Monoclonal antibodies to human estrogen receptor.

Abstract

Extranuclear estrogen receptor protein (estrophilin) of MCF-7 human breast cancer celIs 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 17a position. Elution The preparation of specific antibodies to estrogen receptors has provided a means of recognizing receptor proteins other than by their binding to labeled steroid. Antibodies to calf uterine estrophilin,-generated in either rabbits (1-3) or a goat (3, 4), crossreact with estrogen receptors, but not with androgen or progesterone receptors, from a wide variety of animal species. The hormone specificity and crossreactivity of the rabbit and goat antiestrophilins make them attractive as probes for the study of receptor structure and function, but their use in immunochemical procedures for receptor purification, assay, and cellular localization is limited by the heterogeneity of these antibody preparations. Monoclonal antiestrophilin antibodies secreted by hybridoma cells, obtained by fusion of mouse myeloma cells with splenic lymphocytes from a Lewis rat immunized with calf uterine estrophilin, have been described (4, 5) but, like the immunoglobulin in the serum of the parent rat, they reacted specifically with calf estrophilin and thus are unsuitable for the study of receptors from species other than calf.This Preparation of Estradiol Affinity Adsorbent. 17a-Allylestradiol 3-acetate, prepared by the reaction of estrone with allylmagnesium chloride (9) followed by acetylation of the phenolic group (10), was converted to the side-chain epoxide by treatment with m-chloroperoxybenzoic acid (11). Reaction of the epoxide with reduced thiopropyl-Sepharose (2-hyAbbreviations: E*R, [3H]estradiol-estrophilin complex; i-Ig, immunoglobulin secreted by hybridomas derived from immunized rat; n-Ig, immunoglobulin from serum of nonimmunized rat; HAT, hypoxanthine/amethopterin/thymidine; P3, P3-X63-Ag8; Sp2/0, Sp2/0-Agl4. 5115The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Partial purification of estradiol receptor from Xenopus laevis liver and levels of receptor in relation to estradiol concentration.

“…50%o ammonium sulphate sometimes gave receptor with higher specific activity, but the yields were variable and always low. Affinity chromatography 3-O-acetylestradiol-17ac-propylene oxide was prepared and coupled to thiopropyl-Sepharose 6B (Pharmacia) as described by Greene et al (1980). For each column, 0.1 ml of derivatised gel was diluted with 0.3 ml of Bio-gel A-0.5m (Bio-Rad) and packed in 1 M KCI, 50 mM Tris-HCl pH 7.4.…”

“…The properties of the receptor (Westley and Knowland, 1978) and its behaviour both in adult frogs (Westley and Knowland, 1979) and during tadpole development as vitellogenin inducibility appears (May and Knowland, 1981) all suggest that the receptor is important in regulating vitellogenesis, but so far there have been no attempts to purify it. Here we have taken advantage of a recent improvement in affinity chromatography of estrogen receptors (Greene et al, 1980) to partially purify the receptor, and we have also studied the level of receptor in relation to estradiol concentrations.…”

Neurotensin‐lmmunoreactive Neurons in the Rat Medial Preoptic Area are Oestrogen‐Receptive

“…The production and specificity of both antibodies have been documented previously (see refs. 2,[18][19][20]. For single staining, the sections were incubated in diluted primary antibodies for 40 h at 4 "C. For the anti-oestrogen receptor antibody, affinity purified biotinylated sheep anti-rat immunoglobulins (Amersham; diluted 1/200) followed by steptavidin-biotin-peroxidase (Amersham; Ij200) served as immunolabels and for neurotensin-antibodies, goat anti-rabbit immunoglobulins (Biosys; Ij200) followed by peroxidase-anti-peroxidase (Dakopatts; 1/200) were used; they were each applied for 2 h. All antibodies were diluted in TBS containing 0.25% bovine serum albumin and 0.3% Triton X-100.…”

Immunophenotype of the lymphoid cell infiltrates in breast carcinomas of low oestrogen receptor content

“…ER immunohistology Cryostat sections were cut at 4-6pm and stained using an immunoperoxidase procedure as described elsewhere (Giri et al, 1987) with a monoclonal antibody (Abbott Laboratories Ltd.) to the hormone-binding unit of ER (Greene et al, 1980).…”