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).