Monoclonal antibody-mediated solid-phase assay for mammalian O6-alkylguanine DNA alkyltransferase activity

We describe a sensitive, rapid, and simple assay for mammalian O6-alkylguanine DNA alkyltransferase (O6-AGT) utilizing solid-phase DNA as the substrate and a monoclonal antibody (Mab)-based immuno-slotblot (ISB) for quantitation of O6-ethylguanine (O6-EtG). lambda-phage DNA was treated with N-ethyl-N-nitrosourea and immobilized on newly developed hydrophilic latex beads. After incubation with cell extracts to be assayed for O6-AGT activity, the substrate DNA could be isolated easily by a brief centrifugation through 50% glycerol. The amount of O6-EtG retained in the substrate DNA was determined by ISB using the anti-(O6-ethyl-2'-deoxyguanosine) Mab ER-6. As little as 2 fmol of O6-AGT per reaction tube can be reproducibly measured by this procedure, which is suitable for handling large numbers of samples within a short time (e.g., 80 samples within 2 days). In normal and malignant cells, respectively, O6-AGT activity protects against O6-alkylguanine-mediated mutagenesis and oncogenesis following exposure to N-nitroso carcinogens or confers resistance against cytocidal anti-cancer drugs such as chloroethylnitrosoureas and related compounds. The analysis of cellular O6-AGT activity by a highly sensitive, routinely applicable method is, therefore, of particular interest in studies related to carcinogenesis, molecular epidemiology, and clinical oncology.