Nitrate (NO3) and nitrite (NO2) production are typically used as markers of nitric oxide production (NO) production. These analytes by themselves fail to address the eventual fate of NO or the possible adverse effects associated with its excess production and reaction with free radical species in vivo. Active NO metabolites can react with superoxide to form peroxynitrite (ONOO-) a powerful oxidant and nitrating agent. Subsequent reaction of peroxynitrite with proteins results in nitrotyrosine formation. As a stable end product of peroxynitrite mediated oxidation/nitration, Nitrotyrosine can be used as a surrogate index of NO dependent damage in vivo and has been associated with multiple disease states.
The NWLSS™ Nitrotyrosine ELISA test is a simple "sandwich" ELISA using a plate bound capture antibody (anti nitrated KLH) to nitrotyrosine and a biotinylated secondary tracer antibody. Addition of streptavidin-peroxidase followed by tetramethylbenzidine (TMB) facilitates color development directly proportional to the nitrotyrosine present in the sample. The reaction is stopped using a citric acid solution and the assay is read on a plate reader at 450 nm.