Article abstract
Nature Methods 5, 1039 - 1045 (2008)
Published online: 23 November 2008 | doi:10.1038/nmeth.1272
Epitope mapping of antibodies using bacterial surface display
Johan Rockberg1,2, John Löfblom1,2, Barbara Hjelm1, Mathias Uhlén1 & Stefan Ståhl1
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.
- Department of Molecular Biotechnology, School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, SE-106 91 Stockholm, Sweden.
- These authors contributed equally to this work.
Correspondence to: Mathias Uhlén1 e-mail: mathias@biotech.kth.se
