Advantages of the NWLSS™ Dityrosine ELISA:
- Provides a non-invasive method for evaluating oxidative stress effect on proteins (eg protein oxidation) in urine.
- Specifically detects dityrosine as low as 50 nM.
- Suitable for detecting dityrosine in samples from all species.
Introduction
Under conditions of oxidative stress, certain amino acids are more or less susceptible to alterations by reactive oxygen species (ROS) and reactive nitrogen species (RNS). Among them, Cysteine, Methionine and Tyrosine have been widely investigated. Unlike, byproducts of Cysteine and Methionine oxidation, there are no biological mechanisms to repair oxidatively modified tyrosine so these byproducts are of particular interest in many diseases and aging. Nitrotyrosine, dityrosine, and dibromotyrosine have all been identified as potential protein related biomarkers of oxidative stress. Dityrosine, the subject of this new assay forms as a result of crosslinking of modified proteins. It has recently been shown to form preferentially over nitrotyrosine in the presence of low levels of peroxynitrite (ONOO-) and as such may prove to be another surrogate biomarker for ONOO-mediated damage. Dityrosine has been and continues to be investigated for its role in many biological processes including aging and age related diseases such as atherosclerosis, cataract formation and neurodegenerative diseases such as Parkinson’s and Alzheimer’s.
Test Principle
The NWLSS™ Dityrosine ELISA assay uses a competitive format wherein plate bound dityrosine and sample dityrosine are allowed to compete for binding with a primary antibody (murine anti-Dityrosine). Accordingly, higher concentrations of sample or calibrator leads to reduced binding of the primary antibody to the dityrosine coated plate. An anti-murine secondary antibody conjugated to horse radish peroxidase (Secondary Antibody/HRP Conjugate) is used to facilitate detection of of the bound primary antibody. Subsequent Addition of 3,3',5,5'tetramethylbenzidine (TMB Substrate) results in blue color development proportional to the amount of anti Dityrosine antibody bound to the plate and inversely proportional to the concentration Dityrosine in original samples or calibrators applied to the plate. The reaction is terminated by addition of sulphuric acid (Stop Solution) producing yellow color with measurable absorbance at 450 nm.
