Affinity analysis of the scFvs was done by ELISA as described previously (43). Briefly, different dilutions of BG505:SOSIP.664 gp140 (10–800 nM) were incubated with fixed concentration of the scFvs (3.125 nM) for 16 h at 4°C. Next day, 100 µl of the equilibrated solution was incubated with BG505:SOSIP.664 gp140 coated ELISA plates (500 ng/well) for 20 min at RT to capture the free scFvs. Bound scFv was detected with primary anti-His tag antibody (raised in mouse, Sigma) at 1:1,000 dilution and with secondary anti-mouse HRP conjugated antibody (Sigma) at 1:2,000 dilution as described before. Saturation binding curve was plotted using non-linear regression method and KD values were calculated. Competition ELISA was performed as described in Ref. (13, 43) by using biotinylated VRC01 and BG505:SOSIP.664 gp140 antigen. All the ELISAs were repeated twice and data was analyzed by using GraphPad Prism software 5.0.