Diluted whole blood assays

Diluted whole blood assays were conducted as previously described using MTBK_24820 as the M. tb Beijing/K-specific antigen.²⁷ The protein antigen was separately purified and mixed in a tube for whole blood assay experiments. PHA and M. tb purified protein derivatives (PPD) were used as positive controls for immunocompetence and reactivity to mycobacterial antigens, respectively. RPMI1640 medium was used as a negative control. Diluted blood was added into each well including antigens at 5 µg/mL. After 6 days of incubation at 37℃, the culture supernatant was harvested, and cytokine/chemokine responses were determined by multiplex bead array.