ELISAs to determine epitope specificities of scFv monoclonals. (A) Purified scFv monoclonals were checked for binding reactivity with HXB2 gp120 WT and its D368R mutant by ELISA. Here, anti-HIV-1 bnAbs VRC01 and b12 were used as positive controls and anti-V3 mAb 447-52D, anti-Parvovirus mAb 1418 with anti-hepatitis scFv HepB were used as negative controls. Mean binding titers were compared using unpaired t-test. P values < 0.05 were considered significant and shown by asterisk (*) symbol, (**) if p < 0.01 and (***) if p < 0.001. Here, ns is designated for “not significant.” (B) Competition ELISA of biotinylated VRC01 (at fixed concentration of 50 ng/ml) to BG505:SOSIP.664 D-7324 with decreasing concentrations of scFvs (2B10 and 2E4) and mAbs (2G12, VRC01). 2G12 bnAb was used as negative control, and the bnAb VRC01 was used as positive control for the assay. Dilution used for antibodies was 5 µg/ml to 0.1526 µg/ml.