Optimization for the detection of hepatitis C virus (HCV) antigens in the liver

To optimize the detection of hepatitis C viral antigens in liver tissue, cryostat and formalin-fixed, paraffin-embedded liver sections from 21 patients with chronic hepatic C viral infection were sudied. Fro cryostat sections, six different fixatives were compared. Sixteen primary antibodies were tested: nine different mouse monoclonal anti-hepatitis C virus-core antobodies, a human monoclonal anti-hepatitis C virus-non-structural 4, and six rabbit polyclonals directed against synthetic peptides of the hepatitis C virus core, endelope, and non-sructural 3, non-structural 4, non structural 5. Three detection systems, 3- and 5-step peroxidase-antiperoxidase and avidin-biotin complex, were examined. In cryostat sections, acetone/chloroform formation consistently produced the best signal-to-background ratio. Five anti-hepatitis C virus-core monoclonals which recognize amino acid sequence 26-45 of the hepatitis C virus-core region consistently detected the viral antigen, but not che monoclonals directed against 39-74 of the hepatitis C virus-core region. The human anti-hepatitis C virus-non structural 4, which reacts to amino acid sequence 1700-1705, also regularly detected viral antigen. The rabbit polyclonals produced either negative or nonspecific staining. The 5-step peroxidase-antaiperoxidase provided the strongest signal and the avidin-biotin system produced high background consistently. Overall, hepatitis C virus core and non-structural 4 antigens were detected in 71% and 57% of the patients studied. Of the 16 patients seropositive for hepatitis C virus RNA, 75% and 69% had detectable hepatitis C virus core and non structural 4, in contrast to 60% and 20% of the five hepatitis C virus RNA seronegative patients.