Isotype Determination of Rodent-Derived Monoclonal Antibodies Using Sandwich ELISA

Abstract

For research groups needing to isotype a significant number of antibodies on a fairly regular basis, the protocol outlined here provides a cost-effective option. In this simple sandwich ELISA, the monoclonal antibody is first captured with antibodies that discriminate between heavy-chain isotypes and then is detected with antibodies specific for each light chain. This combination ensures that free light chain secreted by hybridomas does not yield a false-positive result, because in a true positive both the capture and detecting antibodies bind the monoclonal antibody.