A standard ELISA was used to determine binding of mAbs to the panel of HIV-1 Envs. Briefly, ELISA plates were coated with each Env antigen, blocked with 10% FCS in PBS, incubated with human mAbs and washed. Bound mAbs were detected by incubation with AP-conjugated goat anti-human IgG (Southern Biotech). Plates were then washed, substrate (p-NPP, Sigma) was added and plates were read at 405 nm. The relative affinities of mAbs binding to respective coated antigens were determined with ELISA by measuring the concentration of each mAb required to achieve 50% maximal binding at saturation (K 50 ). The ability of mAbs to inhibit binding of sCD4 to gp120 or gp140 was evaluated by ELISA. Serial dilutions of mAbs were pre-incubated with gp120 (or gp140) and added to plates pre-coated with sCD4. After 1 h plates were washed and incubated with sheep polyclonal antibody D7324 (Aalto Bio-Reagents) followed by washing, incubation with AP-conjugated rabbit anti-sheep IgG antibody (Abcam, Cambridge, UK), extensive washing and detection as above.