Chemiluminescence enzyme immunoassay of 8-oxoguanine in DNA.
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 microg sample of DNA.
Bruskov
V I
VI
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, 142292, Russia. bruskov@venus.iteb.serpukov.su
Masalimov
Z K
ZK
Usacheva
A M
AM
D000818:Animals; D003101:Collodion; D004247:DNA / Q000737:chemistry* / Q000528:radiation effects; D004249:DNA Damage; D005720:Gamma Rays; D006147:Guanine / Q000031:analogs & derivatives* / Q000032:analysis; D007124:Immunoenzyme Techniques; D008163:Luminescent Measurements; D008751:Methylene Blue / Q000737:chemistry; D012474:Salmon; D012680:Sensitivity and Specificity
C024829:8-hydroxyguanine; D006147:Guanine; D003101:Collodion; D004247:DNA; D008751:Methylene Blue