title: Generation of novel recombinant antibodies against nitrotyrosine by antibody phage display.
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.
authors.affiliation: University Hospital Zurich, Institute for Clinical Chemistry, Switzerland. danielle.hof@usz.ch
authors.forename: Danielle
authors.initials: D
authors.lastname: Hof
authors.forename: Seraina
authors.initials: S
authors.lastname: Cooksley-Decasper
authors.forename: Sandra
authors.initials: S
authors.lastname: Moergeli
authors.forename: Arnold
authors.initials: A
authors.lastname: von Eckardstein
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000276:immunology*; D000918:Antibody Specificity / Q000235:genetics / Q000276:immunology*; D000941:Antigens / Q000235:genetics / Q000276:immunology*; D001665:Binding Sites; D019921:Biotinylation; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D015151:Immunoblotting; D018796:Immunoconjugates / Q000235:genetics / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D019151:Peptide Library; D030421:Peroxynitrous Acid / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology*; D054852:Small Molecule Libraries / Q000737:chemistry / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000276:immunology / Q000378:metabolism
chemical_list: D000906:Antibodies; D000941:Antigens; D018796:Immunoconjugates; D019151:Peptide Library; D011994:Recombinant Proteins; D054852:Small Molecule Libraries; D030421:Peroxynitrous Acid; C002744:3-nitrotyrosine; D014443:Tyrosine