Nitrotyrosine in plasma of celiac disease patients as detected by a new sandwich ELISA

Inflammation is characterized by increased nitric oxide production. Nitrotyrosine has recently been suggested to be useful as a marker for NO-mediated tissue damage. In context of the development of an ELISA for detection of nitrotyrosine in plasma, monoclonal anti-nitrotyrosine antibodies were developed by injecting mice with nitrated keyhole limpet hemocyanin. The specificity of the antibodies was determined by binding to nitrated BSA, lack of binding to unmodified BSA, tyrosine, 3-chlorotyrosine or phenylalanine and inhibition of binding by nitrotyrosine. The antibodies developed are useful for Western blot analysis and immunohistochemical staining. Using these antibodies a nitrotyrosine sandwich ELISA was developed with a lower detection limit of approximately 0.2 nM. The intra- and interassay variance were 2.4% and 11.9%, respectively. Using this newly developed ELISA, 1.27 ± 1.03 μM nitrotyrosine was detected in plasma samples of celiac disease patients whereas nitrotyrosine was undetectable in control samples. Elevated nitrotyrosine levels were paralleled by an increase in plasma concentrations of NO-oxidation products (NOX), nitrite and nitrate from 15.1 ± 6.1 μM in controls to 61.0 ± 28.2 μM in celiac disease patients. Both nitrotyrosine and NOX levels declined when the patients were on a gluten-free diet, suggesting a relation between intestinal inflammation and plasma nitrotyrosine and NOX levels.