Immunohistochemical Differentiation of Hepatitis D Virus Genotypes

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Abstract
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 mass screening in routine laboratories. This study was designed to develop genotype–specific antibodies against hepatitis delta antigen (HDAg) and to apply these antibodies for immunohistochemical differentiation of HDV genotypes in formalin–fixed, paraffin–embedded liver biopsies of patients. Divergence in the carboxyl–terminal 19 amino acids of the large HDAg between genotypes I and II is more than 70%. Peptides covering these residues were conjugated to keyhole limpet hemocyanin and were used for immunization. The generated antibodies were confirmed for their specificity by binding to type–specific HDAgs expressed in DNA–transfected Huh–7 hepatoma cells. Liver biopsies from 6 patients who had dominant genotype I HDV and 33 patients who had 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 staining was nearly perfect (κ = 0.83). In summary, the carboxyl–terminal 19 amino acids of the large HDAg can be used as immunogens to generate genotype–specific antibodies. These antibodies were proven to be useful in immunohistochemical differentiation of HDV genotypes in liver biopsies.