Immunodetection of 3-nitrotyrosine in the liver of zymosan-treated rats with a new monoclonal antibody: Comparison to analysis by HPLC

Zymosan-induced peritonitis is associated with an increased production of r eactive nitrogen oxides that may contribute to the often-observed failure o f multiple organ systems in this model of acute inflammation. Quantitative biochemical evidence is provided for a marked 13-fold increase in protein-b ound 3-nitrotyrosine (NTyr), a biomarker of reactive nitrogen oxides, in li ver tissue of zymosan-treated rats. In order to investigate the localizatio n of NTyr in this affected tissue, a monoclonal antibody, designated 39B6, was raised against 3-(4-hydroxy-3-nitrophenylacetamido) propionic acid-bovi ne serum albumin conjugate and its performance characterized. 39B6 was judg ed by competition ELISA to be approximate to2 orders of magnitude more sens itive than a commercial anti-NTyr monoclonal antibody. Binding characterist ics of 39B6 were similar, but not identical, to that of a commercial affini ty-purified polyclonal antibody in ELISA and immunohistochemical analyses. Western blot experiments revealed high specificity of 39B6 against NTyr and increased immunoreactivity of specific proteins from liver tissue homogena tes of zymosan-treated rats. Immunohistochemical analysis of liver sections indicated a marked zymosan-induced increase in immunofluorescent staining, which was particularly intense in or adjacent to nonparenchymal cells, but not in the parenchymal cells of this tissue. Quantitative analysis of frac tions enriched in these cell populations corroborated the immunofluorescent data, although the relative amounts detected in response to zymosan treatm ent was greatly reduced compared to whole liver tissue. These results demon strate the high specificity of the newly developed antibody and its usefuln ess in Western blot and immunohistochemical analysis for NTyr, confirm the presence of NTyr by complementary methods, and suggest the possible involve ment of reactive nitrogen oxides in hepatic vascular dysfunction. (C) 2001 Elsevier Science Inc.