The binding activity of anti-V3 mAbs against gp120 proteins and peptides (including the V3 overlapping peptides) were tested by enzyme-linked immunosorbent assay (ELISA) as described [18] . Briefly, ELISA plates were coated overnight with protein or peptide at 1.0 μg/ml, blocked with 2% bovine serum albumin (BSA) in PBS, and then incubated with mAbs at a concentration ranging from 10 to 0.00003 μg/ml (12 dilutions). The bound mAbs were detected by incubation with alkaline phosphatase-conjugated goat anti-human IgG (γ specific) (SouthernBiotech, Birmingham, AL) followed by adding substrate to develop color and the plates were read at 405 nm. The relative affinities of mAbs were determined by measuring the concentration of mAbs required for 50% maximal binding (Max50), defined when the binding curve reached the saturation level as described [70] .