The SDS-PAGE and Western blot analysis of the purified scFv clones 2B10 and 2E4 confirmed that both scFv proteins were more than 95% pure and showed the presence 32 kDa scFv bands (Figure S4 in Supplementary Material). The purified 2B10 and 2E4 scFvs were tested further in ELISA binding assays using HXBc2 gp120 and its mutant D368R. Both the scFvs showed binding to HXBc2 gp120 protein, with no significant binding to its corresponding HXBc2 D368R mutant, as is expected of CD4bs directed Abs (Figure 3A). This was further confirmed by competition ELISA with biotinylated VRC01 (Figure 3B). Moreover, both scFv monoclonals did not show any binding with V3 and MPER peptides (data not shown). In addition, the neutralization activity of both the scFv monoclonals was assessed for their V1V2 and V3-glycan reactivity by using CAP256 WT and its V3-glycan N332A mutant pseudoviruses (Table 3) and 16055 WT and its V1/V2 mutants N156K, N160K, K169A, and K171A pseudoviruses (Table 4). We observed, neither of the two scFvs exhibit V1/V2 or V3-glycan reactivity.