Bradfield C A, Poland A
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Mol Pharmacol. 1988 Nov;34(5):682-8.
A sensitive competitive binding assay for the detection of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other ligands of the Ah receptor was developed using a stable preparation of the Ah receptor, the 40-55% ammonium sulfate fraction of liver cytosol from C57BL/6J mice, and the radioligand [125I]2-iodo-7,8-dibromodibenzo-p-dioxin (specific radioactivity, 2176 Ci/mmol, and binding affinity, KD = 6.5 pM). Conditions are described which maximize assay precision and sensitivity, while minimizing sample counting time, ensuring ligand solubility, and permitting attainment of binding equilibrium for competing ligands. Assay conditions were developed to allow calculation of the binding affinity for competing ligands and to ensure that an unknown competitor could be quantified in terms of "TCDD binding equivalents." Standard assay conditions consisted of incubation of 8 pM radioligand and 18-20 pM Ah receptor with 5-1000 pM TCDD, in a 1-ml volume, for 16 hr at 4 degrees. Statistical analysis of the standard curve of bound radioligand versus the log of the concentration of competing TCDD indicated the minimal detectable concentration of TCDD to be 10 pM (3.2 pg in a 1-ml assay alpha less than or equal to 0.01). The simplicity, sensitivity, and reproducibility of this competitive binding assay should prove useful as a screen to detect planar halogenated aromatic hydrocarbons and other ligands of the Ah receptor. The availability of this 125I-labeled dioxin congener also permitted the characterization of Ah receptor-ligand binding over a range of ligand and receptor concentrations not possible with currently available 3H-ligands.
利用稳定制备的芳烃受体（Ah受体）、C57BL/6J小鼠肝脏胞质溶胶中40 - 55%硫酸铵分级分离物以及放射性配体[125I]2-碘-7,8-二溴二苯并对二恶英（比放射性为2176 Ci/mmol，结合亲和力KD = 6.5 pM），开发了一种用于检测2,3,7,8-四氯二苯并对二恶英（TCDD）及Ah受体其他配体的灵敏竞争性结合测定法。文中描述了一些条件，这些条件能使测定的精密度和灵敏度最大化，同时将样品计数时间减至最短，确保配体的溶解性，并使竞争配体达到结合平衡。所开发的测定条件可用于计算竞争配体的结合亲和力，并确保能根据“TCDD结合当量”对未知竞争者进行定量。标准测定条件包括在1 ml体积中，将8 pM放射性配体和18 - 20 pM Ah受体与5 - 1000 pM TCDD一起在4℃孵育16小时。对结合的放射性配体标准曲线与竞争TCDD浓度对数进行统计分析表明，TCDD的最低可检测浓度为10 pM（在1 ml测定中为3.2 pg，α≤0.01）。这种竞争性结合测定法的简便性、灵敏度和可重复性应会证明其作为检测平面卤代芳烃及Ah受体其他配体的筛选方法很有用。这种125I标记的二恶英同系物的可得性还使得能够在一系列配体和受体浓度范围内表征Ah受体 - 配体结合情况，而这是目前可用的3H配体无法做到的。
