Bright glo luciferase reagent

Neutralization titers of monoclonal Abs were determined using a luciferase-based assay in TZM.bl cells as previously described.50 (link), 51 (link) Briefly, mAb samples were tested using a primary concentration of 25 μg/mL with 5-fold serial dilutions against a panel of 10 HIV-1 Env pseudoviruses that were selected for being either 3BNC117 sensitive/PGT121 resistant or 3BNC117 resistant/PGT121 sensitive. This allowed for the measurement of neutralizing activity of single Abs in samples containing a mixture of both 3BNC117 and PGT121. Antibody titrations were incubated with HIV-1 Env pseudoviruses for 1 h at 37°C, and TZM.bl cells were then added in growth media containing DEAE-dextran at a final concentration of 11 μg/mL. Assay plates were incubated for 48 h at 37°C, 5% CO₂, and luciferase reporter gene expression was measured using Bright-Glo luciferase reagent (Promega) and a Victor 3 luminometer (Perkin Elmer). Neutralization titers (50% and 80% inhibitory concentrations, IC50 and IC80, respectively) were calculated as the mAb concentration at which relative luciferase units (RLU) were reduced by 50% or 80% compared to RLU in virus control wells after subtraction of background RLU in cell control wells. All assays were performed in a laboratory meeting good current laboratory practice standards.

To assess viral replication, macrophages (2.5 × 10⁵ cells/well in 24-well plates) were incubated with VSV-G-pseudotyped HIV-luciferase reporter virus at a multiplicity of infection (MOI) of 0.1 for 4 h at 37°C. Cells were washed four to five times with PBS to remove unbound virus, and then cultured in growth medium. Following 48 h of culture, cells were treated with TLR ligands or vehicle, as indicated in the text and figure legends. After 18 h, the cells were washed twice with PBS and lysed in PBS-0.02% Triton X-100. Luciferase activity was measured using BrightGlo luciferase reagent (Promega) and an MSII luminometer.