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.
This assay features a “competitive” ELISA format in which standards and samples are first incubated with a biotynylated Tracer Antibody in a separate U-bottom microplate. After incubation, the standards and samples are transferred to a microplate precoated with nitrated-HSA and allowed to incubate for an additional hour.
During this step unbound Biotinylated Tracer Antibody binds to stationary phase NO-HAS. A subsequent wash removes excess Tracer and Tracer/protein complex. Addition of streptavidin-peroxidase followed by tetramethylbenzidine (TMB) facilitates color development inversely proportional to the nitrotyrosine present in the sample. The reaction is stopped using an oxalic acid solution and the assay is read on a plate reader at 450 nm.