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.
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D001564:Benzo(a)pyrene / Q000276:immunology / Q000378:metabolism*; D001580:Benzopyrenes / Q000378:metabolism; D004247:DNA / Q000276:immunology / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D051379:Mice; D008854:Microscopy, Electron
D000906:Antibodies; D001580:Benzopyrenes; D001564:Benzo(a)pyrene; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D004247:DNA