A Competitive Binding Assay for 2,3,7,8-tetrachlorodibenzo-p-dioxin and Related Ligands of the Ah Receptor

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.