Goat anti mouse igg2a ap

A standard indirect enzyme-linked immunosorbent assay (ELISA) protocol was used for detection of the antigen-specific IgG in the immunized mouse sera. Briefly, Nunc MaxiSorp 96-well plates were coated with 5 μg/ml of purified His₆-ESAT-6-Pet-BP protein or S. Typhimurium LPS (Sigma) in 0.1 M carbonate buffer, pH 9.6 (overnight, 4°C). The plates were washed three times in wash buffer (PBS, 0.05% Tween 20), incubated with PBS-1% bovine serum albumin (BSA) for 1 h at 37°C, and washed as described above. The plates were incubated for 1 h at 37°C with 3-fold serial dilutions of mouse sera in dilution buffer (PBS, 0.05% Tween 20, 1% BSA), followed by three washes with wash buffer. The plates were incubated with a relevant AP-conjugated secondary antibody in dilution buffer for 1 h at 37°C (goat anti-mouse IgG–AP [1/10,000 dilution; Sigma] for protein- and LPS-specific total IgG or goat anti-mouse IgG1–AP, goat anti-mouse IgG2a–AP, or goat anti-mouse IgG2b–AP [1/2,000; Southern Biotech] for protein-specific IgG isotypes). The plates were washed as described above and developed using SigmaFAST p-nitrophenyl phosphate substrate (Sigma); 405-nm readings were taken at regular intervals by use of a Labsystems Multiscan MS plate reader. The IgG titers were estimated using the specified threshold OD method.