Comparison of direct and indirect two-site binding enzyme immunoassay

Abstract

Rabbit IgG, directed against HBsAg, was purified by positive and by negative affinity chromatography and applied in horseradish peroxidase labelled as well as in unlabelled form in the direct and indirect two-site binding enzyme immunoassay (EIA). Comparing direct and indirect assay the latter is more sensitive and less conjugate consuming. In contrast to the indirect assay in which antibodies, purified by positive affinity chromatography, do not alter detection limit, a 4- to 8-fold higher sensitivity was achieved in the direct EIA in contrast to antibodies, purified by negative affinity chromatography. In the indirect EIA unlabelled second and labelled third antibodies were incubated successively as well as simultaneously. The latter procedure shortened the assay time but needed antibodies purified by positive affinity chromatography and a 10-fold higher conjugate concentration. Greatest sensitivity was obtained in the indirect EIA by the use of labelled second and labelled third antibodies (20–30 ng/1 HBsAg).