Identification of optimal conditions for the detection of benzo[a]pyrene-DNA adducts by enzyme-linked immunoadsorbent assays (ELISA).
The aim of the present report was to establish the optimal conditions for the detection of polycyclic aromatic hydrocarbon adducted to DNA by enzyme-linked immunoadsorbent assays (ELISA). Racemic 7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene ((+/-)-anti-BPDE) modified DNA samples were produced in vitro, by reacting (+/-)-anti-BPDE with calf thymus DNA, and in vivo in Swiss female mice by single i.p. injection of benzo[a]pyrene (B[a]P) (200 mg/kg body weight dissolved in tricaprylin). The BPDE adduct content in vitro and in liver and lung modified DNA was detected by direct and competitive ELISA using serial dilutions of the samples in unmodified calf thymus DNA, and polyclonal rabbit immunoglobulin-G elicited toward BPDE-DNA and BPDE-gelatin, both produced in our laboratory. The carcinogen-macromolecule conjugate in which adducts were sought could be used as an immunogen to produce a specific and potent antibody. Moreover, the modification level of the ELISA standards should be as close to the range as of the biological samples to correctly calculate the adducts, since different binding efficiency between antibody and BPDE-modified DNA is dependent on the BPDE modification level (33). Appropriate extraction of the in vitro modified samples is also necessary to guarantee the exact covalent modification level, eliminating noncovalently linked BPDE. Under these conditions, our results confirm that competitive ELISA is much more sensitive than the direct method, mainly because of the limitations caused by the coating of the antigen in each well (max 5 micrograms DNA/well), whereas the amount of DNA (modified or not) that can be employed for adduct detection by competitive ELISA increases 20-fold. The sensitivity obtained was 0.5 fmol B[a]P/microgramDNA (1.6 adducts/10(7) nucleotides).
Laboratory of Biochemistry, Regina Elena Institute for Cancer Research, Rome, Italy.
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Bucci
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Galati
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Zito
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Falasca
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Federico
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Verdina
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000032:analysis*; D000818:Animals; D001564:Benzo(a)pyrene / Q000032:analysis* / Q000378:metabolism / Q000493:pharmacokinetics*; D002274:Carcinogens, Environmental / Q000032:analysis; D002417:Cattle; D018736:DNA Adducts / Q000032:analysis* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D008099:Liver / Q000378:metabolism; D008168:Lung / Q000378:metabolism; D051379:Mice; D015203:Reproducibility of Results; D013056:Spectrophotometry, Ultraviolet / Q000379:methods
D002274:Carcinogens, Environmental; D018736:DNA Adducts; C043931:benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide-DNA; C056612:benzo(a)pyrene-DNA adduct; D001564:Benzo(a)pyrene; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide