HIV-1 Pseudovirus Neutralization Assay

TZM-bl assays were adapted from Wei et al., 2003 and Montefiori, 2005 [78 (link), 79 (link)]. HIV-1 pseudovirus stocks were generated by transfecting HEK-293T cells. For neutralization assays bNAbs were diluted to 20 µg/mL and a three-fold serial dilution in triplicates was performed in flat-bottom 96 well plates. Pseudovirus at a dilution translating into 20 × RLU of the background control were added to each well, except the cells-only control. After 1 h incubation, 10⁴ TZM-bl cells, containing DEAE dextran, were added to each well and plates were incubated (37 °C, 5% CO₂). After 48 h the supernatant was removed, and cells were washed with PBS prior to adding lysis buffer (Promega, cat. #A8261). The plate was kept at − 80 °C overnight to ensure complete virus inactivation. After thawing, the cell lysate was mixed 1:1 with Bright-Glo luciferase substrate (Promega Luciferase Assay System, cat. #E2610) in a black flat bottom 96 well plate. Luminescence was measured using a GloMax plate reader (96 Microplate Luminometer, Promega, USA). IC₅₀s were compared to published data available in the CATNAP database [34 (link)].