Monoclonal antibodies to rabbit progesterone receptor: crossreaction with other mammalian progesterone receptors.

Abstract

A mouse was immunized with purified rabbit uterine cytosolic progesterone receptor (specific activity: 3 nmol of steroid bound per mg of protein). After fusion of its spleen cells with Sp2-OAg myeloma cells, supernatants of 11 hybrid cultures were found to react in both an immunoenzymatic test and a double-immunoprecipitation test with the progesterone receptor. Clones were obtained from the five hybrid cells that gave the strongest response in both tests. Antibodies from cell culture supernatants and ascitic fluids were characterized. Three are of the IgGl and two of the IgG2a isotype. Their apparent affinity for the progesterone receptor was measured by immunoprecipitation in physiological salt conditions. The equilibrium dissociation constants were between 0.1 and 4 nM. All five monoclonal antibodies crossreacted with the rabbit nuclear receptor, the human cytosolic receptor, and other mammalian (rat, guinea pig) but not avian (chicken) cytosolic progesterone receptors. There was no interaction with the glucocorticoid receptor and corticosteroid binding globulin.The study of steroid hormone receptors has been hampered for many years by difficulties in the purification of these proteins and by the impossibility of using immunological tools for their detection and quantification. Initial progress in this field has been made for the estrogen receptor (1, 2). In the case of the progesterone receptor, preparation of polyclonal antibodies against mammalian receptors (3) and recently against avian receptors (4) has been reported. However for such antigens, which occur in low concentrations and are difficult to purify, the possibility remains of misinterpretations caused by the presence of antibodies directed against proteins contaminating the receptor preparation.To solve this problem we have undertaken the preparation of monoclonal antibodies against the rabbit progesterone receptor. MATERIALS AND METHODSPurification of the Rabbit Uterine Progesterone Receptor. Receptor was purified as described (3). However, to concentrate the receptor and increase the purity a final purification step was added. Receptor eluted from the hydroxyapatite column with 0.2 M sodium phosphate, pH 7.4/30% (vol/vol) glycerol buffer was diluted 1:3.2 in 1 mM sodium phosphate, pH 7.4/30% glycerol buffer. It was applied to a small (0.7-ml) calf thymus DNA-cellulose column. After washing with 10 mM Tris-HCl/1.5 mM EDTA, pH 7.4/30% glycerol buffer (10 ml) and with the same buffer but containing 0.1 M NaCl (10 ml) and finally 5 mM pH 7.4 sodium phosphate buffer (10 ml) the receptor was eluted in 0.8 ml of 5 mM.sodium phosphate/0.5 M NaCl buffer, pH 8.3. The specific activity of the receptor preparation that was used for immunization was 3 nmol of steroid bound per mg of protein. Receptor concentration was 750 pmol/ 0.8 ml.Immunization. A 3-month-old BALB/c mouse received subcutaneous injections of 375 pmol of receptor. The receptor solution was concentrated 2-fold by lyophilization and emulsified with an equal volume (0.2 ml) of complet...

Processing of the precursors of the human thyroid‐stimulating hormone receptor in various eukaryotic cells (human thyrocytes, transfected L cells and baculovirus‐infected insect cells)

“…The membrane pellet was then resuspended in the solubilization buffer: 20 mM Tris/HCl pH 7.5, containing 0.4M NaC1, 1 O m M EDTA, lOmM ATP, 5 mM N-ethylmaleimide (Sigma) and 1.2% Triton X-100 (Sigma) [20]. After centrifugation for 1 h at 1OOOOOXg the supernatant was rotated successively with 25 pl Affi-Gel 10 (Biorad) saturated with a non-specific monoclonal antibody (Mi60 [21]) and 25 pl Affi-Gel 10 saturated with the specific T3-365 monoclonal antibody. The Affi-Gel was then washed extensively as previously described [16] and the receptor was eluted [15].…”

Biochemical and Immunocytochemical Assessment of Neural Progestin Receptors Following Estradiol Treatments that Eliminate the Sex Difference in Progesterone‐Facilitated Lordosis in Guinea‐Pigs

“…In several species the PR has been hypothesized to contain two subunits, an A and B chain, both capable of binding ligand and DNA (34-36). The monoclonal antibodies used in these studies appear to bind sites on the B chain only: PR6, directed against chick oviduct PR (37) and mPR I1 directed against rabbit PR (38), which is thought to contain one B-like subunit (39). If males possessed a population of cells in the VLH and MPOA in which the synthesis of the B, but not the A, chain of the PR were responsive to a single injection of EB, these would appear as increased numbers of PR-IR cells detected with our ICC technique.…”

Specific binding of progesterone receptor to progesterone‐responsive elements does not require prior dimerization

“…The monoclonal antibody for the progesterone receptor, Let 126 [13], was added to the supernatant at a final concentration of 300 pg/ ml and incubated for 2 h at 4"C, with gentle agitation. The resulting comulexes were then incubated with an 80-fold expared in 10 mM Tris, pH 7 [14]. The pellets were solubilized in Laemmli buffer (62.6mM Tris, pH6.8, 2% SDS, 5% 2-mercaptoethanol) [15], before analysis by Western blotting, using the monoclonal antibody Mi60 specific for the progesterone receptor [13].…”