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Exploring epitopes of antibodies toward the human tryptophanyl-tRNA synthetase
KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
KTH, School of Biotechnology (BIO), Molecular Biotechnology.ORCID iD: 0000-0001-9423-0541
KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.ORCID iD: 0000-0002-9282-0174
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2010 (English)In: NEW BIOTECHNOL, ISSN 1871-6784, Vol. 27, no 2, 129-137 p.Article in journal (Refereed) Published
Abstract [en]

There is a need to characterize the epitopes of affinity reagents to develop high quality affinity reagents for research, diagnostics and therapy. Here, we describe the analysis of epitopes of antibodies generated toward human tryptophanyl-tRNA synthetase (WARS) using both combinatorial bacterial display and suspension bead array. The bacterial display revealed that the polyclonal antibody binds to three separate epitopes and peptide scanning using 15-mers revealed binding to a 13 amino acid consensus sequence (ELINRIERATGQR). A mouse monoclonal antibody was generated and the mapping approach revealed binding toward a slightly shifted position of the same epitope. Structural analysis showed that the antibodies bind to a-helical regions on the surface of the target protein. An alanine-scanning experiment showed binding to four specific residues. The implications for the systematic analysis of antibody epitopes on the basis of these results are discussed.

Place, publisher, year, edition, pages
2010. Vol. 27, no 2, 129-137 p.
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-27262DOI: 10.1016/j.nbt.2009.11.001ISI: 000279133600008Scopus ID: 2-s2.0-77952239690OAI: oai:DiVA.org:kth-27262DiVA: diva2:379161
Note
QC 20101217Available from: 2010-12-17 Created: 2010-12-09 Last updated: 2012-01-11Bibliographically approved
In thesis
1. Epitope mapping of antibodies towards human protein targets
Open this publication in new window or tab >>Epitope mapping of antibodies towards human protein targets
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis, based on five research papers, presents results from development and evaluation ofmethods for identifying the interaction site of antibodies on their antigens and the functional investigation of these in different assays. As antibodies have proven to be invaluable tools in diagnostics, therapy and basic research, the demand of characterizing these binding molecules has increased. Techniques for epitope mapping in a streamlined manner are therefore needed, particularly in high throughput projects as the Human Protein Atlas that aims to systematically generate two antibodies with separate epitopes towards all human proteins. 

In paper I we describe an approach to map the epitopes of polyclonal and monoclonal antibodies for the first time using staphylococcal display. This method was combined with peptide scanning and alanine scanning using suspension bead arrays, to create a streamlined approach of highresolution characterization of epitopes recognized by antibodies as demonstrated in paper II. Single epitopes were identified for the monoclonal antibodies and several (one to five) separate epitopes scattered throughout the antigen sequence were determined for each polyclonal antibody. Further, antibodies of different species origin showed overlapping binding epitopes.

In paper III we studied the epitope patterns of polyclonal antibodies generated with the same antigen in different animals. Although common epitope regions could be identified the exact epitope pattern was not repeated, as some epitopes did not reoccur in the repeated immunizations. In paper IV, a potential biomarker for colon cancer, RBM3, was investigated using validated antibodies by epitope mapping and siRNA analysis.

Finally, in paper V, a method for generating epitope-specific antibodies based on affinity purification of a polyclonal antibody is described. The generated antibodies were used in several immunoassays and showed a great difference in functionality. Paired antibodies with separate epitopes were successfully generated and could be used in a sandwich assay or to validate each other in immunohistochemistry.

Taken together, in these studies we have demonstrated valuable concepts for the characterization of antibody epitopes.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2011. xi, 46 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2012:1
Keyword
antibody, antibody validation, biomarker, epitope mapping, peptide array, proteomics, RBM3, staphylococcal surface display
National Category
Medical Biotechnology Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-59529 (URN)978-91-7501-222-3 (ISBN)
Public defence
2012-01-27, FD5, Albanova University Center, Roslagstullsbacken 21, Stockholm, 10:00 (English)
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Supervisors
Note
QC 20120111Available from: 2012-01-11 Created: 2012-01-11 Last updated: 2012-01-11Bibliographically approved

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Löfblom, JohnStåhl, StefanUhlen, Mathias

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