Poellinger L, Lund J, Dahlberg E, Gustafsson J A
Anal Biochem. 1985 Feb 1;144(2):371-84. doi: 10.1016/0003-2697(85)90130-7.
A "batch" hydroxylapatite assay for the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) receptor that does not require detergents is described. The receptor could be assayed in rat target tissues using either of the cytochrome P1-450 inducers [3H]TCDD or [3H]3-methylcholanthrene as radioligands. A phosphate buffer washing procedure was developed on the basis of chromatographic data and optimized to separate nonspecifically and specifically bound ligand. The assay was characterized with respect to washing efficiency, binding specificity, competition, adsorption time, amount of hydroxylapatite required to bind receptor complexes, sensitivity, and effects of detergents. Equilibrium binding parameters were determined. Receptor extracted with phosphate from hydroxylapatite was analyzed on sucrose gradients and was found to exhibit the same sedimentation properties as the receptor in crude cytosol. Furthermore, the applicability of the assay has been demonstrated in cytosolic preparations from three different target tissues: liver, lung, and thymus.
本文描述了一种用于检测2,3,7,8-四氯二苯并对二恶英（TCDD）受体的“批量”羟基磷灰石检测法，该方法无需使用去污剂。可以使用细胞色素P1 - 450诱导剂[3H]TCDD或[3H]3 - 甲基胆蒽作为放射性配体，在大鼠靶组织中检测该受体。基于色谱数据开发了一种磷酸盐缓冲液洗涤程序，并进行了优化，以分离非特异性结合和特异性结合的配体。对该检测法在洗涤效率、结合特异性、竞争性、吸附时间、结合受体复合物所需的羟基磷灰石量、灵敏度以及去污剂的影响等方面进行了表征。测定了平衡结合参数。用磷酸盐从羟基磷灰石中提取的受体在蔗糖梯度上进行分析，发现其沉降特性与粗胞质溶胶中的受体相同。此外，该检测法在来自三种不同靶组织（肝脏、肺和胸腺）的胞质制剂中的适用性也得到了证实。
