A new southwestern chemistry-based ELISA for detection of aryl hydrocarbon receptor transformation: application to the screening of its receptor agonists and antagonists.
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.
Fukuda
Itsuko
I
Department of Life Science, Graduate School of Science and Technology, Kobe University, Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501, Japan.
Nishiumi
Shin
S
Yabushita
Yoshiyuki
Y
Mukai
Rie
R
Kodoi
Rie
R
Hashizume
Kaoru
K
Mizuno
Masashi
M
Hatanaka
Yutaka
Y
Ashida
Hitoshi
H
D000818:Animals; D000906:Antibodies / Q000276:immunology; D026501:Blotting, Southwestern / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D006846:Hydrocarbons, Halogenated / Q000032:analysis* / Q000378:metabolism; D011084:Polycyclic Aromatic Hydrocarbons / Q000032:analysis* / Q000378:metabolism; D018336:Receptors, Aryl Hydrocarbon / Q000032:analysis* / Q000276:immunology / Q000378:metabolism*; D012680:Sensitivity and Specificity
D000906:Antibodies; D006846:Hydrocarbons, Halogenated; D011084:Polycyclic Aromatic Hydrocarbons; D018336:Receptors, Aryl Hydrocarbon