Original Contribution IMMUNODETECTION OF 3-NITROTYROSINE IN THE LIVER OF ZYMOSAN- TREATED RATS WITH A NEW MONOCLONAL ANTIBODY: COMPARISON TO ANALYSIS BY HPLC ISABELLE GIRAULT,* ALEXANDER E. KARU, † MANUELA SCHAPER, ‡ MARY HELEN BARCELLOS-HOFF, § TORY HAGEN,* DAVID S. VOGEL, † BRUCE N. AMES,* ¶ STEPHAN CHRISTEN, ‡ and MARK K. SHIGENAGA* ¶ *Division of Biochemistry and Molecular Biology, University of California, Berkeley CA, USA; † Immunochemistry Facility, College of Natural Resources, University of California, Berkeley CA, USA; ‡ Institute for Infectious Diseases, University of Berne, Berne, Switzerland; § Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley CA, USA; and ¶ Children’s Hospital Oakland Research Institute, Oakland, CA, USA (Received 25 May 2001; Accepted 23 August 2001) Abstract—Zymosan-induced peritonitis is associated with an increased production of reactive nitrogen oxides that may contribute to the often-observed failure of multiple organ systems in this model of acute inflammation. Quantitative biochemical evidence is provided for a marked 13-fold increase in protein-bound 3-nitrotyrosine (NTyr), a biomarker of reactive nitrogen oxides, in liver tissue of zymosan-treated rats. In order to investigate the localization of NTyr in this affected tissue, a monoclonal antibody, designated 39B6, was raised against 3-(4-hydroxy-3-nitrophenylacetamido) propionic acid-bovine serum albumin conjugate and its performance characterized. 39B6 was judged by competition ELISA to be 2 orders of magnitude more sensitive than a commercial anti-NTyr monoclonal antibody. Binding characteristics of 39B6 were similar, but not identical, to that of a commercial affinity-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 homogenates of zymosan-treated rats. Immuno- histochemical 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 fractions enriched in these cell populations corroborated the immunofluorescent data, although the relative amounts detected in response to zymosan treatment was greatly reduced compared to whole liver tissue. These results demonstrate the high specificity of the newly developed antibody and its usefulness in Western blot and immunohistochemical analysis for NTyr, confirm the presence of NTyr by complementary methods, and suggest the possible involvement of reactive nitrogen oxides in hepatic vascular dysfunction. © 2001 Elsevier Science Inc. Keywords—Antibody, 3-Nitrotyrosine, Tyrosine nitration, Nonparenchymal cells, Free radicals, Zymosan, Inflamma- tion INTRODUCTION • NO and its reactive byproducts, which include peroxyni- trite [1,2], • NO 2 [3], and ClNO 2 [4], can damage cellular constituents in vitro. There is considerable evidence that cellular injury during inflammation [5–7] and a number of pathological and degenerative conditions [8,9] result from overproduction of these reactive species in vivo. NTyr, a stable endproduct of the reaction between nitrat- ing species and Tyr, is regarded as a diagnostic marker for these agents [10] and thus is useful in identifying their formation in vivo. Immunohistochemical detection of NTyr with anti- bodies raised against peroxynitrite-treated keyhole lim- pet hemocyanin [11,12] has been employed in numerous studies designed to detect NTyr in tissues affected by reactive nitrogen oxides such as peroxynitrite. Evidence linking NTyr immunoreactivity to sites of tissue injury associated with the elaboration of reactive nitrogen ox- ides suggests that NTyr may be deleterious or serve as an indicator of endogenous biochemical processes associ- Address correspondence to: Dr. Mark Shigenaga, Children’s Hospi- tal, Oakland Research Institute (CHORI), 5700 Martin Luther King Way, Oakland, CA 94609, USA; Tel: (510) 428-3885, x2882; Fax: (510) 450-7910; E-Mail: mshigenaga@chori.org or shigen@uclink4. berkeley.edu. Free Radical Biology & Medicine, Vol. 31, No. 11, pp. 1375–1387, 2001 Copyright © 2001 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/01/$–see front matter PII S0891-5849(01)00712-2 1375