ELISA for TcpA-Specific IgG Antibodies in Serum

Antibodies specific for TcpA in antisera against TCP-expressing V. cholerae strains were quantified by ELISA as previously described [37]. Briefly, ELISA plates were coated with 100 μl His-tagged TcpA-ET (5 μg/ml) or His-tagged TcpA-cla (2.5 μg/ml) per well. Serum was applied from 1:500 to 1:32,000 (2-fold serial dilutions). Following a wash step, 100 μl HRP-conjugated secondary goat anti-mouse IgG, diluted 1:5,000 was applied. Plates were read spectrophotometrically at 450 nm.