally cross-linked with divinylbenzene, grafted co-poly beads, polyacrylamide beads, latex beads, dimethylacrylamide beads optionally crosslinked with N-N'-bis-acryloylethylenediamine, and glass particles coated with a hydrophobic polymer. If desired, the molecules to be added to the solid support can readily be functionalized to create styrene or acrylate moieties, thus enabling the incorporation of the molecules into polystyrene, polyacrylate or other polymers such as polyimide, polyacrylamide, polyethylene, polyvinyl, polydiacetylene, polyphenylene-vinylene, polypeptide, polysaccharide, polysulfone, polypyrrole, polyimidazole, polythiophene, polyether, epoxies, silica glass, silica gel, siloxane, polyphosphate, hydrogel, agarose, cellulose, and the like. In one context, a solid support is first reacted with the HCV antigens (collectively called "the solid-phase components" herein), under suitable binding conditions such that the molecules are sufficiently immobilized to the support. Sometimes, immobilization to the support can be enhanced by first coupling the antigen and/or antibody to a protein with better solid phase-binding properties. Suitable coupling proteins include, but are not limited to, macromolecules such as serum albumins including bovine serum albumin (BSA), keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, and other proteins well known to those skilled in the art. Other reagents that can be used to bind molecules to the support include polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and the like. Such molecules and methods of coupling these molecules to antigens, are well known to those of ordinary skill in the art. See, e.g., Brinkley, M.A. (1992) Bioconjugate Chem. 3:2-13; Hashida et al. (1984) J. Appl. Biochem. 6:56-63; and Anjaneyulu and Staros (1987) International J. of Peptide and Protein Res. 30:117-124. After reacting the solid support with the solid-phase components, any nonimmobilized solid-phase components are removed from the support by washing, and the support-bound components are then contacted with a biological sample suspected of containing HCV antibodies (collectively called "ligand molecules" herein) under suitable binding conditions. If HCV antibodies are present in the sample, they will form a complex with the HCV antigens. After washing to remove any nonbound ligand molecules, detectably labeled anti-xenogenic (e.g., anti-human) antibodies, which recognize an epitope on anti-HCV antibodies, is added. These antibodies bind due to complex formation. In a homogeneous format, the test sample is incubated with the combination of antigens in solution. For example, it may be under conditions that will precipitate any antigen-antibody complexes which are formed. Both standard and competitive formats for homogeneous assays are also known in the art. In a standard format, the amount of HCV antibodies forming the antibody- antigen complex is directly monitored. This may be accomplished by determining whether labeled anti-xenogenic (e.g., anti-human) antibodies which recognize an epitope on anti-HCN antibodies will bind due to complex formation. In a competitive format, the amount of HCN antibodies in the sample is deduced by monitoring the competitive effect on the binding of a known amount of labeled antibody (or other competing ligand) in the complex. More particularly, complexes formed comprising anti-HCN antibody (or, in the case of competitive assays, the amount of competing antibody) are detected by any of a number of known techniques, depending on the format. For example, unlabeled HCN antibodies in the complex may be detected using a conjugate of antixenogeneic Ig complexed with a label, (e.g., an enzyme label). In an immunoprecipitation or agglutination assay format, the reaction between the HCN antigens and the antibody forms a network that precipitates from the solution or suspension and forms a visible layer or film of precipitate. If no anti-HCN antibody is present in the test specimen, no visible precipitate is formed. The above-described assay reagents, including the immunoassay solid support with bound antibodies and antigens, as well as antibodies and antigens to be reacted with the captured sample, can be provided in kits, with suitable instructions and other necessary reagents, in order to conduct immunoassays as described above. The kit will normally contain in separate containers the combination of antigens (either already bound to a solid matrix or separate with reagents for binding them to the matrix), control antibody formulations (positive and/or negative), labeled antibody when the assay format requires same and signal generating reagents (e.g., enzyme substrate) if the label does not generate a signal directly. Instructions (e.g., written, tape, VCR, CD-ROM, etc.) for carrying out the assay usually will be included in the kit. The kit can also contain, depending on the particular irnmunoassay used, other packaged reagents and materials (i.e. wash buffers and the Hke). Standard immunoassays, such as those described above, can be conducted using these kits. HI. Experimental Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope ofthe present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for. EXAMPLE 1 Construction of MEFA 7 and MEFA 7.1 The following example illustrates the preparation of a polyprotein cassette of multiple HCV epitopes. The polyprotein expressed from the multiple epitope cassette is referred to herein as a Multiple Epitope Fusion Antigen (MEFA). Preferably, where an epitope is repeated, the extra copy or copies are tandemly arrayed in the same orientation. It is understood that the region of a viral coding sequence used as an epitope may be varied slightly and still retain antigenic activity, and that the amino acid numbering designation may vary from strain to strain. Thus, the repeated epitopes may vary one from another in amino acid sequence due to strain sequence variations and/or numbering designation. Preferably, the amino acid sequences of repeated epitopes within a MEFA are at least 30% homologous at the amino acid level, more preferably at least 40% homologous at the amino acid level. Unique restriction enzyme sites were introduced in order to connect the epitopes in the prescribed order and enhance the usefulness ofthe invention by facilitating modifications in design of a chimeric antigen. The choice of restriction enzyme sites and cloning procedures are readily determined by one of ordinary skill in the art of recombinant DNA technology. Preferably, the epitope junctions (amino acid sequences created between epitopes due to cloning) do not generate non-specific epitopes. Nonspecific epitopes are, for example, non-HCV sequences which do not exist adjacent to the HCV epitopes in nature. Non-specific epitopes may bind antibodies in a test sample causing false positive assay results. Preferably, the multiple epitope fusion protein is tested for false positive results due to such sequences generated at the epitope junctions. To avoid non-specific interactions with the MEFA due to junction sequences, the DNA sequence encoding the junction may, for example, be mutated such that non-specific interactions with the mutant amino acid sequence are reduced, and cloning ofthe epitope fragments is possible. The HCV MEFA 7 and 7.1 expression cassettes were constructed by cloning the coding nucleotide sequences containing major epitopes in a tandem array as shown in Figure 4. A major epitope was chosen based on antibody reaction frequency and reaction intensity (titer) to the epitope (Chein, D.Y. et al. (1994) Viral Hepatitis and Liver Disease, pp. 320-324). The various DNA segments coding for the HCV epitopes were constructed by PCR amplification or by synthetic oligonucleotides. The amino acids in each segment are set forth in Table 2 above and shown in Figures 5 A-5F. The complete HCV-1 amino acid sequence (3011 amino acids) was determined by Choo, et al. (1991) Proc. Natl. Acad. Sci. USA 88:2451-2455. Oligonucleotides capable of binding to HCV are described in U.S. Patent No. 5,350,671. The numbering ofthe amino acids in epitopes ofthe invention follows the numbering designation provided in Choo, et al., supra, in which amino acid number 1 is the first methionine encoded by the coding sequence ofthe core region, unless otherwise specified. For example, one epitope segment from NS5 is represented by amino acids 2278 to 2313 ofthe HCV polyprotein. An epitope from the El region is represented by amino acids 303 to 320, numbered relative to the HCV-1 polyprotein. MEFA 7 and 7.1 each contain epitopes from HCV-1, HCV-2 and HCV-3, allowing for detection of multiple types of a virus in a single assay. Methods of determining HCV serotype are found in WO 96/27153. For example, epitopes from the 5-1-1 region have been found to vary between serotypes of HCV. A copy of each ofthe HCV type-specific 5-1-1 epitopes present in the MEFAs described herein allows binding of any ofthe HCV types that may be present in the test biological sample. The MEFA 7 and 7.1 constructs were genetically engineered for expression in Saccharomyces cerevisiae, utilizing the yeast expression vector pBS24.1 which contains 2μ sequences for autonomous replication in yeast and the yeast genes leu2-d and URA3 as selectable markers. The β-lactamase gene and the ColEl origin of replication, required for plasmid replication in bacteria, were also present in this expression vector. The yeast expression vector for MEFA 7, ps.MEFA7, was constructed first. Subsequently, the plasmid was modified in the coding region for the HCV core epitopes to create the plasmid ps.MEFA7.1 , encoding the MEFA 7.1 antigen. In particular, as shown in Figures 7A through 7D, a yeast expression plasmid for MEFA 7 was constructed as follows. First, a BamΗUHindlll fragment of 1896 bp, encoding the ADH2/GAPDH hybrid promoter, hSOD (amino acids 1-156), followed by an El epitope (amino acids 303-320, HCV1 strain), was isolated from ps.MEFA6, the expression plasmid encoding MEFA 6, described in International Publication No. WO 97/44469. Next, a Hindlll! Sphl synthetic DNA fragment of 269 bp which contains the coding sequence for E2 HVRla consensus epitope (amino acids 390-410, HCV-1), E2 HVR1+2 consensus epitope (amino acids 384-414, HCVl+2) and the 5' end ofthe helicase domain (amino acids 1193-1229, HCV-1) was created. An SphVEcIXl fragment of 1264 bp, encoding the remainder ofthe helicase domain (amino acids 1230- 1651, HCV-1), was gel-purified from p-Tαc5/He/I plasmid DNA. The Hindϊl/Sphl synthetic DNA fragment and the Sphl/EcIXI 126 bp fragment were ligated into vector pSP72new.HtrccfflI/EcZXI vector, to produce pSP72new.H -ΛII/EcZXI/e2.helicase. This vector was derived from pSP72 an E. coli vector commercially available from Promega, Madison, WI (see, GenBank/ΕMBL Accession Number X65332). In particular, to facilitate the subcloning of several MΕFA 7 epitopes, a new multiple cloning site (MCS) polylinker was introduced, via synthetic oligos, between the Sphl and BgHl sites of pSP72. This new plasmid, named pSP72new, was digested with Hindlll and Ec/XI (also known as EagT), which have unique sites in the MCS. It was then dephosphorylated and gel-purified. E. coli ΗB101 competent cells were transformed with the plasmid, and plated on Luria agar plates containing lOOμg/ml ampicillin. Desired clones were identified using miniprep DNA analysis. After sequence verification, the plasmid pSP72new.H -iTII/Ec XI/e2.heΗcase subclone #4 was digested with Hindlll and Ec XI(ΕagI) to generate a 1534 bp fragment. The HindHlIEcTKl fragment was gel- purified and ligated with EclXUSphl oligonucleotides, encoding the last amino acids of the helicase domain (amino acids 1651-1658, HCN-1), into a pGEM7 HindHVSphl vector. HB101 competent cells were transformed and plated on Luria-ampicillin (lOOμg/ml). After identification of the desired clones and sequence confirmation, pGΕM.7 Hindlll/ Sphl subclone #9 was digested with Hindlll and Sphl to generate a 1560 bp fragment, which was gel purified (see, Figure 7A). To assemble the 3' end portion of MEFA 7, the following steps were performed. The 5-1-1 epitopes (amino acids 1689-1735) from HCN-1, HCN-3 and HCN-2 (in this order) were gel-isolated from ps.MEFA6, the expression plasmid encoding MEFA 6, described in International Publication No. WO 97/44469, as an Sphϊ/Avaϊ fragment of 441 bp. This fragment was ligated with synthetic Avail Xbal oligonucleotides encoding the clOO epitope (amino acids 1901-1936) into a pSP72new.Sj?/zI/ bαI vector. After HBIOI transformation, clone identification, and sequence verification, pSP72newSXi subclone #6 was digested ii Xbal and Notl to prepare a pSP72new bαI/NotI vector. Additionally, an XbάllNcol fragment of 221 bp, which encoded a double repeat of an ΝS5 epitope (amino acids 2278-2313, HCV-1), was isolated from ps.MEFA6. The XbalNcόl fragment was ligated with Ncol/Notϊ oligonucleotides, encoding the first amino acids ofthe HCN-1 core epitope, amino acids 9-17, in which the Lys at position 9 was changed to Arg, and the Asn at position 11 was changed to Thr, into the pSP72new bαI/NotI vector prepared above. HB101 transformants were analyzed and their plasmid DΝA sequenced. A subclone, termed pSP72newSX/XΝi #3, was digested with Notl/Sall to prepare a vector for subsequent subcloning (see, Figure 7B). To complete the assembly ofthe 3' end of MEFA 7, a double repeat ofthe sequence encoding a core epitope with amino acids 9-53 from HCV-1, plus two genotype-specific epitopes ofthe core region (amino acids 64-88, HCV-1 and amino acids 67-84, HCV-2) were subcloned as follows into Noil-Sail digested pSP72newSX/XNi subclone #3. First, a Notl/Xm l fragment of 92 bp encoding amino acids 18-51 of a core epitope was isolated from pd.Corel91RT clone #20. Plasmid pd.Corel91RT was constructed by ligating into the pBS24.1 BamHl-Sall yeast expression vector, a 1365bp BamHl-Ncol fragment for the ADH2/GAPDH promoter, and a 615 bp Ncol-SaR fragment encoding the first 191 amino acids of HCN-1 core with amino acid 9 mutated from Lys to Arg and amino acid 11 mutated from Asn to Thr. The 615 bp Ncoϊ-Sall fragment was derived from an E. coli expression vector in which the core sequence for amino acids 1-191, with the same two mutations described above, had been cloned. The 92 bp Notl/Xmnl fragment was ligated with a pSP72newNotlKpn vector and with Xmnϊ/Kpnl oligonucleotides which encode the 3' end ofthe complete core epitope. After sequence verification ofthe positive clones, pSP72newΝKi subclone #4 was digested with Notl and Kpnl, and a 224 bp fragment was gel-isolated. This NotVKpήl fragment was ligated with 284 bp of oligonucleotides (Kpnl-SαU. ends) encoding a complete repeat ofthe core epitope described above into the pSP72newSX/XΝi NotllSαR. vector described above. After HBIOI transformation, clone identification and sequence verification, pSP72newSX/XN/NSi subclone #18 was digested with Sphl and Sail and a fragment of 1317 bp was gel-isolated (see, Figure 7C). Lastly, the following fragments, described above, were ligated into the pBS24.1 BamBI/Sall yeast expression vector to create ps.MΕFA7 (see, Figure 7D): the BamΕSJ Hindlll fragment of 1896 bp (Figure 7A) the HindHllSphl fragment of 1560 bp (Figure 7A) the Sphl/Sall fragment of 1317bp (Figure 7C) S. cerevisiae strain AD3 was transformed with ps.MEFA7 and single transformants were checked for expression after depletion of glucose in the medium. The recombinant protein was expressed at high levels in yeast, as detected by Coomassie blue staining. In particular, yeast cells were transformed with the MEFA expression plasmid using a lithium acetate protocol. Ura " transformants were streaked for single colonies and patched onto LeuJ8% glucose plates to increase plasmid copy number. Leu starter cultures were grown for 24 hours at 30°C and then diluted 1 :20 in YEPD (yeast extract bactopeptone 2% glucose) media. The cells were grown for 48 hours at 30 °C and harvested. To test for expression ofthe MEFA 7 recombinant antigen, an aliquot ofthe cells was lysed with glass beads in lysis buffer (10 mM Tris-Cl pH 7.5, 1 mM EDTA, 10 mM DTT). The lysate was centrifuged at high speed. The supernatant and insoluble pellet were analyzed on SDS protein gels. MEFA 7 was highly enriched in the insoluble pellet fraction. The MEFA 7 antigen was purified as follows. S. cerevisiae cells expressing MEFA 7 were harvested as described above. The cells were suspended in lysis buffer (50 mM Tris, 0.15 M NaCl, 1 mM EDTA, 1 mM PMSF, pH 8.0) and lysed in a Dyno- Mill (Wab Willy A. Bachofon, Basel, Switzerland) or equivalent apparatus using glass beads. The lysate was centrifuged at low speed conditions (3,000 to 5,000 rpm, 15 min) and the pellet containing the insoluble protein fraction was washed with increasing concentrations of urea (1 M, 2 M, 3 M) in lysis buffer. Protein was solubilized from the centrifugation pellet with 0.1 N NaOH, 4 M urea in lysis buffer. Cell debris was removed by low speed centrifugation at 3,000 to 5,000 rpm, 15 min. The supernatant was adjusted to pH 8.0 with 6 N HC1 to precipitate proteins insoluble under these conditions. The precipitate was removed by centrifugation and the supernatant was adjusted to 2.3% SDS, 50 mM DTT, pH 8.0 and boiled for 3 min. Proteins in the mixture were fractionated by gel filtration on a Pharmacia Sephacryl S-400 in phosphate buffered saline containing 0.1 % SDS, 1 mM EDTA and adjusted to pH 7.4. Column eluate fractions containing MEFA 7 were collected, pooled, and concentrated on an Amicon YM-30 membrane. Gel filtration was repeated on the pooled fractions using the same column and conditions. During the analysis of MEFA 7 in a trial assay, it was discovered that a monoclonal antibody used as a detection conjugate reacted with a specific sequence of the core epitope (amino acids 33-38). Thus, ps.MEFA7.1 was designed to eliminate amino acids 33-38 from the core epitope region. A yeast expression vector for MEFA 7.1 was made as follows. First, the double repeat ofthe core epitope at the 3' end of ps.MEFA7 was modified. To do so, an Ncol/Kpήl synthetic fragment of 206 bp, encoding the first core epitope repeat (amino acids 9-32, 39-42 and 64-88 of HCN-1, and amino acids 67-82 of HCN-2), and a Kpnl/Sall synthetic fragment of 233 bp encoding amino acids 83 and 84 of HCV-2, followed by the second core epitope repeat (amino acids 9-32, amino acids 39-42 and amino acids 64-88, HCV-1, amino acids 67-84, HCV-2) were subcloned respectively into a pSP72new.NcoI/R nI vector and a pSP72new nl/Sα/I vector. After HB 101 transformation, clone identification and sequence confirmation, pSP72newΝKi clone #21 was digested with Ncol and Kpnl to isolate the NcoVKpήl fragment of 206 bp and pSP72newKSi clone #32 was digested with Kpnl and Sail to isolate the KpnllSaR fragment of 233 bp. Plasmid ps.MEFA7.1 was assembled by Hgating the following fragments into the pBS24.1 BamH /Sall yeast expression vector (see, Figure 8) : the BamHl/Hindϊll fragment of 1896 bp, described above for ps.MEFA7 ; the Hindlll/Sphl fragment of 1560 bp described above for ps.MEFA7; an SphllNcόl fragment of 776 bp isolated from ps.MEFA7 encoding the 5-1-1 epitopes, clOO epitope and ΝS5 epitope; the Ncol/Kpnl fragment of 206 bp; and KpnJJSall fragment of 233 bp. S. cerevisiae strain AD3 was transformed withps.MEFA7.1 and single transformants were checked for expression after depletion of glucose in the medium, as described above. The recombinant protein was expressed at high levels in yeast, as detected by Coomassie blue staining. The MEFA 7.1 antigen was purified as follows. S. cerevisiae cells expressing MEFA 7.1 were harvested as described above. The cells were suspended in lysis buffer (50 mM Tris, 0.15 M NaCl, ImM EDTA, pH 8.0) and lysed in a Dyno-MiU (Wab Willy A. Bachofen, Basel, Switzerland) or equivalent apparatus using glass beads. The lysate was centrifuged at 10,000 rpm, 30 min in a JA-10 rotor, and the pellet containing the insoluble protein fraction was washed with increasing concentrations of Urea (1 M, 2 M, 3 M) in lysis buffer. Protein was solubilized from the centrifugation pellet with 0.1 N NaOH, 4 M Urea, 50 mM DTT in lysis buffer. Cell debris was removed by centrifugation at 14,000 rpm, 20 min in a JA-14 rotor. The supernatant was adjusted to pH 8.0 with 6 N HC1 to precipitate proteins insoluble under these conditions. The precipitate was removed by centrifugation at 14,000 rpm, 20 min in a JA-14 rotor. The supernatant was adjusted to 2.3% SDS and heated to 70-75° C in boiling water then cooled to room temperature. Proteins in the mixture were fractionated by gel filtration on a Pharmacia Sephacryl S-400 HR in PBS containing 0.1 % SDS, 1 mM EDTA and adjusted to pH 7.4. Column eluate fractions containing MEFA 7.1 were collected, pooled, and concentrated on an Amicon YM-30 membrane. Pooled gel filtration fractions were adjusted to 2.3 % SDS, 50 mM DTT, and heated/cooled as above. This pool was subjected to a second gel filtration step on a Pharmacia Sephacryl S-300 HR column under the same conditions as the first gel filtration step. EXAMPLE 2 Recombinant Production of an NS3/4a Conformational Epitope A conformational epitope of NS3/4a was obtained as follows. This epitope has the sequence specified in Figures 3 A through 3D and differs from the native sequence at positions 403 (amino acid 1428 ofthe HCV-1 full-length sequence) and 404 (amino acid 1429 ofthe HCV-1 full-length sequence). Specifically, the Thr normally occurring at position 1428 ofthe native sequence has been mutated to Pro and Ser which occurs at position 1429 ofthe native sequence has been mutated to He. In particular, the yeast expression vector used was pBS24.1, described above. Plasmid pd.hcvl a.ns3ns4aPI, which encoded a representative NS3/4a epitope used in the subject immunoassays, was produced as follows. A two step procedure was used. First, the following DNA pieces were ligated together: (a) synthetic oligonucleotides which would provide a 5' Hindlll cloning site, followed by the sequence ACAAAACAAA, the initiator ATG, and codons for HCV la, beginning with amino acid 1027 and continuing to a BgH site at amino acid 1046; (b) a 683 bp BgH-Clal restriction fragment (encoding amino acids 1046-1274) from pAcHLTns3ns4aPI; and (c) a pSP72 vector (Promega, Madison, WI, GenBank/EMBL Accession Number X65332) which had been digested with HindΩI and CM, dephosphorylated, and gel-purified. Plasmid pAcHLTns3ns4aPI was derived from pAcHLT, a baculovirus expression vector commercially available from BD Pharmingen (San Diego, CA). In particular, a pAcHLT EcoRI-Rstl vector was prepared, as well as the following fragments: EcoRI- Alwήl, 935 bp, corresponding to amino acids 1027-1336 ofthe HCV-1 genome; Alwήl-Sacϊl, 247 bp, corresponding to amino acids 1336-1419 ofthe HCV-1 genome; Hinfl-BgR, 175 bp, corresponding to amino acids 1449-1509 ofthe HCV-1 genome; BgH-Pstl, 619 bp, corresponding to amino acids 1510-1711 of the HCV-1 genome, plus the transcription termination codon. A SacU -Hinfl synthetically generated fragment of 91 bp, coπesponding to amino acids 1420-1448 ofthe HCV-1 genome and containing the PI mutations (Thr-1428 mutated to Pro, Ser-1429 mutated to He), was ligated with the 175 bp Hinfl-BgR fragment and the 619 bp BgH-Pstl fragment described above and subcloned into a pGΕM-5Zf(+) vector digested with SαcII and Pstl. pGEM-5Zf(+) is a commercially available E. coli vector (Promega, Madison, WI, GenBank/EMBL Accession Number X65308). After transformation of competent HBIOI cells, miniscreen analysis of individual clones and sequence verification, an 885 bp Sacll-Pstl fragment from pGEM5.PI clone2 was gel- purified. This fragment was ligated with the EcoRI-.4/w«1 935 bp fragment, the Alwήl- SαcII 247 bp fragment and the pAcHLT EcoRI-Pstϊ vector, described above. The resultant construct was named pAcHLTns3ns4aPI. The ligation mixture above was transformed into HBIOI -competent cells and plated on Luria agar plates containing 100 μg/ml ampicillin. Miniprep analyses of individual clones led to the identification of putative positives, two of which were amplified. The plasmid DNA for pSP72 laHC, clones #1 and #2 were prepared with a Qiagen Maxiprep kit and were sequenced. Next, the following fragments were ligated together: (a) a 761 bp Hindlll-Clal fragment from pSP721aHC #1 (pSP72.1aHC was generated by ligating together the following: pSP72 which had been digested with Hindlll and Claϊ, synthetic oligonucleotides which would provide a 5 ' Hindlll cloning site, followed by the sequence ACAAAACAAA, the initiation codon ATG, and codons for HCVla, beginning with amino acid 1027 and continuing to aBglll site at amino acid 1046, and a 683 bp Bgl∑l-Clal restriction fragment (encoding amino acids 1046-1274) from pAcHLTns3ns4aPI); (b) a 1353 bp BamHI-Hinά ll fragment for the yeast hybrid promoter ADH2/GAPDH; (c) a 1320 bp Clal-Sal fragment (encoding HCVla amino acids 1046-1711 with Thr 1428 mutated to Pro and Ser 1429 mutated to He) from pAcHLTns3ns4aPI; and (d) the pBS24.1 yeast expression vector which had been digested with RαmFfl and Sail, dephosphorylated and gel-purified. The ligation mixture was transformed into competent HBIOI and plated on Luria agar plates containing 100 μg/ml ampicillin. Miniprep analyses of individual colonies led to the identification of clones with the expected 3446 bp BamHl-Sall insert which was comprised ofthe ADH2/GAPDH promoter, the initiator codon ATG and HCVla NS3/4a from amino acids 1027-1711 (shown as amino acids 1-686 of Figures 3A-3D), with Thr 1428 (amino acid position 403 of Figures 3A-3D) mutated to Pro and Ser 1429 (amino acid position 404 of Figures 3A-3D) mutated to He. The construct was named pd.HCVla.ns3ns4aPI (see, Figure 9). S. cerevisiae strain AD3 was transformed with pd.HCVla.ns3ns4aPI and single transformants were checked for expression after depletion of glucose in the medium. The recombinant protein was expressed at high levels in yeast, as detected by Coomassie blue staining and confirmed by immunoblot analysis using a polyclonal antibody to the helicase domain of NS3. EXAMPLE 3 Purification of NS3/4a Conformational Epitope The NS3/4a conformational epitope was purified as follows. S. cerevisiae cells from above, expressing the NS3/4a epitope were harvested as described above. The cells were suspended in lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 0.1 μM pepstatin, 1 μM leupeptin) and lysed in a Dyno-Mill (Wab Willy A. Bachofon, Basel, Switzerland) or equivalent apparatus using glass beads, at a ratio of 1:1:1 cells:buffer:0.5 mm glass beads. The lysate was centrifuged at 30100 x g for 30 min at 4°C and the pellet containing the insoluble protein fraction was added to wash buffer (6 ml/g start cell pellet weight) and rocked at room temperature for 15 min. The wash buffer consisted of 50 mM NaPO 4 pH 8.0, 0. 3 M NaCl, 5 mM β-mercaptoethanol, 10% glycerol, 0.05% octyl glucoside, 1 mM EDTA, 1 mM PMSF, 0.1 μM pepstatin, 1 μM leupeptin. Cell debris was removed by centrifugation at 30100 x g for 30 min at 4°C. The supernatant was discarded and the pellet retained. Protein was extracted from the pellet as follows. 6 ml/g extraction buffer was added and rocked at room temperature for 15 min. The extraction buffer consisted of 50 mM Tris pH 8.0, 1 M NaCl, 5 mM β-mercaptoethanol, 10% glycerol, 1 mM EDTA, 1 mM PMSF, 0.1 μM pepstatin, 1 μM leupeptin. This was centrifuged at 30100 x g for 30 min at 4°C. The supernatant was retained and ammonium sulfate added to 17.5% using the following formula: volume of supernatant (ml) multiplied by x% ammonium sulfate/(l - x% ammonium sulfate) = ml of 4.1 M saturated ammonium sulfate to add to the supernatant. The ammonium sulfate was added dropwise while stirring on ice and the solution stirred on ice for 10 min. The solution was centrifuged at 17700 x g for 30 min at 4°C and the pellet retained and stored at 2°C to 8°C for up to 48 hrs. The pellet was resuspended and run on a Poly U column (Poly U Sepharose 4B, Amersham Pharmacia) at 4°C as follows. Pellet was resuspended in 6 ml Poly U equilibration buffer per gram of pellet weight. The equilibration buffer consisted of 25 mM HEPES pH 8.0, 200 mM NaCl, 5 mM DTT (added fresh), 10% glycerol, 1.2 octyl glucoside. The solution was rocked at 4°C for 15 min and centrifuged at 31000 x g for 30 min at 4°C. A Poly U column (1 ml resin per gram start pellet weight) was prepared. Linear flow rate was 60 cm/hr and packing flow rate was 133% of 60 cm/hr. The column was equilibrated with equilibration buffer and the supernatant ofthe resuspended ammonium sulfate pellet was loaded onto the equiHbrated column. The column was washed to baseline with the equilibration buffer and protein eluted with a step elution in the following Poly U elution buffer: 25 mM HEPES pH 8.0, 1 M NaCl, 5 mM DTT (added fresh), 10% glycerol, 1.2 octyl glucoside. Column eluate was run on SDS-PAGE (Coomassie stained) and aliquots frozen and stored at -80 °C. The presence ofthe NS3/4a epitope was confirmed by Western blot, using a polyclonal antibody directed against the NS3 protease domain and a monoclonal antibody against the 5-1-1 epitope (HCV 4a). Additionally, protease enzyme activity was monitored during purification as follows. An NS4A peptide (KKGSVVIVGRJNLSGKPAJJPKK), and the sample containing the ΝS3/4a conformational epitope, were diluted in 90 μl of reaction buffer (25 mM Tris, pH 7.5, 0.15M NaCl, 0.5 mM EDTA, 10% glycerol, 0.05 n-Dodecyl B-D- Maltoside, 5 mM DTT) and allowed to mix for 30 minutes at room temperature. 90 μl ofthe mixture were added to a microtiter plate (Costar, Inc., Corning, NY) and 10 μl of HCV substrate (AnaSpec, Inc., San Jose CA) was added. The plate was mixed and read on a Fluostar plate reader. Results were expressed as relative fluorescence units (RFU) per minute. Using these methods, the product ofthe 1 M NaCl extraction contained 3.7 RFU/min activity, the ammonium sulfate precipitate had an activity of 7.5 RFU/min and the product ofthe Poly U purification had an activity of 18.5 RFU/min. EXAMPLE 4 Coating Solid Support with the HCV Antigens The HCN ΝS3/4a conformational epitope and MEFA 7.1 antigen were coated onto plates as follows. HCV coating buffer (50 mM NaPO4 pH 7.0, 2 mM EDTA and 0.1% Chloroacetamide) was filtered through a 0.22 μ filter unit. The following reagents were then added sequentially to the HCV coating buffer and stirred after each addition: 2 μg/ml BSA-Sulfhydryl Modified, from a 10 mg/ml solution (Bayer Corp. Pentex, Kankakee, Illinois); 5 mM DTT from a 1 M solution (Sigma, St. Louis, MO); 0.45 μg/ml NS3/4a (protein concentration of 0.3 mg/ml); 0.375 μg/ml MEFA 7.1 (protein concentration of 1 mg/ml). The final solution was stirred for 15 minutes at room temperature. 200 μl ofthe above solution was added to each well of a Costar high binding, flat bottom plate (Corning Inc., Corning, New York) and the plates were incubated overnight in a moisture chamber. The plates were then washed with wash buffer (1 x PBS, 0.1% TWEEN-20), Tapped dry and 285 μl Ortho Post-Coat Buffer (1 x PBS, pH 7.4, 1% BSA, 3% sucrose) added. The plates were incubated for at least 1 hour, tapped and dried overnight at 2-8°C. The plates were pouched with desiccants for future use. EXAMPLE 5 Early Seroconversion Studies The performance ofthe NS3/4a and MEFA 7.1 antigens in a combination assay (HCV 4.0) was compared to other HCV assays to test the seroconversion detection limits and to compare these limits to those obtained in other commercially available assays. Panels of commercially available human blood samples were used which were HCV- infected. The PHV panels shown in the tables below were purchased from Boston Biomedica, Inc., West Bridgewater, MA (BBI). The 6212 panels were purchased from Bioclinical Partners, Franklin, MA (BCP). The SC panels were purchased from North American Biologies, Inc., BocoRatan, FL (NABI). The day on which the blood samples was obtained is indicated in the tables. The HCV 4.0 assay was conducted as follows. 200 μl of specimen diluent buffer (1 g/1 casein, 100 mg/1 recombinant human SOD, 1 g/1 chloracetamide, 10 g/1 BSA, 500 mg/1 yeast extract, 0.366 g/1 EDTA, 1.162 g/1 KPO 4 , 5 ml/1 Tween-20, 29.22 g/1 NaCl, 1.627 g/1 NaPO 4 , 1% SDS) was added to the coated plates. 20 μl of sample was then added. This was incubated at 37°C for one hour. The plates were washed with wash buffer (1 x PBS, pH 7.4, 0.1% Tween-20). 200 μl conjugate solution (a mouse anti- human IgG-HRP, such as mouse anti-human IgG-HRP diluted 1:22,000 in ORTHO HCV 3.0 ELISA Test System with Enhanced SAVe bulk conjugate diluent (Ortho- Clinical Diagnostics, Raritan, New Jersey) was added and incubated for 60 minutes at 37°C. This was washed as above, and 200 μl substrate solution (1 OPD tablet/10ml) was added. The OPD tablet contains o-phenylenediamine dihydrochloride and hydrogen peroxide for horse radish peroxidase reaction color development. This was incubated for 30 minutes at room temperature in the dark. The reaction was stopped by addition of 50 μl 4N H 2 SO 4 and the plates were read at 492 nm, relative to absorbance at 690 nm as control. The other assays used in the study were as follows: The Abbott PRISM assay (Abbott Laboratories, Abbott Park, IL), is commercially available and is an antibody-based detection assay. The assay was performed using the manufacturer's instructions. The ORTHO HCN Version 3.0 ELISA Test System (HCV 3.0) (Ortho Clinical Diagnostics, Raritan, New Jersey) is an antibody-based detection assay. The assay was conducted using the manufacturer's instructions. The Pasteur MONOLISA anti-HCV Plus Version 2 assay (Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France) is an antibody-based detection assay. The assay was performed using the manufacturer's instructions. The performance of the HCV 4.0 assay was compared to the HCV 3.0, PRISM and Pasteur assays (see, Tables 3 and 4). HCV antibodies present in the blood panels (anti-c33c or anti-c22) are set forth in Table 4. In particular, 17 seroconversion panels from the three commercial sources set forth above were assayed using the techniques above. As can be seen, for the c33c panels, HCV 4.0 showed earlier detection (by 1-3 bleeds) than HCV 3.0 in 9 out of 9 c33c panels, and earlier detection than PRISM in 6 out of 9 panels and equivalent detection as compared with PRISM in 3 ofthe 9 panels. For the c22 panels, HCV 4.0 showed earlier detection than HCV 3.0 in 3 of 8 panels and equivalent detection in the other 5 panels. HCV 4.0 also showed earlier detection than PRISM in 2 of 8 panels and equivalent detection in 6 of 8 panels. The range of improvement seen was 2-14 days over both the HCV 3.0 and PRISM assays. ITS β\ © υ Q- © r-- 90 9\ TABLE 3 (continued) Ortho Abbott © 00 TABLE 4 EXAMPLE 6 HCN 4.0 Genotype Sensitivity The genotype sensitivity ofthe HCN 4.0 assay was compared to the HCN 3.0 and Pasteur assays, described above. In particular, samples from 10 different HCN genotypes, specified in Table 5, were diluted as indicated in the table (2-fold or 10-fold depending on initial sample titering) and used in the three assays, using the procedures described above. All three tests were run simultaneously. The data is shown as signal or raw O.D. The data suggests that the HCN 4.0 prototype is more sensitive in detecting diluted genotype samples. CM r Ifl (β N r (O IO r- TJ- CO CO r 00 U) O (O CO S N T4- r-. r-. io N co en N w tn w ιq - o o T; O O T- j •*- O N ω « Q Q Q Q ■ — a O O CD O T^ d d d CM d d d z z z z O Q. o d d c o cό -oc T- O t^ IO CM O cn IO f- cn IO ∞ co t^ co r- t^ rt CO o O LO CO co O cn co TT" CO co IO T™* n CM T cn rt cn O CO > - CO t- CM CD CD CD CD o • r T- o ■ rt CO CO o CM ιq O O CO X O CD O CD d CD d d d d d d T- d d d CO O CM d O cό o r«. O cn IO IO CO CM O t» co en T- rt CO 1^ co £ r- co cn T- CO O cn u> rf cn o CO co T- O CM IO O r- CO CO rt T- o > t. o co T- o M T- O CD Tf T— o o lO CM o o T— O o O o X O T~ CD O o o o d d d O d d o O o o o d O o o (1) α. " • <o rf cn O o TΓ- IO Tt CM f^ ιo CO o CM CO co CO t^ ^- to rt O N. cn o O IO CM t^ IO T— cn o CM r» n o O IO IO >υ o (0 CO O o Tt Tt ιq CM CD c cn O r- I O 00 co CM rt T™ o X o CM CM T- d T- d d d d d o CM T— o o T- d O o Q. c O o o o o o o o O o o o O O o O o o O o o o o o o O o o O o o o O o O ϊ o O o o o o o o IO o o O o o o _ 2 -ϊ o o CM o o o IO o IO T" o o IO o o o IO o o o CM CM IO o ▼- o IO o CM CM •o T- CM CM IO TT- CM CM IO T- CM EXAMPLE 7 Competition Studies The following competition study was conducted in order to assess whether the NS3/4a conformational epitope detected different antibodies than other HCV antigens. In particular, the NS3/4a antigen was compared with the c200 antigen as follows. 0.5 μg and 1.0 μg of NS3/4a, produced as described above, or c200 (Hepatology (1992) 15:19-25, available in the ORTHO HCV Version 3.0 ELISA Test System, Ortho- Clinical Diagnostics, Raritan, New Jersey), were mixed with 20 μl of sample PHV914-5 (an early seroconversion bleed obtained from blood of an infected individual) in a total volume of 220 μl (1 x PBS). The mixture was incubated for 1 hour in micro wells at 37°C. The mixture was then transferred to NS3/4a-coated plates and incubated for 1 hour at 37°C. Plates were washed and assayed as follows. 1 μg of c200 antigen was added to 10 μl of sample PHN914-5 in a total volume of about 220 μl. The mixture was incubated for 1 hour in a micro well at 37°C and 200 μl transferred to an ΝS3/4a-coated plate (100 ng/assay) and incubated for 1 hour at 37°C. Plates were washed five times with 1 x PBS, 0.1% Tween-20. 200 μl of conjugate solution (described above) were added, and the plates incubated and assayed as described in Example 4 for the HCN 4.0 assay. Controls which consisted of PHN914-5 and 1 x PBS (without antigen) were also treated as above. Results are shown in Table 6. Percent inhibition results shown in column 4 are calculated as column 3 minus (column 2 divided by column 3 times 100). As can be seen, the data show that ΝS34a is neutralized by early seroconversion antibodies and c200 is not. A strong signal was achieved when antibodies in PHN914-5 c33c early seroconversion panel member reacted with the ΝS34a coated on the plate. The c200 antigen was not neutralized by these antibodies. This is shown in the top panel of Table 6. When NS34a was mixed with the PHV914-5 sample, it was neutralized and therefore no antibodies were present in the sample to react with NS34a that was coated on the microplate. The data indicate that NS34a may be detecting a different class of antibodies than is detected by c200. Competition Studies to Show NS34a Antigen Detects Different Antibodies in Early c33c Seroconversion Panel Compared to c200 Antigen TABLE 6 EXAMPLE 8 Stability Studies of NS3/4a Conformational Epitope To assess the role of stability ofthe NS3/4a epitope to assay performance, the following study was done to determine NS3/4a immunoreactivity versus time at room temperature. Small aliquots of stock NS3/4a were allowed to sit at room temperature and then frozen at intervals as shown in Table 7. All vials were coated simultaneously and tested against two early NS3 seroconversion panels. Assays were conducted as described above in Example 5 for HCV 4.0. As can be seen in Table 7, the NS3/4a stock is not stable and immunoreactivity decreases with time. In addition, maintaining NS3/4a conformation is necessary for immunoreactivity. Further stability studies were conducted as follows. Two conformational monoclonal antibodies made against NS3/4a using standard procedures were substituted for anti-HCN early seroconversion panels. Stock ΝS3/4a vials were stored at room temperature at time intervals 3, 6 and 24 hours. The NS3/4a from the frozen vials was coated at 90 ng/ml and assayed using the procedure described above. Results suggested that the two monoclonals were indeed conformational and their reactivity was sensitive to the handling of stock NS3/4a antigen at room temperature. The reactivity of a positive control monoclonal antibody did not change. TABLE 7 EXAMPLE 9 Immunoreactivity of NS3/4a Conformational Epitope Verus Denatured NS3/4a The immunoreactivity ofthe NS3/4a conformational epitope, produced as described above, was compared to NS3/4a which had been denatured by adding SDS to the NS3/4a conformational epitope preparation to a final concentration of 2%. The denatured NS3/4a and conformational NS3/4a were coated onto microtiter plates as described above. The c200 antigen (Hepatology (1992) 15:19-25, available in the ORTHO HCV Version 3.0 ELISA Test System, Ortho-Clinical Diagnostics, Raritan, New Jersey) was also coated onto microtiter plates. The c200 antigen was used as a comparison it is presumed to be non-conformational due to the presence of reducing agent (DTT) and detergent (SDS) in its formulation. The immunoreactivity was tested against two early HCV seroconversion panels, PHV 904 and PHV 914 (commercially available human blood samples from Boston Biomedica, Inc., West Bridgewater, MA), using the ELISA assay procedure described above. The results are shown in Table 8. The data suggests that the denatured or linearized form of NS3/4a (as well as c200) does not detect early seroconversion panels as early as the NS3/4a conformational epitope. TABLE 8 Immunoreactivity ofthe conformational epitope was also tested using ' monoclonal antibodies to NS3/4a, made using standard procedures. These monoclonal antibodies were then tested in the ELISA format described above against NS3/4a and denatured NS3/4a and c200 antigen. The data show that anti-NS3/4a monoclonals react to the NS3/4a and denatured NS3/4a in a similar manner to the seroconversion panels shown in Table 9. This result also provides further evidence that the NS3/4a is conformational in nature as monoclonal antibodies can be made which are similar in reactivity to the early c33c seroconversion panels . Accordingly, novel HCV detection assays have been disclosed. From the foregoing, it will be appreciated that, although specific embodiments ofthe invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope thereof.

Claims: Claims 1. An immunoassay solid support consisting essentially of at least one HCV NS3/4a conformational epitope and a multiple epitope fusion antigen, bound to the support, wherein said NS3/4a epitope and/or said multiple epitope fusion antigen react specifically with anti-HCV antibodies present in a biological sample from an HCV- infected individual. 2. The immunoassay solid support of claim 1, wherein said NS3/4a epitope comprises the amino acid sequence depicted in Figures 3A-3D, or an amino acid sequence with at least 80% sequence identity thereto which has protease activity. 3. The immunoassay solid support of claim 1, wherein said NS3/4a epitope comprises the amino acid sequence depicted in Figures 3 A-3D, or an amino acid sequence with at least 90% sequence identity thereto which has protease activity. 4. The immunoassay solid support of claim 1, wherein said NS3/4a epitope comprises the amino acid sequence depicted in Figures 3 A-3D, or an amino acid sequence with at least 98% sequence identity thereto which has protease activity. 5. The immunoassay solid support of claim 1, wherein said NS3/4a epitope consists ofthe amino acid sequence depicted in Figures 3A-3D. 6. The immunoassay solid support of claim 1, wherein said multiple epitope fusion antigen comprises the amino acid sequence depicted in Figures 5 A-5F, or an amino acid sequence with at least 80% sequence identity thereto which reacts specifically with anti-HCV antibodies present in a biological sample from an HCV- infected individual. 7. The immunoassay solid support of claim 1, wherein said multiple epitope fusion antigen comprises the amino acid sequence depicted in Figures 5 A-5F, or an amino acid sequence with at least 90% sequence identity thereto which reacts specifically with anti-HCV antibodies present in a biological sample from an HCV- infected individual. 8. The immunoassay solid support of claim 1, wherein said multiple epitope fusion antigen comprises the amino acid sequence depicted in Figures 5 A-5F, or an amino acid sequence with at least 98% sequence identity thereto which reacts specifically with anti-HCV antibodies present in a biological sample from an HCV- infected individual. 9. The immunoassay solid support of claim 1, wherein said multiple epitope fusion antigen consists ofthe amino acid sequence depicted in Figures 5A-5F. 10. An immunoassay solid support consisting essentially of at least one HCV NS3/4a conformational epitope and a multiple epitope fusion antigen, bound to the support, wherein said NS3/4a conformational epitope comprises the amino acid sequence depicted in Figures 3A-3D, or an amino acid sequence with at least 80% sequence identity thereto which has protease activity, and said multiple epitope fusion antigen comprises the amino acid sequence depicted in Figures 5 A-5F, or an amino acid sequence with at least 80% sequence identity thereto which reacts specifically with anti- HCV antibodies present in a biological sample from an HCV-infected individual. 11. The immunoassay solid support of claim 10, wherein said NS3/4a conformational epitope comprises the amino acid sequence depicted in Figures 3 A-3D, or an amino acid sequence with at least 90% sequence identity thereto which has protease activity, and said multiple epitope fusion antigen comprises the amino acid sequence depicted in Figures 5 A-5F, or an amino acid sequence with at least 90% sequence identity thereto which reacts specifically with anti-HCV antibodies present in a biological sample from an HCV-infected individual. 12. The immunoassay solid support of claim 10, wherein said NS3/4a conformational epitope comprises the amino acid sequence depicted in Figures 3 A-3D, or an amino acid sequence with at least 98% sequence identity thereto which has protease activity, and said multiple epitope fusion antigen comprises the amino acid sequence depicted in Figures 5 A-5F, or an amino acid sequence with at least 98% sequence identity thereto which reacts specifically with anti-HCV antibodies present in a biological sample from an HCV-infected individual. 13. The immunoassay solid support of claim 10, wherein said NS3/4a conformational epitope consists ofthe amino acid sequence depicted in Figures 3A-3D, and said multiple epitope fusion antigen consists ofthe amino acid sequence depicted in Figures 5A-5F. 14. An immunoassay solid support consisting essentially of at least one HCV NS3/4a conformational epitope and a multiple epitope fusion antigen, bound to the support, wherein saidNS3/4a conformational epitope consists ofthe amino acid sequence depicted in Figures 3 A-3D, and said multiple epitope fusion antigen consists of the amino acid sequence depicted in Figures 5A-5F. 15. An immunodiagnostic test kit comprising the immunoassay solid support of any of claims 1-14, and instructions for conducting the immunodiagnostic test. 16. A method of producing an immunoassay solid support, comprising: (a) providing a solid support; and (b) binding to the solid support at least one HCV NS3/4a conformational epitope and a multiple epitope fusion antigen, wherein said NS3/4a epitope and/or said multiple epitope fusion antigen react specifically with anti-HCV antibodies present in a biological sample from an HCV-infected individual. 17. The method of claim 16, wherein said NS3/4a conformational epitope comprises the amino acid sequence depicted in Figures 3 A-3D, or an amino acid sequence with at least 80% sequence identity thereto which has protease activity, and said multiple epitope fusion antigen comprises the amino acid sequence depicted in Figures 5A-5F, or an amino acid sequence with at least 80% sequence identity thereto which reacts specifically with anti-HCV antibodies present in a biological sample from an HCV-infected individual. 18. The method of claim 17, wherein said NS3/4a conformational epitope consists ofthe amino acid sequence depicted in Figures 3A-3D, and said multiple epitope fusion antigen consists ofthe amino acid sequence depicted in Figures 5A-5F. 19. A method of producing an immunoassay solid support, comprising: (a) providing a solid support; and (b) binding to the solid support at least one HCV NS3/4a conformational epitope and a multiple epitope fusion antigen, wherein said NS3/4a conformational epitope consists ofthe amino acid sequence depicted in Figures 3A-3D, and said multiple epitope fusion antigen consists ofthe amino acid sequence depicted in Figures 5A-5F. 20. A multiple epitope fusion antigen comprising the amino acid sequence depicted in Figures 5A-5F, or an amino acid sequence with at least 80% sequence identity thereto which reacts specifically with anti-HCV antibodies present in a biological sample from an HCV-infected individual. 21. The multiple epitope fusion antigen of claim 20, wherein said multiple epitope fusion antigen comprises the amino acid sequence depicted in Figures 5A-5F, or an amino acid sequence with at least 90% sequence identity thereto which reacts specifically with anti-HCV antibodies present in a biological sample from an HCV- infected individual. 22. The multiple epitope fusion antigen of claim 20, wherein said multiple epitope fusion antigen comprises the amino acid sequence depicted in Figures 5 A-5F, or an amino acid sequence with at least 98% sequence identity thereto which reacts specifically with anti-HCV antibodies present in a biological sample from an HCV- infected individual. 23. The multiple epitope fusion antigen of claim 20, wherein said multiple epitope fusion antigen consists ofthe amino acid sequence depicted in Figures 5A-5F. 24. A polynucleotide comprising a coding sequence for the multiple epitope fusion antigen of any of claims 20-23. 25. The polynucleotide of claim 24, wherein said polynucleotide comprises the nucleic acid sequence depicted in Figures 5A-5F, or a nucleic acid sequence with at least 80% sequence identity thereto which encodes a polypeptide that reacts specifically with anti-HCV antibodies present in a biological sample from an HCV-infected individual. 26. The polynucleotide of claim 24, wherein said polynucleotide comprises the nucleic acid sequence depicted in Figures 5 A-5F, or a nucleic acid sequence with at least 90% sequence identity thereto which encodes a polypeptide that reacts specifically with anti-HCV antibodies present in a biological sample from an HCV-infected individual. 27. The polynucleotide of claim 24, wherein said polynucleotide comprises the nucleic acid sequence depicted in Figures 5 A-5F, or a nucleic acid sequence with at least 98% sequence identity thereto which encodes a polypeptide that reacts specifically with anti-HCV antibodies present in a biological sample from an HCV-infected individual. 28. A recombinant vector comprising: (a) a polynucleotide according to any of claims 24-27; (b) and control elements operably linked to said polynucleotide whereby the coding sequence can be transcribed and translated in a host cell. 29. A host cell transformed with the recombinant vector of claim 28. 30. A method of producing a recombinant multiple epitope fusion antigen comprising: (a) providing a population of host cells according to claim 29; and (b) culturing said population of cells under conditions whereby the multiple epitope fusion antigen encoded by the coding sequence present in said recombinant vector is expressed. 31. Use of an immunoassay solid support according to any of claims 1 - 14 for the manufacture of a diagnostic reagent for use in a method of detecting hepatitis C virus (HCV) infection in a biological sample. 32. A method of detecting hepatitis C virus (HCV) infection in a biological sample, said method comprising: (a) providing an immunoassay solid support according to any of claims 1-14; (b) combining a biological sample with said solid support under conditions which allow HCV antibodies, when present in the biological sample, to bind to said NS3/4a epitope and/or said multiple epitope fusion antigen to form a first immune complex; (c) adding to the solid support from step (b) under complex forming conditions a detectably labeled antibody, wherein said labeled antibody is reactive with said immune complex; (d) detecting second immune complexes formed between the detectably labeled antibody and the first immune complex, if any, as an indication of HCV infection in the biological sample.

Inventors: ARCANGEL PHILLIP, CHIEN DAVID Y, COIT DORIS, GEORGE-NASCIEMENTO CARLOS, MEDINA-SELBY ANGELICA, TANDESKE LAURA, ARCANGEL, PHILLIP, CHIEN, DAVID, Y., COIT, DORIS, GEORGE-NASCIEMENTO, CARLOS, MEDINA-SELBY, ANGELICA, TANDESKE, LAURA
Applicants: ARCANGEL PHILLIP, CHIEN DAVID Y, CHIRON CORP, COIT DORIS, GEORGE NASCIEMENTO CARLOS, MEDINA SELBY ANGELICA, TANDESKE LAURA, ARCANGEL, PHILLIP, CHIEN, DAVID, Y., CHIRON CORPORATION, COIT, DORIS, GEORGE-NASCIEMENTO, CARLOS, MEDINA-SELBY, ANGELICA, TANDESKE, LAURA
Application year: 2001
Main classification: G01N33
IPC codes: C07K14, C07K19, C12M1, C12N1, C12N5, C12N15, C12P21, G01N33