Sensitive radioimmunoassays for O6-n-butyldeoxyguanosine, O2-n-butylthymidine and O4-n-butylthymidine

Abstract

Radioimmunoassays have been developed using monoclonal antibodies from hybridomas raised against bovine serum albumin conjugates of O⁶-n-butylguanosine, O²-n-butylthymidine riboside and O⁴-n-butylthymidine riboside. The assays showed 50% inhibition of binding of specific [³H]butyl-deoxynucleosides by 0.044, 0.069 and 0.45 pmole of cold O⁶-n-butyldeoxyguanosine, O²-n-butylthymidine and O⁴-n-butylthymidine respectively, corresponding to affinity constants of 2.7 × 10¹⁰, 1.1 × 10¹⁰ and 8.8 × 10⁸ respectively. In competitive radioimmunoassays a similar degree of inhibition required ∼10⁷-fold higher concentrations of normal deoxy-nucleotides. From the relative inhibitions produced by a wide range of alkylated and normal nucleosides and bases we conclude that each antibody primarily recognises butylation, as compared to other alkylations, at the sites specified by the immunogen. The radioimmunoassays should be suitable for the detection of these potentially promutagenic lesions in digests of DNA exposed to low (biological) levels of butylation.