Quantitative study of stereospecific binding of monoclonal antibody to anti-benzo(a)pyrene diol epoxide-N-2-dG adducts by capillary electrophoresis immunoassay

摘要

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 (K-b: 3.57 x 10(8) M-1)among all four investigated anti-BPDE-dG mononucleoside adducts, and the cis-(-)-anti-BPDE-dG displays lowest affinity (K-b: 1.14 x 10(7) 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 (K-b: 6.36 +/- 0.54 x 10(8) M-1) than trans-(-)-anti-BPDE-90mers (K-b: 4.52 +/- 0.52 x 10(8) M-1). The mAb 8E11 also displays high affinity with BPDE-dG adducts in genomic DNA (K-b: 3.74 x 10(8) M-1), indicating its promising applications for sensitive immuno-detection of BPDE-DNA adducts in genomic DNA. (C) 2010 Elsevier B.V. All rights reserved.