Rabbit IgM ELISA

In this assay the IgM present in samples reacts with the anti-IgM antibodies, which have been adsorbed to the surface of polystyrene microtiter wells. After the removal of unbound proteins by washing, anti-IgM antibodies conjugated with horseradish peroxidase (HRP) are added. These enzyme-labeled antibodies form complexes with the previously bound IgM. Following another washing step, the bound enzyme is assayed by the addition of a chromogenic substrate, 3,3’,5,5’-tetramethylbenzidine (TMB). The quantity of bound enzyme varies directly with the concentration of IgM in the sample tested; thus, the absorbance, at 450 nm, is a measure of the concentration of IgM in the test sample. The quantity of IgM in the test sample can be interpolated from the standard curve constructed from the standards and corrected for sample dilution.