Mouse Serum ELISA for Antibody Detection

Mouse serum samples were collected for an enzyme-linked immunosorbent assay (ELISA), which was performed as previously described [23 (link)]. In brief, microtiter plate wells (Corning Incorporated, New York City, NY, USA) were coated with mHI_N2, MntC, mSEB or SpA5 (200 ng per well) in 0.05 M carbonate buffer (pH 9.5) overnight at 4 °C. The primary antibodies were diluted serum samples, and the secondary antibodies were HRP-conjugated goat anti-mouse IgG, anti-mouse IgG1, anti-mouse IgG2a or anti-mouse IgG2b (Sigma). The optical density was measured at 450 nm, and the titres were defined as the highest dilution that yielded an absorbance value of more than twice the value of the pre-immune serum.