Screening of cross-neutralizing plasma (CNP) samples for the presence of anti-CD4 binding site (CD4bs) antibodies. (A) Nine CNP samples were tested for the presence of anti-CD4bs antibodies using CD4bs specific probes RSC3 core wild-type (WT) and its mutant Δ371I/P363N at 2 µg/ml. (B) CNP samples were further tested for the presence of anti-CD4bs antibodies using HXBc2 WT and its mutant HXBc2 D368R gp120. Reciprocal Max50 binding titers were calculated using the least square regression method. The ELISA was repeated twice in triplicates. HD was the plasma of one healthy seronegative donor used as negative control. Mean reciprocal Max50 binding titers were compared using unpaired t-test for each plasma sample for RSC3 core WT and its mutant or HXBc2 WT and its mutant D368R. P values <0.05 were considered significant and shown by asterisk (*) symbol, (**) if p < 0.01 and (***) if p < 0.001. (C) The binding of RSC3 core WT and its mutant Δ371I/P363N was checked with anti-CD4bs mAb VRC01 and b12 (positive controls) and anti-V3 mAb 447-52D, anti-MPER mAb 2F5 and anti-Parvovirus mAb 1418 were used as negative controls. (D) The binding of HXB2 gp120 WT and its D368R mutant was tested with mAbs.