ELISA for Antigen-Specific Immunoglobulin

ELISA for total and antigen-specific immunoglobulin was performed as previously described^{22 (link)}. Plates (Costar) were coated ON with 5 μg/ml anti-Ig antibodies or NP₂₀-BSA and NP₄-BSA (Biosearch Technologies) in PBS at 4°C. Plates were blocked with 1% BSA/PBS, serial dilutions of sera were applied and incubated at 20°C (room temperature) for 2–3 h. Subsequently, plates were washed (with PBS/0.05% Tween20) and incubated with alkaline phosphatase (AP)-conjugated anti-mouse Ig isotype-specific antibodies followed by pNPP substrate (Sigma-Aldrich) as detection reagents. Absorbance was measured at 405_nm with a microplate reader (Molecular Devices) and values were calculated according to Ig standards or to reference serum from wild-type mice immunized twice with NP₁₉-OVA (expressed in arbitrary units, AU). Ratios between NP_4- versus NP₂₀-binding antibodies were calculated as an estimate for affinity maturation. Fecal IgA ELISA were performed as previously described^{50 (link)}: fresh stools were collected, dissolved in PBS (100 μl/0.01 g), centrifuged, and IgA concentrations determined by ELISA on the recovered supernatants. Antibodies and immunoglobulin standards were purchased through Southern Biotech.