A COLORIMETRIC METHOD FOR QUANTIFICATION OF OXIDATIVE DAMAGE TO DNA USING ELISA-LIKE ASSAY.

Abstract

DNA damage induced by reactive oxygen species has attracted considerable interest in recent years and has generated extensive discussion on the relevance of spontaneous DNA damage to aging and to cancer in humans. Although several methods have been used to measure the amount of DNA damage, they are either laborious, use radioactivity or are hard to replicate. A better candidate for an easy and sensitive way to detect DNA damage is a novel reagent prepared by reacting 0- (carboxymethyl)hydroxylamine with biotin hydrazide in the presence of carbodimide. The product of this reaction is a reagent called Aldehyde Reactive Probe (ARP). One of the many damages from reactive oxygen species due to attack by hydroxyl radical on the deoxyribose moiety is a base loss in the form of an apurinic (AP site). During Enzyme immunoassay, the ARP reagent acts as a bridge as it tags the damaged DNA molecule's AP site through its amino terminal and the enzyme through its avidin/biotin complex. The number of AP sites can then be determined by biotin/avidin system followed by enzymatic colorimetric detection. This system can measure the number of AP sites as low as 1 AP/10⁵ base pairs.