Introduction

Oxidative stress is known to play an important role in the development of various diseases and aging process. Under conditions of oxidative stress,8-hydroxy-2'-deoxyguanosine (8-OHdG) is formed when DNA is oxidatively damaged by reactive oxygen species (ROS). 8-OHdG is one of the most sensitive biomarkers for oxidative stress and can be detected in urine, plasma and/or DNA isolated from cells and tissues in humans and animals. The figure below depicts the oxidation 2'-deoxyguanosine (2'dG) to form the 8-OHdG biomarker.

Summary of Test

The NWLSS™ 8-OHdG ELISA assay uses a competitive format wherein a murine monoclonal antibody to 8-OHdG (Primary Antibody) and sample or standard are added to a microtiter plate which has been precoated with 8-OHdG. During an overnight incubation, sample or calibrator 8-OHdG competes with plate-bound 8-OHdG for binding with the antibody. Accordingly, higher concentrations of sample or calibrator lead to reduced binding of the antibody to the 8-OHdG coated on the plate. A subsequent wash step removes any free 8-OHdG/antibody adduct leaving stationary plate bound 8-OHdG complexed to antibody for later detection. Anti-murine antibody conjugated to horse radish peroxidase (HRP-Conjugate) is then added to the plate. HRP-conjugate binds to remaining murine anti-8-OHdG and unbound HRP-conjugate is removed in another wash step. Addition of 3,3',5,5'tetramethylbenzidine (TMB Substrate) results in blue color development proportional to the amount of anti 8OHdG antibody bound to the plate and inversely proportional to the concentration 8-OHdG in original samples or calibrators applied to the plate. The reaction is terminated by addition of phosphoric acid (Stop Solution) producing yellow color with measurable absorbance at 450 nm.