The amino acid sequences of core and non-structural proteins(NS3, NS4 and NSS) of Hepatitis C virus (HCV) were used for the selection of peptides to be synthesized. For the HCV core protein, the whole regin was tested using ten sequential peptides. The epitopes in HCV non-structural proteins were predicted by the combined analyses of the hydropathicity, acrophilicity, flexibility, and antigenicity parameters.
Peptides corresponding to the selected regions were synthesized by a solid-phase method with automatic peptide synthesizer. The synthesized peptides were purified with preparative reverse-phase high-performance liquid chromatography (HPLC) with a silicated octadecyl($C_18$) column. The majority of the peptides were isolated as a single peak and analyzed its purity using analytical HPLC. Purified peptides were used as test antigens for ELISA.
Antigenic reactivity was compared between free peptide, peptide-carrier conjugate and streptavidin-biotinylated peptide antigen. The result indicated that free peptide antigens showed the low reactivity on both C22 and NS4-1924 peptide and that peptide-carrier conjugate antigens showed the variation in reactivity depending on the peptides used, whereas the application of stretavidin-biotinylated peptide antigen significantly enhanced the reactivity, not influenced by any kinds of antigenic peptides.
The relative sensitivity of free peptides, peptide-carrier conjugates, or streptavidin-biotinylated peptides antigens was also investigated. The results demonstrate that the streptavidin-biotinylated peptide antigen is the most efficient one for detecting antibodies in HCV-infected sera and setup for peptide-based identification of immunodominant epitopes.
To determine the immunodominant regions of the core and non-structural protein of HCV, thirty-two synthetic peptides were tested for their reactivities against HCV antibody positive sera. Among them, 15 peptides (8 corresponding to the core peptides, 4 corresponding to th...