Hrp conjugated anti mouse igm

We developed an ELISA system to monitor the amount of recombinant antibodies in various samples. Maxisorp plates were coated with 100 μl of 0.5 μg/ml anti-mouse kappa light chain (Sigma-Aldrich, St. Louis, MO). Plates were blocked at RT for 3 hours with TBS containing 10% adult bovine serum (Sigma), 5% sucrose and 0.05% sodium azide, dried at RT for 2 hours, stored at 4 °C and used with 1 month. Antibody samples were diluted in TBS containing 2% adult bovine serum and 0.01% Tween 20. Diluted samples were incubated with the plates at RT for 1 hour and washed 4 times with TBS containing 0.05% Tween 20. The plates were incubated with 1:4,000 dilution of HRP-conjugated anti-mouse IgM (Santa Cruz Biotechnology, Santa Cruz, CA) or 1: 20,000 anti-mouse IgG (Nacalai Tesque, Kyoto, Japan) at RT for 1 hour. After washing, color was developed with TMB. Reactions were stopped with 0.5 M HCl and the optical density was read with plate reader at 450 nm. Antibody amounts were estimated with the built-in software of the plate reader using appropriate Ig as standard ranging from 3.125 to 200 ng/ml.