ELISA binding reactivity of 2B10 and 2E4 scFvs with HIV-1 monomeric and native-like trimeric envelope glycoproteins. ELISA was done to determine binding reactivity of the scFvs with (A) native-like HIV-1 BG505-SOSIP.664 D-7324 gp140 trimeric protein at 2 µg/ml. The anti-HIV-1 mAbs VRC01 and 2G12 were used as positive controls and anti-HIV-1 gp41 (MPER) 2F5, anti-V3 mAb 447-52D, anti-parvovirus mAb 1418, and anti-hepatitis scFv HepB were used as negative controls. (B) The binding analysis of scFvs with subtype C 96ZM651 gp120 monomer. The anti-HIV-1 gp120 mAbs VRC01 and 447-52D were used as positive controls and mAb 2F5, HepB scFv, and mAb 1418 were used as negative controls. Mean binding titers were compared with negative control using unpaired t-test. P values <0.05 were considered as significant, (**) if p < 0.01 and (***) if p < 0.001.