The isolation of monoclonal antibodies selected for the detection of imidazole ring-opened N7-ethylguanine in purified DNA and in cells in situ. Crossreaction with methyl, 2-hydroxethyl and sulphur mustard adducts

Monoclonal antibodies have been obtained against imidazole ring-openedN7-ethylguanine (RON7-EtGua) in DNA. The antibodies were selectedfor good performance in the ELISA with either DNA or nucleatedblood cells as immobilized antigen. Antibodies thus selectedwere studied for their suitability for the in situ detectionof RON7-EtGua in the nuclei of cells by means of immunofluorescencemicroscopy (IFM). Two antibodies have been characterized indetail with respect to specificity and sensitivity. CompetitiveELISA demonstrated that the antibodies recognize not only RON7-EtGuabut also the corresponding methyl and 2-hydroxyethyl components,with efficiencies that vary with the chemical environment (base,nucleoside or DNA), the nature of the alkyl group and the antibody.They have a clear specificity for the ring-opened alkyl adductsand show an at least 100-fold stronger preference for such structuresin DNA when compared to the free nucleoside adducts. Furthermore,they hardly bind to non-alkylated DNA, and do not bind to guanosineor N1- or O⁶-ethylguanosine. Analysis by DNA-ELISA showed thatthe binding preference of antibody N7E-026 for ring-opened alkyladducts is methyl = ethyl > 2-hydroxyethyl sulphur mustard,while that of N7E-102 is 2-hydroxyethyl > ethyl > methyl= sulphur mustard. Analysis of RON7-EtGua in DNA with competitiveELISA, DNA-ELISA and IFM showed that in all cases the lowestdetection limit can be reached with antibody N7E-026. CompetitiveELISA was the most sensitive method, followed by DNA-ELISA andIFM, with detection limits of 2.2, 16 and 23 RON7-EtGua/10⁶nudeotides respectively. In the DNA-ELISA, 12 methyl adducts/10⁶nucleotides can be detected with N7E-026 and 11 2-hydroxyethyladducts/10⁶ nudeotides with N7E-102.