Blotto

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.

Uterine tissues were pulverized on dry ice and total protein isolated using TPER (Thermo Fisher). Protein concentration was assessed using a Qubit protein assay (Thermo Fisher). Samples (5 μg) were loaded on a 10% TGX gel (Bio-Rad), run at 150 V, and transferred to PVDF membrane using the Trans-Blot Turbo transfer system (Bio-Rad). The blot was blocked in 5% Blotto (Thermo Fisher) in tris buffered saline plus 0.1% Tween-20 (TBS-T) for 1 h at RT. Rabbit monoclonal anti-OLFM4 (Cell Signaling Technology) was diluted to 0.6 μg/mL in 5% blocking solution and applied to the blot overnight at 4°C. The blot was washed 3 times for 15 min with TSB-T, and incubated with donkey anti-rabbit IgG diluted 1:25,000 in 1% blocking buffer for 1 h at RT. Following 3 washes in TBS-T for 15 min each, immunoreactive bands were visualized using Super Signal West Femto reagents (Thermo Fisher) following the manufacturer’s instructions. Images were captured using a ChemiDoc Touch Gel Doc system (Bio-Rad). Antibodies were stripped with Restore (Thermo Fisher) for 30 min at 37°C. Peroxidase conjugated mouse monoclonal anti-β-actin (Sigma) at a dilution of 1:10,000 in 5% blocking solution was applied for 1 h at RT and visualized as described above.

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.