Goat anti mouse igm μ chain

ELISA for total IgM and IgG in mice serum were performed by sandwich ELISA. 96-well plates were coated with goat anti–mouse IgG (H+L) antibody (1.3 µg/ml; Dianova) followed by blocking with 1% BSA. Sequentially diluted mouse sera (1:1,000–1:150,000) were applied to the plates. Plates were washed and Ig binding was detected by HRP-conjugated antibodies: goat anti–mouse IgM μ-chain (EMD Millipore) and goat anti–mouse IgG γ-chain (EMD Millipore). ELISA for Ova-specific antibodies in mouse serum was performed by indirect ELISA. Plates were coated with Ova (5 µg/ml in 0.1 M NaHCO₃, pH 8.2) followed by blocking with 1% BSA. Diluted serum samples (1:4,000) were applied on the plate and detected by HRP-conjugated antibodies: goat anti–mouse IgM μ-chain (EMD Millipore); goat anti–mouse IgG γ-chain (EMD Millipore); goat anti–mouse IgG1 γ1-chain (SouthernBiotech); and rabbit anti–mouse IgG2b γ2b-chain (Invitrogen). Enzyme reaction was done with 3,3′,5,5′-Tetramethylbenzidine substrate (Sigma-Aldrich) and stopped with 0.5 M H₂SO₄, and absorbance was measured by plate reader (VICTOR3; Perkin Elmer) at 450 nm.