The invention relates to a method of detecting HCV infection in a biological sample, the method comprising providing an immunoassay solid support, comprising an HCV anti-core antibody, an antigen comprising an HCV NS3/4a epitope, and an HCV multiple epitope fusion antigen, that can detect both HCV antigens and antibodies present in a sample. The invention also includes polynucleotides encoding multiple epitope fusion antigens for use in the assay, recombinant vectors and host cells comprising such polynucleotides, and methods of producing the multiple epitope fusion antigens.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention is based in part, on the finding that HCV seroconversion antibodies are typically anti-core and anti-NS3 (helicase). Accordingly, the invention provides an HCV core antigen and NS3 antibody combination assay that can detect both HCV antigens and antibodies present in a sample using a single solid matrix.

Accordingly, in one embodiment, the subject invention is directed to an immunoassay solid support comprising at least one HCV anti-core antibody and at least one isolated HCV NS3/4a epitope bound thereto. The antibody and NS3/4a epitope can be any of the herein described molecules. Additionally, the solid support may include any of the multiple epitope fusion antigens described herein, such as the multiple epitope fusion antigen comprising the amino acid sequence depicted in FIGS. 7A-7F (see Original Patent).

In certain embodiments, the solid support comprises at least two HCV anti-core antibodies bound thereto. Moreover, the anti-core antibody may be a monoclonal antibody. Additionally, the NS3/4a epitope may be a conformational epitope, such as a conformational NS3/4a epitope comprising the amino acid sequence depicted in FIGS. 4A-4D (see Original Patent).

In another embodiment, the invention is directed to an imununoassay solid support comprising at least two HCV anti-core monoclonal antibodies and at least one HCV NS3/4a conformational epitope comprising the amino acid sequence depicted in FIGS. 4A-4D, bound thereto.

In still a further embodiment, the invention is directed to a method of detecting HCV infection in a biological sample. The method comprises: (a) providing an immunoassay solid support as described above; (b) combining a biological sample with the solid support under conditions which allow HCV antigens and antibodies, when present in the biological sample, to bind to the at least one anti-core antibody and the NS3/4a epitope, respectively; (c) adding to the solid support from step (b) under complex forming conditions (i) a first detectably labeled antibody, wherein the first detectably labeled antibody is a detectably labeled HCV anti-core antibody, wherein the labeled anti-core antibody is directed against a different HCV core epitope than the at least one anti-core antibody bound to the solid support; (ii) an antigen that reacts with an HCV antibody from the biological sample reactive with the NS3/4a epitope; and (iii) a second detectably labeled antibody, wherein the second detectably labeled antibody is reactive with the antigen of (ii); and (d) detecting complexes formed between the antibodies and antigens, if any, as an indication of HCV infection in the biological sample. The NS3/4a epitope may be a conformational epitope, such as a conformational epitope having the NS3/4a sequence depicted in FIGS. 4A-4D (see Original Patent).

In yet another embodiment, the invention is directed to a method of detecting HCV infection in a biological sample. The method comprises: (a) providing an immunoassay solid support with at least two HCV anti-core antibodies bound thereto, as described above; (b) combining a biological sample with the solid support under conditions which allow HCV antigens and antibodies, when present in the biological sample, to bind to the at least two anti-core antibodies and the NS3/4a epitope, respectively; (c) adding to the solid support from step (b) under complex forming conditions (i) a first detectably labeled antibody, wherein the first detectably labeled antibody is a detectably labeled HCV anti-core antibody, wherein the labeled anti-core antibody is directed against a different HCV core epitope than the anti-core antibodies bound to the solid support; (ii) an epitope from the c33c region of the HCV polyprotein fused to an hSOD amino acid sequence; and (iii) a second detectably labeled antibody, wherein the second detectably labeled antibody is reactive with the hSOD amino acid sequence; and (d) detecting complexes formed between the antibodies and antigens, if any, as an indication of HCV infection in the biological sample. The NS3/4a epitope may be a conformational epitope, such as a conformational epitope having the NS3/4a sequence depicted in FIGS. 4A-4D.

In any of the above embodiments, the anti-core antibody may be directed against an N-terminal region of the HCV core antigen, such as against amino acids 10-53 of HCV, numbered relative to the HCV1 polyprotein sequence, and/or the detectably labeled HCV anti-core antibody may be directed against a C-terminal region of the HCV core antigen, such as amino acids 120-130 of HCV, numbered relative to the HCV1 polyprotein sequence. Moreover, the antigen that reacts with an HCV antibody from the biological sample may be from the NS3 region, such as an epitope from the c33c region of the HCV polyprotein and can be fused with a human superoxide dismutase (hSOD) amino acid sequence. In this embodiment, the second detectably labeled antibody is reactive with the hSOD amino acid sequence.

In another embodiment, the invention is directed to a method of detecting HCV infection in a biological sample. The method comprises: (a) providing an immunoassay solid support including two HCV anti-core monoclonal antibodies and a conformational epitope comprising the amino acid sequence depicted in FIGS. 4A-4D; (b) combining a biological sample with the solid support under conditions which allow HCV antigens and antibodies, when present in the biological sample, to bind to the at least two anti-core antibodies and the NS3/4a conformational epitope, respectively; adding to the solid support from step (b) under complex forming conditions (i) a first detectably labeled antibody, wherein the first detectably labeled antibody is a detectably labeled HCV anti-core antibody, wherein the labeled anti-core antibody is directed against a different HCV core epitope than the at least two anti-core antibodies bound to the solid support; (ii) an epitope from the c33c region of the HCV polyprotein fused to an hSOD amino acid sequence; and (iii) a second detectably labeled antibody, wherein the second detectably labeled antibody is reactive with said hSOD amino acid sequence; detecting complexes formed between the antibodies and antigens, if any, as an indication of HCV infection in the biological sample.

In certain embodiments, the at least two anti-core antibodies are directed against an N-terminal region of the HCV core antigen, such as against amino acids 10-53 of HCV, numbered relative to the HCV1 polyprotein, and the detectably labeled HCV anti-core antibody is directed against a C-terminal region of the HCV core antigen, such as against amino acids 120-130 of HCV, numbered relative to the HCV polyprotein sequence.

In another embodiment, the invention is directed to a method of detecting HCV infection in a biological sample. The method comprises: (a) providing an immunoassay solid support which includes a multiple epitope fusion antigen; (b) combining a biological sample with the solid support under conditions which allow HCV antigens and antibodies, when present in the biological sample, to bind to the at least one anti-core antibody, the NS3/4a epitope, and the multiple epitope fusion antigen; (c) adding to the solid support from step (b) under complex forming conditions (i) a first detectably labeled antibody, wherein the first detectably labeled antibody is a detectably labeled HCV anti-core antibody, wherein the labeled anti-core antibody is directed against a different HCV core epitope than the at least one anti-core antibody bound to the solid support; (ii) first and second antigens that react with an HCV antibody from the biological sample reactive with the NS3/4a epitope and the multiple epitope fusion antigen, respectively; and (iii) a second detectably labeled antibody, wherein the second detectably labeled antibody is reactive with the antigens of (ii); (d) detecting complexes formed between the antibodies and antigens, if any, as an indication of HCV infection in the biological sample.

The anti-core antibody may be directed against an N-terminal region of the HCV core antigen and said first detectably labeled HCV anti-core antibody may be directed against a C-terminal region of the HCV core antigen, as described above. Moreover, the first antigen that reacts with an HCV antibody from the biological sample may comprise an epitope from the c33c region of the HCV polyprotein, and may be fused with an hSOD amino acid sequence. In this context, the second detectably labeled antibody is reactive with the hSOD amino acid sequence. Additionally, the second antigen that reacts with an HCV antibody from the biological sample may comprise an epitope from the c22 region of the HCV polyprotein, such as an epitope comprising amino acids Lys.sub.10 to Ser.sub.99 of the HCV polyprotein, with a deletion of Arg47 and a substitution of Leu for Trp at position 44, numbered relative to the HCV1 polyprotein sequence. The epitope may be fused with an hSOD amino acid sequence. If so, the second detectably labeled antibody is reactive with the hSOD amino acid sequence. The multiple epitope fusion antigen may comprise the amino acid sequence depicted in FIGS. 7A-7F (see Original Patent).

In yet a further embodiment, the invention is directed to a method of detecting HCV infection in a biological sample, said method comprising: (a) providing an immunoassay solid support which comprises two HCV anti-core monoclonal antibodies, an HCV NS3/4a conformational epitope comprising the amino acid sequence depicted in FIGS. 4A-4D, and a multiple epitope fusion antigen comprising the amino acid sequence depicted in FIGS. 7A-7F, bound thereto; (b) combining a biological sample with the solid support under conditions which allow HCV antigens and antibodies, when present in the biological sample, to bind to the at least two anti-core antibodies, the NS3/4a conformational epitope, and the multiple epitope fusion antigen, respectively; (c) adding to the solid support from step (b) under complex forming conditions (i) a first detectably labeled antibody, wherein the first detectably labeled antibody is a detectably labeled HCV anti-core antibody, wherein the labeled anti-core antibody is directed against a different HCV core epitope than the at least two anti-core antibodies bound to the solid support; (ii) an epitope from the c33c region of the HCV polyprotein fused to an hSOD amino acid sequence and an epitope from the c22 region of the HCV polyprotein fused to an hSOD amino acid sequence; and (iii) a second detectably labeled antibody, wherein said second detectably labeled antibody is reactive with said hSOD amino acid sequences; (d) detecting complexes formed between the antibodies and antigens, if any, as an indication of HCV infection in the biological sample.

In this embodiment, the at least two anti-core antibodies may be directed against an N-terminal region of the HCV core antigen, such as against amino acids 10-53 of HCV, numbered relative to the HCV1 polyprotein, and the detectably labeled HCV anti-core antibody is directed against a C-terminal region of the HCV core antigen, such as against amino acids 120-130 of HCV, numbered relative to the HCV1 polyprotein sequence. Moreover, the epitope from the c22 region may comprise amino acids Lys.sub.10 to Ser.sub.99 of the HCV polyprotein, with a deletion of Arg47 and a substitution of Leu for Trp at position 44, numbered relative to the HCV1 polyprotein sequence.

In other embodiments, the invention is directed to immunodiagnostic test kits comprising the immunoassay solid support described above, and instructions for conducting the immunodiagnostic test.

In still further embodiments, the invention is directed to methods of producing an immunoassay solid support, comprising: (a) providing a solid support; and (b) binding at least one HCV anti-core antibody, such as one or two or more, and at least one isolated HCV NS3/4a epitope thereto, and optionally, a multiple epitope fusion antigen thereto. The anti-core antibodies, NS3/4a epitopes and multiple epitope fusion antigens are as described above.

In additional embodiments, the invention is directed to a multiple epitope fusion antigen comprising the amino acid sequence depicted in FIGS. 7A-7F (see Original Patent), or an amino acid sequence with at least 80% sequence identity, such as 90% or more sequence identity, thereto which 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 of the amino acid sequence depicted in FIGS. 5A-5F (see Original Patent).

In further embodiments, the invention is directed to a polynucleotide comprising a coding sequence for the multiple epitope fusion antigen above, a recombinant vectors comprising the polynucleotides, host cells transformed with the recombinant vectors, and methods of producing a recombinant multiple epitope fusion antigen comprising: (a) providing a population of host cells as above; and (b) culturing the population of cells under conditions whereby the multiple epitope fusion antigen encoded by the coding sequence present in the recombinant vector is expressed.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, recombinant DNA techniques and immunology, within the skill of the 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 and N. Kaplan eds., Academic Press, Inc.).

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

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 of the 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 of the 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 antibodies and antigens which are present during the early stages of HCV seroconversion, thereby increasing detection accuracy and reducing the incidence of false results. The methods can be conveniently practiced in a single assay format.

More particularly, the assay is conducted on a solid support to which has been bound one or more HCV anti-core antibodies (directed against either the same or different HCV core epitopes) and an epitope derived from the NS3/4a region of the HCV polyprotein. Examples of particular anti-core antibodies useful in the present invention include, but are not limited to, antibody molecules such as monoclonal antibodies, directed against epitopes in the core region found between amino acids 10-53; amino acids 10-45; amino acids 67-88; amino acids 120-130, or antibodies directed against any of the core epitopes identified in, e.g., Houghton et al., U.S. Pat. No. 5,350,671; 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; and commonly owned, allowed U.S. patent application Ser. Nos. 08/403,590 and 08/444,818, the disclosures of which are incorporated herein by reference in their entireties.

The NS3/4a region of the HCV polyprotein has been described and the amino acid sequence and overall structure of the 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. Pat. No. 5,843,752, incorporated herein by reference in its entirety. 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 of the antigen with antibodies in a biological sample from an HCV-infected subject, and a loss of the epitope's immunoreactivity upon denaturation of the 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 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. A particularly convenient assay for testing protease activity is set forth in the examples below.

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 of the 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.

Representative amino acid sequences for NS3/4a polypeptides are shown in FIG. 3 and FIGS. 4A through 4D (see Original Patent). The bolded alanine occurring at position 182 of FIG. 3 (see Original Patent) is substituted for the native serine found at this position in order to prevent autocatalyisis of the molecule that might otherwise occur. The amino acid sequence shown at positions 2-686 of FIGS. 4A through 4D 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 FIG. 4) is mutated to Pro, and the Ser normally occurring at position 1429 of HCV-1 (amino acid position 404 of FIG. 4) is mutated to Ile. 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. Pat. No. 5,843,752 and Zhang et al., U.S. Pat. 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, positions 2-182 of FIG. 4. The structure of the 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 of the active site of the molecule. Particularly, it is desirable to maintain amino acids 1- or 2-155 of FIG. 4, 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 FIG. 4 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 FIG. 4 (see Original Patent)). Thus, if helicase activity is desired, this portion of the 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 solid support may also comprise other antigens. For example, multiple epitope fusion antigens (termed "MEFAs"), as described in International Publication No. WO 97/44469, may be bound to the solid support for use in the subject assays. Such MEFAs include multiple epitopes derived from two or more of the various viral regions shown in FIG. 1 and Table 1 (see Original Patent). In particular, as shown in FIG. 1 and Table 1, An HCV polyprotein, upon cleavage, produces at least ten distinct products, in the order of NH.sub.2-Core-E1-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, E1 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 of the 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 of the 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, found about positions 1658-1711), two proteins (NS4b found at about positions 1712-1972, and NS5a found at about positions 1973-2420), and an RNA-dependent RNA polymerase (NS5b found at about positions 2421-3011).

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 of the epitopes is different than their linear order in the genome in which they occur. The linear order of the sequences of the 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-L- ys-Gln-Asn (SEQ ID NO:6), which represents a consensus sequence for amino acids 390-410 of the HCV type 1 genome. A representative E2 epitope present in a MEFA of the invention can comprise a hybrid epitope spanning 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 of the virus but are slightly different from one viral strain to another. For example, HCV includes the antigenic determinant known as 5-1-1 (See, FIG. 1). This particular antigenic determinant appears in three different forms on the three different viral strains of HCV. Accordingly, in a preferred embodiment of the 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 of the present invention can also include multiple copies which are exact copies of the same epitope.

Representative MEFAs for use with the present assays are described in International Publication No. WO 97/44469. Additional representative MEFAs for use herein include those termed MEFA 12, MEFA 13 and MEFA 13.1. It is to be understood that these MEFAs are merely representative and other epitopes derived from the HCV genome will also find use with the present assays and may be incorporated into these or other MEFAs.

The DNA sequence and corresponding amino acid sequence of MEFA 12 is shown in FIGS. 7A through 7F. The general structural formula for MEFA 12 is shown in FIG. 6 (see Original Patent) and is as follows: hSOD-E1(type 1)-E2 HVR consensus (type 1a)-E2 HVR consensus (types 1 and 2)-c33c short (type 1)-5-1-1(type 1)-5-1-1(type 3)-5-1-1(type 2)-c100(type 1)-NS5(type 1)-NS5(type 1)-core (types 1+2)-core (types 1+2). This multiple copy epitope includes the following amino acid sequence, numbered relative to HCV-1 (the numbering of the 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 of the core region): amino acids 1-69 of superoxide dismutase (SOD, used to enhance recombinant expression of the protein); amino acids 303 to 320 of the polyprotein from the E1 region; amino acids 390 to 410 of the polyprotein, representing a consensus sequence for the hypervariable region of HCV-1a E2; amino acids 384 to 414 of the polyprotein from region E2, representing a consensus sequence for the E2 hypervariable regions of HCV-1 and HCV-2; amino acids .sup.121I-1457 of the 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 of the polyprotein; two exact copies of an epitope from the NS5 region of HCV-1, each with amino acids 2278 to 2313 of the HCV polyprotein; and two copies of three epitopes from the core region, two from HCV-1 and one from HCV-2, which copies are equivalent antigenic determinants represented by amino acids 9 to 53 and 64-88 of HCV-1 and 67-84 of HCV-2.

Table 2 (see Original Patent) shows the amino acid positions of the various epitopes in MEFA 12 with reference to FIGS. 7A through 7F herein (see Original Patent). The numbering in the tables is relative to HCV-1. See, Choo et al. (1991) Proc. Natl. Acad. Sci. USA 88:2451-2455. MEFAs 13 and 13.1 also share the general formula specified above for MEFA 12, with modifications as indicated in Tables 3 and 4 (see Original Patent), respectively.

In one assay format, the sample is combined with the solid support, as described further below. If the sample is infected with HCV, core antigens, as well as HCV antibodies to those epitopes present on the solid support, will bind to the solid support components. A detectably labeled anti-core antibody is then added. The labeled anti-core antibody is directed against a different epitope than the anti-core antibody that is bound to the solid support. This anti-core antibody binds the core antigen captured by the anti-core antibodies on the solid support.

An antigen that reacts with the captured HCV antibody from the biological sample, which captured sample HCV antibody is reactive with the NS3/4a epitope, is also added. This antigen is preferably an epitope derived from the NS3 region of the HCV polyprotein. This antigen binds the captured HCV antibody from the sample. A number of antigens including such epitopes are known, including, but not limited to antigens derived from the c33c and c100 regions, as well as fusion proteins comprising an NS3 epitope, such as c25. These and other NS3 epitopes are useful in the present assays and are known in the art and described in, e.g., Houghton et al, U.S. Pat. No. 5,350,671; 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; and commonly owned, allowed U.S. patent application Ser. Nos. 08/403,590 and 08/444,818, the disclosures of which are incorporated herein by reference in their entireties.

A second labeled antibody, directed against the antigen described above, is added. This antibody can be directed against any epitope included in the antigen. For example, the antibody can be directed against the NS3 region present in the antigen. Alternatively, if the antigen above is expressed as a fusion protein, the second labeled antibody can be directed against the fusion partner. Additional antigens and antibodies can be added to the assay, particularly if the solid support includes a MEFA. These assay formats are explained further below.

A representative assay under the invention is depicted in FIG. 2 (see Original Patent). As shown in the figure, the solid support includes two anti-core monoclonal antibodies, termed c11-3 and c11-7. These antibodies are directed against an epitope found in the N-terminal region of the core protein at amino acids 10-53, numbered relative to the HCV1 polyprotein sequence. The solid support also includes an epitope to NS3/4a. The biological sample is added to the solid support. HCV core antigen, as well as antibodies directed against the NS3/4a epitope, both present in the sample, will bind the capture reagents on the solid support.

Horse radish peroxidase (HRP)-labeled anti-core monoclonal antibody c11-14, directed against a C-terminal region of the core found at amino acid positions 120-130, numbered relative to the HCV1 polyprotein sequence, is then added. A fusion protein, comprising a sequence from human SOD (hSOD) and an epitope from the c33c region is added, as is a second HRP-labeled antibody, directed against the SOD portion of the fusion protein. The SOD-c33c fusion will bind to the anti-NS3 antibody and the anti-SOD antibody will, in turn, bind the SOD-c33c fusion protein. Detection of the label indicates the presence of HCV infection.

Another representative assay under the invention is depicted in FIG. 8 (see Original Patent). The antibody assay configuration is an antigen-antibody-antigen sandwich capture assay using both NS3/4a and MEFA 12. The solid support includes the two anti-core monoclonal antibodies described above, an epitope to NS3/4a, as well as a representative MEFA, MEFA 12, which includes a truncated version of human SOD. As with the assay above, the biological sample is added to the solid support. HCV core antigen, as well as antibodies directed against the NS3/4a epitope and epitopes of the MEFA, present in the sample, will bind the capture reagents on the solid support. Two antigens, one reactive with sample antibodies that bind NS3/4a (as described above) and one reactive with sample antibodies that bind MEFA 12, are added. In FIG. 8, the antigen reactive with the MEFA 12/sample antibody complex, is a fusion between an SOD molecule and c22ks .DELTA.47-L44W. The c22ks antigen is from the core region and includes amino acids Lys.sub.10 to Ser.sub.99 of the polyprotein, as well as a deletion of Arg47 normally present and a substitution of Leu for Trp at position 44. The antibody detection conjugate is the second HRP-labeled monoclonal anti-SOD antibody, described above.

The above-described antigen/antibody combination assays are particularly advantageous as both the HCV core antigen and antibodies to NS3/4a and/or core may be detected by the same support in the same assay. Moreover, as described above, additional HCV epitopes, such as SOD-fused to c100, 5-1-1, NS5 antigens, as well as 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 combination cocktail to cover other non-structural epitopes of HCV.

In order to further an understanding of the invention, a more detailed discussion is provided below regarding production of antibodies for use in the subject immunoassays; production of polypeptides for use in the immunoassays; and methods of conducting the immunoassays.

Production of Antibodies for Use in the HCV Immunoassays

As explained above, the assay utilizes various antibodies which are bound to a solid support (e.g., one or more anti-core antibodies), and that detect antigen/antibody complexes formed when HCV infection is present in the sample. These antibodies may be polyclonal or monoclonal antibody preparations, monospecific antisera, human antibodies, or may be hybrid or chimeric antibodies, such as humanized antibodies, altered antibodies, F(ab').sub.2 fragments, F(ab) fragments, Fv fragments, single-domain antibodies, dimeric or trimeric antibody fragment constructs, minibodies, or functional fragments thereof which bind to the antigen in question.

Antibodies are produced using techniques well known to those of skill in the art and disclosed in, for example, U.S. Pat. Nos. 4,011,308; 4,722,890; 4,016,043; 3,876,504; 3,770,380; and 4,372,745. For example, polyclonal antibodies are generated by immunizing a suitable animal, such as a mouse, rat, rabbit, sheep or goat, with an antigen of interest. In order to enhance immunogenicity, the antigen can be linked to a carrier prior to immunization. Such carriers are well known to those of ordinary skill in the art. Immunization is generally performed by mixing or emulsifying the antigen in saline, preferably in an adjuvant such as Freund's complete adjuvant, and injecting the mixture or emulsion parenterally (generally subcutaneously or intramuscularly). The animal is generally boosted 2-6 weeks later with one or more injections of the antigen in saline, preferably using Freund's incomplete adjuvant. Antibodies may also be generated by in vitro immunization, using methods known in the art. Polyclonal antiserum is then obtained from the immunized animal. See, e.g., Houghton et al., U.S. Pat. No. 5,350,671, for a description of the production of anti-HCV polyclonal antibodies.

Monoclonal antibodies are generally prepared using the method of Kohler and Milstein (1975) Nature 256:495-497, or a modification thereof. Typically, a mouse or rat is immunized as described above. However, rather than bleeding the animal to extract serum, the spleen (and optionally several large lymph nodes) is removed and dissociated into single cells. If desired, the spleen cells may be screened (after removal of nonspecifically adherent cells) by applying a cell suspension to a plate or well coated with the antigen. B-cells, expressing membrane-bound immunoglobulin specific for the antigen, will bind to the plate, and are not rinsed away with the rest of the suspension. Resulting B-cells, or all dissociated spleen cells, are then induced to fuse with myeloma cells to form hybridomas, and are cultured in a selective medium (e.g., hypoxanthine, aminopterin, thymidine medium, "HAT"). The resulting hybridomas are plated by limiting dilution, and are assayed for the production of antibodies which bind specifically to the immunizing antigen (and which do not bind to unrelated antigens). The selected monoclonal antibody-secreting hybridomas are then cultured either in vitro (e.g., in tissue culture bottles or hollow fiber reactors), or in vivo (e.g., as ascites in mice).

The production of various anti-HCV monoclonal antibodies has been described in, e.g., Houghton et al., U.S. Pat. No. 5,350,671; Chien et al., International Publication No. WO 93/00365; commonly owned, allowed U.S. patent application Ser. Nos. 08/403,590 and 08/444,818; and Kashiwakuma et al., U.S. Pat. No. 5,871,904, incorporated herein by reference in their entireties.

As explained above, antibody fragments which retain the ability to recognize the antigen of interest, will also find use in the subject immunoassays. A number of antibody fragments are known in the art which comprise antigen-binding sites capable of exhibiting immunological binding properties of an intact antibody molecule. For example, functional antibody fragments can be produced by cleaving a constant region, not responsible for antigen binding, from the antibody molecule, using e.g., pepsin, to produce F(ab').sub.2 fragments. These fragments will contain two antigen binding sites, but lack a portion of the constant region from each of the heavy chains. Similarly, if desired, Fab fragments, comprising a single antigen binding site, can be produced, e.g., by digestion of polyclonal or monoclonal antibodies with papain. Functional fragments, including only the variable regions of the heavy and light chains, can also be produced, using standard techniques such as recombinant production or preferential proteolytic cleavage of immunoglobulin molecules. These fragments are known as F.sub.v. See, e.g., Inbar et al. (1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al. (1976) Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096.

A single-chain Fv ("sFv" or "scFv") polypeptide is a covalently linked V.sub.H-V.sub.L heterodimer which is expressed from a gene fusion including V.sub.H- and V.sub.L-encoding genes linked by a peptide-encoding linker. Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85:5879-5883. A number of methods have been described to discern and develop chemical structures (linkers) for converting the naturally aggregated, but chemically separated, light and heavy polypeptide chains from an antibody V region into an sFv molecule which will fold into a three dimensional structure substantially similar to the structure of an antigen-binding site. See, e.g., U.S. Pat. Nos. 5,091,513, 5,132,405 and 4,946,778. The sFv molecules may be produced using methods described in the art. See, e.g., Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85:5879-5883; U.S. Pat. Nos. 5,091,513, 5,132,405 and 4,946,778. Design criteria include determining the appropriate length to span the distance between the C-terminus of one chain and the N-terminus of the other, wherein the linker is generally formed from small hydrophilic amino acid residues that do not tend to coil or form secondary structures. Such methods have been described in the art. See, e.g., U.S. Pat. Nos. 5,091,513, 5,132,405 and 4,946,778. Suitable linkers generally comprise polypeptide chains of alternating sets of glycine and serine residues, and may include glutamic acid and lysine residues inserted to enhance solubility.

"Mini-antibodies" or "minibodies" will also find use with the present invention. Minibodies are sFv polypeptide chains which include oligomerization domains at their C-termini, separated from the sFv by a hinge region. Pack et al. (1992) Biochem 31:1579-1584. The oligomerization domain comprises self-associating .alpha.-helices, e.g., leucine zippers, that can be further stabilized by additional disulfide bonds. The oligomerization domain is designed to be compatible with vectorial folding across a membrane, a process thought to facilitate in vivo folding of the polypeptide into a functional binding protein. Generally, minibodies are produced using recombinant methods well known in the art. See, e.g., Pack et al. (1992) Biochem 31:1579-1584; Cumber et al. (1992) J Immunology 149B: 120-126.

Production of Antigens for Use in the HCV Immunoassays

As explained above, the molecules of the present invention are generally produced recombinantly. Thus, polynucleotides encoding HCV 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 cDNA and genomic libraries 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. Pat. 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; Nambair 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. In 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 of the ligated nucleotide sequence via PVR. 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.sub.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. Pat. 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 of the expression of the 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 of the constructs. Examples include the SV40 early gene enhancer (Dijkema et al. (1985) EMBO J. 4:761), the enhancer/promoter derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus (Gorman et al. (1982) Proc. Natl. Acad. Sci. USA 79:6777) and elements derived from human CMV (Boshart et al. (1985) Cell 41:521), such as elements included in the CMV intron A sequence (U.S. Pat. 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 of the coding sequence with respect to the control sequences being such that the coding sequence is transcribed under the "control" of the control sequences (i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence). Modification of the 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. Pat. No. 5,843,752 and Zhang et al., U.S. Pat. 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 of the 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, inter alia, Invitrogen, San Diego Calif. ("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 inter 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. Pat. 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 of the appropriate growth conditions and recovery methods are within the skill of the art.

The recombinant production of various HCV antigens has been described. See, e.g., Houghton et al., U.S. Pat. No. 5,350,671; 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.

Immunodiagnostic Assays

Once produced, the above anti-core antibodies and NS3/4a antigens are placed on an appropriate solid support for use in the subject immunoassays. 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 anti-core antibodies and NS3/4a epitope (collectively called "the solid-phase components" herein), and optionally, one or more MEFAs, 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 and antigens (collectively called "ligand molecules" herein) under suitable binding conditions. After washing to remove any nonbound ligand molecules, a second anti-core antibody, directed against a different epitope than the anti-core antibody bound to the support, is added under suitable binding conditions. The added anti-core antibody includes a detectable label, as described above, and acts to bind any core antigen that might be present in the sample which has reacted with the support-bound anti-core antibody. Also added are one or more antigens that can react with antibodies present in the sample that have, in turn, reacted with the NS3/4A epitope. As explained above, the antigen is typically derived from the NS3 region of the HCV polyprotein, and particularly from the c33c region of HCV. See, Houghton et al, U.S. Pat. No. 5,350,671; Chien et al., Proc. Natl. Acad. Sci. (1989) 89:10011-10015; International Publication No. WO 93/00365; and commonly owned, allowed U.S. patent application Ser. Nos. 08/403,590 and 08/444,818, for a description of this region and epitopes derived therefrom. A labeled antibody directed against this antigen is also added. The antibody will therefore bind the antigen, which has reacted with anti-NS3 antibodies present in the sample. For this purpose, the c33c epitope can be conveniently provided as a fusion between c33c and human superoxide dismutase (hSOD), produced recombinantly e.g., by methods described in Houghton et al., U.S. Pat. No. 5,350,671. The nucleotide and amino acid sequences for human SOD are known and reported in Hallewell et al., U.S. Pat. No. 5,710,033. A labeled antibody directed against human SOD can therefore be used to detect the presence of complexes formed between the NS3/4a epitope, any antibodies in the sample which react with this epitope, and HCV polypeptides which in turn bind the antibody in the sample.

If a MEFA is present on the solid support, one or more additional antigens, reactive with antibodies from the biological sample which are bound to antigens present on the MEFA, may also be added to the assay. Particularly useful in this context is an antigen derived from the core region of HCV, and more particularly, from the c22 antigen which includes 119 N-terminal core amino acids of the HCV polyprotein. One particular antigen derived from c22 is c22ks .DELTA.47-L44W which includes amino acids Lys.sub.10 to Ser.sub.99 of the polyprotein, as well as a deletion of Arg47 normally present and a substitution of Leu for Trp at position 44. As with the c33c epitope described above, this antigen can be provided as a fusion with hSOD and the same labeled antibody, directed against human SOD, can be used to detect the presence of complexes formed between antibodies present in the sample and the NS3/4a epitope and/or the MEFA, which complexes are also bound with the HCV antigens (e.g., c33c and c22).

More particularly, an ELISA method can be used, wherein the wells of a microtiter plate are coated with the solid-phase components. A biological sample containing or suspected of containing ligand molecules is then added to the coated wells. After a period of incubation sufficient to allow ligand-molecule binding to the immobilized solid-phase component, the plate(s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule (labeled anti-core antibody), an NS3 epitope-containing molecule, and an antibody directed against the NS3 epitope-containing molecule added. These molecules are allowed to react with any captured sample antigen and antibody, the plate washed and the presence of the labeled antibodies detected using methods well known in the art.

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 can also contain, depending on the particular immunoassay used, suitable labels and other packaged reagents and materials (i.e. wash buffers and the like). Standard immunoassays, such as those described above, can be conducted using these kits.
 

Claim 1 of 5 Claims

1. A polynucleotide comprising a coding sequence for a multiple epitope fusion antigen consisting of the amino acid sequence of SEQ ID NO:5.

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