Antibodies to rabbit progesterone receptor: crossreaction with human receptor.

Logeat, Frédérique; Hai, M. T. Vu; Milgröm, Edwin

doi:10.1073/pnas.78.3.1426

Cited by 54 publications

(30 citation statements)

References 8 publications

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“…Receptor was purified as described (3). However, to concentrate the receptor and increase the purity a final purification step was added.…”

Section: Methodsmentioning

confidence: 99%

“…Nonspecific immunoprecipitation was measured by performing parallel incubations but with an unrelated monoclonal antibody. The concentration of steroid-receptor was measured by the dextran/charcoal adsorption method (10 (3). The affinity of these antibodies towards the different mammalian receptors in various ionic strength conditions has not yet been studied in detail.…”

Section: Methodsmentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

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

Logeat¹,

Hai²,

Fournier³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

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132

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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...

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“…Receptor was purified as described (3). However, to concentrate the receptor and increase the purity a final purification step was added.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

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

Logeat¹,

Hai²,

Fournier³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

132

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“…Gradients were centrifuged at 365,000 x g in a vertical rotor (Beckman, VTi 65) for 3 h. Fractions were collected from the bottom of the tube and submitted to radioactive counting. react with other intra-or extracellular steroid binders, as has previously been described by Logeat et al (20). Hyperimmune goat sera (anti-rabbit uteroglobin) were used as control (21).…”

Section: Sucrose Gradient Centrifugation and Immunoprecipitation Behamentioning

confidence: 99%

Oestrogen‐Insensitive Progestin Receptors in the Central Nervous System: Physicochemical and Immunoreactive Characteristics

Cerbón¹,

Martineíz²,

Pérez‐Palacios³

1989

J Neuroendocrinology

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The intracellular effects of progesterone in the central nervous system are exerted via two distinct receptors: The classical oestrogen-regulated progestin receptor and the non-oestrogen-inducible progestin receptor. To assess whether the oestrogeninsensitive receptor is related to the oestrogen-dependent receptor or whether it is a different binding macromolecule, its physicochemical and immunoreactive characteristics (immunoprecipitation with polyclonal anti-uterine progesterone receptor antisera) were studied in neural tissues of the female rat. The results disclosed that the dissociation constant (Kdr 0.4-0.5 x M), stereospecificity and sedimentation coefficient (7-8 S ) in linear sucrose gradients of the oestrogen-insensitive progestin receptor were identical to those reported for the oestrogen-regulated progestin receptor, although its saturation binding capacity was significantly lower. Results of in vifro nuclear acceptor assays revealed that the progestin receptor complexes from cerebellum and cerebral cortex, were able to specifically bind to cell nuclei preparations in a fashion similar to that observed with the uterine progestin receptor, although to a lesser extent. Interestingly, a similar nuclear uptake of receptor complexes was noticed when standardized cerebellum and uterus cytosol preparations with a similar receptor content were used. The anti-uterine progesterone receptor immunoglobulins used in the immunoprecipitation studies were able to specifically recognize the progestin receptor populations of the anterior pituitary and hypothalamus (oestrogen-regulated receptors) as well as those of the cerebellum and cerebral cortex (oestrogen-insensitive receptors). The results presented show that both the oestrogen-sensitive and the oestrogeninsensitive cytosol progestin receptors in brain bind the same progesterone-like molecules used as radioligands and also react with the same antibody when tested in an immunoprecipitation radioassay. The striking similarities found in binding kinetics, physicochemical characteristics and immunoreactive behaviour in the two progestin receptors studied demonstrated that both macromolecules belong to the same family of proteins in spite of their different sensitivity to oestrogens. The overall data seem to suggest a common origin of the two progestin receptor populations in brain but with different mechanisms of hormonal regulation.

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“…The isolation of polyclonal and monoclonal antibodies to estrogen (1,2), glucocorticoid (3)(4)(5), and progestin (6)(7)(8) receptors in recent years, however, has provided alternative means to study these hormone receptors. An antibody against androgen receptors, however, has not been produced, mainly because of the difficulty in obtaining androgen receptors in sufficiently pure form and in amounts needed for efficient immunization.…”

mentioning

confidence: 99%

Autoimmune anti-androgen-receptor antibodies in human serum.

Liao¹,

Witte²

1985

Proc. Natl. Acad. Sci. U.S.A.

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Circulating autoantibodies to human and rat androgen receptors are present at high titers in the blood sera of some patients with prostate diseases. The antibodies from some serum samples were associated with a purified IgG fraction and interacted with the 3.8S cytosolic androgen-receptor complexes of rat ventral prostate to form 9-to 12S units. Other serum samples, however, formed 14-to 19S units, suggesting that other immunoglobulins might be involved. In the presence of an anti-human immunoglobulin as a second antibody, the androgen-receptor-antibody complexes could be immunoprecipitated. The antibodies interacted with the nuclear and the cytosolic androgen-receptor complexes, either the DNA-binding or the nonbinding form, but not with receptors for estradiol, progestin, or dexamethasone from a variety of sources. Human testosterone/estradiol-binding globulin, rat epididymal androgen-binding protein, or rat prostate a-protein (a nonreceptor steroid-binding protein) also did not interact with the antibodies to form immunoprecipitates. About 37% of male and 3% of female serum samples screened had significant antibody titer. The chance ofrinding serum with a high titer is much better in males older than 66 years than in the younger males or females at all ages. The presence of the high-titer antibodies may make it possible to prepare monoclonal antibodies to androgen receptors without purification of the receptors for immunization.Steroid receptors are usually characterized by their ability to bind radioactive ligands. The isolation of polyclonal and monoclonal antibodies to estrogen (1, 2), glucocorticoid (3-5), and progestin (6-8) receptors in recent years, however, has provided alternative means to study these hormone receptors. An antibody against androgen receptors, however, has not been produced, mainly because of the difficulty in obtaining androgen receptors in sufficiently pure form and in amounts needed for efficient immunization. In this report, we show that anti-androgen receptor antibodies are present in high titers in blood sera of some male patients having clinical prostate problems. MATERIALS AND METHODSMaterials. 5a- [1,2,3,4,5,6,16,17-3H(N)

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Antibodies to rabbit progesterone receptor: crossreaction with human receptor.

Cited by 54 publications

References 8 publications

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

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

Oestrogen‐Insensitive Progestin Receptors in the Central Nervous System: Physicochemical and Immunoreactive Characteristics

Autoimmune anti-androgen-receptor antibodies in human serum.

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