Production and characterization of an estrogen receptor beta subtype-specific mouse monoclonal antibody

An important step in differentiating the unique physiological roles of the alpha and beta forms of estrogen receptor is to determine the precise expre ssion pattern of each of these receptors. We report the generation and char acterization of a murine IgG1 monoclonal antibody (MAb), ER15.64A that is E R beta subtype-specific and capable of recognizing full-length human ER bet a as well as all of its known protein isoforms. ER15.64A, raised against a ER beta peptide (aa2-18)-keyhole limpet hemocyanine conjugate, reacted to t he immunizing peptide and the full-length E. coli expressed ER beta in ELIS A and BIAcore assays. It also immunostained nuclei of Sf9 insect cells that were infected with an ER beta -baculovirus. In Western analysis, ER15.64A recognized ER beta1 and ER beta2 proteins from a reticulocyte in vitro tran scription/translation preparation. This antibody did not crossreact with re combinant ER alpha in ELISA, BIAcore, immunocytochemistry, or Western blot analysis. The specificity of ER15.64A should make this antibody a useful to ol for monitoring expression of ER beta and its isoforms at the protein lev el and should aid in distinguishing the pattern of ER beta receptor express ion from that of ER alpha.