NITROTYROSINE IN PLASMA OF CELIAC-DISEASE PATIENTS AS DETECTED BY A NEW SANDWICH ELISA

Inflammation is characterized by increased nitric oxide production. Ni trotyrosine has recently been suggested to be useful as a marker for N O-mediated, tissue damage. In context of the development of an ELISA f or detection of nitrotyrosine in plasma, monoclonal anti-nitrotyrosine antibodies were developed by injecting mice with nitrated keyhole lim pet hemocyanin. The specificity of the antibodies was determined by bi nding to nitrated BSA, lack of binding to unmodified BSA, tyrosine, 3- chlorotyrosine or phenylalanine and inhibition of binding by nitrotyro sine. The antibodies developed are useful for Western blot analysis an d immunohistochemical staining. Using these antibodies a nitrotyrosine sandwich ELISA was developed with a lower detection limit of approxim ately 0.2 nM. The intra- and interassay variance were 2.4% and 11.9%, respectively. Using this newly developed ELISA, 1.27 +/- 1.03 mu M nit rotyrosine was detected in plasma samples of celiac disease patients w hereas nitrotyrosine was undetectable in control samples. Elevated nit rotyrosine levels were paralleled by an increase in plasma concentrati ons of NO-oxidation products (NOx), nitrite and nitrate from 15.1 +/- 6.1 mu M in controls to 61.0 +/- 28.2 mu M in celiac disease patients. Both nitrotyrosine and NOx levels declined when the patients were on a gluten-free diet, suggesting a relation between intestinal inflammat ion and plasma nitrotyrosine and NOx levels. (C) 1998 Elsevier Science Inc.