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Proteome-wide Epitope Mapping of Antibodies Using Ultra-dense Peptide Arrays
KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.ORCID-id: 0000-0002-5248-8568
KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
Vise andre og tillknytning
2014 (engelsk)Inngår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 13, nr 6, s. 1585-1597Artikkel i tidsskrift (Fagfellevurdert) 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.

sted, utgiver, år, opplag, sider
2014. Vol. 13, nr 6, s. 1585-1597
HSV kategori
Identifikatorer
URN: urn:nbn:se:kth:diva-147955DOI: 10.1074/mcp.M113.033308ISI: 000337239500016Scopus ID: 2-s2.0-84901938911OAI: oai:DiVA.org:kth-147955DiVA, id: diva2:733777
Merknad

QC 20140711

Tilgjengelig fra: 2014-07-11 Laget: 2014-07-10 Sist oppdatert: 2017-12-05bibliografisk kontrollert
Inngår i avhandling
1. Characterization of antibody specificity using peptide array technologies
Åpne denne publikasjonen i ny fane eller vindu >>Characterization of antibody specificity using peptide array technologies
2014 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Stockholm: KTH Royal Institute of Technology, 2014. s. xi, 49
Serie
TRITA-BIO-Report, ISSN 1654-2312 ; 2014:16
Emneord
Antibody, Epitope mapping, Peptide array, Suspension bead array, Antigen, Specificity, Cross-reactivity, Immunization, Immunogenicity
HSV kategori
Forskningsprogram
Bioteknologi
Identifikatorer
urn:nbn:se:kth:diva-155723 (URN)978-91-7595-316-8 (ISBN)
Disputas
2014-11-28, Gardaulan, Nobels väg 18, Solna, 10:15 (engelsk)
Opponent
Veileder
Merknad

QC 20141111

Tilgjengelig fra: 2014-11-11 Laget: 2014-11-11 Sist oppdatert: 2015-02-18bibliografisk kontrollert

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