All antigens were coated on ELISA plates at a concentration of 2 µg/ml and incubated overnight at 4°C. Plates were blocked with 300 μl/well of 15% FBS in RPMI for 1.5 h, followed by addition of 100 μl/well of heat inactivated plasma at different dilutions (1:30, 1:100, 1:300, 1:1,000, 1:3,000, and 1:10,000) or anti-HIV-1 monoclonal antibodies (VRC01, b12, 447-52D and 2F5) and anti-Parvovirus mAb 1418, each at 10 µg/ml concentration and incubated for 1 h at 37°C. Each of the above steps was followed by washing with 1× PBS (Phosphate Buffered Saline) with 0.1% Tween 20. Next, 100 µl of alkaline phosphatase (AP)-conjugated anti-human IgG Fc antibody [1:2,000 diluted in 1× PBS with 0.05% Tween 20 (Southern Biotech)] was added to the plates and the immune complexes were reacted with AP substrate in 10% DAE buffer (1 mg/ml). Reaction was stopped by adding 6 N NaOH and plates were read at 410 nm. The reported Max50 value is the 50% maximal binding of plasma antibodies that showed saturation. Max50 values were calculated as described previously (45).