Ag85c

Mtb fractions and recombinant Mtb proteins (CFP10, PstS1, Ag85A, Ag85B, Ag85C, MPT32 and ESAT6) were obtained through BEI Resources (Manassas, Virginia). Mtb fractions and antigens were dissolved in either DMSO or PBS and stored as aliquots at -20°C.

Ag85A (catalog number NR-14871; BEI Resources), Ag85B (our purified recombinant), or Ag85C (catalog number NR-14858; BEI Resources) was used to coat wells at 0.5 µg/well in phosphate-buffered saline (PBS) and incubated overnight at 4°C. The plates were washed 3 times with PBS (pH 7.4) with 0.05% Tween 20 and blocked with PBS containing 1.0% BSA for 1 h. A starting concentration of 5 µg/ml of mAb 710, 711, or 712 was serially diluted, and 200 µl/well was incubated at room temperature for 2 h. Plates were washed 5 times and incubated with goat anti-mouse IgG-HRP (MP Biomedicals) for 1 h at room temperature. Plates were washed 7 times and developed with the 3,3′,5,5′-tetramethylbenzidine (TMB) substrate according to the manufacturer’s instructions (BD). The reaction was stopped with 2 M sulfuric acid, and the absorbance was read at 450 nm with a Synergy H1 microplate reader (BioTek).