Immunochemical studies of estrogen receptors.
Fusion of splenic lymphocytes from Lewis rats, immunized with affinity-purified estrogen receptor from the cytosol of MCF-7 human breast cancer cells, with two different mouse myeloma lines, has provided 13 monoclonal hybridoma lines secreting antiestrophilin antibodies, each of which (with one possible exception) recognizes a different antigenic determinant in the human receptor molecule. Of this library of monoclonal antibodies, some react with estrophilin from all sources tested, some react with mammalian but not avian receptors, whereas one preparation appears specific for estrophilin from primate sources. By proteolytic digestion under controlled conditions with mercury-deactivated papain, chymotrypsin, and trypsin, respectively, it is possible to remove sequentially the determinants recognized by one, two or three of the monoclonal antibodies, leaving the epitopes for the six remaining antibodies investigated on the steroid-binding portion of the receptor. The proteolytic fragment containing the epitope most readily removed (by mercuripapain) also contains the DNA-binding domain of the activated receptor molecule. Immunocytochemical staining, using the peroxidase procedure with various monoclonal antibody preparations, of frozen sections of human breast cancer tissue, fixed in ethanol or in picric acid-formaldehyde reagent, shows clearly that the majority of the native receptor, which appears in the cytosol after tissue homogenization, is actually localized within the nuclear compartment in the intact cell. The immunocytochemical technique also permits the identification of mixed populations of receptor-containing and non-containing cells in human breast cancers.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001665:Binding Sites; D001943:Breast Neoplasms / Q000032:analysis; D002352:Carrier Proteins / Q000276:immunology; D003429:Cross Reactions; D003600:Cytosol / Q000032:analysis; D004247:DNA / Q000378:metabolism; D000939:Epitopes / Q000032:analysis; D005260:Female; D006801:Humans; D007124:Immunoenzyme Techniques; D051381:Rats; D011960:Receptors, Estrogen / Q000276:immunology*
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D000939:Epitopes; D011960:Receptors, Estrogen; C010140:estrophilin; D004247:DNA