The use of immobilized estradiol antiserum in the study of receptors and other estradiol-binding proteins

doi:10.1016/0003-2697(79)90361-0

Analytical Biochemistry

Volume 94, Issue 2, 15 April 1979, Pages 278-286

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Abstract

Antiestradiol antibody immobilized on a polymer film is set in competition for tritium-labeled estradiol with estradiol receptor or antiestradiol antibody in solution. Measurement of equilibrium quantity of radiolabel in test solutions in the presence and in the absence of dissolved antibody or receptor gives a data base for evaluating the association constant and the quantity of the estradiol-complexing species in solution. The immobilized antibody method avoids the problems and uncertainties arising in the step of separating the bound from the free hormone which is the pivotal step in all currently used procedures for the determination and characterization of steroid receptors. With the exception of equilibrium dialysis, it is the only procedure presently available to study steroid antibodies and receptors in solution at unperturbed equilibrium. Using the immobilized antibody method at 4°C, 0.86 ± 0.12 × 10¹¹m⁻¹ was the association constant found for estradiol receptor in rat uterine cytosol, and 2.6 ± 0.5 × 10¹¹m⁻¹ was the association constant found for antiestradiol antibody raised in a rabbit to estradiol linked to bovine serum albumin via a C-6 carboxymethyloxime.

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Cited by (4)

Stimulation by estradiol-17β of thymidine kinase activity in the rat uterus
1984, Journal of Steroid Biochemistry
The action of estradiol-17β(E₂) on thymidine kinase (TK) activity was studied in uteri from immature female rats. It was demonstrated that a single injection of E₂ highly stimulated the enzyme activity which reached its maximum level 24 h after hormone administration. Physiological amounts of E₂ were efficient and changes in TK activity were observed exclusively in uterus and liver. A single injection of Tamoxifen produced the same effect as E₂ but repeated administration resulted in the complete inhibition of enzyme activity. Using antibiotics it was demonstrated that E₂ induced the synthesis of new enzyme molecules rather than an increase in enzyme activity. This statement was corroborated by the fact that after hormone administration the increase in TK activity was preceded by an increase in RNA-polymerase activity and followed by that in DNA-polymerase α activity. Moreover, the separation of TK isoenzymes on DEAE-Sephadex and the use of d-CTP as inhibitor of the adult isozyme suggested that E₂ induced the “fetal” form of the enzyme.
In addition, it was demonstrated that TK activity in uteri from ovariectomized adult female rats was enhanced by E₂ administration, and that the increase was due to the stimulation of the fetal isoenzyme.
It was suggested that TK could be used as a marker of the action of estrogens and antiestrogens in target organs.
Filter assay method for the estrogen receptor protein: Detection of both filled and unfilled estrogen binding sites
1982, Analytical Biochemistry
New filter assay methods are presented for quantitating both cytoplasmic and nuclear forms of the estrogen receptor protein. These methods exploit the strong adsorption of this protein to glass-fiber filters, which appears to occur without loss of steroid binding affinity. A “direct assay protocol” is described that detects only unfilled (nonliganded) estrogen binding sites. In addition, a convenient “exchange assay protocol” has been developed that detects, in addition, those receptors present whose binding sites have already bound nonradioactive estradiol. For the exchange assay, an extract containing receptor is adsorbed to a filter, which is washed free of unbound steroid and then equilibrated for a prolonged period with an excess volume of buffer containing radioactive estradiol. After brief washing in steroid-free buffer, the radioactivity adsorbed to the filter is measured to determine the amount of receptor present. These assays can be used at either 4 or 23°C, over a broad range of salt concentrations. The background of nonspecific binding is extremely low, due in part to the almost negligible affinity of free estradiol for the glass-fiber support.
Determination of steroid hormone‐dependency of tumours utilizing tissue sections. Survey of histochemical techniques and their application in surgical pathology
1986, The Journal of Pathology
Comparison of Histochemical and Biochemical Assays for Estrogen Receptor in Human Breast Cancer Cell Lines
1984, Cancer Research

^☆: This work was supported by National Cancer Institute Grant CA 22795 National Institutes of Health, Bethesda, Md.

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The use of immobilized estradiol antiserum in the study of receptors and other estradiol-binding proteins☆

Abstract

Fertil. Steril

J. Biol. Chem

Steroids

J. Steroid Biochem

J. Clin. Endocrinol. Metab

Immunochemistry

J. Clin. Endocrinol. Metab

J. Clin. Endocrinol. Metab

J. Clin. Endocrinol. Metab

Endocrinology

Eur. J. Biochem

J. Clin. Invest

J. Clin. Endocrinol. Metab