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Proteome-wide Epitope Mapping of Antibodies Using Ultra-dense Peptide Arrays
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0002-5248-8568
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
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2014 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 13, no 6, 1585-1597 p.Article in journal (Refereed) Published
Abstract [en]

Antibodies are of importance for the field of proteomics, both as reagents for imaging cells, tissues, and organs and as capturing agents for affinity enrichment in mass-spectrometry-based techniques. It is important to gain basic insights regarding the binding sites (epitopes) of antibodies and potential cross-reactivity to nontarget proteins. Knowledge about an antibody's linear epitopes is also useful in, for instance, developing assays involving the capture of peptides obtained from trypsin cleavage of samples prior to mass spectrometry analysis. Here, we describe, for the first time, the design and use of peptide arrays covering all human proteins for the analysis of antibody specificity, based on parallel in situ photolithic synthesis of a total of 2.1 million overlapping peptides. This has allowed analysis of on-and off-target binding of both monoclonal and polyclonal antibodies, complemented with precise mapping of epitopes based on full amino acid substitution scans. The analysis suggests that linear epitopes are relatively short, confined to five to seven residues, resulting in apparent off-target binding to peptides corresponding to a large number of unrelated human proteins. However, subsequent analysis using recombinant proteins suggests that these linear epitopes have a strict conformational component, thus giving us new insights regarding how antibodies bind to their antigens.

Place, publisher, year, edition, pages
2014. Vol. 13, no 6, 1585-1597 p.
National Category
Biochemistry and Molecular Biology
URN: urn:nbn:se:kth:diva-147955DOI: 10.1074/mcp.M113.033308ISI: 000337239500016ScopusID: 2-s2.0-84901938911OAI: diva2:733777

QC 20140711

Available from: 2014-07-11 Created: 2014-07-10 Last updated: 2014-11-11Bibliographically approved
In thesis
1. Characterization of antibody specificity using peptide array technologies
Open this publication in new window or tab >>Characterization of antibody specificity using peptide array technologies
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Antibodies play an important role in the natural immune response to invading pathogens. The strong and specific binding to their antigens also make them indispensable tools for research, diagnostics and therapy.

This thesis describes the development of methods for characterization of an- tibody specificity and the use of these methods to investigate the polyclonal antibody response after immunization. Paper I describes the development of an epitope-specific serum fractionation technique based on epitope map- ping using overlapping peptides followed by chromatographic separation of polyclonal serum. This technique together with another epitope mapping technique based on bacterial display of protein fragments were then used to generate antibody sandwich pairs (Paper I), investigate epitope variations of repeated immunizations (Paper II) and to determine the ratio of antibodies targeting linear and conformational epitopes of polyclonal antibodies (Paper III). Paper IV describes the optimization of in situ-synthesized high-density peptide arrays for epitope mapping and how different peptide lengths influ- ence epitope detection and resolution. In Paper V we show the development of planar peptide arrays covering the entire human proteome and how these arrays can be used for epitope mapping and off-target binding analysis. In Paper VI we show how polyclonal antibodies targeting linear epitopes can be used for peptide enrichment in a rapid, absolute protein quantification protocol based on mass spectrometry.

Altogether these investigations demonstrate the usefulness of peptide arrays for fast and straightforward characterization of antibody specificity. The work also contributes to a deeper understanding of the polyclonal anti- body response obtained after immunization with recombinant protein frag- ments.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2014. xi, 49 p.
TRITA-BIO-Report, ISSN 1654-2312 ; 2014:16
Antibody, Epitope mapping, Peptide array, Suspension bead array, Antigen, Specificity, Cross-reactivity, Immunization, Immunogenicity
National Category
Biochemistry and Molecular Biology
Research subject
urn:nbn:se:kth:diva-155723 (URN)978-91-7595-316-8 (ISBN)
Public defence
2014-11-28, Gardaulan, Nobels väg 18, Solna, 10:15 (English)

QC 20141111

Available from: 2014-11-11 Created: 2014-11-11 Last updated: 2015-02-18Bibliographically approved

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Forsström, BjornAxnäs, Barbara BislawskaHu, Francis JingxinNilsson, PeterHudson, Elton PaulRockberg, JohanUhlén, Mathias
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Proteomics and NanobiotechnologyScience for Life Laboratory, SciLifeLab
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