Quantifying NF-κB and TNF-α in LTBI and TB

A Total/Phopsho NF-κB p65 (Total/Phospho) Human InstantOne^™ ELISA Kit (Thermo Fisher Scientific), which is a sandwich ELISA-based kit, was used to measure the levels of total and phosphorylated NF-κB, as per the manufacturer’s instructions [30 (link)]. We used cell lysates of blood samples collected from individuals with LTBI (n = 10) and patients with active TB (n = 12) to measure the levels of total and phosphorylated NF-κB. The absorbance was read using an ELISA reader (Multiskan RC/MS/EX Microplate Reader, Thermo LabSystems, Thermo Fisher Scientific) at a wavelength of 450 nm.
To determine the levels of TNF-α, we used culture supernatants collected after blockade and stimulation with CFP + PMA. TNF-α levels were determined in samples from the patients’ samples (9 LTBI and 11 TB) using the BD OptEIA Human TNF Set Kit [28 (link)], as per the manufacturer’s instructions. The optical density (OD) was measured at 450 nm using an ELISA reader (Multiskan RC/MS/EX Microplate Reader, Thermo LabSystems, Thermo Fisher Scientific). The standard curve was calculated from the readings of different concentrations of recombinant cytokines provided in commercial kits (500 pg/mL, 250 pg/mL, 125 pg/mL, 65.5 pg/mL, 31.3 pg/mL, 15.6 pg/mL, 7.8 pg/mL, and 3.9 pg/mL). The actual concentrations were calculated from the standard curve (r² = 0.997) using the obtained OD values.