Peptide-Specific Serum Antibody Detection

The experiments were performed using a modified version of the protocol described previously (14 (link)). Immulon 4HBX microtiter plates (ThermoFisher Scientific, Waltham, MA) were coated with 2 µg/ml of peptides overnight at 4°C and blocked with 1x PBS containing 0.1% Tween 20 with 3% BSA (blocking buffer) for 1 hour at room temperature. Mouse sera diluted 1 to 100 in blocking buffer were added to the plate and incubated for 2 hours. Secondary anti-mouse IgG in blocking buffer was added and incubated for 1hour. Then the substrate o-phenylenediamine (0.05%) (Sigma-Aldrich, St. Louis, MO) and 0.06% hydrogen peroxide in citrate-phosphate buffer, pH 5.0 (100 µl/well) was applied. Reaction was stopped after 15 minutes by the addition of 50 µl/well of 2.5 N sulfuric acid. Absorbance was read at 490 nm with a reference wavelength of 560 nm.