Ultrasensitive and rapid enzyme immunoassay (thin aqueous layer immune complex transfer enzyme immunoassay) for antibody IgG to HIV-1 p17 antigen.

The immune complex transfer enzyme immunoassay for antibody IgG to HIV-1 p17 antigen was performed in two different ways (the present immunoassays I and II) within shorter periods of time than previously reported. In the present (simultaneous) immunoassay I, antibody IgG to HIV-1 p17 antigen in 10 microL of serum samples was incubated simultaneously with 2,4-dinitrophenyl-maltose binding protein-recombinant p17(rp17) fusion protein and rp17-beta-D-galactosidase conjugate in a total volume of 22 microL for 10 min to form the immune complex comprising the three components. The reaction mixture was incubated with a polystyrene bead of 6.35 mm in diameter coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG for 5 min in a styrol test tube (13.3 x 54 mm and 2.1 g) to trap the immune complex. After washing, the polystyrene bead was incubated with 30 microL of epsilonN-2,4-dinitrophenyl-L-lysine solution in a polystyrene tube (12 x 75 mm) coated with affinity-purified (antihuman IgG gamma-chain) IgG for 10 min to transfer the immune complex. In the present (sequential) immunoassay 11, a polystyrene bead of 6.35 mm in diameter coated successively with affinity-purified (anti-2,4-dinitrophenyl group) IgG and 2,4-dinitrophenyl-maltose binding protein-rp17 fusion protein was incubated in a styrol test tube (13.3 x 54 mm and 2.1 g) sequentially with antibody IgG to HIV-1 p17 antigen in 10 microL of serum samples in a total volume of 16 microL for 5 min and subsequently with rp17-beta-D-galactosidase conjugate in a volume of 10 microL for 5 and 10 min. The immune complex formed on the polystyrene bead was transferred to a polystyrene tube coated with affinity-purified (antihuman IgG gamma-chain) IgG for 5 and 10 min in the same way as in the present immunoassay I. During the incubations, the styrol test tubes containing the polystyrene beads and reaction mixtures were shaken, and the polystyrene test tubes were rotated with shaking, so that the polystyrene beads were rotated randomly, and small drops (16 to 30 microL) of the reaction mixtures evenly contacted all parts of the solid phase surfaces during the incubations, though only small parts of the solid phase surfaces were contacted at one time. The intent was to continuously mix thin aqueous layers of the reaction mixtures covering the solid phase surfaces with the rest of the reaction mixtures. (Therefore, these immunoassays were called thin aqueous layer immunoassays.) beta-D-Galactosidase activity bound to the polystyrene tubes was assayed by fluorometry for 30 and 60 min. The present immunoassays I and II, in which only 15 to 25 min were used for the immunoreactions, were as sensitive if not more so than the previous immune complex transfer enzyme immunoassay requiring 150 min for the immunoreactions. In these earlier immunoreactions, the immune complex comprising the three components formed by 30 min incubation was trapped onto two polystyrene beads (3.2 mm in diameter) coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG for 60 min, and was then transferred to two polystyrene beads (3.2 mm in diameter) coated with affinity-purified (antihuman IgG y-chain) IgG for 60 min in a total volume of 150 microL. Furthermore, the present (sequential) immunoassay 11 (and probably I) could become approximately 10 times more sensitive by assaying bound beta-D-galactosidase activity for a longer period of time (10 h), since beta-D-galactosidase activity, bound nonspecifically in the presence of serum samples from HIV-1 seronegative subjects, was considerably low.