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High-resolution mapping of linear antibody epitopes using ultrahigh-density peptide microarrays
KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).ORCID-id: 0000-0002-9977-5724
KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).ORCID-id: 0000-0002-5248-8568
KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101). KTH, Centra, Science for Life Laboratory, SciLifeLab.ORCID-id: 0000-0002-4657-8532
Visa övriga samt affilieringar
2012 (Engelska)Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 11, nr 12, s. 1790-1800Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Antibodies empower numerous important scientific, clinical, diagnostic, and industrial applications. Ideally, the epitope(s) targeted by an antibody should be identified and characterized, thereby establishing antibody reactivity, highlighting possible cross-reactivities, and perhaps even warning against unwanted (e.g. autoimmune) reactivities. Antibodies target proteins as either conformational or linear epitopes. The latter are typically probed with peptides, but the cost of peptide screening programs tends to prohibit comprehensive specificity analysis. To perform high-throughput, high-resolution mapping of linear antibody epitopes, we have used ultrahigh-density peptide microarrays generating several hundred thousand different peptides per array. Using exhaustive length and substitution analysis, we have successfully examined the specificity of a panel of polyclonal antibodies raised against linear epitopes of the human proteome and obtained very detailed descriptions of the involved specificities. The epitopes identified ranged from 4 to 12 amino acids in size. In general, the antibodies were of exquisite specificity, frequently disallowing even single conservative substitutions. In several cases, multiple distinct epitopes could be identified for the same target protein, suggesting an efficient approach to the generation of paired antibodies. Two alternative epitope mapping approaches identified similar, although not necessarily identical, epitopes. These results show that ultrahigh-density peptide microarrays can be used for linear epitope mapping. With an upper theoretical limit of 2,000,000 individual peptides per array, these peptide microarrays may even be used for a systematic validation of antibodies at the proteomic level.

Ort, förlag, år, upplaga, sidor
2012. Vol. 11, nr 12, s. 1790-1800
Nyckelord [en]
antibody affinity, antibody specificity, article, controlled study, DNA sequence, epitope mapping, high throughput screening, human, linear system, molecular size, peptide synthesis, priority journal, protein expression, protein microarray, protein targeting, signal transduction, substitution reaction
Nationell ämneskategori
Cell- och molekylärbiologi
Identifikatorer
URN: urn:nbn:se:kth:diva-110177DOI: 10.1074/mcp.M112.020800ISI: 000313557000023Scopus ID: 2-s2.0-84870659704OAI: oai:DiVA.org:kth-110177DiVA, id: diva2:586283
Forskningsfinansiär
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceEU, FP7, Sjunde ramprogrammet, 222773
Anmärkning

QC 20130111

Tillgänglig från: 2013-01-11 Skapad: 2013-01-10 Senast uppdaterad: 2018-01-11Bibliografiskt granskad
Ingår i avhandling
1. Characterization of antibody specificity using peptide array technologies
Öppna denna publikation i ny flik eller fönster >>Characterization of antibody specificity using peptide array technologies
2014 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Stockholm: KTH Royal Institute of Technology, 2014. s. xi, 49
Serie
TRITA-BIO-Report, ISSN 1654-2312 ; 2014:16
Nyckelord
Antibody, Epitope mapping, Peptide array, Suspension bead array, Antigen, Specificity, Cross-reactivity, Immunization, Immunogenicity
Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
Bioteknologi
Identifikatorer
urn:nbn:se:kth:diva-155723 (URN)978-91-7595-316-8 (ISBN)
Disputation
2014-11-28, Gardaulan, Nobels väg 18, Solna, 10:15 (Engelska)
Opponent
Handledare
Anmärkning

QC 20141111

Tillgänglig från: 2014-11-11 Skapad: 2014-11-11 Senast uppdaterad: 2015-02-18Bibliografiskt granskad

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Rockberg, JohanForsström, BjörnNilsson, PeterUhlén, Mathias
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Proteomik (stängd 20130101)Science for Life Laboratory, SciLifeLab
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Molecular & Cellular Proteomics
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