Optimization for the detection of hepatitis C virus 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 hepatis C viral infection were studied. For cryostat sections, six different fixatives were compared. Sixteen primary antibodies were tested: nine different mouse monoclonal anti-hepatitis C virus-core antibodies, a human monoclonal anti-hepatitis C virus-non-structural 4, and six rabbit polyclonals directed against synthetic peptides of the hepatitis C virus core, envelope, and non-structural 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 the 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-antiperoxidase 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. Hepatitis C virus antigens were detected exclusively in the cytoplasm of hepatocytes. Positive cells were usually found scattered in the hepatic lobules. In paraffin-embedded sections, none of the tested systems detected hepatitis C virus antigens. We conclude that hepatitis C virus core and non-structural 4 antigens can be detected in cryostat liver sections of patients with chronic hepatitis C virus infection. The optimum conditions include acetone/chloroform fixation of cryostat sections, using appropriate monoclonals, and a 5-step peroxidase-antiperoxidase detection system. Exclusive cytoplasmic hepatocyte staining is consistent with cytoplasmic replication of a hepatotropic virus.