The neutralization efficiency of the purified scFv monoclonals was tested with HIV-1 pseudoviruses and primary isolates as described previously (43, 44, 51). Briefly, HIV-1 envelope expression plasmids from a standard panel for subtypes A, B, C, AD, CD, and CRF2 AG were obtained from the NIH AIDS Reagent Program (NIH ARP). Pseudoviruses were generated by co-transfecting HEK293T cells with a plasmid expressing envelope and envelope deficient HIV-1 backbone vector pSG3Δenv as described earlier (51), and primary isolates were derived from PBMCs of HIV-1 infected Indian donors as reported earlier (33, 45, 52). Next, starting with 100 µg/ml of scFv concentration, three-fold serial dilutions were made in 10% DMEM [Dulbecco Modified Eagles’s Medium, HyClone, GE Healthcare supplemented with 10% FBS (Fetal Bovine Serum) and 1% PenStrep (Penicillin and Streptomycin) antibiotics] and incubated with 200 TCID of the pseudoviruses/primary isolates for 1 h at 37°C. Then, 104 cells/well TZM-bl cells were added to the scFv and virus complex along with 25 µg/ml DEAE Dextran (Sigma) (Indinavir (1 mM) was additionally added with primary isolates). After 48 h, 150 µl volume of culture medium was removed and 50 µl of Bright-Glo Luciferase assay reagent (Promega) was added to TZM-bl cells followed by 2-min incubation at room temperature to allow complete cell lysis. Then, luciferase activity was read in 96-well black plate (Costar) using Synergy 2 Multi-Mode Reader (BioTek). The IC50 values for the scFvs were calculated by a dose-response curve fit with non-linear function, with the help of Graph Pad prism software 5.0. Each experiment was repeated twice and performed in duplicates and IC50 titers were calculated.