Characterization of antibody specificity using peptide array technologies
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
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
Biochemistry and Molecular Biology
Research subject Biotechnology
IdentifiersURN: urn:nbn:se:kth:diva-155723ISBN: 978-91-7595-316-8OAI: oai:DiVA.org:kth-155723DiVA: diva2:762237
2014-11-28, Gardaulan, Nobels väg 18, Solna, 10:15 (English)
Templin, Markus, Dr.
Uhlén, Mathias, Professor
QC 201411112014-11-112014-11-112015-02-18Bibliographically approved
List of papers