Tmb elisa chromogenic substrate

MAb binding to AM assay was performed by BLI using the OctectRed system (ForteBio, Pall) (33 (link)). Briefly, purified capsular AM was biotinylated and immobilized onto streptavidin-coated sensors. The sensor was then dipped into wells containing 1 mAb at serial dilutions to create binding curves and calculate the K_D. The binding of AM mAbs to purified capsular AM and intact bacterial surface was further quantified by ELISA (33 (link)). Briefly, purified AM was coated on 96-well microtiter plates (Maxisorp, Thermo Fisher Scientific) at 10 μg/mL in 50 μL. For whole-cell ELISA, M. tuberculosis strains grown in media without detergent were coated on 96-well plates overnight and blocked with BLOTTO (Thermo Fisher Scientific) (33 (link)). Serially diluted mAbs were then added to the antigen- or bacteria-coated wells, and the bound mAb was probed with horseradish peroxidase–conjugated goat anti-human IgG Fc (2048-05, SouthernBiotech), followed by TMB-ELISA chromogenic substrate (Thermo Fisher Scientific). The reaction was stopped by adding an equal volume of 2N sulfuric acid (MilliporeSigma), and the optical densities (ODs) were measured at 450 nm subtracted by 540 nm. A human IgG1 mAb against a non–M. tuberculosis antigen was included as a negative control.

MAb binding to AM assay was performed by BLI using the OctectRed system (ForteBio, Pall) (33 (link)). Briefly, purified capsular AM was biotinylated and immobilized onto streptavidin-coated sensors. The sensor was then dipped into wells containing 1 mAb at serial dilutions to create binding curves and calculate the K_D. The binding of AM mAbs to purified capsular AM and intact bacterial surface was further quantified by ELISA (33 (link)). Briefly, purified AM was coated on 96-well microtiter plates (Maxisorp, Thermo Fisher Scientific) at 10 μg/mL in 50 μL. For whole-cell ELISA, M. tuberculosis strains grown in media without detergent were coated on 96-well plates overnight and blocked with BLOTTO (Thermo Fisher Scientific) (33 (link)). Serially diluted mAbs were then added to the antigen- or bacteria-coated wells, and the bound mAb was probed with horseradish peroxidase–conjugated goat anti-human IgG Fc (2048-05, SouthernBiotech), followed by TMB-ELISA chromogenic substrate (Thermo Fisher Scientific). The reaction was stopped by adding an equal volume of 2N sulfuric acid (MilliporeSigma), and the optical densities (ODs) were measured at 450 nm subtracted by 540 nm. A human IgG1 mAb against a non–M. tuberculosis antigen was included as a negative control.