Characterization of polyclonal antibodies to the Ah receptor prepared by immunization with a synthetic peptide hapten

Abstract

A synthetic peptide based on the N-terminal amino acid sequence of the Ah receptor purified from C57BL/6J mice, linked to keyhole limpet hemocyanin, proved a remarkably good immunogen. All six rabbits that were immunized produced polyclonal antiserum that reacted with the synthetic peptide and the denatured and undenatured Ah receptor. Western blots were especially useful for antibody characterization; hepatic cytosol from C57BL/6J mice, in which the Ah receptor was photoaffinity labeled with 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin, was resolved by gel electrophoresis and electrotransferred to nitrocellulose. Co-incidence of the major band immunochemically stained with immunoaffinity-purified antibodies (with apparent Mr = 95,000) and the radiolabeled band on the autoradiograph indicated the specificity of the antibody. The estimated sensitivity of detection of the Ah receptor on a blot is 60 to 120 pg/200 micrograms of protein/gel lane. On Western blots, the antipeptide antibodies stained the photoaffinity-labeled Ah receptor from all four murine variants and all vertebrate forms examined (chicken, rodents, monkey, human), indicating conservation of these N-terminal epitopes. The immunoaffinity-purified antibodies also immunoprecipitated undenatured photoaffinity-labeled Ah receptor from diluted cytosol.