More sensitive immune complex transfer enzyme immunoassay for antibody IgG to p17 of HIV-1 with shorter incubation time for immunoreactions and larger volumes of serum samples

Abstract

In the previous immune complex transfer enzyme immunoassay for antibody IgG to p17 of HIV-1, the immune complex comprising 2,4-dinitrophenyl-bovine serum albumin-recombinant p17 conjugate, anti-p17 IgG, and recombinant p17-beta-D-galactosidase conjugate was trapped onto polystyrene beads coated with (anti-2,4-dinitrophenyl group) IgG by overnight incubation and was transferred to polystyrene beads coated with (antithuman IgG gamma-chain) IgG by 3 hr incubation in the presence of excess of epsilon N-2,4-dinitrophenyl-L-lysine. These processes were made efficient by incubation with shaking and by using solid phases with larger surface areas. In addition, the volume of serum samples used was increased from 10 microliters to 100 microliters. As a result, the sensitivity was improved 20-30-fold and was approximately 100,000-fold higher than that of Western blotting for p17 band, even when both trapping and transferring of the immune complex were performed for only 30 min. Furthermore, testing many samples became easily possible with higher sensitivity using microplates and a fluororeader.