Figure 5

Binding of sCD4 to CAP treated and untreated gp120. Recombinant gp120 IIIB (5 μg in 400 μl of 0.1 M sodium acetate buffer pH 7.0) was treated with CAP (5 mg/ml) for 5 min at 37°C and then cooled to 0°C. CAP was omitted in control experiments. BSA was added to a final concentration of 25 μg/ml and the samples were filtered using a 2 ml Centricon centrifugal ultrafiltration device with a M_w cutoff of 100,000 and centrifuged at 3,500 × g for 30 min. gp120 retained on the filters was washed with PBS, resuspended in PBS, and serially diluted 5-fold. The diluted samples (corresponding to gp120 quantities indicated on the abscissa) were used to coat wells of 96-well polystyrene plates. Binding to the wells of biotin labeled sCD4 was detected from subsequent binding of HRP-streptavidin.