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Multiplex epitope mapping using bacterial surface display reveals both linear and conformational epitopes.

ABSTRACT: As antibody-based diagnosis and therapy grow at an increased pace, there is a need for methods which rapidly and accurately determine antibody-antigen interactions. Here, we report a method for the multiplex determination of antibody epitopes using bacterial cell-surface display. A protein-fragment library with 10(7) cell clones, covering 60 clinically-relevant protein targets, was created and characterized with massively parallel sequencing. Using this multi-target fragment library we determined simultaneously epitopes of commercial monoclonal and polyclonal antibodies targeting PSMA, EGFR, and VEGF. Off-target binding was observed for one of the antibodies, which demonstrates the methods ability to reveal cross-reactivity. We exemplify the detection of structural epitopes by mapping the therapeutic antibody Avastin. Based on our findings we suggest this method to be suitable for mapping linear and structural epitopes of monoclonal and polyclonal antibodies in a multiplex fashion and could find applicability in serum profiling as well as other protein-protein interaction studies.

SUBMITTER: Hudson EP

PROVIDER: S-EPMC3463815 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Multiplex epitope mapping using bacterial surface display reveals both linear and conformational epitopes.

Hudson Elton P EP Uhlen Mathias M Rockberg Johan J

Scientific reports 20121004

As antibody-based diagnosis and therapy grow at an increased pace, there is a need for methods which rapidly and accurately determine antibody-antigen interactions. Here, we report a method for the multiplex determination of antibody epitopes using bacterial cell-surface display. A protein-fragment library with 10(7) cell clones, covering 60 clinically-relevant protein targets, was created and characterized with massively parallel sequencing. Using this multi-target fragment library we determine ...[more]

PMID: 23050090

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Project description:BackgroundPrecise determination of conformational epitopes of neutralizing antibodies represents a key step in the rational design of novel vaccines. A powerful experimental method to gain insights on the physical chemical nature of conformational epitopes is the selection of linear peptides that bind with high affinities to a monoclonal antibody of interest by phage display technology. However, the structural characterization of conformational epitopes from these mimotopes is not straightforward, and in the past the interpretation of peptide sequences from phage display experiments focused on linear sequence analysis to find a consensus sequence or common sequence motifs.ResultsWe present a fully automated search method, EpiSearch that predicts the possible location of conformational epitopes on the surface of an antigen. The algorithm uses peptide sequences from phage display experiments as input, and ranks all surface exposed patches according to the frequency distribution of similar residues in the peptides and in the patch. We have tested the performance of the EpiSearch algorithm for six experimental data sets of phage display experiments, the human epidermal growth factor receptor-2 (HER-2/neu), the antibody mAb Bo2C11 targeting the C(2) domain of FVIII, antibodies mAb 17b and mAb b12 of the HIV envelope protein gp120, mAb 13b5 targeting HIV-1 capsid protein and 80R of the SARS coronavirus spike protein. In all these examples the conformational epitopes as determined by the X-ray crystal structures of the antibody-antigen complexes, were found within the highest scoring patches of EpiSearch, covering in most cases more than 50% residues of experimental observed conformational epitopes. Input options of the program include mapping of a single peptide or a set of peptides on the antigen structure, and the results of the calculation can be visualized on our interactive web server.AvailabilityUsers can access the EpiSearch from our web server http://curie.utmb.edu/episearch.html.

Project description:IntroductionHuman bone morphogenetic proteins (BMPs) constitute a large family of cytokines related to members of the transforming growth factor-β superfamily, which fulfill biological functions by specificity binding to their cognate type I (BRI) and type II (BRII) receptors through conformational wrist and linear knuckle epitopes, respectively.Methods and resultsWe systematically examined the intermolecular recognition and interaction between the BMP proteins and BRI receptor at structural, energetic and dynamic levels. The BRI-binding site consists of three hotspot regions on BMP surface, which totally contribute ~70% potency to the BMP-BRI binding events and represent the core sections of BMP conformational wrist epitope; the contribution increases in the order: hotspot 2 (~ 8%) < hotspot 3 (~ 20%) < hotspot 1 (~ 40%). Multiple sequence alignment and structural superposition revealed a consensus sequence pattern and a similar binding mode of the three hotspots shared by most BMP members, indicating a high conservation of wrist epitope in BMP family. The three hotspots are natively folded into wellstructured U-shaped,, loop and double-stranded conformations in BMP proteins, which, however, would become largely disordered when splitting from the protein context to derive osteogenic peptides in free state, thus largely impairing their rebinding capability to BRI receptor. In this respect, cyclization strategy was employed to constrain hotspot 1/3-derived peptides into a native-like conformation, which was conducted by adding a disulfide bond across the ending arms of linear peptides based on their native conformations. Fluorescence-based assays substantiated that the cyclization can effectively improve the binding affinities of osteogenic peptides to BRI receptor by 3-6-fold. The cyclic peptides also exhibit a good selectivity for BRI over BRII (> 5-fold), confirming that they can specifically target the wrist epitope-binding site of BRI receptor.ConclusionThe rationally designed cyclic peptides can be regarded as the promising lead entities that should be further chemically modified to enhance their in vivo biological stability for further bioengineering therapeutic osteogenic peptides against chondrocyte senescence and bone disorder.

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Multiplex epitope mapping using bacterial surface display reveals both linear and conformational epitopes.

Publications

Multiplex epitope mapping using bacterial surface display reveals both linear and conformational epitopes.

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