Quantitation of O6-ethyldeoxyguanosine in ENU alkylated DNA by polyclonal and monoclonal antibodies.

Rabbit polyclonal and mouse monoclonal antibodies were developed against O6-ethylguanosine conjugated with keyhole limpet hemocyanin. Radioimmunoassay (RIA) and a modified enzyme-linked immunosorbant assay (ELISA) were established for the determination of antigen-antibody binding and quantitation of potentially mutagenic O6-ethyldeoxyguanosine (O6-EtdGuo) in DNA treated with ethylnitrosourea (ENU) in vitro and in vivo. Optimum as well as reproducible antibody binding could be observed with conjugate concentrations at 0.1 ng/well immobilized by overnight drying at 37 degrees C. RIA was several-fold more sensitive than ELISA in detecting inhibition with O6-EtdGuo requiring 0.1 pmol for the 50% inhibition of tracer-antibody binding. In competitive inhibition assays with polyclonal and monoclonal antibodies, a linear dose resonse relation was obtained with DNA hydrolyzates alkylated in vitro with increasing concentrations of ENU. Significantly lower modification levels, e.g., 16.0 fmol O6-EtdGuo, at an O6-EtdGuo/dGuo molar ratio of 2.6 X 10(-7) in a hydrolyzate of 80 micrograms rat liver DNA ethylated in vivo with 10 micrograms ENU/g body weight was determined immunologically. The rate of elimination of O6-EtdGuo determined in human fetal kidney epithelial cells treated with 0.65 mM ENU showed that 50% of initial O6-EtdGuo was removed within 1 h followed by a slow phase of repair with 23% remaining at 8 h post-treatment.