Quantitation by electron microscopy of the binding of highly specific antibodies to benzo[a]pyrene-DNA adducts

Highly specific antibodies bound to carcinogen adducts in DNAmodified with (+ / - )7ß, 8-dihydroxy-9, 10-epoxy-7,8, 9, 10-tetrahydrobenzo[a]pyrene (BPDE I) were quantitatedby electron microscopy (EM) visualization and these observationswere compared with quantitation of adducts by enzyme-linkedimmunosorbent assay (ELISA). The antiserum, elicited in rabbitsfollowing inoculation with BPDE I-modified DNA, has been foundto be highly specific in its recognition of BPDE I-deoxyguanosinemoieties. Parallel DNA samples prepared for analysis by ELISAand EM quantitation were randomized, encoded, and analyzed todetermine extents of carcinogen modification in double-blindstudies. After levels of modification were determined by immunoassays,DNA samples were prepared for EM analysis by incubation withamounts of anti-BPdG-DNA serum in excess of that necessary forcomplete binding of antibody to antigenic sites. At equilibrium,samples were enzymatically digested with papain in order tocleave anti-BPdG-DNA IgG molecules into Fab fragments in situ.Following column exclusion chromatography, BPdG-DNA-Fab complexeswere incubated with ferritin-labeled Fab‘ fragments ofgoat [anti-rabbit F(ab’)2] IgG in amounts in excess ofthose necessary for complete binding. When DNA samples weremodified to between 0 and 40 fmol adduct/ µg DNA, excellentagreement was obtained between ELISA quantitation and visualizationby EM of antibodies bound to adducts.