Quantifying Mouse Antibody Levels by ELISA

The levels of antibodies in mouse sera were determined by enzyme-linked immunosorbent assay (ELISA) as previously described [25 (link)]. In brief, the microtiter plates (Corning Costar, Cambridge, MA, USA) were coated overnight at 4 °C with 10 μg /ml STAg in 50 mM carbonate buffer pH 9.6 (100 μl per well). After three washes, the plates were blocked with 3 % Bovine Serum Albumin (BSA) for 2 h at 37 °C and subsequently incubated with the mouse sera diluted 1:10 in PBS for 1 h at 37 °C. HRP-conjugated goat anti-mouse IgA, IgM, IgE, IgG, IgG₁, or IgG_2a (SouthernBiotech, Birmingham, AL, USA) was used as the secondary antibody to detect bound antibodies. Finally, the immune complexes were developed by incubation with 3,3,5,5-tetramethylbenzidine (TMB) for 20 min. The reaction was stopped by adding 2 M H₂SO₄, and the absorbance was measured at 450 nm with an automated ELISA reader (Multiskan FC, Thermo scientific, Waltham, MA, USA). All samples were run in triplicate.