Recombinant ag85b

Mycobacteria-, Ag85B- and ESAT-6-specific antibodies in serum were measured by indirect enzyme-linked immunosorbent assay using Mtb H37Rv lysate (BEI Resources), recombinant Ag85B (BEI Resources), or recombinant ESAT-6 (BEI Resources) to coat and AP-labeled anti-mouse IgG, IgG1, and IgG2c for detection (SouthernBiotech).

96-well MaxiSorp polystyrene plates (NUNC, Thermo Fisher Scientific, Rochester, NY) were coated with 2 μg/mL recombinant Ag85B (BEI Resources) in 100 μL PBS and incubated at 4°C overnight. Plates were washed 4 times with PBS containing 0.05% Tween 20 (Sigma-Aldrich, wash buffer) and blocked with 200 μL 5% NFDM at 37°C for 2 hours, washed 4 times with wash buffer and serum samples serially-diluted in PBS/1% FCS (ELISA diluent), were added to wells and incubated at 37°C for 1 hour. Wells incubated with ELISA diluent or naïve mouse serum were used as negative controls. Plates were then washed 6 times and biotinylated goat anti-mouse IgG antibody (Southern Biotech, Birmingham, AL) diluted in ELISA diluent was added, and plates were incubated at room temperature and left for 45 minutes. Plates were washed again 6 times and 100 μL streptavidin-alkaline phosphatase (GE Healthcare UK Limited) diluted in ELISA diluent was added at room temperature for 30 minutes. Plates were subsequently washed 8 times and developed with 100 μL pNPP AP substrate (BioFx Laboratories, Owing Mills, MD) at room temperature for 15–25 minutes. Color development was stopped with 50 μL Stopping Solution (BioFx Laboratories) and plates were read at 405 nm on a Synergy HT Multi-Mode Microplate Reader (BioTek). Data are expressed as mean endpoint antibody titers ± SEM.