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Epitope mapping of antibodies using bacterial surface display
KTH, School of Biotechnology (BIO), Molecular Biotechnology.ORCID iD: 0000-0002-9977-5724
KTH, School of Biotechnology (BIO), Molecular Biotechnology.ORCID iD: 0000-0001-9423-0541
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
KTH, School of Biotechnology (BIO), Molecular Biotechnology.ORCID iD: 0000-0002-9282-0174
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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.

Place, publisher, year, edition, pages
2008. Vol. 5, no 12, 1039-1045 p.
Keyword [en]
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: urn:nbn:se:kth:diva-7848DOI: 10.1038/nmeth.1272ISI: 000261212700018PubMedID: 19029907OAI: oai:DiVA.org:kth-7848DiVA: diva2:12989
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: 2012-01-11Bibliographically approved
In thesis
1. Staphylococcal surface display for protein engineering and characterization
Open this publication in new window or tab >>Staphylococcal surface display for protein engineering and characterization
2007 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Even though our understanding of mechanisms such as protein folding and molecular recognition is relatively poor, antibodies and alternative affinity proteins with entirely novel functions are today generated in a routine manner. The reason for this success is an engineering approach generally known as directed evolution.

Directed evolution has provided researchers with a tool for circumventing our limited knowledge and hence the possibility to create novel molecules that by no means could have been designed today. The approach is based on construction of high-complexity combinatorial libraries from which protein variants with desired properties can be selected. Engineered proteins are already indispensable tools in nearly all areas of life science and the recent advent of mainly monoclonal antibodies as therapeutic agents has directed even more attention to the field of combinatorial protein engineering.

In this thesis, I present the underlying research efforts of six original papers. The overall objective of the studies has been to develop and investigate a new staphylococcal surface display method for protein engineering and protein characterization. The technology is based on display of recombinant proteins on surface of the Gram-positive bacteria Staphylococcus carnosus. In two initial studies, two key issues were addressed in order to improve the protein engineering method in regard to affinity discrimination ability and transformation efficiency. The successful results enabled investigation of the staphylococcal display system for de novo generation of affibody molecules from large combinatorial libraries. In this study, a high-complexity protein library was for the first time displayed on surface of Gram-positive bacteria and by means of fluorescence-activated cell sorting, specific affinity proteins for tumor necrosis factor-alpha were isolated. Moreover, in following papers, the staphylococcal display method was further improved and investigated for affinity determination, soluble protein production and epitope mapping purposes in order to facilitate downstream characterizations of generated affinity proteins.

Taken together, in these studies we have demonstrated that the staphylococcal display system is a powerful alternative to existing technologies for protein engineering and protein characterization.

Place, publisher, year, edition, pages
Stockholm: KHT, 2007. x, 95 p.
Keyword
affibody, combinatorial library, epitope mapping, Gram-positive bacteria, protein engineering, staphylococcal surface display
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-4584 (URN)978-91-7178-834-4 (ISBN)
Public defence
2008-01-11, FD5, Albanova, Roslagstullsbacken 21, Stockholm, 09:00
Opponent
Supervisors
Note
QC 20100809Available from: 2007-12-14 Created: 2007-12-14 Last updated: 2010-08-09Bibliographically approved
2. Methods for Generation and Characterization of Monospecific Antibodies
Open this publication in new window or tab >>Methods for Generation and Characterization of Monospecific Antibodies
2008 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Recent advances in biotechnology have generated possibilities to investigate and measure parts of life previously left for believers to explain. Utilizing the same book of recipes, the genome, our cells produce selections of proteins at a time and thereby niche into a multitude of specialized cell types, tissues and organs comprising our body. Knowledge of the precise protein composition in a given organ at normal and disease condition would be of invaluable importance, both for identification of disease causes and the design of new pharmaceuticals, as well as for a deeper understanding of the processes of life. This doctoral thesis describes the start and progress of a visionary project (HPR) to localize all human proteins in our body, with emphasis on the generation and characterization of antibodies used as protein targeting missiles. To facilitate the identification of one human protein in a complex environment like our body, it is of significant importance to have precise and specific means of detection. The first two papers (I-II), describe software developed for generation of monospecific antibodies satisfying such needs, using a set of rules for antigen optimization. Five years after project start a large amount of antibodies with documented characteristics have been generated. The third paper (III), illustrates an attempt to sieve these antibody characteristics to develop a tool, for further improvement of antigen selection, based on the correlation between antigen sequence and amount of specific antibody generated.Having a panel of protein-specific antibodies is a possession of a great value, not only for localization studies, but also as possible target-directed pharmaceuticals. In such cases, knowledge of the precise epitope recognized by the antibody on its target protein, is an important aid, both for understanding its effect as well as unwanted cross-reactivity. Paper (IV) describes the development of a high-resolution method for epitope mapping of antibodies using staphylococcal display. An application of the method is described in the last paper (V) where it is used to map an anti-HER2 monospecific antibody with growth-inhibiting effects on breast cancer cells. The monospecific antibody was fractionated into separate populations and five novel epitopes related to cancer cell growth-inhibition was determined.Altogether these methods are valuable tools for generation and characterization of monospecific antibodies.

Place, publisher, year, edition, pages
Stockholm: KTH, 2008. xii, 66 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2008:20
Keyword
antibody, epitope mapping, HER2, human protein atlas, immunogenicity, proteomics
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-9338 (URN)978-91-7415-139-8 (ISBN)
Public defence
2008-10-31, F3, KTH, Lindstedtsvägen 26, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
QC 20100907Available from: 2008-10-21 Created: 2008-10-21 Last updated: 2010-09-07Bibliographically approved
3. 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)
Opponent
Supervisors
Note
QC 20120111Available from: 2012-01-11 Created: 2012-01-11 Last updated: 2012-01-11Bibliographically approved

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Löfblom, JohnStåhl, StefanUhlén, Mathias

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