A microELISA for the quantitation of mouse monoclonal IgM in hybridoma culture supernatants

A microELISA has been set up which is able to sensitively quantitate mouse monoclonal IgM in hybridoma culture supernatants in a suitable range of concentrations. This assay uses ion exchange chromatography-purified rabbit anti-mouse IgM polyclonal antibodies, both in coating capture step and conjugated to horseradish peroxidase in revealing step. It was possible to detect concanavalin A-Sepharose affinity-purified and gel filtration-highly purified mouse monoclonal IgM in the range 190 ng/mL to 12360 ng/mL (sensitivity 125.5 ng/mL). This assay showed a variation coefficient of 4.50% for intraplate repeatability and 7.50% for interplate reproducibility. When compared with an alternative ELISA test that uses a commercial goat anti-IgM-alkaline phosphatase conjugate a correlation coefficient r = 0.92 was found. With this assay, the quality control program of the authors’ hybridoma unit is completed, which was developed with their own resources.