Ultrasensitive Enzyme Immunoassay of Antibody IgG to IIIV-1 Reverse Transcriptase by Immune Complex Transfer with Detergents

Abstract

ABSTRACT Antibody IgG to HIV-1 reverse transcriptase (RT) was measured in three different ways. In immunoassay I, a polystyrene bead noncovalently coated with recombinant RT (rRT) was allowed to react with anti-RT IgG and rRT-β-D-galactosidase (GAL). In immunoassay II, a polystyrene bead noncovalently coated with (anti-2, 4-dinitrophenyl group) IgG was allowed to react with 2, 4-dinitrophenyl-bovine serum albumin (BSA)-rRT, anti-RT IgG and rRT-GAL. In immunoassay III, 2, 4-dinitrophenyl-biotinyl-BSA-rRT and (anti-human IgG γ-chain) Fab'-GAL were substituted for the corresponding conjugates in immunoassay II. The immune complex(es) of the three or/and four components formed on the polystyrene bead was quickly (only 2.5 min) eluted with detergents such as Triton X-100, Tween-20 and CHAPS in the absence or presence of ɛN-2, 4-dinitrophenyl-L-lysine and was transferred to a polystyrene bead successively coated with biotinyl-BSA (covalently), streptavidin and biotinyl-(anti-human IgG γ-chain) Fab' (immunoassays I and II) or with biotinyl-BSA (covalently) and streptavidin (immunoassay III ). By immune complex transfer with detergents, the sensitivity to anti-RT IgG was improved 280 to 800-fold in immunoassay I, 1,800 to 2,600-fold in immunoassay II and 100 to 500-fold in immunoassay III over that of immunoassay I without immune complex transfer, that is, a widely used conventional enzyme immunoassay.