Characterization of site-directed monoclonal and polyclonal antipeptide antibodies to human estrogen receptor
Al-Fadhli, Suad M.
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Monoclonal and polyclonal antibodies were developed against a synthetic peptide derived from a sequence in the A/B transactivation domain (residues 140-154) of the human estrogen receptor (hER). These antibodies were characterized with respect to site specificity, receptor specificity and species specificity and ability to bind to the van ous receptor forms. The antibodies were site specific since binding to estrogen receptor (ER) was displaced with the free peptide. The antibodies did not recognize progestrone, and androgen receptors. The antibodies cross-reacted with the ER from calf, rat and mouse uteri and human breast tumors, suggesting that the epitope for these monoclonal antibodies (MAbs) is conserved among vanous spectes. The interaction of the antibody with the functional estrogen receptor has been characterized by sucrose density gradient analysis. The MAbs did not recognize the molybdate-stabilized, untransformed, oligomeric (8S), suggesting that this epitope may be masked by interaction with heat shock proteins or conformationaly inaccessible. The antibodies reacted with the activated (4S) and the transformed (5S) forms of the ER, suggesting that the epitope is accessible in the 4S and the 5S forms of the ER and that dimerization of the 4S ER into 5S ER does not interfere with the epitope accessibility. Immunoblot analysis using polyclonal and monoclonal antibodies demonstrated cross-reactivity with a 55 kDa nuclear protein (NP55). This protein was detected only in the nuclear-KCI extracts but not the cytosolic extracts . It lacked estradiol binding activity, and is expressed in steroid-hormone target tissues from different species. This protein was also detected in some ER positive human breast tumors but was not detected in ER negative tumors. This protein (NP55) was not detected with polyclonal or monoclonal anti bodies developed against different regions of the ER, indicating that it is not a proteolyzed form of the ER. The availability of site-directed antibodies to ER-functional domains represent a valuable tool, particularly, m studying ER in its different forms, and in the analyses of the structural integrity of human breast tumor estrogen receptors. The observation that one of these antibodies recognizes a umque nuclear protein may prove useful to identify this ER-related protein.
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