Expression Of A HCV Multi-epitope Antigen And Study On The Feasibility Of A Double-antigen-sandwich ELISA To Detect Anti-HCV

Objectives: ELISA provide the main approach for the diagnosis of HCV infection, but all the present ELISA kits use a mixture of Core, NS3, NS4 and NS5 as capture antigens, and anti-human IgG-HRP as conjugate. Among populations with a low prevalence (<10%) of infection, the proportion of false-positive results averages 35%, hence, it is important and urgent to develop a more specific method. The above problems may be overcome by using double-antigen-sandwich ELISA method, which showed high sensitivity and specificity.Study design and rusults: To establish a double-antigen-sandwich ELISA system, a multiple-epitope fusion antigen which contains the immunodominant epitopes from the HCV structural (Core, Envelope) and nonstructural (NS3,NS4,NS5) and genotype-specific regions was constructed and expressed in pQE30 vector and Pinpoint?Xa-1 T vector, with its N-terminal connected to a 6 X His tag and a biotinated tag, respectively. Detection with positive sera containing only antibody to a single antigen segment indicated all the epitopes in the recombinant protein retained their antigenicity.Affinity purified 6 X His-tagged multiple-epitope fusion antigen was immobilized as capture antigen, biotin-tagged antigen then reacted with captured anti-HCV in positive sample, with Streptavidin-HRP as conjugate to report the ruselts. 400 samples from healthy blood donors were detected with no false positive. When the double-antigen-sandwich ELISA were used to detect 350 samples which had been reported anti-HCV positive by indirect ELISA, 12 samples showed negative. This 12 samples were tested with more specific methods such as RT-PCR and recombinant immunoblot assay (RIB A), and all of them remained negative. The results indicated that the double-antigen-sandwich ELISA we eatablished was very specific. Conclusion: False-positive results due to rheumatoid factor, hyperglobulinemia andnon-specific antibody to C100 antigen and fusion protein SOD happens occasionally with currently used indirect ELISA. To resolve this problem, a more sensitive and specific double-antigen-sandwich ELISA may be helpful. Since it is difficult to mix the individual antigens in equalmolar amounts and can't label some insoluble recombinant antigen with enzymes, it will be helpful to express a recombinant polyprotein containing all necessary immunodomins. Preliminary studies indicate that the new double-antigen-sandwich ELISA based on the multiple-epitope fusion antigen is very sensitive and specific. Higher sensitivity may be obtained because antibodies other than IgG (particularly IgM) could also be detected by this method, and this may shorten the period from infection to diagnosis.