Developing a monoclonal antibody based capture assay to quantify cytosolic aryl hydrocarbon receptor
Devasthanam, Anand Shankar
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The Aryl Hydrocarbon Receptor (AHR) is a ligand inducible cytosolic transcription factor that up- and down-regulates genes, resulting in a wide range of biological and toxicological responses. The AhR typically responds to polycyclic aromatic hydrocarbons with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), or dioxin being the highest affinity ligand. The discovery of non-typical ligands has spurred interest among AhR researchers since it could help in uncovering some of the endogenous roles of the AhR and thus explain its physiological relevance. Cytosolic AhR level is a good indicator of a cells response to toxiclogical ligands. Although several studies have assessed AHR mRNA levels following exposure to polycyclic aromatic hydrocarbons and other AHR ligands, an antibody-based assay to quantify cytosolic AHR has yet to be established. Anti-AHR monoclonal antibodies to conserved peptide sequences at the N- and C-terminal of the AHR were used to capture AHR in hepatic cytosol fractions using an indirect enzyme immunoassay approach. Mouse Hepa-1 cytosol, cited to have the most abundant source of AHR, was used as one source. Cytosol preparations were also obtained from liver tissue samples of mice (C57BL/6 and BALB/c), AHR -/- mice, rat (adult Sprague Dawley) and human. Western blotting confirmed good species cross-reactivity of the monoclonal antibodies, with no signal present in cytosol from AHR -/- mice. Immunohistochemistry data was obtained from developing C57BL/6 mouse prostate tissue before and after TDCC treatment. The capture assay utilized either monoclonal anti-AHR developed to the N-terminal peptide, or monoclonal anti-AHR developed to the C-terminal peptide as the capture antibody. The capture assay was extended to establish an Ah receptor quantitation assay. The AhR monoclonal antibodies and capture assay for cytosolic AhR will provide valuable tools for future studies on the function and regulation of the Ah receptor.