Development of Methods for the Production of Monoclonal Antibodies Specific for Depurination Adducts of Benzo[a]pyrene and for Their Use in a Competitive Enzyme-linked Immunosorbent Assay

Nearly 80% of benzo[a]pyrene (BP)-DNA adducts are lost by depurination and excreted in the urine. These adducts offer a unique opportunity for biomonitoring BP exposure, although their hydrophobic nature poses challenges for methods development. We have implemented a competitive enzyme-linked immunosorbent assay (ELISA) for detection of 7-(benzo[a]pyren-6-yl)guanine (BP-6-N7Gua), a major depurination adduct of BP. Linkage of the adduct to keyhole limpet hemocyanin and bovine serum albumin via the heterobifunctional linker succinimidyl-4-(N-maleimido-methyl)-cyclohexane-l-carboxylate produced an effective immunogen and capture complex, respectively. Two cycles of hybridoma production yielded 23 monoclonal antibodies (MAb) specific for the capture complex. One of these MAb was used to optimize the competitive ELISA for BP-6-N7Gua. Conditions of the reaction between specific MAb and the free adduct were extensively modified to produce an assay that detects 50 fmol free adduct/mL reaction mixture.