PRODUCTION OF ENGINEERED IGM-BINDING SINGLE-CHAIN ANTIBODIES IN ESCHERICHIA-COLI

Two single-chain antibodies were engineered and tested as novel binding proteins with specificity for immunoglobulin M. Genes for the two single-chain Fv proteins were assembled from the variable light chain cDNA and variable heavy chain cDNA of monoclonal antibodies DA4.4 and Bet 2, which specifically bind human ISM and mouse IgM, respectively. Both single-chain Fv proteins were designed with a 14-amino acid linker which bridged the variable light chain and variable heavy chain domains. The two proteins were expressed in Escherichia coli, purified and assayed for IgM-binding activity. Both proteins demonstrate a binding specificity for their corresponding IgM which is similar to the monoclonal antibodies from which they were derived. These small ISM-binding proteins may have applications in the investigation of the immune response and in the detection and purification of monoclonal antibodies, cell-associated antibodies, and IgM from serum.