ELISA for detection of secreted anti-HEL IgM

HEL Ag was dissolved in a carbonate buffer (pH 9.5) at 20μg/ml and coated on 96-well plates (Thermo scientific) for at least 12 hours at 4˚C. Coated plates were blocked with 200μl/well blocking buffer (2% BSA in PBS) for 2 hours at room temperature (RT). Culture media collected from stimulated B cells were first diluted at 1:5 with blocking buffer, then serially diluted to 1:15, 1:45, and 1:135. Diluted samples (60μl/well), in duplicate, were loaded onto plates and incubated at RT for 2 hours. Plates were washed with PBS-T. HEL-specific IgM was detected by HRP conjugated goat anti-mouse IgM. Plates were washed 6 times with PBS-T, and HRP substrate (1-Step Ultra TMB-ELISA, Thermo scientific) was added. Plates were incubated for 5-60 minutes to allow color development. The HRP substrate reaction was stopped with 2M H₂SO₄. OD value was read by Nanoquant infinite M200 (Switzerland) at wavelength 450nm. All plates were normalized to a reference serum sample. Culture medium serves as the negative control. The modified OD values for each sample were averaged between duplicate wells, subtracted by the OD of negative control and multiplied by the dilution factor to calculate the relative unit.