The immune complex transfer enzyme immunoassay for HIV-1 p24 antigen was performed in three different ways (in the present immunoassays I, II, and III) within much shorter periods of time than previously reported. In the present (simultaneous) immunoassay I, p24 antigen was incubated simultaneously with 2,4-dinitrophenyl-biotinyl-bovine serum albumin-affinity-purified rabbit anti-p24 Fab' conjugate and monoclonal mouse anti-p24 Fab'-beta-D-galactosidase conjugate in a total volume of 19 microL for 15 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 to trap the immune complex. After washing, the polystyrene bead was incubated with 35 microL of epsilonN-2,4-dinitrophenyl-L-lysine for 15 min to elute the immune complex (the first eluate) and, after removing the first eluate, with an additional 35 microL of epsilonN-2,4-dinitrophenyl-L-lysine for 1 min (the second eluate). The first and second eluates were incubated with a polystyrene test tube (12 x 75 mm) coated with streptavidin for 15 min. In the present (sequential) immunoassay II, a polystyrene bead of 6.35 mm in diameter successively coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG and 2,4-dinitrophenyl-biotinyl-bovine serum albumin-affinity-purified rabbit anti-p24 Fab' conjugate was incubated with p24 antigen in a total volume of 20 microL for 5 min and subsequently with monoclonal mouse anti-p24 Fab'-beta-D-galactosidase conjugate in a volume of 5 microL for 20 min. The immune complex formed on the polystyrene bead was transferred to a polystyrene test tube coated with streptavidin as described above. In the present (sequential) immunoassay III, p24 antigen was incubated with monoclonal mouse anti-p24 Fab'-beta-D-galactosidase conjugate in a total volume of 19 microL for 10 min and with a polystyrene bead of 6.35 mm in diameter coated successively with affinity-purified (anti-2,4-dinitrophenyl group) IgG and 2,4-dinitrophenyl-biotinyl-bovine serum albumin-affinity-purified rabbit anti-p24 Fab' conjugate for 20 min. The immune complex formed on the polystyrene bead was transferred as described above. The incubations were performed at room temperature either by shaking the polystyrene beads (one/assay) and the reaction mixtures in styrol test tubes (13.3 x 54 mm and 2.1 g) so as to randomly rotate the polystyrene beads or by rotating the polystyrene test tubes (12 x 75 mm) containing the reaction mixtures, so that small drops (19 to 70 microL) of the reaction mixtures evenly contacted all parts of the solid phase surfaces during the incubations (although they contacted only small parts of the solid phase surfaces at a time) to continuously mix thin aqueous layers covering the solid phase surfaces with the rest of the reaction mixtures. (Therefore, these immunoassays are called thin aqueous layer immunoassays.) The detection limits of p24 antigen by 1 hr assay of bound beta-D-galactosidase activity in the present immunoassays I, II, and III were 0.1, 0.2 and 0.1 amol/assay, respectively, and were slightly higher than or equal to that by the previously reported immune complex transfer enzyme immunoassay, in which the immune complex was formed for 4 hr, was trapped for 16 hr, and was transferred for 3 hr followed by 1-hr assay of bound beta-D-galactosidase activity. By 20-hr assay of bound beta-D-galactosidase activity, the detection limit of p24 antigen was further lowered to 10 zmol/assay in the present (simultaneous) immunoassay I and to 3 zmol/assay in the present (sequential) immunoassay III. However, the nonspecific reaction(s) with serum samples from HIV-1 seronegative subjects hampered the improvement of the detection limit by 20-hr assay of bound beta-D-galactosidase activity.