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 DNA modified with (+/-)7 beta,8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE I) were quantitated by electron microscopy (EM) visualization and these observations were compared with quantitation of adducts by enzyme-linked immunosorbent assay (ELISA). The antiserum, elicited in rabbits following inoculation with BPDE I-modified DNA, has been found to be highly specific in its recognition of BPDE I-deoxyguanosine moieties. Parallel DNA samples prepared for analysis by ELISA and EM quantitation were randomized, encoded, and analyzed to determine extents of carcinogen modification in double-blind studies. After levels of modification were determined by immunoassays, DNA samples were prepared for EM analysis by incubation with amounts of anti-BPdG-DNA serum in excess of that necessary for complete binding of antibody to antigenic sites. At equilibrium, samples were enzymatically digested with papain in order to cleave anti-BPdG-DNA IgG molecules into Fab fragments in situ. Following column exclusion chromatography, BPdG-DNA-Fab complexes were incubated with ferritin-labeled Fab' fragments of goat [anti-rabbit F(ab')2] IgG in amounts in excess of those necessary for complete binding. When DNA samples were modified to between 0 and 40 fmol adduct/micrograms DNA, excellent agreement was obtained between ELISA quantitation and visualization by EM of antibodies bound to adducts.