OPTIMAL CONDITIONS OF IMMUNE-COMPLEX TRANSFER ENZYME-IMMUNOASSAY FOR P24 ANTIGEN OF HIV-1

In the immune complex transfer enzyme immunoassay for HIV-1 p24 antige n, different preparations of anti-p24 Fab'-beta-D-galactosidase conjug ate, various periods of time for immunoreactions involved, and shaking for incubations with polystyrene beads were tested. On the basis of t he results of these experiments, p24 antigen was measured as follows. The antigen was reacted simultaneously with 2,4-dinitrophenyl-biotinyl -bovine serum albumin-affinity-purified rabbit anti-p24 Fab' conjugate and highly polymerized monoclonal mouse anti-p24 Fab'-beta-D-galactos idase conjugate at 37 degrees C for 2 hr. The immune complex formed co mprising the three components was trapped onto colored poly styrene be ads coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG f or 1.5 hr and was transferred to white polystyrene beads coated with s treptavidin in the presence of epsilon N-2,4-dinitrophenyl-L-lysine fo r 1.5 hr. The incubations with polystyrene beads were performed at roo m temperature with shaking. P-D-Galactosidase activity bound to the wh ite polystyrene beads was assayed by fluorometry at 30 degrees C for 2 hr. The detection limit of p24 antigen (0.1 amol/tube and 10 amor (0. 24 pg)/ml of serum) was equal to that obtained when the formation, tra pping, and transferring of the immune complex were performed for 4, 16 , and 3 hr, respectively, by incubation without shaking. Namely, the p eriod of time required for the immune complex transfer enzyme immunoas say of p24 antigen was markedly shortened (25.5-7 hr) without loss of the sensitivity. By the improved immune complex transfer enzyme immuno assay, p24 antigen was detected 12-20 days earlier than the detection of antibodies to H-V-I, i.e., seroconversion by the conventional ELISA . (C) 1998 Wiley-Liss, Inc.