ABSTRACT
Monoclonal antibodies have been obtained against imidazole ring-opened N7-ethylguanine (RON7-EtGua) in DNA. The antibodies were selected for good performance in the ELISA with either DNA or nucleated blood cells as immobilized antigen. Antibodies thus selected were studied for their suitability for the in situ detection of RON7-EtGua in the nuclei of cells by means of immunofluorescence microscopy (IFM). Two antibodies have been characterized in detail with respect to specificity and sensitivity. Competitive ELISA demonstrated that the antibodies recognize not only RON7-EtGua but 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 adducts and show an at least 100-fold stronger preference for such structures in DNA when compared to the free nucleoside adducts. Furthermore, they hardly bind to non-alkylated DNA, and do not bind to guanosine or N1- or O6-ethylguanosine. Analysis by DNA-ELISA showed that the binding preference of antibody N7E-026 for ring-opened alkyl adducts is methyl approximately ethyl greater than 2-hydroxyethyl much greater than sulphur mustard, while that of N7E-102 is 2-hydroxyethyl greater than ethyl greater than methyl approximately sulphur mustard. Analysis of RON7-EtGua in DNA with competitive ELISA, DNA-ELISA and IFM showed that in all cases the lowest detection limit can be reached with antibody N7E-026. Competitive ELISA was the most sensitive method, followed by DNA-ELISA and IFM, with detection limits of 2.2, 16 and 23 RON7-EtGua/10(6) nucleotides respectively. In the DNA-ELISA, 12 methyl adducts/10(6) nucleotides can be detected with N7E-026 and 11 2-hydroxyethyl adducts/10(6) nucleotides with N7E-102.
Subject(s)
Antibodies, Monoclonal/isolation & purification , DNA/analysis , Guanine/analogs & derivatives , Mustard Gas/metabolism , Animals , Antibodies, Monoclonal/immunology , Antibody Specificity , Cross Reactions , Enzyme-Linked Immunosorbent Assay , Guanine/analysis , Guanine/immunology , Guanine/metabolism , Humans , Methylation , Mice , Mice, Inbred BALB C , RatsABSTRACT
A method has been developed for the determination of N7-(2-hydroxyethyl)-guanine (N7-EtOHGua) via HPLC with electrochemical detection (EC). N7-EtOHGua is the major base adduct formed in DNA upon exposure to ethylene oxide. N7-EtOHGua, released from DNA, was separated from the unmodified nucleobases by chromatography on a reversed-phase column. For electrochemical detection, an amperometric detector cell was used with a glassy carbon working electrode, set at 1.35 V relative to an Ag/AgCl reference electrode. With purified N7-EtOHGua a linear dose-response relation was observed in the range between 0.11 and 13 pmol. The signal-to-noise ratio during analysis of 0.11 pmol N7-EtOHGua was about 8 to 1. Determination of adducts in a series of DNA samples treated with 0.16-10 mM ethylene oxide showed a linear dose-dependent increase in the level of N7-modifications. For DNA samples, the detection limit of this HPLC-EC analysis is 1 N7-EtOHGua per 6 x 10(6) nucleotides.
