Quantifying mAb Binding to HIV-1 Env

Titrating amounts of mAbs (100 µg mL⁻¹ in 4-fold titrations) were added to HIV-1 env-transfected 293T cells (as described above) 2 days post-transfection and incubated for 1 h at 4 °C in 1× PBS^{52 (link)}. After washing, cells were fixed with 2% para-formaldehyde (PolySciences) for 20 min at room temperature. Cells were then washed and stained with a 1:200 dilution of PE-conjugated goat anti-human IgG (Southern Biotech, #2040-09) for 1 h at room temperature. Binding was analyzed using flow cytometry by determining mean fluorescence intensities (MFIs) of PE⁺-stained cell populations. FlowJo v10.4 was used for data analysis and interpretation.