Nitrotyrosine has been identified as a marker of inflammation and NO production. Nitrotyrosine is formed in presence of the active metabolite NO. Various pathways including the formation of peroxinitrite lead to nitrotyrosine production. Since nitrotyrosine is a stable endproduct of peroxynitrite oxidation, assesment of its plasma concentration may be useful as a marker of NO-dependent damage in vivo. Since NOx is only an indicator for enhanced NO production, protein associated nitrotyrosine might be a more suitable marker for damage induced by reactive nitrogen intermediates derived from NO. Furthermore, most proteins have a longer half life in the circulation than NOx levels. The presence of nitrotyrosine has been detected in various inflammatory processes including atherosclerotic placques, celiac disease, rheumatoid arthritis, chronic renal failure and septic shock. In normal plasma low, undetectable, levels of nitrotyrosine are present. Nitrosylation of the aminoacid tyrosine occurs both for free tyrosine and for protein bound tyrosine. The ELISA detects nitrotyrosine containing proteins. Since the assay detects a modified aminoacid the assay is useful for proteins of all species.
The Nitrotyrosine ELISA kit is to be used for the in vitro quantitative determination of nitrotyrosine in plasma and other biological samples.
The Nitrotyrosine ELISA is a ready-to-use solid-phase enzyme-linked immunosorbent assay based on the sandwich principle with a working time of 3½ hours.
The efficient format of a plate with twelve disposable 8-well strips allows free choice of batch size for the assay.
Samples and standards are incubated in microtiter wells coated with antibodies recognizing nitrotyrosine.
Biotinylated tracer antibody will bind to captured nitrotyrosine.
Streptavidin-peroxidase conjugate will bind to the biotinylated tracer antibody.
Streptavidin-peroxidase conjugate will react with the substrate, tetramethylbenzidine (TMB).
The enzyme reaction is stopped by the addition of citric acid.
The absorbance at 450 nm is measured with a spectrophotometer. A standard curve is obtained by plotting the absorbance (linear) versus the corresponding concentrations of the nitrotyrosine standards (log).
The nitrotyrosine concentration of samples, which are run concurrently with the standards, can be determined from the standard curve.