Quantitative study of stereospecific binding of monoclonal antibody to anti-benzo(a)pyrene diol epoxide-N2-dG adducts by capillary electrophoresis immunoassay

The stereospecific binding of monoclonal antibody (mAb) 8E11 to anti-benzo(a)pyrene diol epoxide (BPDE)-dG adducts in single nucleoside, long oligonucleotide, and genomic DNA were quantitatively evaluated using noncompetitive and competitive capillary electrophoresis (CE) immunoassays. Two single-stranded TMR-BPDE-90mers containing a single anti-BPDE-dG adduct with defined stereochemistry and a fluorescent label at 5′-end were used as fluorescent probes for competitive CE immunoassay. To quantitatively evaluate the binding affinity through competitive CE immunoassays, a series of equations were derived according to the binding stoichiometry. The binding of mAb 8E11 to trans-(+)-anti-BPDE-dG displays strongest affinity (Kb: 3.57 × 108 M−1) among all four investigated anti-BPDE-dG mononucleoside adducts, and the cis-(−)-anti-BPDE-dG displays lowest affinity (Kb: 1.14 ×107 M−1). The binding of monoclonal antibody (mAb) 8E11 to BPDE-dG adducts in long DNA (90mer) preferentially forms the complex with a stoichiometry of 1:1, and that mAb 8E11 displays a slightly higher affinity with trans-(+)-anti-BPDE-90mers (Kb: 6.36 ± 0.54 × 108 M−1) than trans-(−)-anti-BPDE-90mers (Kb: 4.52 ± 0.52 × 108 M−1). The mAb 8E11 also displays high affinity with BPDE-dG adducts in genomic DNA (Kb: 3.74 × 108 M−1), indicating its promising applications for sensitive immuno-detection of BPDE-DNA adducts in genomic DNA.