Immunohistochemical differentiation of hepatitis D virus genotypes

Determination of hepatitis D virus (HDV) genotypes is epidemiologically and clinically important. Phylogenic analysis based on sequencing analysis of multiple HDV strains isolated from sera of patients is not convenient for m ass screening in routine laboratories. This study was designed to develop g enotype-specific antibodies against hepatitis delta antigen (HDAg) and to a pply these antibodies for immunohistochemical differentiation of HDV genoty pes in formalin-fixed, paraffin-embedded liver biopsies of patients, Diverg ence in the carboxyl-terminal 19 amino acids of the large HDAg between geno types I and II is more than 70%. Peptides covering these residues were conj ugated to keyhole limpet hemocyanin and were used for immunization. The gen erated antibodies were confirmed for their specificity by binding to type-s pecific HDAgs expressed in DNA-transfected Huh-7 hepatoma cells. Liver biop sies from 6 patients who had dominant genotype I HDV and 33 patients who ha d dominant genotype II HDV in sera were stained with these antibodies, The accuracy for these antibodies was 94.9%, and the agreement between dominant HDV genotypes in serum and dominant hepatic HDV genotypes based on HDAg st aining was nearly perfect (kappa = 0.83). In summary, the carboxyl-terminal 19 amino acids of the large HDAg can be used as immunogens to generate gen otype-specific antibodies. These antibodies were proven to be useful in imm unohistochemical differentiation of HDV genotypes in liver biopsies.