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Schematic overview of the epitope mapping methods used in the study.

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posted on 2012-12-19, 00:32 authored by Barbara Hjelm, Björn Forsström, John Löfblom, Johan Rockberg, Mathias Uhlén

(A) Epitope mapping using bacterial display, in which the target gene is fragmented and a library of clones is expressed on S. carnosus. The cell displayed peptide library is assayed for binding to the antibody using a flow cytometer Binding clones are sorted, sequenced and aligned back to the antigen sequence in order to conclude epitopes. (B) The sequencing of the flow-sorted libraries is used to determine the epitope regions. To the left, a typical FACS dot plot showing sorting of a cell displayed library incubated with the investigated antibody. The colored bars to the right show aligned binding sequences from the different sorted populations and on top the consensus epitopes derived from the alignment. (C) Epitope mapping using peptide bead arrays with overlapping peptides, spanning the antigen sequence, coupled to color coded beads. Antibody binding towards the peptides is evaluated using a flow cytometer instrument and epitope regions are identified on the antigen. (D) A schematic view of how binding profiles are used to determine epitope regions.

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