Dissociation of hepatitis B surface antigen (HBsAg) from immobilised anti-HBsAg antibody in a two site immunoradiometric assay: its relevance to hook effect and recycled assay.

The mechanism of the hook effect in a two site immunometric assay of hepatitis B surface antigen (HBsAg) was studied using a novel method. The results showed that HBsAg dissociated from immobilized anti-HBsAg at low as well as high doses of antigen (Ag). This probably was not due to lack of immobilized antibody (Ab) or dissociation of HBsAg non-specifically bound to the immobilized Ab. The dissociated Ag sequestered the labelled Ab in the second reaction and contributed to the decrease in percent binding (B%) of the assay and thus the hook effect. The dissociated Ag could contaminate the recovered pooled labelled Ab in recycled assay and give rise to high non-specific binding. Prolonged washing decreased the side reaction but also the B% of the assay.