Radioimmunoassay of O6-methyldeoxyguanosine in DNA of cells alkylated in vitro and in vivo.

Abstract

Mouse monoclonal and rabbit polyclonal antibodies have been prepared against O6-methylguanosine complexed with bovine serum albumin. In radioimmunoassay 50% inhibition of binding of [3H]O6-methyldeoxyguanosine (O6-MedG) was obtained with 0.3 pmol or 0.16 pmol unlabelled O6-MedG using monoclonal or polyclonal antibodies having affinity constants of 3.0 X 10(9) and 5.9 X 10(9) I mol-1 respectively. Cross-reactivity with normal nucleosides and other adducts was determined. Sensitivity and reproducibility were improved by chromatographic separation using an Aminex A6 column eluted with 10 mM NH4HCO3 buffer which allowed quantitation of one molecule O6-MedG per 10(7) molecules dG in 2 mg calf thymine DNA. Values for O6-MedG from 14 samples of rat liver and kidney DNA analysed by the method described and by radiochromatography on Sephadex G10 were almost identical (correlation coefficient, 0.98). A modified procedure for the purification of 5-25 micrograms DNA from 1-5 X 10(6) fibroblasts applied to polycarbonate filters was used in providing further validation of the RIA system by measuring the persistence of O6-MedG in cell lines of known repair capacity.