Preparation and application of monoclonal antibodies against hepatitis C virus nonstructural proteins

To prepare hybridoma cell lines that secrete monoclonal antibodies against hepatitis C virus (HCV) recombinant proteins NS3 and NS5 and to evaluate their use in the study of HCV NS3 and NS5 antigen distribution in human liver tissue.

Hybridoma cell lines were generated using spleen cells from BALB/C mice immunized with recombinant NS3 and NS5 proteins, following conventional protocols. Antibody-secreting cells were screened by solid phase ELISA and cloned by limited dilution. The specificity of the monoclonal antibodies was determined by testing hybridoma culture supernatants by Western blots of E. coli expressing the recombinant HCV proteins and ELISA with HCV core and hepatitis B virus (HBV) antigens. The monoclonal antibodies were employed in immunohistochemistry studies to determine the distribution of HCV NS5 and NS3 antigens in 51 paraffin embedded human liver tissue samples.

Eight hybridoma cell lines secreting monoclonal antibodies against HCV NS3 and NS5 proteins were generated and named 2B6, 2F3, 3D8, 3D9, 8B2, 6F11, 4C6 and 7D9. Only one of them, 2B6 (secreting antibodies against NS3 protein), cross-reacted with the C7 polypeptide, a different recombinant NS3 polypeptide. The rest of the cell lines showed no cross-reactivity with HCV core or HBV antigens. In addition, monoclonal antibodies against NS3 antigens did not cross-react with NS5 antigens, and vice versa. In immunohistochemistry studies, these monoclonal antibodies did not detect HCV antigens in specimens from patients infected only with HBV (n = 20). In HCV-infected specimens (n = 31), the rates of positive detection of NS3 and NS5 antigens were 51.6% (16/31) and 54.9% (17/31), respectively. Six of these 31 specimens were from patients infected only with HCV and half of them were positive for HCV NS3 and NS5 antigens. In specimens from patients co-infected with HBV and HCV (n = 25), the rates of NS3 and NS5 antigen positive detection were 52% (13/25) and 56% (14/25), respectively, which are similar to those obtained in samples from patients infected only with HCV. In specimens from chronic active cirrhosis patients, the rates of HCV NS3 and NS5 antigen detection were 70.6% (12/17) and 76.5% (13/17), respectively.

We successfully prepared monoclonal antibodies that are specific against recombinant HCV NS3 and NS5 proteins and could be useful for clinical immunohistochemistry diagnosis.