The 2B10 and 2E4 scFvs were next tested for their ability to bind native-like HIV-1 trimeric envelope glycoprotein BG505:SOSIP.664 D-7324, and we observed high binding of both the scFvs with the trimeric antigen, which is known to bind only to bnAbs with high affinity and negligible binding with non-NAbs (Figure 4A). Further, binding analysis of both scFv monoclonals with recombinant HIV-1 96ZM651 subtype C protein confirmed their ability to bind with subtype C viral envelopes (Figure 4B). The affinity dissociation constant (KD) of both 2B10 scFv-BG505:SOSIP.664 gp140 complex and 2E4 scFv-BG505:SOSIP.664 gp140 complex was determined by ELISA and saturation binding curve was plotted through non-linear regression analysis (Figure 5). The Kd value for 2B10 scFv is 9.2 ± 1.4 × 10−8 M (R2 = 0.9709) and for 2E4 scFv is 1.4 ± 0.2 × 10−7 M (R2 = 0.9662). In binding analysis experiments, HepB, a scFv directed against hepatitis B antigen (54), was used as negative control and anti-CD4bs bnAb VRC01 was used as positive control.