Quantifying Serum Antibody Levels

Antibody titers of SRBC-specific Ig and total Ig in serum were measured by ELISA, similar to previously reported [26 (link)]. For anti-SRBC Ab analysis, 96 well Nunc-Immuno plates were coated with SRBC membrane protein overnight at 4°C. For anti-mouse IgA, anti-mouse total IgG or anti-mouse IgM Abs, directly peroxidase-conjugated secondary Abs (Sigma-Aldrich) were used. For analysis of IgG1 (A85-1, BD Biosciences), IgG2b (R12-3, BD Biosciences), IgG2c (Abcam) and IgG3 (R40-82, BD Biosciences), biotin-labeled secondary Abs were used along with Avidin-HRP (BD Biosciences). For total Ig isotype analysis, 96 well Nunc-Immuno plates were coated with anti-Ig kappa light chain Ab (187.1, BD Biosciences) diluted in PBS overnight at 4°C. Wells were blocked with 10% FCS and diluted serum was added and incubated at room temperature for 2 h.