Hrp conjugated anti mouse fc antibodies

ELISA was used to evaluate the humoral immune response elicited by OmpAVac vaccination. In brief, flat-bottomed 96-well ELISA plates were coated with purified OmpA (200 ng/well) or synthesized peptides encoding loop1, loop2, loop3, and loop4 (600 ng/well) and incubated overnight at 4°C. Then, the antigen-coated plates were blocked with 200 μL of blocking buffer [PBS containing 0.05% Tween 20 and 2% BSA, pH 7.5 (PBST)] for 60 minutes at 37°C. Serially diluted (two-fold) sera (100 μL) were then added to each well, followed by incubation for 60 min at 37°C. After rinsing three times with PBST, the plates were incubated with HRP-conjugated anti-mouse Fc antibodies (Abcam) for 1 h at 37°C. Finally, color was developed with TMB (Sigma), and the reaction was stopped by adding 0.1 M sulfuric acid. The optical density (OD) was determined at 450 nm. The subtype of anti-OmpAVac IgG was also determined via ELISA. The major difference in these assays was that the sera from OmpAVac-immunized mice were diluted 1:2000 and used as primary antibodies, while goat anti-mouse IgG1, IgG2a, and IgG2b mAbs (Abcam) were used as secondary antibodies. The remaining steps were the same as those described for determination of the titer of OmpAVac antibodies.

ELISA was used to evaluate the humoral immune response elicited by vaccination. In brief, 96-well ELISA plates were coated with purified Vo (3 µg/ml) and incubated overnight at 4 °C. Then, the antigen-coated plates were blocked with 200 µl of blocking buffer [PBS containing 0.01% Tween 20 and 2% BSA, pH 7.5 (PBST)] overnight at 4 °C. Serially diluted (twofold) sera (100 µl) were then added to each well, followed by incubation for 60 min at 37 °C. After three rinses with PBST, the plates were incubated with HRP-conjugated anti-mouse Fc antibodies (Abcam) for 45 min at 37 °C. Finally, the color was developed with TMB (Sigma), and the reaction was stopped by adding 0.1 M sulfuric acid. The optical density (OD) was determined at 450 nm. The subtype of anti-Vo IgG was also determined via ELISAs.