Detection of Oxidative DNA Base Damages
Antibodies to a variety of oxidized DNA bases have been generated in a number of laboratories including our own (see Table I). Most of these antibodies have been elicited using protein-conjugated haptens of interest. In general, the antibodies have reasonable affinity such that appropriate sensitivity in the various assays can be achieved. The difficulty with antibodies that recognize oxidized DNA bases is that the oxidized bases do not differ largely from their unoxidized derivatives. Thus, the specificity for detecting lesions in DNA must be high especially when one considers the low level of damaged compared to undamaged bases. Even if the sensitivity of the assay can be amplified, cross-reactivity of the antibody with the unoxidized base in DNA remains an obstacle to successful detection of low levels of the oxidized base. This particular problem is not seen as often when antibodies are elicited to the chemical adducts that have features that vary quite dramatically from the unadducted base. An additional consideration is that the lesion must be stable to the procedures used during preparation of the immunogen and during DNA denaturation. The latter is usually necessary since the antibodies often do not recognize the lesion as well in duplex DNA. Despite these shortcomings, antibodies to oxidized bases have been effectively utilized to detect these lesions in oxidized or ionizing radiation-treated DNA in vitro.
KeywordsCompetitive ELISA Isoamyl Alcohol Immunochemical Detection Guanidinium Hydrochloride Thymine Glycol
Unable to display preview. Download preview PDF.
- Barbas, C. F., III, and Lerner, R. A. (1991). Combinatorial immunoglobulin libraries on the surface of phage (Phabs):Rapid selection of antigen-specific Fabs, in:Methods: A Companion to Methods in Enzymology, Vol. 2 (R. A. Lerner and D. R. Burton, eds.), Academic Press, Orlando, pp. 119–124.Google Scholar
- Bespalov, I. A., Purmal, A. A., Erlanger, B. F., Wallace, S. S., and Melamede, R. J. (1996a). Antibodies to oxidized pyrimidines selected from combinatorial phage display libraries. Submitted for publication.Google Scholar
- Degan, P., Shigenaga, M. K., Park, E. M., Alperin, P. E., and Ames, B. N. (1991). Immunoaffinity isolation of urinary 8-hydroxy-2′-deoxyguanosine and 8-hydroxyguanine and quantitation of 8-hydroxy-2′-deoxyguano-sine in DNA by polyclonal antibodies. Carcinogenesis 12:865–871.PubMedCrossRefGoogle Scholar
- Fuciarelli, A. F., Shum, F. Y., and Raleigh, J. A. (1987). Intramolecular cyclization in irradiated nucleic acids: Correlation between high-performance liquid chromatography and an immunochemical assay for 8,5′-cycloadenosine in irradiated poly(A). Radiat. Res. 110:35–44.PubMedCrossRefGoogle Scholar
- Ide, H., Kow, Y. W, Chen, B.-X., Erlanger, B. R., and Wallace, S.S. (1996). Properties of antibodies elicited to 7-hydro-8-oxopurines and their reaction with X-irradiated DNA. Submitted for publication.Google Scholar
- Leadon, S. A. (1988). Immunological probes for lesions and repair patches in DNA, in:DNA Repair. A Laboratory Manual of Research Procedures (E. C. Friedberg and P.C. Hanawalt, eds.), Dekker, New York, pp. 311–326.Google Scholar
- Loft, S., Astrup, A., Buemann, B., and Poulsen, H. E. (1994). Oxidative DNA damage correlates with oxygen consumption in humans. FASEB J. 536:534–537.Google Scholar
- Park, E. M., Shigenaga, M. K., Degan, P., Korn, T. S., Kitzler, J. W, Wehr, C. M., Kolachana, P., and Ames, B. N. (1992). Assay of oxidized DNA lesions: Isolation of 8-oxoguanine and its nucleoside derivatives from biological fluids with a monoclonal antibody column. Proc. Natl. Acad. Sci. USA 89:3375–3378.PubMedCrossRefGoogle Scholar
- Shigenaga, M. K., Aboujaoude, E. N., Chen, Q., and Ames, B. N. (1994). Assays of oxidative DNA damage biomarkers 8-oxo-2′-deoxyguanosine and 8-oxoguanine in nuclear DNA and biological fluids by high-performance liquid chromatography with electrochemical detection, in:Methods in Enzymology: Oxygen Radicals in Biological Systems, Part D (L. Packer, ed.), Academic Press, San Diego, CA, pp. 16–33.CrossRefGoogle Scholar