Ultra leaf anti human ifn γ antibody

Whole blood was collected from healthy donors enrolled in the CUBS study (ethics approved by BREC# BE022/13 at UKZN). Informed consent was obtained from all participants. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by centrifugation on Ficoll-Paque (Sigma), resuspended in RPMI 1640 medium (Thermo Fisher) containing 10% Fetal Calf serum, 1% ampicillin and seeded at 2 × 10⁵ cells/well in 96 well plates. Cells were cocultured with Dynabeads Human T-Activator CD3/CD28 (Invitrogen) to stimulate T cells at 37 °C in 5% CO₂ for 48 h. Supernatants were recovered by centrifugation. IFN-γ was depleted from the supernatant with 5 μg/mL Ultra-LEAF anti-human IFN-γ antibody (Biolegend, clone B27).

Binding of IFN-γ to Mtb was assessed by mixing varying concentrations of IFN-γ with whole bacterial cells fixed in 4% PFA and incubating overnight at 4 °C. Bacteria were pelleted and resuspended in 0.1% Tween in PBS before seeding onto microtiter wells for 2 h at 37 °C. After washing, Ultra-LEAF anti-human IFN-γ antibody (Biolegend, clone B27) at 1 μg/mL was added to the wells which were incubated for a further 2 h at room temperature. Anti-mouse HRP at 1:5000 was then added for 1 h at room temperature. TMB solution (Sigma Aldrich) was used as a substrate and 1 M sulfuric acid as stop solution. Optical density was read at 450 nm measured with GloMax Discover microplate reader (Promega). For flow cytometry, after overnight incubation with IFN-γ, bacterial pellets were stained with human anti-IFN-γ Brilliant Violet 421 (Biolegend, clone 4 S.B3) or anti-TNF-α Alexa Fluor 700 (BD, clone MAb11) at 1:20 for 1 h at room temperature. Data was acquired using BD Aria Fusion cytometer and analyzed using FlowJo Software v.10.