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Generation of novel recombinant antibodies against nitrotyrosine by antibody phage display


Hof, D; Cooksley-Decasper, S; Moergeli, S; von Eckardstein, Arnold (2011). Generation of novel recombinant antibodies against nitrotyrosine by antibody phage display. Human Antibodies, 20(1-2):15-27.

Abstract

Nitrotyrosine is a posttranslational protein modification that occurs under oxidative and nitrosative stress, and plays an important role in numerous pathological conditions. To analyse nitrotyrosine formation several commercial monoclonal and polyclonal antibodies reacting with 3-nitrotyrosine have been developed which however do not work properly in all required assays. Here, antibody phage display was used to select recombinant antibodies that specifically react with nitrotyrosine in various protein contexts. Nine initial selections were carried out, using synthetic peptides, peroxynitrite-modified proteins and conjugated proteins as antigens. Four antibodies were isolated that each exhibited a characteristic binding reactivity that greatly depended on the antigens that were used for their selections. In general, the selections using small, synthetic and biotinylated peptides were the most successful approach. Subsequently, antibody 11B1 was affinity matured by error prone mutagenesis, resulting in the isolation of two antibodies, designated 47A7 and 47B1. Competition ELISA and immunoblotting after treatment with sodium dithionite further demonstrated the specificity of antibody 47B1 for nitrotyrosine. The results presented here demonstrate that antibody phage display is a useful method to isolate antibodies against posttranslational modifications, which are powerful tools in the proteomic era.

Abstract

Nitrotyrosine is a posttranslational protein modification that occurs under oxidative and nitrosative stress, and plays an important role in numerous pathological conditions. To analyse nitrotyrosine formation several commercial monoclonal and polyclonal antibodies reacting with 3-nitrotyrosine have been developed which however do not work properly in all required assays. Here, antibody phage display was used to select recombinant antibodies that specifically react with nitrotyrosine in various protein contexts. Nine initial selections were carried out, using synthetic peptides, peroxynitrite-modified proteins and conjugated proteins as antigens. Four antibodies were isolated that each exhibited a characteristic binding reactivity that greatly depended on the antigens that were used for their selections. In general, the selections using small, synthetic and biotinylated peptides were the most successful approach. Subsequently, antibody 11B1 was affinity matured by error prone mutagenesis, resulting in the isolation of two antibodies, designated 47A7 and 47B1. Competition ELISA and immunoblotting after treatment with sodium dithionite further demonstrated the specificity of antibody 47B1 for nitrotyrosine. The results presented here demonstrate that antibody phage display is a useful method to isolate antibodies against posttranslational modifications, which are powerful tools in the proteomic era.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > University Hospital Zurich > Institute of Clinical Chemistry
Dewey Decimal Classification:610 Medicine & health
540 Chemistry
Language:English
Date:2011
Deposited On:15 Jan 2012 10:18
Last Modified:05 Apr 2016 15:20
Publisher:IOS Press
ISSN:1093-2607
Publisher DOI:https://doi.org/10.3233/HAB-2011-0238
PubMed ID:21558620

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