Quantitative study of stereospecific binding of monoclonal antibody to anti-benzo(a)pyrene diol epoxide-N(2)-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-90 mers 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 x10(7) M(-1)). The binding of monoclonal antibody (mAb) 8E11 to BPDE-dG adducts in long DNA (90 mer) preferentially forms the complex with a stoichiometry of 1:1, and that mAb 8E11 displays a slightly higher affinity with trans-(+)-anti-BPDE-90 mers (K(b): 6.36+/-0.54 x 10(8)M(-1)) than trans-(-)-anti-BPDE-90 mers (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.
Wang
Chao
C
State Key Laboratory of Environmental Chemistry and Eco-toxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Haidian Distr., Beijing 100085, China.
Li
Tao
T
Wang
Zhixin
Z
Feng
Feng
F
Wang
Hailin
H
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001667:Binding, Competitive; D045744:Cell Line, Tumor; D018736:DNA Adducts / Q000378:metabolism*; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D005453:Fluorescence; D015894:Genome, Human / Q000235:genetics; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007834:Lasers; D016014:Linear Models; D012235:Rhodamines / Q000378:metabolism; D013237:Stereoisomerism
D000911:Antibodies, Monoclonal; D018736:DNA Adducts; D012235:Rhodamines; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; C005358:tetramethylrhodamine; C040060:7,8-dihydroxy-9,10-epoxide-7,8,9,10-tetrahydrobenzo(a)pyrene-10-deoxyguanosine; D003849:Deoxyguanosine