A microassay for the determination of binding parameters of estrogen and androgen receptors employing affinity immobilization on Cibacron blue 3GA-Sepharose 6B

Abstract

The problems of currently available ligand-binding assays for sex-steroid receptor proteins include the relatively large mass of tissue required, the interference by sex hormone-binding globulin (SHBG), and use in the androgen receptor (AR) assay of the unstable synthetic ligand methyltrienolone. To overcome these difficulties the stabilizing effect of the dye Cibacron blue 3GA on AR and estrogen receptor (ER) proteins, and its ability to bind to these proteins, was utilized in developing an assay system for each receptor that could be applied to small samples. Use of the affinity gel Cibacron blue 3GA-Sepharose 6B (Blue gel) for the immobilization of AR, ER, and the steroid ligands bound to these receptors in the standard two-tier column assay system enabled the use of a 1:100 (original tissue weight:volume) concentration, making possible full (5–7 point) Scatchard analysis on tissue specimens of a mass as low as 15–20 mg. Significant stabilization of AR and ER was observed and association constants for these receptors were of a similar order of magnitude to those obtained either by Sephadex LH-20 gel filtration or the dextran-coated charcoal adsorption technique. Inactivation by dilution was shown to be largely prevented based on results obtained with cytosol concentrations from 1:5 to 1:100 (original tissue weight:volume). Because Blue gel does not bind SHBG, the natural steroid 5α-androstan-17β-ol-3-one (DHT) may be employed as a ligand in the AR assay. An additional advantage of SHBG not being retained by the Blue gel was that simultaneous determination of DHT binding to this protein in the column eluates could be performed. Intraassay and interassay variations were 4.6 and 8.9% for ER and 7.9 and 12.0% for AR, respectively.