Epitope mapping of antibodies using bacterial surface display
2008 (English)In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 5, no 12, 1039-1045 p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
2008. Vol. 5, no 12, 1039-1045 p.
ephrin B3; epidermal growth factor receptor 2; epitope; monoclonal antibody; polyclonal antibody; proteome; transcription factor; transcription factor SATB2; unclassified drug; antibody affinity; antibody labeling; antibody specificity; antigen antibody complex; article; bacterial cell; binding affinity; binding site; controlled study; cross reaction; epitope mapping; flow cytometry; host cell; ligand binding; nonhuman; priority journal; protein targeting; Staphylococcus carnosus; Antibodies; Biological Assay; Cell Membrane; Epitope Mapping; Protein Engineering; Staphylococcus; Bacteria (microorganisms)
Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:kth:diva-7848DOI: 10.1038/nmeth.1272ISI: 000261212700018PubMedID: 19029907OAI: oai:DiVA.org:kth-7848DiVA: diva2:12989
QC 20100809. Uppdaterad från Submitted till Published. Titel ändrad, tidigare titel: "Combinatorial epitope mapping of antibodies using staphylococcal surface display" 20100809.2007-12-142007-12-142012-01-11Bibliographically approved