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Parallel Immunizations of Rabbits Using the Same Antigen Yield Antibodies with Similar, but Not Identical, Epitopes
KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).ORCID iD: 0000-0002-5248-8568
KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).ORCID iD: 0000-0001-9423-0541
KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).ORCID iD: 0000-0002-9977-5724
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2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 12, e45817- p.Article in journal (Refereed) Published
Abstract [en]

A problem for the generation of polyclonal antibodies is the potential difficulties for obtaining a renewable resource due to batch-to-batch variations when the same antigen is immunized into several separate animals. Here, we have investigated this issue by determining the epitopes of antibodies generated from parallel immunizations of rabbits with recombinant antigens corresponding to ten human protein targets. The epitopes were mapped by both a suspension bead array approach using overlapping synthetic 15-mer peptides and a bacterial display approach using expression of random fragments of the antigen on the surface of bacteria. Both methods determined antibody binding with the aid of fluorescent-based analysis. In addition, one polyclonal antibody was fractionated by peptide-specific affinity capture for in-depth comparison of epitopes. The results show that the same antigen immunized in several rabbits yields polyclonal antibodies with similar epitopes, but with larger differences in the relative amounts of antibodies to the different epitopes. In some cases, unique epitopes were observed for one of the immunizations. The results suggest that polyclonal antibodies generated by repeated immunizations do not display an identical epitope pattern, although many of the epitopes are similar.

Place, publisher, year, edition, pages
2012. Vol. 7, no 12, e45817- p.
Keyword [en]
animal experiment, antigen binding, article, breast cancer, cancer tissue, controlled study, epitope mapping, human, human tissue, immunization, immunohistochemistry, nonhuman, prediction, protein structure, rabbit, Staphylococcus carnosus, Western blotting
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-111858DOI: 10.1371/journal.pone.0045817ISI: 000312694300002PubMedID: 23284606Scopus ID: 2-s2.0-84871314636OAI: oai:DiVA.org:kth-111858DiVA: diva2:587397
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceKnut and Alice Wallenberg FoundationSwedish Research CouncilVinnova
Note

QC 20130115

Available from: 2013-01-14 Created: 2013-01-14 Last updated: 2017-12-06Bibliographically 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.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2014:16
Keyword
Antibody, Epitope mapping, Peptide array, Suspension bead array, Antigen, Specificity, Cross-reactivity, Immunization, Immunogenicity
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
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)
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Note

QC 20141111

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

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Forsström, BjörnLöfblom, JohnUhlén, Mathias

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