Journal of Immunological Methods, 144 (1991)87-91
87
© 1991 Elsevier Science Publishers B.V. All rights reserved 0022-1759/91/$03.50
JIM 07005
A rapid method for the quantitation of estrogen receptors in small amounts of tissue E z i o Bettini a n d A d r i a n a Maggi Milano Molecular Pharmacology Lab, Institute of Pharmacological Sciences, University of Milan, Milan, Italy
(Received 12 November 1990,revised received 20 May 1991, accepted 8 July 1991)
A rapid and highly reproducible protocol which permits the detection of quantities of estrogen receptor as low as 5 f m o l / m g protein is described. The separation of free and receptor-bound hormone is achieved by specific immunoprecipitation of the hormone-receptor complex. This procedure can be performed without perturbing the equilibrium of the binding reaction. Key words: Estrogen receptor; Steroid receptor quantitation; Estrogen receptor immuno-quantitation
Introduction The quantitation of soluble receptors such as steroid receptors has traditionally been hampered by the technical difficulties encountered in the separation of free and bound ligands. The various approaches adopted to date involve the adsorption of either the free ligand or the hormone-receptor complex to an insoluble matrix. Because of its inexpensive nature and ease of application, the method currently most widely utilized for the measurement of steroid receptors involves the adsorption of free steroids to dextran-coated charcoal. In this case, the quantitation is based on the measurement of the labeled hormone bound to its cognate receptor (Korenman et al., 1969; Mester et al., 1970; Sanborn et al., 1975). This method has two major limitations: (a) at low protein concentration the charcoal may Correspondence to: A. Maggi, Milano Molecular Pharmacology Lab, Institute of Pharmacological Sciences, University of Milan, Piazza Durante 11, 20131 Milan, Italy (Tel.: 39/2/2884567; Fax: 39/2/2820603).
also adsorb some of the hormone-receptor complex, (b) the charcoal may strip some of the steroid from the steroid-receptor complex. Both shortcomings can lead to an underestimation of the receptor concentration (Clark et al., 1988) and, in addition, the latter can alter the equilibrium of the reaction. Several other alternative methodologies have been described. These are based on the adsorption of receptor-steroid complex on charged surfaces such as hydroxylapatite (Erdos et al., 1970), D E A E cellulose filters (Santi et al., 1973) or protamine sulfate (Chamness et al., 1985). Although these methods are rapid and generally highly reproducible they suffer from very high levels of non-specific binding and consequent impairment of the measurement of low receptor concentrations. The application of the above-mentioned methodologies is therefore restricted to tissues rich in steroid receptors. In tissues such as the brain for example, where these receptors are present in low concentrations, alternative methodologies have been utilized. In this case the
88 method of choice for the quantitation of steroid receptors involves the separation of free hormone from the complex by chromatography on Sephadex-LH columns. However, this method, is time consuming and can be applied to only a limited number of samples at the same time. Autoradiography after injection of labeled steroid is the methodology generally used to establish the pattern of steroid receptor distribution in target regions of the central nervous system (McEwen et al., 1979). Despite its sensitivity and high resolution (Loy et al., 1988), technical difficulties limit its use. Finally, over the last 6 years, many immunological methods for the determination of steroid receptors have been reported (Jensen et al., 1982; Pertschuk et al., 1985; Thorpe et al., 1986; Press and Greene, 1988). However, none of these methods retain the valuable feature of the biochemical assay, measurement of the binding affinity of the receptor. In this paper, we present a method involving immunoprecipitation, which measures low concentrations of receptor against a very small background of non-specific binding and, furthermore, gives a value for the dissociation constant of the ligand receptor interaction.
Materials and methods
Animals Female Sprague-Dawley rats (Charles River, Calco, Italy) were used as a source of tissue for all experiments. The ovaries were removed from all rats 15 days before killing.
Antibodies Mouse ascites fluid containing the specific monoclonal antibody JS 34/32 (Moncharmont et al., 1982) was kindly provided by Dr. B. Moncharmont. The antibody belongs to IgG subclass and binds to protein A.
Cytosol preparation The cytosol was prepared essentially as previously described (Maggi et al., 1989). Briefly, animals were killed by decapitation, tissues rapidly excised and immediately homogenized in TED buffer (10 mM Tris pH 7.4, 1.5 mM EDTA and
1.0 mM DTT) (100 mg fresh tissue/ml). After centrifugation at 850 × g for 10 min, the supernatant was collected and centrifuged for 60 min at 100,000 × g. This second supernatant was immediately assayed. The protein content of the cytosolic fraction was determined by the Bradford method (1976).
Binding assay Each determination was performed on 300/zl of cytosol. The incubation with the 3H-/3-estradiol (3HE2) (Amersham, Buckinghamshire, England, 68 Ci/mmol) was carried out for 16 h at 4°C (with gentle shaking) to reach equilibrium. 10 ~1 of mouse ascites fluid containing the specific antiER antibody (Ab) were diluted to a protein concentration of 1 /~g//zl and then added to the binding solution. A second incubation was performed for 3 h at 4°C in order to allow the formation of the complex between the antibody and the receptor bound to the hormone (3HE2ER-Ab). The 3HEz-ER-Ab was precipitated by the addition of 150/xl of a protein A-Sepharose (Pharmacia, Uppsala, Sweden) suspension (500 /zg/ml TED buffer) and centrifugation at 6000 rpm in an Eppendorf centrifuge for 10 min at 4°C. The supernatant containing the unbound 3HE2 was discarded. The resin was washed three times with 1 ml of TED buffer by successive resuspensions and centrifugations. Finally, the 3HE2 bound was extracted with 2 ml of ethanol which were added to 10 ml of scintillation cocktail (Atomlight, New England Nuclear, Florence, Italy) for the quantitation. In a few experiments the same amount of scintillation cocktail was directly added to the pellet containing the 3HEzER-Ab complex with no loss in counting efficiency. For single point analysis, total binding was determined with a saturating concentration of 3HE2 (2.5 nM). For the saturation curve the following concentrations of 3HE2 were used: 0.1, 0.3, 0.8, 2.2, 6.1 nM. Non-specific binding was assessed in the presence of 10 ~M /3-estradiol.
Immunoenzymatic quantification of the ER For the immunoenzymatic assay of ER the Abbott kit (Abbott Diagnostic Products, Rome, Italy) was used, with a cytosol volume of 100 ~zl.
89
The samples of each animal were tested in duplicate.
TABLE I INTRA-ASSAY VARIABILITY OF M E T H O D F O R THE QUANTITATION OF E S T R O G E N RECEPTORS Binding a (cpm)
Results
Coefficient of variation
(%) A saturation curve was performed by incubating the cytosol from rat hypothalamus with increasing concentrations of 3HE2 in the presence or absence of an excess of cold hormone (10 tzM). Scatchard analysis of the data obtained indicated the presence of a single binding site (Fig. 1). The calculated apparent affinity ( K d = 0.37 nM) and the number of binding sites (Bma x = 8.4 f m o l / m g prot.) were in agreement with previous reports in which the rat brain estrogen receptor was quantified by different methodolo-
A
1.000 8o0
2oo
o.moleolelgj..e°eIo.°lllalt eel°*~l
0
d~e.,,..4 ............... III ............. 0 2 4 [Sd-ESTRADIOL] (nM)
8
25
B 2O
i,o 5
2
4 6 BOUND (fmoloe/mg proL)
8
Fig. 1. 3HE2 binding in cytosol of rat hypothalamus. A: different concentrations of 3HE 2 (0.1, 0.3, 0.8, 2.2, 6.1 nM) were used in the absence (total binding, B) or presence (non-specific binding, A) of 10 ~ M cold /3-estradiol. Specific binding (o) was calculated as the difference between total and non-specific binding. B: Scatchard plot of specifically bound 3HE 2. Bmax = 8.4 f m o l / m g prot. K d = 0.37 nM.
Total binding Non-specific binding
4,258 _+397 290 + 143
8.8 49
The binding was performed with 2.5 nM 3H fl-estradiol in the absence (total binding) or presence of 10/~M /3-estradiol (non-specific binding). The cytosol was prepared from the uterus of a single rat. Data represent the means+_standard deviation of ten determinations. a
gies (Anderson et al., 1973). The non-specific binding was remarkably low, being about 6% of the total binding. The proportion of 3HE e bound with respect to the 3HE2 added varied in proportion to the concentration of receptor in the cytosol. With nervous tissue, where the content of receptor is known to be low, the percentage of 3HE2 bound was 1 - 4 % of the total, with uterine tissue the percentage increased to 5 - 1 5 % depending on the amount of total radioactivity added. Receptor quantitation was also highly reproducible as indicated in Table I which shows data resulting from ten determinations performed on uterine cytosol. In this experiment, the calculated standard deviation was less than 10% of the specific binding. The sensitivity of the assay was assessed by a dilution test in which the cytosol from rat uterus was diluted several times to obtain E R concentrations ranging between 4.5 and 145 fmol/ml. In the various samples the total protein concentrations ranged between 0.09 and 2.9 m g / m l . The values of E R assessed in the dilution series were strictly correlated with the degree of dilution (regression coefficient, r = 0.99) indicating that the determination of E R by this method is linear between 4.5 and 145 fmol of steroid receptor and can be used to measure E R in solutions containing only a few fmol of the ligand (Fig. 2). E R was then assayed in the cytosol of several organs and in different areas of the CNS. As indicated in Table II, the values of 3HE2 binding activity measured with the immunoprecipitation method
90
only in those known to be a target for the hormone (Maggi et al., 1989). E O
E
Discussion
"O ,100
0
0.25
0.5
0.75
1
l/dilution factor
Fig. 2. Dilution test: 3H-estradiol specific binding was determinated on serial dilutions (1/1; 1/2; 1/4; 1/8; 1/16; 1/32) of uterine cytosol. The correlation coefficient of the curve was r = 0.99.
described correlated closely with determinations performed using an EIA-ER kit and with the binding data reported in the literature. In fact, high binding activities were detected in uterus and liver, well known target organs for estrogen; no binding was found in psoas muscle or in lung. With regard to the analysis of several brain regions: 3HE2 binding activity could be detected
The binding procedure described in this paper represents a new, rapid and highly sensitive method to quantify ER. The specificity of the receptor-antibody interaction guarantees the quantitative and specific isolation of ER in tissues rich in other steroid receptors. The major advantage of the present methodology is its rapidity and the separation of the free hormone is therefore achieved without alteration of the equilibrium of the binding reaction. This procedure is, therefore, of particular interest for the detection of receptors with a lowered affinity for the ligand (e.g., mutated receptors, products of transfected genes, etc.). Furthermore, the low levels of non-specific binding permit the quantitation of very low concentrations of steroid receptors. Because of its high sensitivity the method should become of choice for the quantitation of ER in mammalian brain.
Acknowledgements TABLE II COMPARISON BETWEEN 3HE2 BINDING AND IMMUNOLOGICAL QUANTITATION OF ESTROGEN RECEPTOR IN VARIOUS RAT ORGANS OR REGIONS OF THE CNS The binding assay was performed on cytosolic extracts as described in the materials and methods section. The data represent the mean _+standard deviation of measurements performed on five different rats Tissue
3HE2 binding (fmol/mg prot.)
ER protein (fmol/mg prot.)
Uterus Liver Lung Muscle Brain:Hypothalamus Hippocampus Cortex Brainstem Cerebellum
296 +76 51 _+ 5 N.D. a N.D. 11.4_+ 1.5 5.3_+ 0.9 0.5_+ 0.1 N.D. N.D.
260 _+55 59 + 6 N.D. 0.4+ 0.1 8.8_+ 1.7 8.9_+ 1.7 0.6_+ 0.1 N.D. N.D.
a Below levels of detection.
We are grateful to Dr. Bruno Moncharmont for a generous gift of ER-specific monoclonal antibodies and to Ms. Anna Menegatti for her excellent secretarial help. This work has been supported by the Pharmaceutical Company Hoffmann-La Roche and by grants from the National Council of Research (Target project on Biotechnology and Bioinstrumentation and Genetic Engineering C.N.R.).
References Anderson, J.N., Peck, E.J. and Clark, J.H. (1973) Nuclear receptor estrogen complex: accumulation, retention and localization in the hypothalamus and pituitary. Endocrinology 93, 711-717. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254.
91 Chamness, G.C., Huff, K. and McGuire, W.L. (1975) Protamine-precipitated estrogen receptor: a solid-phase ligand exchange assay. Steroids 25, 627-635. Clark, J.H., Peck, E.J. and MarKaverich, B.M. (1988) In: W.T. Schrader and B.W. O'Malley (Eds.), Laboratory Methods Manual for Hormone Action and Molecular Endocrinology, 12th edn. Houston Biological Assoc., Houston, pp. 1-28.
Erdos, T., Best-Belpomme M. and Bessada, R. (1970) A rapid assay for binding estradiol to uterine receptor(s). Anal. Biochem. 37, 244-252. Jensen, E.V., Greene, G.L., Closs, L.E., De Sombre, E.R. and Nadji, M. (1982) Receptor reconsidered: a 20 year perspective. Rec. Prog. Hor. Res. 38, 1-40. Korenman, S.C., Perrin, L.E. and McCallum, T.P. (1969) A radio-ligand binding assay system for estradiol measurement in human plasma. J. Clin. Endocrinol. 29, 879-883. Loy, R., Gerlach, J.L. and McEwen, B.S. (1988) Autoradiographic localization of estradiol-binding neurons in the rat hippocampal formation and entorhinal cortex. Dev. Brain Res. 39, 245-251. Maggi, A., Susanna, L., Bettini, E., Mantero, G. and Zucchi, I. (1989) Hippocampus: a target for estrogen action in mammalian brain. Mol. Endocrinol. 3, 1165-1170. McEwen, B.S., Davis, P.G., Parson, P. and Pfaff, D.W. (1979) The brain as a target for steroid hormone action. Annu. Rev. Neurosci. 2, 65-112.
Mester, J., Robertson, D.M., Feherty, P. and Kellie, A.E. (1970) Determination of high-affinity oestrogen receptor sites in uterine supernatant preparations. Biochem. J. 120, 831-836. Moncharmont, B., Su, J.-L. and Parikh, I. (1982) Monoclonal antibodies against estrogen receptor: interaction with different molecular forms and functions of the receptor. Biochemistry 21, 6916-6921 Pertschuk, L.P., Eisenberg, K.B., Carter, A.C. and Felman, J.G. (1985) Immunohistologic localization of estrogen receptors in breast cancer with monoclonal antibodies. Cancer 55, 1513-1518. Press, M.F. and Greene, G.L. (1988) Localization of progesterone receptor with monoclonal antibodies to the human progestin receptor. Endocrinology 122, 1165-1175. Sanborn, B.M., Steinberger, A.S., Meistrich, M.L. and Steinberger, E. (1975) Androgen binding sites in testis cell fractions as measured by a nuclear exchange assay. J. Steroid Biochem. 6, 1459-1468. Santi, D.V., Sibley, C.H., Perriard, E.R., Tomkins, G. and Baxter, J.D. (1973) A filter assay for steroid hormone receptors. Biochemistry 12, 2412-2416. Thorpe, S.M., Lykkesfeldt, A.E., Vinterby, A. and Lonsdorferm, M. (1986) Quantitative immunological detection of estrogen receptors in nuclear pellets from human breast cancer biopsies. Cancer 46 (Suppl.), 42515-42555.
87
© 1991 Elsevier Science Publishers B.V. All rights reserved 0022-1759/91/$03.50
JIM 07005
A rapid method for the quantitation of estrogen receptors in small amounts of tissue E z i o Bettini a n d A d r i a n a Maggi Milano Molecular Pharmacology Lab, Institute of Pharmacological Sciences, University of Milan, Milan, Italy
(Received 12 November 1990,revised received 20 May 1991, accepted 8 July 1991)
A rapid and highly reproducible protocol which permits the detection of quantities of estrogen receptor as low as 5 f m o l / m g protein is described. The separation of free and receptor-bound hormone is achieved by specific immunoprecipitation of the hormone-receptor complex. This procedure can be performed without perturbing the equilibrium of the binding reaction. Key words: Estrogen receptor; Steroid receptor quantitation; Estrogen receptor immuno-quantitation
Introduction The quantitation of soluble receptors such as steroid receptors has traditionally been hampered by the technical difficulties encountered in the separation of free and bound ligands. The various approaches adopted to date involve the adsorption of either the free ligand or the hormone-receptor complex to an insoluble matrix. Because of its inexpensive nature and ease of application, the method currently most widely utilized for the measurement of steroid receptors involves the adsorption of free steroids to dextran-coated charcoal. In this case, the quantitation is based on the measurement of the labeled hormone bound to its cognate receptor (Korenman et al., 1969; Mester et al., 1970; Sanborn et al., 1975). This method has two major limitations: (a) at low protein concentration the charcoal may Correspondence to: A. Maggi, Milano Molecular Pharmacology Lab, Institute of Pharmacological Sciences, University of Milan, Piazza Durante 11, 20131 Milan, Italy (Tel.: 39/2/2884567; Fax: 39/2/2820603).
also adsorb some of the hormone-receptor complex, (b) the charcoal may strip some of the steroid from the steroid-receptor complex. Both shortcomings can lead to an underestimation of the receptor concentration (Clark et al., 1988) and, in addition, the latter can alter the equilibrium of the reaction. Several other alternative methodologies have been described. These are based on the adsorption of receptor-steroid complex on charged surfaces such as hydroxylapatite (Erdos et al., 1970), D E A E cellulose filters (Santi et al., 1973) or protamine sulfate (Chamness et al., 1985). Although these methods are rapid and generally highly reproducible they suffer from very high levels of non-specific binding and consequent impairment of the measurement of low receptor concentrations. The application of the above-mentioned methodologies is therefore restricted to tissues rich in steroid receptors. In tissues such as the brain for example, where these receptors are present in low concentrations, alternative methodologies have been utilized. In this case the
88 method of choice for the quantitation of steroid receptors involves the separation of free hormone from the complex by chromatography on Sephadex-LH columns. However, this method, is time consuming and can be applied to only a limited number of samples at the same time. Autoradiography after injection of labeled steroid is the methodology generally used to establish the pattern of steroid receptor distribution in target regions of the central nervous system (McEwen et al., 1979). Despite its sensitivity and high resolution (Loy et al., 1988), technical difficulties limit its use. Finally, over the last 6 years, many immunological methods for the determination of steroid receptors have been reported (Jensen et al., 1982; Pertschuk et al., 1985; Thorpe et al., 1986; Press and Greene, 1988). However, none of these methods retain the valuable feature of the biochemical assay, measurement of the binding affinity of the receptor. In this paper, we present a method involving immunoprecipitation, which measures low concentrations of receptor against a very small background of non-specific binding and, furthermore, gives a value for the dissociation constant of the ligand receptor interaction.
Materials and methods
Animals Female Sprague-Dawley rats (Charles River, Calco, Italy) were used as a source of tissue for all experiments. The ovaries were removed from all rats 15 days before killing.
Antibodies Mouse ascites fluid containing the specific monoclonal antibody JS 34/32 (Moncharmont et al., 1982) was kindly provided by Dr. B. Moncharmont. The antibody belongs to IgG subclass and binds to protein A.
Cytosol preparation The cytosol was prepared essentially as previously described (Maggi et al., 1989). Briefly, animals were killed by decapitation, tissues rapidly excised and immediately homogenized in TED buffer (10 mM Tris pH 7.4, 1.5 mM EDTA and
1.0 mM DTT) (100 mg fresh tissue/ml). After centrifugation at 850 × g for 10 min, the supernatant was collected and centrifuged for 60 min at 100,000 × g. This second supernatant was immediately assayed. The protein content of the cytosolic fraction was determined by the Bradford method (1976).
Binding assay Each determination was performed on 300/zl of cytosol. The incubation with the 3H-/3-estradiol (3HE2) (Amersham, Buckinghamshire, England, 68 Ci/mmol) was carried out for 16 h at 4°C (with gentle shaking) to reach equilibrium. 10 ~1 of mouse ascites fluid containing the specific antiER antibody (Ab) were diluted to a protein concentration of 1 /~g//zl and then added to the binding solution. A second incubation was performed for 3 h at 4°C in order to allow the formation of the complex between the antibody and the receptor bound to the hormone (3HE2ER-Ab). The 3HEz-ER-Ab was precipitated by the addition of 150/xl of a protein A-Sepharose (Pharmacia, Uppsala, Sweden) suspension (500 /zg/ml TED buffer) and centrifugation at 6000 rpm in an Eppendorf centrifuge for 10 min at 4°C. The supernatant containing the unbound 3HE2 was discarded. The resin was washed three times with 1 ml of TED buffer by successive resuspensions and centrifugations. Finally, the 3HE2 bound was extracted with 2 ml of ethanol which were added to 10 ml of scintillation cocktail (Atomlight, New England Nuclear, Florence, Italy) for the quantitation. In a few experiments the same amount of scintillation cocktail was directly added to the pellet containing the 3HEzER-Ab complex with no loss in counting efficiency. For single point analysis, total binding was determined with a saturating concentration of 3HE2 (2.5 nM). For the saturation curve the following concentrations of 3HE2 were used: 0.1, 0.3, 0.8, 2.2, 6.1 nM. Non-specific binding was assessed in the presence of 10 ~M /3-estradiol.
Immunoenzymatic quantification of the ER For the immunoenzymatic assay of ER the Abbott kit (Abbott Diagnostic Products, Rome, Italy) was used, with a cytosol volume of 100 ~zl.
89
The samples of each animal were tested in duplicate.
TABLE I INTRA-ASSAY VARIABILITY OF M E T H O D F O R THE QUANTITATION OF E S T R O G E N RECEPTORS Binding a (cpm)
Results
Coefficient of variation
(%) A saturation curve was performed by incubating the cytosol from rat hypothalamus with increasing concentrations of 3HE2 in the presence or absence of an excess of cold hormone (10 tzM). Scatchard analysis of the data obtained indicated the presence of a single binding site (Fig. 1). The calculated apparent affinity ( K d = 0.37 nM) and the number of binding sites (Bma x = 8.4 f m o l / m g prot.) were in agreement with previous reports in which the rat brain estrogen receptor was quantified by different methodolo-
A
1.000 8o0
2oo
o.moleolelgj..e°eIo.°lllalt eel°*~l
0
d~e.,,..4 ............... III ............. 0 2 4 [Sd-ESTRADIOL] (nM)
8
25
B 2O
i,o 5
2
4 6 BOUND (fmoloe/mg proL)
8
Fig. 1. 3HE2 binding in cytosol of rat hypothalamus. A: different concentrations of 3HE 2 (0.1, 0.3, 0.8, 2.2, 6.1 nM) were used in the absence (total binding, B) or presence (non-specific binding, A) of 10 ~ M cold /3-estradiol. Specific binding (o) was calculated as the difference between total and non-specific binding. B: Scatchard plot of specifically bound 3HE 2. Bmax = 8.4 f m o l / m g prot. K d = 0.37 nM.
Total binding Non-specific binding
4,258 _+397 290 + 143
8.8 49
The binding was performed with 2.5 nM 3H fl-estradiol in the absence (total binding) or presence of 10/~M /3-estradiol (non-specific binding). The cytosol was prepared from the uterus of a single rat. Data represent the means+_standard deviation of ten determinations. a
gies (Anderson et al., 1973). The non-specific binding was remarkably low, being about 6% of the total binding. The proportion of 3HE e bound with respect to the 3HE2 added varied in proportion to the concentration of receptor in the cytosol. With nervous tissue, where the content of receptor is known to be low, the percentage of 3HE2 bound was 1 - 4 % of the total, with uterine tissue the percentage increased to 5 - 1 5 % depending on the amount of total radioactivity added. Receptor quantitation was also highly reproducible as indicated in Table I which shows data resulting from ten determinations performed on uterine cytosol. In this experiment, the calculated standard deviation was less than 10% of the specific binding. The sensitivity of the assay was assessed by a dilution test in which the cytosol from rat uterus was diluted several times to obtain E R concentrations ranging between 4.5 and 145 fmol/ml. In the various samples the total protein concentrations ranged between 0.09 and 2.9 m g / m l . The values of E R assessed in the dilution series were strictly correlated with the degree of dilution (regression coefficient, r = 0.99) indicating that the determination of E R by this method is linear between 4.5 and 145 fmol of steroid receptor and can be used to measure E R in solutions containing only a few fmol of the ligand (Fig. 2). E R was then assayed in the cytosol of several organs and in different areas of the CNS. As indicated in Table II, the values of 3HE2 binding activity measured with the immunoprecipitation method
90
only in those known to be a target for the hormone (Maggi et al., 1989). E O
E
Discussion
"O ,100
0
0.25
0.5
0.75
1
l/dilution factor
Fig. 2. Dilution test: 3H-estradiol specific binding was determinated on serial dilutions (1/1; 1/2; 1/4; 1/8; 1/16; 1/32) of uterine cytosol. The correlation coefficient of the curve was r = 0.99.
described correlated closely with determinations performed using an EIA-ER kit and with the binding data reported in the literature. In fact, high binding activities were detected in uterus and liver, well known target organs for estrogen; no binding was found in psoas muscle or in lung. With regard to the analysis of several brain regions: 3HE2 binding activity could be detected
The binding procedure described in this paper represents a new, rapid and highly sensitive method to quantify ER. The specificity of the receptor-antibody interaction guarantees the quantitative and specific isolation of ER in tissues rich in other steroid receptors. The major advantage of the present methodology is its rapidity and the separation of the free hormone is therefore achieved without alteration of the equilibrium of the binding reaction. This procedure is, therefore, of particular interest for the detection of receptors with a lowered affinity for the ligand (e.g., mutated receptors, products of transfected genes, etc.). Furthermore, the low levels of non-specific binding permit the quantitation of very low concentrations of steroid receptors. Because of its high sensitivity the method should become of choice for the quantitation of ER in mammalian brain.
Acknowledgements TABLE II COMPARISON BETWEEN 3HE2 BINDING AND IMMUNOLOGICAL QUANTITATION OF ESTROGEN RECEPTOR IN VARIOUS RAT ORGANS OR REGIONS OF THE CNS The binding assay was performed on cytosolic extracts as described in the materials and methods section. The data represent the mean _+standard deviation of measurements performed on five different rats Tissue
3HE2 binding (fmol/mg prot.)
ER protein (fmol/mg prot.)
Uterus Liver Lung Muscle Brain:Hypothalamus Hippocampus Cortex Brainstem Cerebellum
296 +76 51 _+ 5 N.D. a N.D. 11.4_+ 1.5 5.3_+ 0.9 0.5_+ 0.1 N.D. N.D.
260 _+55 59 + 6 N.D. 0.4+ 0.1 8.8_+ 1.7 8.9_+ 1.7 0.6_+ 0.1 N.D. N.D.
a Below levels of detection.
We are grateful to Dr. Bruno Moncharmont for a generous gift of ER-specific monoclonal antibodies and to Ms. Anna Menegatti for her excellent secretarial help. This work has been supported by the Pharmaceutical Company Hoffmann-La Roche and by grants from the National Council of Research (Target project on Biotechnology and Bioinstrumentation and Genetic Engineering C.N.R.).
References Anderson, J.N., Peck, E.J. and Clark, J.H. (1973) Nuclear receptor estrogen complex: accumulation, retention and localization in the hypothalamus and pituitary. Endocrinology 93, 711-717. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254.
91 Chamness, G.C., Huff, K. and McGuire, W.L. (1975) Protamine-precipitated estrogen receptor: a solid-phase ligand exchange assay. Steroids 25, 627-635. Clark, J.H., Peck, E.J. and MarKaverich, B.M. (1988) In: W.T. Schrader and B.W. O'Malley (Eds.), Laboratory Methods Manual for Hormone Action and Molecular Endocrinology, 12th edn. Houston Biological Assoc., Houston, pp. 1-28.
Erdos, T., Best-Belpomme M. and Bessada, R. (1970) A rapid assay for binding estradiol to uterine receptor(s). Anal. Biochem. 37, 244-252. Jensen, E.V., Greene, G.L., Closs, L.E., De Sombre, E.R. and Nadji, M. (1982) Receptor reconsidered: a 20 year perspective. Rec. Prog. Hor. Res. 38, 1-40. Korenman, S.C., Perrin, L.E. and McCallum, T.P. (1969) A radio-ligand binding assay system for estradiol measurement in human plasma. J. Clin. Endocrinol. 29, 879-883. Loy, R., Gerlach, J.L. and McEwen, B.S. (1988) Autoradiographic localization of estradiol-binding neurons in the rat hippocampal formation and entorhinal cortex. Dev. Brain Res. 39, 245-251. Maggi, A., Susanna, L., Bettini, E., Mantero, G. and Zucchi, I. (1989) Hippocampus: a target for estrogen action in mammalian brain. Mol. Endocrinol. 3, 1165-1170. McEwen, B.S., Davis, P.G., Parson, P. and Pfaff, D.W. (1979) The brain as a target for steroid hormone action. Annu. Rev. Neurosci. 2, 65-112.
Mester, J., Robertson, D.M., Feherty, P. and Kellie, A.E. (1970) Determination of high-affinity oestrogen receptor sites in uterine supernatant preparations. Biochem. J. 120, 831-836. Moncharmont, B., Su, J.-L. and Parikh, I. (1982) Monoclonal antibodies against estrogen receptor: interaction with different molecular forms and functions of the receptor. Biochemistry 21, 6916-6921 Pertschuk, L.P., Eisenberg, K.B., Carter, A.C. and Felman, J.G. (1985) Immunohistologic localization of estrogen receptors in breast cancer with monoclonal antibodies. Cancer 55, 1513-1518. Press, M.F. and Greene, G.L. (1988) Localization of progesterone receptor with monoclonal antibodies to the human progestin receptor. Endocrinology 122, 1165-1175. Sanborn, B.M., Steinberger, A.S., Meistrich, M.L. and Steinberger, E. (1975) Androgen binding sites in testis cell fractions as measured by a nuclear exchange assay. J. Steroid Biochem. 6, 1459-1468. Santi, D.V., Sibley, C.H., Perriard, E.R., Tomkins, G. and Baxter, J.D. (1973) A filter assay for steroid hormone receptors. Biochemistry 12, 2412-2416. Thorpe, S.M., Lykkesfeldt, A.E., Vinterby, A. and Lonsdorferm, M. (1986) Quantitative immunological detection of estrogen receptors in nuclear pellets from human breast cancer biopsies. Cancer 46 (Suppl.), 42515-42555.
