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- Title
Epitope mapping of antibodies using bacterial surface display.
- Authors
Rockberg, Johan; Löfblom, John; Hjelm, Barbara; Uhlén, Mathias; Ståhl, Stefan
- 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.
- Publication
Nature methods, 2008, Vol 5, Issue 12, p1039
- ISSN
1548-7105
- Publication type
Journal Article
- DOI
10.1038/nmeth.1272