Development of a Monoclonal Antibody Recognizing Benzo[c]phenanthrenediol Epoxide-DNA Adducts:  Application to Immunohistochemical Detection of DNA Damage

Abstract

A monoclonal antibody was developed against (+/-)-anti-benzo[c]phenanthrenediol epoxidemodified DNA, and sensitivity and specificity were determined by competitive enzyme-linked immuno-sorbent assay (ELISA). Antibody 10F9 has 50% inhibition in the ELISA at 50 fmol of B[c]PhDE-DNA adducts. There was weak cross-reactivity with DNA modified by (+/-)-anti-benzo[a]pyrenediol epoxide (50% inhibition at 150 pmol). Testing of oligonucleotides containing either (+)- or (-)-trans-anti-B[c]PhDE-adenine adducts indicated similar recognition of both stereoisomers. A quantitative immunoperoxidase technique with antibody 10F9 was developed using 10T1/2 cells treated with B[c]PhDE then piloted on exfoliated oral cells from five smokers and five nonsmokers. Mean staining in smokers (184 +/- 11) was 1.64-fold higher than in nonsmokers (112 +/- 9, p < 0.0001). This antibody should be useful for the detection and quantitation of B[c]PhDE-DNA adducts in cell culture and animal studies and in humans with environmental or occupational exposure to polycyclic aromatic hydrocarbons.

DNA adducts: Formation, biological effects, and new biospecimens for mass spectrometric measurements in humans

“…Unfortunately, the biomonitoring of DNA adducts in FFPE tissue, by MS‐based measurements, has not advanced because of the difficulties in recovering high quality DNA that is fully reversed of crosslinks under mild conditions, and completely digestible, by nucleases, for quantitative chemical measurements . Thus, the analysis of DNA damage in FFPE tissue has been largely conducted by IHC techniques, for which only a limited set of antibodies are available . Recently, vendors of commercial kits for cancer genomics have reported that the cross‐links of DNA in FFPE biospecimens can be unraveled under mild temperature and pH conditions, and the DNA can serve as templates for high fidelity PCR.…”