ELISA plates (Nunc, MaxiSorb) were coated with gp120 (3 μg/ml in carbonate buffer 0.1 M NaHCO 3 , 0.1 M Na 2 CO 3 , pH 9.6). To each well of a 96-well plate 100 μl of the gp120 solution was added and the plates were sealed with adhesive foil (Sarstedt) and incubated at 8°C overnight. Subsequently 250 μl of a BSA solution (20 μg/ml PBS) was added to each well and the plates incubated for 1 h at room temperature. After blocking, the plates were washed 5-times with PBS containing 0.05% Tween 20 (PBST). Modified serum albumin was added to the wells in a total volume of 50 μl PBS. After incubation for 30 min. rCD4 or antibody was added to each well in a total volume of 50 μl PBS. The plates were incubated for 1 h at room temperature and then washed 5-times with PBST. To detect gp120-bound rCD4 we used the anti-CD4 mouse monoclonal antibody NCL-CD4-1F6 (Novocastra). Bound anti-CD4 antibody was detected by a secondary anti-mouse HRP-antibody conjugate diluted 1:500 in PBS (BioRad). Bound gp120-specific monoclonal antibody was monitored directly using the anti-mouse-HRPantibody conjugate (BioRad). After binding of the secondary antibody, the plates were washed 5 times with PBST and 5 times with PBS. To each well 100 μl of a commercially available one-component ready to use solution of 3,3',5,5'-tetramethylbenzidine (TMB, Sigma Aldrich) was added, the reaction stopped with 1 M H 2 SO 4 , and the plate read at 450 nm.