Pseudovirus Neutralization Assay Protocol

The neutralization sensitivities of pseudoviruses were measured following a previously described protocol [35 ]. Briefly, serial dilutions of MAbs, sCD4, or fusion inhibitors in DMEM with 10 % FCS were placed in 96-well cell culture plates in triplicate. After dilution, 200 TCID₅₀ of each respective pseudovirus suspension was added to each well. Each plate had triplicates of the respective viral control (without any inhibitor) and a cell control (without any virus). The virus-MAb mixture was incubated for 1 h (37 °C, 5 % CO₂). After incubation, 10⁴ TZM-bl cells in suspension with 30 µg/mL of DEAE-dextran were added to each well and incubated for 48 h (37 °C, 5 % CO₂). Finally, the cells were washed, lysed, and the firefly luciferase activity was measured using a Galacto-star system (Life Technologies, Carlsbad, CA, USA). The percent inhibition by MAb, sCD4, or fusion inhibitor was determined by comparing the RLU in the presence and absence of an inhibitor. Each assay was repeated at least three times and validated according to the pass/fail criteria for TZM-bl cell based neutralization assay described by Sarzotti-Kelsoe et al. [36 (link)].