title: Epitope mapping of antibodies using bacterial surface display.
abstract: We describe a method for mapping the epitopes recognized by antibodies, based on bacterial surface expression of antigen protein fragments followed by antibody-based flow-cytometric sorting. We analyzed the binding sites of both monoclonal and polyclonal antibodies directed to three human protein targets: (i) the human epidermal growth factor receptor 2 (HER2), (ii) ephrin-B3 and (iii) the transcription factor SATB2. All monoclonal antibodies bound a single epitope, whereas the polyclonal antibodies showed, in each case, a binding pattern with one to five separate epitopes. A comparison of polyclonal and monoclonal antibodies raised to the same antigen showed overlapping binding epitopes. We also demonstrated that bacterial cells with displayed protein fragments can be used as affinity ligands to generate epitope-specific antibodies. Our approach shows a path forward for systematic validation of antibodies for epitope specificity and cross-reactivity on a whole-proteome level.
authors.affiliation: Department of Molecular Biotechnology, School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, SE-106 91 Stockholm, Sweden.
authors.forename: Johan
authors.initials: J
authors.lastname: Rockberg
authors.forename: John
authors.initials: J
authors.lastname: Löfblom
authors.forename: Barbara
authors.initials: B
authors.lastname: Hjelm
authors.forename: Mathias
authors.initials: M
authors.lastname: Uhlén
authors.forename: Stefan
authors.initials: S
authors.lastname: Ståhl
mesh_terms: D000906:Antibodies / Q000235:genetics / Q000276:immunology*; D001681:Biological Assay / Q000379:methods*; D002462:Cell Membrane / Q000235:genetics / Q000276:immunology*; D018604:Epitope Mapping / Q000379:methods*; D015202:Protein Engineering / Q000379:methods; D013210:Staphylococcus / Q000235:genetics / Q000276:immunology*
chemical_list: D000906:Antibodies