Figure 3. Depletion and competition of plasma antibodies from cross-neutralizing plasma.

The depletion and competition of antibodies from three cross-neutralizing plasma (CNP) (AIIMS206, AIIMS239 and AIIMS249) was carried out with V3 (35 mer), MPER (25 mer) and IDR (19 mer) specific peptides. The antibodies from CNP were depleted by passing plasma samples (at 1∶30 dilution) over antigen coated ELISA plates (peptide coating concentration, 10 µg/ml) six times and the percent depletion of antibodies to each region was calculated as: percent depletion = 100−[100×(OD₄₀₅ at the last passage/OD₄₀₅ at the first passage)]. The percentage antibody depletion for CNP against each peptide is shown (A). As control, mock depletion of CNP was carried out on the uncoated plates which showed minimal effect on antibody depletion (A). For competition, CNP were preincubated 30 minutes with the same peptides at a final peptide concentration of 20 µg/ml. Both depleted (designated as ‘D’) and competed (designated as ‘+’) plasma were subsequently tested for neutralization with all eight viruses (data shown in Table 3). To validate the competition assay, we tested 447-52D an anti-V3 antibody known to neutralize SF162 (a tier 1 sensitive subtype-B virus) along with two peptides corresponding to consensus-C and B V3 sequences (at a final concentration, 10 µg/ml) (B).