Serological detection of hepatitis B viral infection by a panel of solid-phase enzyme-linked immunosorbent assays (ELISA)

Abstract

Immunoassays for the detection of hepatitis B virus (HBV) in biological samples were developed. Using recombinant HBV antigens (Ags) and HBV-specific antibodies (Abs), we designed and evaluated a panel of enzyme-linked immunosorbent assays (ELISAs) detecting the main hepatitis B-related viral markers, namely HBV surface Ag (HBsAg), HBV e Ag (HBeAg), Abs to HBsAg (anti-HBs), Abs to HBV core Ag (anti-HBc) and Abs to HBeAg (anti-HBe), in blood serum. The ELISAs were validated using a panel of prescreened, by commercial tests, serum samples. In principle, HBV Ags or anti-HBV monoclonal antibodies (mAbs) were immobilised on microplate wells. Horseradish peroxidase (HRP) or biotin were used to prepare labeled Abs. Specifically for the determination of HBsAg and HBeAg, two-site sandwich immunoenzymometric assays were developed. The useful range was estimated at 20-500 ng/ml and human serum samples assayed were diluted 10- and 4-fold for HBsAg and HBeAg, respectively, with phosphate buffered saline (PBS) containing Tween 20 and gelatin. For the detection of Abs to HBs an indirect ELISA was formulated. Sera were similarly 4-fold diluted in the same buffer. Finally, competitive ELISAs were used for detecting anti-HBc and anti-HBe and sera tested were diluted 20- and 5-fold, respectively. All selected dilutions resulted in the accurate and reliable determination of HBV Ags and anti-HBV Abs. Taken altogether, these ELISAs are highly specific and equally sensitive to the circulating tests. However, their design could be very useful for research and/or preclinical studies of selected HBV-infected individuals.