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Parallel Immunizations of Rabbits Using the Same Antigen Yield Antibodies with Similar, but Not Identical, Epitopes
KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).ORCID-id: 0000-0002-5248-8568
KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).ORCID-id: 0000-0001-9423-0541
KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).ORCID-id: 0000-0002-9977-5724
Vise andre og tillknytning
2012 (engelsk)Inngår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, nr 12, s. e45817-Artikkel i tidsskrift (Fagfellevurdert) 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.

sted, utgiver, år, opplag, sider
2012. Vol. 7, nr 12, s. e45817-
Emneord [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
HSV kategori
Identifikatorer
URN: urn:nbn:se:kth:diva-111858DOI: 10.1371/journal.pone.0045817ISI: 000312694300002PubMedID: 23284606Scopus ID: 2-s2.0-84871314636OAI: oai:DiVA.org:kth-111858DiVA, id: diva2:587397
Forskningsfinansiär
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceKnut and Alice Wallenberg FoundationSwedish Research CouncilVinnova
Merknad

QC 20130115

Tilgjengelig fra: 2013-01-14 Laget: 2013-01-14 Sist oppdatert: 2017-12-06bibliografisk 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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