Chemiluminescence Enzyme Immunoassay of 8-Oxoguanine in DNA

V. I. Bruskov^1,2*, Zh. K. Masalimov², and A. M. Usacheva¹

¹Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, 142292 Russia

²Pushchino State University, Pushchino, Moscow Region, 142492 Russia; fax: (0967) 79-0553; E-mail: bruskov@venus.iteb.serpukhov.su

^* To whom correspondence should be addressed.

Received November 3, 1998; Revision received December 29, 1998

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A test system has been developed to determine 8-oxoguanine in DNA, the most important biomarker of damage to DNA bases by reactive oxygen species. The system is based on a chemiluminescence enzyme immunoassay with the use of monoclonal antibodies (mcAB) against 8-oxoguanine. The test involves several stages: 1) immobilization of DNA on nitrocellulose membrane filters using an efficient technique with preliminary formation of a complex with protamine sulfate; 2) formation of antigen--antibody complexes (mcAB with 8-oxoguanine in DNA) with secondary antibodies and with a peroxidase--antiperoxidase complex (PAP method); 3) detection of increased chemiluminescence in a solution of hydrogen peroxide, luminol, and p-iodophenol. The increased chemiluminescence is determined with a conventional liquid scintillation counter for measuring beta-radioactivity. The system was tested by determining 8-oxoguanine formation in DNA upon gamma-irradiation and upon photosensitized oxidation of guanine under visible light in the presence of methylene blue. A linear dose dependence of 8-oxoguanine formation in DNA was shown for gamma-irradiation. The radiation-chemical yield of 8-oxoguanine (G = 0.57 molecule per 100 eV) is convenient to use for calibration of the amount of 8-oxoguanine formed under other conditions. The sensitivity of the method permits the detection of several femtomoles of 8-oxoguanine in a 40 µg sample of DNA.

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KEY WORDS: 7,8-dihydro-8-oxoguanine (8-hydroxyguanine), DNA damage, reactive oxygen species, enzyme immunoassay, enhanced chemiluminescence, monoclonal antibodies

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