The PBMCs from 9 pediatric cross-neutralizers were pooled and an anti-HIV-1 pediatric scFv phage library was constructed, as described in the methodology section. The variable genes for heavy chain (VH) and light chains kappa and lambda (VLκ and VLλ) were successfully amplified showing desired 400 bp bands, except VLκ3 and VLλ2 (Figures S1A–C in Supplementary Material) followed by successful construction of scFv gene (800 bp) by pull-through PCR (Figure S1D in Supplementary Material). Next, colony PCR of 20 random clones from the unselected phage library (before biopanning) showed two clones with no scFv inserts (Figure S2 in Supplementary Material) thus, confirming 90% ligation efficiency. DNA sequencing of 18 colony PCR positive clones confirmed that sequence of all scFv inserts were distinct (Table S1 in Supplementary Material). On the basis of these results, it is inferred that, diversity of the constructed phage library was 90% with 9 × 108 diverse clones. Biopanning of the library was performed using RSC3 protein coated on magnetic beads. The integrity of coated RSC3 protein on the beads was checked by flow cytometry by using anti-CD4bs bnAb VRC01 and more than 99% beads were found coated with RSC3 core protein (Figure S3 in Supplementary Material). After five rounds of biopanning, 150 scFv clones were randomly selected, their binding specificity was checked and eight scFv clones were identified which showed highest binding with the RSC3 protein and a decrease in binding of at least twofold with RSC3 Δ 371I/P363N protein, demonstrating CD4bs specificity (Figure 2).