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Epitope mapping of antibodies towards human protein targets
KTH, School of Biotechnology (BIO), Proteomics.
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 [en]
antibody, antibody validation, biomarker, epitope mapping, peptide array, proteomics, RBM3, staphylococcal surface display
National Category
Medical Biotechnology Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-59529ISBN: 978-91-7501-222-3 (print)OAI: oai:DiVA.org:kth-59529DiVA: diva2:475959
Public defence
2012-01-27, FD5, Albanova University Center, Roslagstullsbacken 21, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
QC 20120111Available from: 2012-01-11 Created: 2012-01-11 Last updated: 2012-01-11Bibliographically approved
List of papers
1. Epitope mapping of antibodies using bacterial surface display
Open this publication in new window or tab >>Epitope mapping of antibodies using bacterial surface display
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2008 (English)In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 5, no 12, 1039-1045 p.Article in journal (Refereed) Published
Abstract [en]

We describe a method for mapping the epitopes recognized by antibodies, based on bacterial surface expression of antigen protein fragments followed by antibody-based flow-cytometric sorting. We analyzed the binding sites of both monoclonal and polyclonal antibodies directed to three human protein targets: (i) the human epidermal growth factor receptor 2 (HER2), (ii) ephrin-B3 and (iii) the transcription factor SATB2. All monoclonal antibodies bound a single epitope, whereas the polyclonal antibodies showed, in each case, a binding pattern with one to five separate epitopes. A comparison of polyclonal and monoclonal antibodies raised to the same antigen showed overlapping binding epitopes. We also demonstrated that bacterial cells with displayed protein fragments can be used as affinity ligands to generate epitope-specific antibodies. Our approach shows a path forward for systematic validation of antibodies for epitope specificity and cross-reactivity on a whole-proteome level.

Keyword
ephrin B3; epidermal growth factor receptor 2; epitope; monoclonal antibody; polyclonal antibody; proteome; transcription factor; transcription factor SATB2; unclassified drug; antibody affinity; antibody labeling; antibody specificity; antigen antibody complex; article; bacterial cell; binding affinity; binding site; controlled study; cross reaction; epitope mapping; flow cytometry; host cell; ligand binding; nonhuman; priority journal; protein targeting; Staphylococcus carnosus; Antibodies; Biological Assay; Cell Membrane; Epitope Mapping; Protein Engineering; Staphylococcus; Bacteria (microorganisms)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-7848 (URN)10.1038/nmeth.1272 (DOI)000261212700018 ()19029907 (PubMedID)
Note
QC 20100809. Uppdaterad från Submitted till Published. Titel ändrad, tidigare titel: "Combinatorial epitope mapping of antibodies using staphylococcal surface display" 20100809.Available from: 2007-12-14 Created: 2007-12-14 Last updated: 2017-12-14Bibliographically approved
2. Exploring epitopes of antibodies toward the human tryptophanyl-tRNA synthetase
Open this publication in new window or tab >>Exploring epitopes of antibodies toward the human tryptophanyl-tRNA synthetase
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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.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-27262 (URN)10.1016/j.nbt.2009.11.001 (DOI)000279133600008 ()2-s2.0-77952239690 (Scopus ID)
Note
QC 20101217Available from: 2010-12-17 Created: 2010-12-09 Last updated: 2012-01-11Bibliographically approved
3. Immunizations of inbred rabbits using the same antigen yield antibodies with similar, but not identical, epitopes
Open this publication in new window or tab >>Immunizations of inbred rabbits using the same antigen yield antibodies with similar, but not identical, epitopes
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

A problem for the generation of polyclonal antibodies is the potential difficulties to obtain 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 in-bred 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 gene 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 in-bred 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.

National Category
Medical Biotechnology Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-59559 (URN)
Note
QS 2012Available from: 2012-01-11 Created: 2012-01-11 Last updated: 2014-11-11Bibliographically approved
4. High nuclear RBM3 expression is associated with an improved prognosis in colorectal cancer
Open this publication in new window or tab >>High nuclear RBM3 expression is associated with an improved prognosis in colorectal cancer
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2011 (English)In: Proteomics. Clinical applications, ISSN 1862-8354, Vol. 5, no 11-12, 624-35 p.Article in journal (Refereed) Published
Abstract [en]

Purpose: In this study, we investigated the prognostic impact of human RBM3 expression in colorectal cancer using tissue microarray-based immunohistochemical analysis. Experimental design: One polyclonal antibody and four monoclonal anti-RBM3 antibodies were generated and epitope mapped using two different methods. Bacterial display revealed five distinct epitopes for the polyclonal antibody, while the four mouse monoclonal antibodies were found to bind to three of the five epitopes. A peptide suspension bead array assay confirmed the five epitopes of the polyclonal antibody, while only one of the monoclonal antibodies could be mapped using this approach. Antibody specificity was confirmed by Western blotting and immunohistochemistry, including siRNA-mediated knock-down. Two of the antibodies (polyclonal and monoclonal) were subsequently used to analyze RBM3 expression in tumor samples from two independent colorectal cancer cohorts, one consecutive cohort (n=270) and one prospectively collected cohort of patients with cancer of the sigmoid colon (n=305). RBM3-expression was detected, with high correlation between both antibodies (R=0.81, p<0.001). Results: In both cohorts, tumors with high nuclear RBM3 staining had significantly prolonged the overall survival. This was also confirmed in multivariate analysis, adjusted for established prognostic factors. Conclusion and clinical relevance: These data demonstrate that high tumor-specific nuclear expression of RBM3 is an independent predictor of good prognosis in colorectal cancer.

National Category
Biomedical Laboratory Science/Technology
Identifiers
urn:nbn:se:kth:diva-59105 (URN)10.1002/prca.201100020 (DOI)000298334000006 ()21956899 (PubMedID)2-s2.0-84855183064 (Scopus ID)
Funder
Knut and Alice Wallenberg FoundationScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note
QC 20120110Available from: 2012-01-10 Created: 2012-01-10 Last updated: 2012-06-15Bibliographically approved
5. Generation of monospecific antibodies based on affinity capture of polyclonal antibodies
Open this publication in new window or tab >>Generation of monospecific antibodies based on affinity capture of polyclonal antibodies
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2011 (English)In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 20, no 11, 1824-1835 p.Article in journal (Refereed) Published
Abstract [en]

A method is described to generate and validate antibodies based on mapping the linear epitopes of a polyclonal antibody followed by sequential epitope-specific capture using synthetic peptides. Polyclonal antibodies directed towards four proteins RBM3, SATB2, ANLN, and CNDP1, potentially involved in human cancers, were selected and antibodies to several non-overlapping epitopes were generated and subsequently validated by Western blot, immunohistochemistry, and immunofluorescence. For all four proteins, a dramatic difference in functionality could be observed for these monospecific antibodies directed to the different epitopes. In each case, at least one antibody was obtained with full functionality across all applications, while other epitope-specific fractions showed no or little functionality. These results present a path forward to use the mapped binding sites of polyclonal antibodies to generate epitope-specific antibodies, providing an attractive approach for large-scale efforts to characterize the human proteome by antibodies.

Keyword
epitope mapping, monospecific antibody, affinity chromatography
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-47969 (URN)10.1002/pro.716 (DOI)000296273700009 ()2-s2.0-80054717087 (Scopus ID)
Funder
Knut and Alice Wallenberg FoundationScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note
QC 20111117Available from: 2011-11-17 Created: 2011-11-15 Last updated: 2017-12-08Bibliographically approved

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