Development of monoclonal antibodies recognizing 7-(2-hydroxyethyl)guanine and imidazole ring-opened 7-(2-hydroxyethyl)guanine

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Abstract
7-(2-Hydroxyethyl)guanine (7HEG) is of biological interest because it is formed in vivo by reaction of DNA with ethylene oxide (EO). Furthermore, the major DNA adduct of vinyl chloride, 7-(2-oxyethyl)guanine, can be converted to this adduct by reduction. Two monoclonal antibodies (9E2, 4A5) recognizing 7HEG have been developed from BALB/c mice immunized with the adduct coupled to keyhole limpet hemocyanin. In addition, another antibody (8E10) was developed against the imidazole ring-opened form of the adduct (ro-7HEG). ELISAs were used to determine the sensitivity and specificity of these antibodies. With antibody 9E2, 50% inhibition of antibody binding in the competitive ELISA was at 54 pmol of the modified base 7HEG/well and 67 pmol 7HEGR/well, while with antibody 4A5, the values were 3.6 pmol 7HEG/well and 6.7 pmol 7HEGR/well. Antibody 8E10 gave 50% inhibition at 48 pmol ro-7HEGR/well. Neither antibody 9E2 nor 8E10 cross-reacted with unmodified DNA or with the normal nudeosides at the highest concentration tested. However, antibody 4A5 had a low affinity for deoxyguanosine (50% inhibition at 31 000 pmol). Sensitivity of adduct measurement can be increased 3- to 10-fold using an ELISA with fluorescence endpoint detection. These antibodies have been used to determine the level of adducts in DNA modified in vitro with [3H]- or [14C]EO. Because of the cross-reactivity of the most sensitive antibody, 4A5, with deoxyguanosine, a combined HPLC/immunoassay method was developed to quantitate 7HEG in DNA. The limit of sensitivity of this method is dependent upon the amount of DNA available for analysis. Using 30 fmol as the lowest detectable amount (20% inhibition) in the fluorescent ELISA with antibody 4A5 and 100 μg of DNA assayed per well, adduct levels of 1/107 nucleotide can be determined. This method was applied to DNA adduct detection in EO-treated myeloma cells and whole blood. Antibody 8E10 was also used in immunohistochemical studies to visualize ring-opened adducts in cells treated with EO followed by high pH. These antibodies will be used for the detection and quantitation of adducts in human samples.