Antigen-Specific IgG, IgG1, or IgG2a Determination by ELISA

Serum samples were obtained from peripheral blood withdrawn from each mouse on day 42 for analysis of antibody and cytokines (n=6/group). After coagulation (60 min, 37 °C), sera were collected by centrifugation (15 min, 3000 rpm) and stored at −80 °C until use. ELISA was used to assay the antibody titers against OMV or Bb whole-cell lysates in sera.¹⁹ OMV and the whole protein (10 μg/mL and 2 μg/mL) from Bb bacteria were dissolved in 0.05 M carbonate buffer, respectively. The resulting protein solution (100 μL/mL) was used to coat the 96-well plates (Corning) and incubated at 37 °C, overnight. Subsequently, the plates were washed four times with PBST, followed by blocking with 5% non-fat milk-PBS at 37 °C for 2 h. After three washes, 100 μL of the diluted serum sample (1:10) was added to each well. The plates were then incubated at 37 °C in the dark for 1 h. Next, horseradish peroxidase-conjugated goat anti-mouse IgG, IgG1, and IgG2a (Abcam) were added to each well, respectively, and incubated at 37 °C for 1 h. Finally, ELISA TMB (ready-to-use) (Multi Sciences) was added, and after incubation at 37 °C in the dark for 15 min, the reaction was stopped with 100 μL/well of stop reagent (0.5 M H₂SO₄). The absorbance was measured at 450 nm on a microplate reader.