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Epitope mapping of antibodies using bacterial surface display

Nature Methods volume 5, pages 1039–1045 (2008)Cite this article

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.

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Figure 1: Schematic outline of the epitope mapping approach.
Figure 2: Schematic representation of recombinant surface protein and analysis of functional surface expression.
Figure 3: Epitope mapping of antibodies toward human HER2 and the Flag peptide.
Figure 4: Epitope mapping of monospecific antibodies toward human ephrin-B3.
Figure 5: Epitope mapping of antibodies toward SATB2.

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Acknowledgements

We are grateful to P.-Å. Nygren and J. Lundeberg for comments and advice. M. Hansson and H. Johannesson at Atlas Antibodies AB are acknowledged for funding the generation of SATB2 monoclonals. This work was supported by funding from the ProNova center (project B3) and the Knut and Alice Wallenberg foundation. J.L. was partially supported by grant 621-2003-2876 from the Swedish Research Council.

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Author notes

  1. Johan Rockberg and John Löfblom: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Biotechnology, School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, Stockholm, SE-106 91, Sweden

    Johan Rockberg, John Löfblom, Barbara Hjelm, Mathias Uhlén & Stefan Ståhl

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  1. Johan Rockberg
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  2. John Löfblom
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  3. Barbara Hjelm
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  4. Mathias Uhlén
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Correspondence to Mathias Uhlén.

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Supplementary Figures 1–3, Supplementary Methods (PDF 1241 kb)

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Rockberg, J., Löfblom, J., Hjelm, B. et al. Epitope mapping of antibodies using bacterial surface display. Nat Methods 5, 1039–1045 (2008). https://doi.org/10.1038/nmeth.1272

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