Monoclonal antibodies against a component related to soluble estrogen receptor

Mice were immunized with 1 to 3 micrograms of cytoplasmic estrogen receptor fragment purified from human myometrium by affinity chromatography. Two RE-antibody-secreting clones were detected from one fusion that were capable of precipitating cytosol RE. Monoclonal antibody D5 (subclass IgG1) reacts with an antigen that is related to RE from immunoprecipitation studies but which can be separated from the hormone binding unit. In the presence of anti-mouse serum, D5 precipitates labeled human cytoplasmic RE complexes from breast tumor, fibroid, myometrial, and endometrial preparations but does not react with nuclear RE from human endometrium or cytoplasmic RE from other species tested. Conversely, antibody C3 (Class IgM) precipitates human cytoplasmic RE and nuclear RE complexes as well as labeled cytoplasmic RE from rat and calf uterus and chick oviduct. Neither antibody reacts with progesterone receptor or androgen receptor from human breast tumor, SHBG from human plasma, or rat alpha-fetoprotein. With D5, steroid labeling of cytoplasmic RE at 25 degrees increased the RE immune complex precipitated. D5 precipitates molybdate-stabilized RE from myometrial cytosol when labeled at 25 degrees but not at 4 degrees. C5 precipitates molybdate-stabilized RE whether cytosol was steroid-labeled at 4 degrees or 25 degrees. For D5, optimal precipitation of RE from human breast tumor was observed when cytosol was steroid-labeled at 25 degrees in buffers of pH range 5 to 6. Immunochemical studies indicate that D5 is associated with a Mr 29,000 component in RE-positive cytosols. Electrofocusing and sucrose density gradient analysis confirmed that D5 antigen is a non-hormone-binding component related to cytosolic RE from breast tumor and myometrium.