The 293-F Ag85A elicits weaker immune responses than native Ag85A. Serum IgG reactivities of mice immunized with adjuvant alone, native Ag85A, or 293-F Ag85A against (A) native Ag85A, (B) 293-F Ag85A, or (C) gamma irradiated Mtb, determined by ELISA. Serum IgG titers were reported as the reciprocal dilution that results in an optical density of 0.5 at 450 nm. (D) Intact Mtb binding of immune sera analyzed by flow cytometry. Overlaid histograms represent data from four technical repeats. (E) Flow cytometry analysis for opsonophagocytic activities of immune sera coincubated with FITC-labeled Mtb and J774 mouse macrophages. Splenic mononuclear cells harvested from mice immunized with native Ag85A (F) or 293-F Ag85A (G) were assessed for their IL-2 production upon stimulation with native, 293-F, or N246Q Ag85A by ELISA. IL-2 concentrations were normalized to media. (H) Serum IgG reactivities of mice immunized with native Ag85A against native, 293-F, or N246Q Ag85A were determined in a dot blot analysis, and dots were quantified with ImageJ software. Data shown are from the same membrane with the same exposure time. Statistical significance was determined with the two-tailed Student’s t test. ****P < 0.0001; ***P < 0.001; **P < 0.01; ns, not significant.