A new southwestern chemistry-based ELISA for detection of aryl hydrocarbon receptor transformation: application to the screening of its receptor agonists and antagonists

Abstract

Halogenated aromatic hydrocarbons (HAHs), such as 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), produce a wide variety of biological and toxic effects mainly through the aryl hydrocarbon receptor (AhR)-dependent mechanism. After the binding of HAHs, the AhR subsequently transforms its form in order to interact with a specific DNA sequence, the dioxin responsive element (DRE). Thus, detection of the transformed AhR is a target for estimation of the biological and toxic potency of ligands. In this study, we have developed a simple method for quantitative assessment of the transformation state of AhR based on an enzyme-linked immunosorbent assay (ELISA) combined with southwestern chemistry technique (SW–ELISA) that detects the complex of transformed AhR:fluorescein isothiocyanate (FITC)-labeled DRE probe. SW–ELISA has shown the response to HAHs including TCDD and other known agonists in a dose-dependent manner. In the case of TCDD, SW–ELISA has revealed a minimum detection limit (MDL) of 2 pM (0.026 pg/assay), a median effective concentration (EC 50) value of 0.125 nM (1.6 pg/assay), and a maximum response at 10 nM (129 pg/assay). Furthermore, SW–ELISA provides the confirmation that flavonoids, the potent antagonists for AhR as reported previously, show the inhibitory effects on TCDD-induced AhR transformation. These results indicate that SW–ELISA is a new and straightforward method for the detection of AhR transformation and will be useful in screening of agonists or antagonists for AhR.