Figure 2.

Multivalent anti-HIV-1 nanobodies show increased binding towards HIV-1 Env trimers from global panel. (A) SDS-PAGE protein separation followed by coomassie blue staining of the purified anti-HIV-1 nanobody-constructs under reducing conditions. Molecular weight (MW) is indicated in kilodalton (kDa). (B) Average area under the curve (AUC) of binding curves of J3, 2E7 and 1F10 nanobody variants to SOSIP.v9.0 trimers from the global panel (CNE55, Ce1176_A3, 25710-2.43, CH119.10, BJOX002000.03.2, Ce7030 and 246-F3_C10) as determined by ELISA using OD₄₅₀. Average AUC of OD450 was determined by doing a definite integral between the start and end point using GraphPad. Friedman matched comparison (three groups) and Wilcoxon matched-pairs signed rank tests (two groups) was used to compare the groups with significance indicated as *p <0.05. Data shown are the average of three independent experiments. (C) Binding of nanobodies and nanobody-IgG1 constructs to Ce1176 as determined by ELISA. *The binding of the nanobody and nanobody-IgG1 constructs to the concerned epitopes was determined using an anti-VHH-HRP secondary antibody, starting concentration 500 nM.** The binding of the nanobody-IgG1 constructs to the concerned epitopes was determined using a Goat-anti-Human-HRP secondary antibody, starting concentration 50 nM. Kd values for BG505 SOSIP.664 as determined by BLI, based on the average association of different antibody concentrations. ND, not determined; NB, non-binding. Data are representative of at least two independent experiments.