ELISA of purified scFvs was done by using HIV-1 monomeric and trimeric envelope glycoproteins (HIV-1 96ZM651 gp120 monomer subtype C, BG505 SOSIP.664 D7324 trimeric antigen and HxBc2 gp120 and HxBc2 D368R mutant gp120 monomers) as described previously (2, 43). Briefly, all antigens were coated on ELISA plates at 2 µg/ml concentration, blocked with 15% FBS, 2% BSA in a RPMI media; threefold serial dilutions of scFvs and mAb controls (VRC01, b12, 447-52D, 2G12, 2F5 and 1418) were added followed by detection with 1:1,000 dilution of primary antibody (Anti-His tag for scFvs and HRP conjugated anti-human Fc antibody for mAb controls) in 2% milk phosphate-buffer saline (MPBS). For scFvs, 1:2,000 diluted anti-mouse HRP-conjugated secondary antibody in 2% MPBS was added. Each of the above steps was followed by washing the plate with 1× PBS with 0.1% Tween 20. Immediately after washing, 100 µl of TMB substrate (BioLegend) was added and incubated at RT till the color developed. Reaction was stopped by adding 8 N H2SO4 and the absorbance was read at 450 nm.