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 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-b...
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| Veröffentlicht in: | Free radical biology & medicine 2001-12, Vol.31 (11), p.1375-1387 |
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| creator | Girault, Isabelle Karu, Alexander E Schaper, Manuela Barcellos-Hoff, Mary Helen Hagen, Tory Vogel, David S Ames, Bruce N Christen, Stephan Shigenaga, Mark K |
| description | 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. 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 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. |
| doi_str_mv | 10.1016/S0891-5849(01)00712-2 |
| format | Article |
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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. 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 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.</description><identifier>ISSN: 0891-5849</identifier><identifier>EISSN: 1873-4596</identifier><identifier>DOI: 10.1016/S0891-5849(01)00712-2</identifier><identifier>PMID: 11728809</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>3-Nitrotyrosine ; Animals ; Antibodies, Monoclonal - immunology ; Antibody ; Antibody Specificity ; Blotting, Western ; Chromatography, High Pressure Liquid ; Fluorescent Antibody Technique ; Free radicals ; Haptens - chemistry ; Haptens - immunology ; Immunoassay ; Immunohistochemistry ; Inflammation ; Liver - chemistry ; Liver - drug effects ; Male ; Mice ; Nonparenchymal cells ; Rats ; Rats, Inbred F344 ; Tissue Distribution ; Tyrosine - analogs & derivatives ; Tyrosine - analysis ; Tyrosine - immunology ; Tyrosine nitration ; Zymosan ; Zymosan - pharmacology</subject><ispartof>Free radical biology & medicine, 2001-12, Vol.31 (11), p.1375-1387</ispartof><rights>2001 Elsevier Science Inc.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c392t-3a6054d9b8f4a85c4c642cf77968a38336799fe7253b6ca915570120bc236ed43</citedby><cites>FETCH-LOGICAL-c392t-3a6054d9b8f4a85c4c642cf77968a38336799fe7253b6ca915570120bc236ed43</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/S0891584901007122$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,776,780,3537,27901,27902,65306</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/11728809$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Girault, Isabelle</creatorcontrib><creatorcontrib>Karu, Alexander E</creatorcontrib><creatorcontrib>Schaper, Manuela</creatorcontrib><creatorcontrib>Barcellos-Hoff, Mary Helen</creatorcontrib><creatorcontrib>Hagen, Tory</creatorcontrib><creatorcontrib>Vogel, David S</creatorcontrib><creatorcontrib>Ames, Bruce N</creatorcontrib><creatorcontrib>Christen, Stephan</creatorcontrib><creatorcontrib>Shigenaga, Mark K</creatorcontrib><title>Immunodetection of 3-nitrotyrosine in the liver of zymosan-treated rats with a new monoclonal antibody: comparison to analysis by HPLC</title><title>Free radical biology & medicine</title><addtitle>Free Radic Biol Med</addtitle><description>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. 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 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.</description><subject>3-Nitrotyrosine</subject><subject>Animals</subject><subject>Antibodies, Monoclonal - immunology</subject><subject>Antibody</subject><subject>Antibody Specificity</subject><subject>Blotting, Western</subject><subject>Chromatography, High Pressure Liquid</subject><subject>Fluorescent Antibody Technique</subject><subject>Free radicals</subject><subject>Haptens - chemistry</subject><subject>Haptens - immunology</subject><subject>Immunoassay</subject><subject>Immunohistochemistry</subject><subject>Inflammation</subject><subject>Liver - chemistry</subject><subject>Liver - drug effects</subject><subject>Male</subject><subject>Mice</subject><subject>Nonparenchymal cells</subject><subject>Rats</subject><subject>Rats, Inbred F344</subject><subject>Tissue Distribution</subject><subject>Tyrosine - analogs & derivatives</subject><subject>Tyrosine - analysis</subject><subject>Tyrosine - immunology</subject><subject>Tyrosine nitration</subject><subject>Zymosan</subject><subject>Zymosan - pharmacology</subject><issn>0891-5849</issn><issn>1873-4596</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2001</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkc-KFDEQh4Mo7jj6CEpOsh7azd9O4kVkUHdhwAX1HNLpajbSnYxJZpfeB_C57dkZ9Lingqqv6gf1IfSakveU0PbiO9GGNlILc07oO0IUZQ17glZUK94IadqnaPUPOUMvSvlFCBGS6-fojFLFtCZmhf5cTdM-ph4q-BpSxGnAvImh5lTnnEqIgEPE9QbwGG4hH-b385SKi03N4Cr0OLta8F2oN9jhCHd4SjH5MUU3Yhdr6FI_f8A-TTuXQ1kialr6bpxLKLib8eX1dvMSPRvcWODVqa7Rzy-ff2wum-23r1ebT9vGc8Nqw11LpOhNpwfhtPTCt4L5QSnTasc1560yZgDFJO9a7wyVUhHKSOcZb6EXfI3eHu_ucvq9h1LtFIqHcXQR0r5YxTglrXgcpJoJyZbINZJH0C_fKhkGu8thcnm2lNiDKftgyh40WELtgynLlr03p4B9N0H_f-ukZgE-HgFY_nEbINviA0QPfciLK9un8EjEX1ZopBo</recordid><startdate>20011201</startdate><enddate>20011201</enddate><creator>Girault, Isabelle</creator><creator>Karu, Alexander E</creator><creator>Schaper, Manuela</creator><creator>Barcellos-Hoff, Mary Helen</creator><creator>Hagen, Tory</creator><creator>Vogel, David S</creator><creator>Ames, Bruce N</creator><creator>Christen, Stephan</creator><creator>Shigenaga, Mark K</creator><general>Elsevier Inc</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7U7</scope><scope>C1K</scope><scope>7X8</scope></search><sort><creationdate>20011201</creationdate><title>Immunodetection of 3-nitrotyrosine in the liver of zymosan-treated rats with a new monoclonal antibody: comparison to analysis by HPLC</title><author>Girault, Isabelle ; Karu, Alexander E ; Schaper, Manuela ; Barcellos-Hoff, Mary Helen ; Hagen, Tory ; Vogel, David S ; Ames, Bruce N ; Christen, Stephan ; Shigenaga, Mark K</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c392t-3a6054d9b8f4a85c4c642cf77968a38336799fe7253b6ca915570120bc236ed43</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2001</creationdate><topic>3-Nitrotyrosine</topic><topic>Animals</topic><topic>Antibodies, Monoclonal - immunology</topic><topic>Antibody</topic><topic>Antibody Specificity</topic><topic>Blotting, Western</topic><topic>Chromatography, High Pressure Liquid</topic><topic>Fluorescent Antibody Technique</topic><topic>Free radicals</topic><topic>Haptens - chemistry</topic><topic>Haptens - immunology</topic><topic>Immunoassay</topic><topic>Immunohistochemistry</topic><topic>Inflammation</topic><topic>Liver - chemistry</topic><topic>Liver - drug effects</topic><topic>Male</topic><topic>Mice</topic><topic>Nonparenchymal cells</topic><topic>Rats</topic><topic>Rats, Inbred F344</topic><topic>Tissue Distribution</topic><topic>Tyrosine - analogs & derivatives</topic><topic>Tyrosine - analysis</topic><topic>Tyrosine - immunology</topic><topic>Tyrosine nitration</topic><topic>Zymosan</topic><topic>Zymosan - pharmacology</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Girault, Isabelle</creatorcontrib><creatorcontrib>Karu, Alexander E</creatorcontrib><creatorcontrib>Schaper, Manuela</creatorcontrib><creatorcontrib>Barcellos-Hoff, Mary Helen</creatorcontrib><creatorcontrib>Hagen, Tory</creatorcontrib><creatorcontrib>Vogel, David S</creatorcontrib><creatorcontrib>Ames, Bruce N</creatorcontrib><creatorcontrib>Christen, Stephan</creatorcontrib><creatorcontrib>Shigenaga, Mark K</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Toxicology Abstracts</collection><collection>Environmental Sciences and Pollution Management</collection><collection>MEDLINE - Academic</collection><jtitle>Free radical biology & medicine</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Girault, Isabelle</au><au>Karu, Alexander E</au><au>Schaper, Manuela</au><au>Barcellos-Hoff, Mary Helen</au><au>Hagen, Tory</au><au>Vogel, David S</au><au>Ames, Bruce N</au><au>Christen, Stephan</au><au>Shigenaga, Mark K</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Immunodetection of 3-nitrotyrosine in the liver of zymosan-treated rats with a new monoclonal antibody: comparison to analysis by HPLC</atitle><jtitle>Free radical biology & medicine</jtitle><addtitle>Free Radic Biol Med</addtitle><date>2001-12-01</date><risdate>2001</risdate><volume>31</volume><issue>11</issue><spage>1375</spage><epage>1387</epage><pages>1375-1387</pages><issn>0891-5849</issn><eissn>1873-4596</eissn><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. 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 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.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>11728809</pmid><doi>10.1016/S0891-5849(01)00712-2</doi><tpages>13</tpages></addata></record> |
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| ispartof | Free radical biology & medicine, 2001-12, Vol.31 (11), p.1375-1387 |
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| subjects | 3-Nitrotyrosine Animals Antibodies, Monoclonal - immunology Antibody Antibody Specificity Blotting, Western Chromatography, High Pressure Liquid Fluorescent Antibody Technique Free radicals Haptens - chemistry Haptens - immunology Immunoassay Immunohistochemistry Inflammation Liver - chemistry Liver - drug effects Male Mice Nonparenchymal cells Rats Rats, Inbred F344 Tissue Distribution Tyrosine - analogs & derivatives Tyrosine - analysis Tyrosine - immunology Tyrosine nitration Zymosan Zymosan - pharmacology |
| title | Immunodetection of 3-nitrotyrosine in the liver of zymosan-treated rats with a new monoclonal antibody: comparison to analysis by HPLC |
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