Preparation, identification, and clinical application of anti-HBs monoclonal antibody that binds both wild-type and immune escape mutant HBsAgs

Abstract

Using a standard cellular fusion technique and indirect enzyme-linked immunosorbent assay (ELISA), a hybridoma cell line strain secreting anti-HBs monoclonal antibody (mAb) (defined G6 mAb) was obtained. The cells grew and secreted mAb stably. Antibody titers in the culture supernatant and ascites were 2.048×106 and 4.096×106, respectively. By applying the anti-HBs G6 mAb and horseradish peroxidase (HRP)-labeled goat anti-HBs antibody, we developed a sandwich ELISA (defined G6m ELISA) for detecting both wild-type and immune escape mutant HBsAgs (IEM HBsAg). The assay was performed to detect 17 species of genome recombinant expression HBsAg, including two wild-type species and 15 IEM HBsAg species, which varied in the "a" determinant, in a group of patients infected with hepatitis B virus (HBV). The patients previously had a lower ELISA detection signal [(absorbance of patients/absorbance of normal people (P/N): 1.0-4.5)]. The results demonstrated that the sensitivity of this assay to wild-type HBsAg was no less than 0.125 μg/L; 12 of 15 IEM HBsAg species (P/N≥2.5) were positive for G6 mAb. Of the positive IEM HBsAg species, two had a low absorbance value at 450 nm (A450), one had an intermediate A450 value and nine had a high A450 value, which was 7.55%(mean), 59.4%and 92.1%-109.4% of the wild-type A450 value, respectively. The two species with low A450 value and the three negative species mutated at the bases 120-124 in the first loop of the HBV "a" determinant. Using the G6 ELISA and two commercial ELISA kits (A and B), 177 patients were tested. The G6 ELISA had a significantly higher detection rate than either commercial ELISAs (19.21% vs 14.89% and 6.21%, respectively; P <0.01, P <0.05, respectively). © Higher Education Press and Springer-Verlag GmbH 2009.