MONOCLONAL-ANTIBODIES THAT RECOGNIZE THE NATIVE HUMAN THYROTROPIN RECEPTOR

Monoclonal antibodies have been produced that recognize the native hum an thyrotropin receptor by using a sensitive screening protocol based on flow cytofluorimetry combined with recombinant eukaryotic cells exp ressing high levels of the full-length functional receptor. The more s tandard screening method of ELISA preferentially selected antibodies t hat only reacted with the denatured receptor. Mice were immunized with recombinant receptor produced in either eukaryotic or prokaryotic sys tems; after screening and cloning, three stable hybridoma lines were e stablished. An IgM antibody (7B5) produced in response to the eukaryot ic material recognized only the native receptor (by flow cytofluorimet ry) and did not react with denatured material on ELISA or immunoblotti ng, suggesting that its epitope is conformational. In contrast, two Ig G1 antibodies (2C11 and 3B12) produced in response to the prokaryotic material recognized both native and denatured receptor (by flow cytofl uorimetry, immunoprecipitation and immunoblotting). The use of differe nt recombinant constructs in the immunoblotting procedure allowed the epitopes for both of the IgG1 antibodies to be assigned to the region 125-369. None of the antibodies stimulated production of cAMP by recom binant cells expressing the full-length functional receptor, but one o f the IgG1 antibodies (2C11) did inhibit binding of radiolabelled thyr otropin to these same cells. These antibodies, and others that can now be produced with this screening protocol, will help define the relati onship between structure and function of this important receptor.