Methods and materials for detection of estrophilin

#The present invention relates generally to antibodies for estrogen receptor protein and the in vitro detection and quantification of such protein through use of such antibodies. More specifically, the invention provides novel antibody preparations which have specific reactivity with estrogen recepetor and improved test methods and reagents for detection and quantification of receptor protein by means of immunological reactions.

#It is generally recognized that specific estrogen-binding proteins, called "estrogen receptors" or, generically, "estrophilin" are responsible for the uptake of estrogenic hormones by certain tissues. The hormones are believed to interact with extranuclear estrophilin present in hormone dependent or "target" cells, with the "activated" estrogen-receptor complex so formed being translocated to the cell nucleus where it binds to the chromatin and in some way enhances the ability of the nucleus to synthesize certain types of RNA.

#It has been determined that certain tissues, notably certain human breast cancer tissues, are estrogenic hormone "dependent" in the sense that systemic deprivation of supportive estrogen will result in regression of tissue growth and cell proliferation. As one example of this dependence, bilateral adrenalectomy can effect striking remission of advanced breast cancer in postmenopausal women and similar remissions are observed after hypophysectomy. Estrogen deprivation by surgical ablation of tissue responsible for estrogen production and/or endocrine additive therapy afford the most effective treatments presently available for advanced breast cancer. Unfortunately, less than one-half of the premenopausal patients and even a smaller fraction of postmenopausal patients respond to this type of therapy--indicating that breast cancer tissue is not always of the cellular type which is estrogenic hormone dependent. Consequently, it is significant to the prognosis and treatment of human breast cancer to be able to ascertain whether excised tumor tissue of a breast cancer patient is comprised predominantly of estrogen dependent cell types. On the basis of such information, a reasonable ablation response prediction may be made. Surgicial removal of estrogen producing glands for the purpose of estrogen deprivation may be restricted to those patients most likely to be helped by the procedure. Correlatively, other breast cancer patients can be spared the trauma of essentially useless surgery and may be placed immediately into alternative therapeutic programs such as radiation or chemotherapy.

#The estradiol-receptor protein in the form of an estradiol-receptor complex which is capable of inducing formation of an antibody having a specific reactivity with the estradiol-receptor protein in an immunological reaction was prepared according to a modification of the method of Gorell, et al., "Purification of Nuclear Estrogen Receptors" in Proceedings of the Fifth International Congress of Endocrinology, (Excerpta Medica Foundation, Amsterdam) Vol. 1, pp. 467-472 (1977). Unless otherwise noted, all procedures were carried out at 0.degree.-2.degree. C.

#A calf uterine homogenate in T.sub.10 buffer was centrifuged at 10,000.times.g to separate the cytosol from crude nuclear sediment. The cytosol was treated with 30 nM E* (5.7 Ci nmol.). One-fifth of the sediment was washed successively with K.sub.400 T.sub.10, pH 7.9, and T.sub.10, pH 7.5, and then resuspended in the cytosol. After incubation of the stirred mixture at 25.degree. C. for 60 minutes, the nuclear sediment was collected by centrifugation, washed with T.sub.10 buffer and extracted with K.sub.400 T.sub.10 buffer. The extracted receptor complex was precipitated with ammonium sulfate (30% of saturation) and redissolved in K.sub.400 T.sub.25, pH 7.5, (0.1 volume of the original extract) by gentle homogenization followed by stirring for 20 minutes. After clarification by centrifugation at 98,000.times.g for 60 minutes, the solution was subjected to gel filtration on Sephadex G-200 (Regular grade, Pharmacia) in K.sub.400 T.sub.25 buffer. The estrogen receptor complex (determined by radioactivity assay) separated from the bulk of other proteins (determined by optical density at 280 nm) which eluted in the void volume. The pooled receptor-containing fractions were concentrated first by ultrafiltration with XM-50 membrane (Amicon) and further by salt precipitation with ammonium sulfate (30% of saturation). The receptor complex was redissolved in K.sub.30 T.sub.10, pH 7.5, and subjected to polyacryamide gel electrophoresis using 0.7 centimeter diameter, 5% or 7% acryamide gel with 2.5% cross-linkage prepared in Tris-HCl buffer pH 8.9. The electrophoresis is carried out with a current of 3 milliamps per gel, using electrode buffers of Tris-glycine, pH 8.3, or Tris-borate, pH 8.6 The receptor protein was isolated by slicing the gels, extracting the pooled gel slices with 10 nM sodium phosphate buffer, pH 7.4, (0.1 ml per slice) and combining the fractions with the highest radioactivity (R.sub.f =0.45 for 7% gels; R.sub.f =0.60 for 5% gels). The pooled extract was dialyzed against K.sub.10 T.sub.10 (azide-free), pH 7.5, and lyophilized to dryness.

#The product has an isoelectric point of about 6.0, shows a single stained protein band on acrylamide gel electrophoresis, and analytical ultracentrifugation indicates a single ultraviolet absorbing macromolecular species. Elution from a calibrated Sephadex G-200 column indicates a Stokes radius of 36.5, corresponding to a molecular weight of about 66,000 daltons.