Soluble phage ELISA was performed as described previously (43, 49, 50), with few modifications. The ELISA plates were coated with 100 µl of RSC3 core and its mutant Δ371I/P363N at 2 µg/ml in 0.1 M NaHCO3 (pH 8.6) and incubated overnight at 4°C. The anti-CD4bs bnAbs VRC01 and b12 was used as positive controls; anti-CD4bs non-neutralizing antibody (non-nAb) b6, anti-V3 mAb 447-52D, anti-Parvovirus mAb 1418, and scFv against hepatitis virus HepB scFv were used as negative controls at 1 µg/ml concentration in these assays. The phage clones were taken as positive by ELISA, if the absorbance of RSC3 core coated wells was at least two times higher than RSC3 core mutant delta 37I/P363N and at least three times higher than binding of phage clones to unrelated antigens (unrelated peptide pool, BSA).