Monoclonal antibodies to human estrogen receptor

Abstract

Extranuclear estrogen receptor protein (estrophilin) of MCF-7 human breast cancer cells was purified by passage of the cytosol fraction of a cell homogenate through an affinity column of estradiol linked to Sepharose by a substituted di-n-propyl sulfide bridge in the 17 alpha position. Elution with 50 micro M [3H]estradiol in 10% (vol/vol) dimethyl formamide/0.5 M sodium thiocyanate gave 40% recovery of [3H]estradiol-estrophilin showing 14% of the specific radioactivity expected for the pure complex. Serum from a Lewis rat immunized with this partially purified estradiol-receptor complex contained antiestrophilin antibodies that reacted not only with nuclear and extranuclear estradiol-receptor complexes from MCF-7 cells but also with estrophilin from rat, calf, and monkey uterus, hen oviduct, and human breast cancers. Splenic lymphocytes from the immunized rat were fused with cells of two different mouse myeloma lines (P3-X63-Ag8 and Sp2/0-Ag14) to yield hybridoma cultures, 2% of which produced antibodies to estrophilin. After cloning by limiting dilution, three hybridoma lines secreting antiestrophilin were expanded in suspension culture and as ascites tumors in athymic mice to provide substantial quantities of monoclonal antibodies that recognize mammalian but not avian estrophilin and that show different degrees of reactivity with receptor from nonprimate sources. By growing the clone from Sp2/0 in the presence of [35S]methionine, radiolabeled monoclonal IgG has been prepared. These monoclonal antibodies should prove useful in the study of estrogen receptors of human reproductive tissues, in particular for the radioimmunochemical assay and immunocytochemical localization of receptors in breast cancers.

PIP: MCF-7 human breast cancer cell estrophilin was purified by passing the cytosol fraction of a cell homogenate through an affinity column of estradiol linked to Sepharose by a substituted di-n-propyl sulfide bridge in the 17 alpha-position. When eluted with 50 mcM radiolabeled estradiol in 10% dimethyl formamide-.5 M sodium thiocynate, a 40% recovery of the radiolabeled estradiol-estrophilin occured along with 14% of the specific radio activity expected from the pure complex. Lewis rats immunized with this partially purified estradiol receptor complex had serum that contained antiestrophilin antibodies that reacted with nuclear and extranuclear receptor complexs from MCF-7 cells and with the estrophilin from rat, calf, and monkey uterus, hen oviduct, and other human breast cancers. Hybridoma cultures of 2 different mouse myeloma lines (P3-X63-Ag8 and Sp2/0-Ag14) yielded antibodies to estrophilin 2% of the time. After cloning, 3 hybridoma lines that secreted antiestrophilin were expanded to provide quantities of monoclonal antibodies that recognize mammalian but not avian estrophilin and that show different degrees of reactivity with receptor from nonprimate sources. by growing the clone from Sp2/0 in the presence of radiolabeled methionine, radiolabeled monoclonal immunoglobulin G was prepared and should be useful in the study of estrogen receptors in human reproductive tissues.