Recombinant HCV type 2a Nonstructural Protein 5 (a.a. 2212-2313) [GST]

Different isolates of hepatitis C virus (HCV) show nucleotide sequence variability throughout the genome. Detection of antibodies to recombinant proteins derived from hepatitis C virus genotype 1, the prototype HCV clone HCV-PT, constitutes the main method for screening HCV infection. The influence of the genomic variability on the serological diagnosis of HCV by enzyme immunoassay remains poorly defined. The aim of this study was to assess the serological reactivity of a panel of well characterized French HCV isolates typed by sequence analysis from patients with chronic hepatitis. The 73 sera samples were tested in three third generation EIA tests and three confirmatory assays. HCV isolates were determined by RT-PCR and sequencing in NS5B region of the genome. The 73 sera were positive in the three EIA tests. The three confirmatory tests showed a weaker reactivity with NS5 protein whatever the genotype, and a lower reactivity in NS4 antigens of non-type 1 sequences, particularly for genotype 3. Even though the reactivity of the antigens differed among the HCV isolates, the 73 isolates from genotype 1-6 were reactive with the three commercial screening assays. These results demonstrate that using a single test is adequate in the routine diagnosis of HCV infection in clinical laboratory, as recommended by the last French and Europeen consensus conference.

Based on B cell epitope predictions, a recombinant antigen with multiple epitopes from four Hepatitis C Virus fragments (C, NS3, NS4 and NS5) were engineered. The recombinant gene was then highly expressed in E. coli. The non-modified and C-terminal-modified recombinant proteins were used for coating and biotin labeling, respectively, to establish the double-antigen sandwich ELISA. Ten positive reference samples confirmed by the CHIRON RIBA HCV 3.0 SIA kit were detected positive, Forty one plasma samples were positive among samples from 441 volunteers, which indicated that the recombinant antigen could readily react well with plasma HCV antibody. As critical reagents of double-antigen sandwich ELISA, the recombinant multi-epitope antigen and the C-terminal-modified and biotin-conjugated antigen show good antigenicity. In this study, we provide a simple approach to produce multiple epitopes within one recombinant protein in order to avoid the costly expression of less-effective pools of multiple proteins, which is the conventional strategy of diagnostic antigen production for HCV antibody detection.