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 b y enzyme-linked immunoadsorbent assays (ELISA). Racemic xy-t-9,10-epox y-7,8,9,10-tetrahydro-benzo[a]pyrene ((+/-)-anti-BPDE) modified DNA sa mples were produced in vitro, by reacting (+/-)-anti-BPDE with calf th ymus 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 tricapryli n). The BPDE adduct content in vitro and in liver and lung modified DN A was detected by direct and competitive ELISA using serial dilutions of the samples in unmodified calf thymus DNA, and polyclonal rabbit im munoglobulin-G elicited toward BPDE-DNA and BPDE-gelatin, both produce d in our laboratory. The carcinogen-macromolecule conjugate in which a dducts were sought could be used as an immunogen to produce a specific and potent antibody. Moreover, the modification level of the ELISA st andards should be as close to the range as of the biological samples t o correctly calculate the adducts, since different binding efficiency between antibody and BPDE-modified DNA is dependent on the BPDE modifi cation level (33). Appropriate extraction of the in vitro modified sam ples is also necessary to guarantee the exact covalent modification le vel, eliminating noncovalently linked BPDE. Under these conditions, ou r results confirm that competitive ELISA is much more sensitive than t he direct method mainly because of the limitations caused by the coati ng of the antigen in each well (max 5 mu g DNA/well), whereas the amou nt 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/mu gDNA (1.6 adducts/10(7) nucleotides).