Determination of serum antibody titers via ELISA

A solid-phase antibody capture ELISA was used to monitor antibody titer responses after each booster in vaccinated animals. ELISA plates were coated with COC-BSA conjugate, and a range of serial dilutions of antisera was added in triplicate. Biotin-labeled anti-mouse secondary antibody (Jackson Immunoresearch, West Grove, PA, USA), coupled to an OPD system (Sigma – Aldrich, St. Louis, MO, USA) as chromogenic substrate, was used to detect immunopositive signals. Antibody titers were initially defined as the inverse of the serum dilution that gave 50% of the maximal response.