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Characterization of PrEST-based antibodies towards human Cytokeratin-17
KTH, School of Biotechnology (BIO), Proteomics.
KTH, School of Biotechnology (BIO), Proteomics.
KTH, School of Biotechnology (BIO), Proteomics.ORCID iD: 0000-0001-8141-8449
KTH, School of Biotechnology (BIO), Proteomics.
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2009 (English)In: JIM - Journal of Immunological Methods, ISSN 0022-1759, E-ISSN 1872-7905, Vol. 342, 20-32 p.Article in journal (Refereed) Published
Abstract [en]

Antibody-based proteomics efforts depend on validated antibodies to ensure correct annotation of analyzed proteins. We have previously argued that a low sequence identity to other proteins is a key feature for antigens used in antibody generation. Thus, a major challenge for whole-proteome studies is how to address families of highly sequence related proteins within the context of generating specific antibodies. In this study, two non-overlapping parts of human Cytokeratin-17, a protein belonging to the intermediate filament family of highly sequence-related proteins, were selected as a model system to study the specificity and cross reactivity of antibodies generated towards such a target. These recombinantly produced Protein Epitope Signature Tags (PrESTs) were immunized in five rabbits each and the batch-to-batch variations in the obtained immune responses were studied by mapping of linear epitopes using synthetic overlapping peptides. The obtained results showed a similar but not identical immune response in the respective antibody groups with a limited number of epitopes being identified. Immunohistochemical analysis of the affinity purified monospecific antibodies on tissue micro arrays resulted in a general recognition of human cytokeratins for all analyzed binders whereas antibodies identified as binding to the most unique parts of the PrESTs showed the most Cytokeratin-17 like staining. The data presented here support the strategy to use sequence identity scores as the main criteria for antigen selection but also indicate the possibility to instead produce a single antibody recognizing a defined group of proteins when the intended targets overall sequence identity score is too high. This type of group-specific antibodies would be an important tool for antibody-based projects aiming for a complete coverage of the human proteome.

Place, publisher, year, edition, pages
2009. Vol. 342, 20-32 p.
Keyword [en]
Cytokeratin-17; PrEST; Epitope mapping; Antibody
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-11430DOI: 10.1016/j.jim.2008.11.013ISI: 000264685300003OAI: oai:DiVA.org:kth-11430DiVA: diva2:276023
Note
QC 20100712Available from: 2009-11-10 Created: 2009-11-10 Last updated: 2011-01-11Bibliographically approved
In thesis
1. Generation and characterization of antibodies for proteomics research
Open this publication in new window or tab >>Generation and characterization of antibodies for proteomics research
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Specific antibodies are invaluable tools for proteomics research. The availability of thoroughly validated antibodies will help to improve our understanding of protein expression, localization and function; fundamental processes and features of all living organisms. The objectives of the studies in this thesis were to develop high-throughput methods to facilitate the generation and purification of monospecific antibodies, and to address problems associated with antigen selection for difficult target proteins and subsequent validation issues.

In the first of the studies, it was demonstrated that antibodies specific to human proteins could be generated in a high-throughput manner using protein epitope signature tags (PrESTs) as both antigens and affinity ligands. A previously developed purification process was adapted to a high-throughput format and this, in combination with the development of a protein microarray assay, resulted in monospecific antibodies that were used for profiling protein expression in 48 human tissues. Data obtained in these analyses suggest that a complete Human Protein Atlas should be attainable within the next ten years. In order to reduce the number of animals needed for such a massive project, and improve the cost-efficiency of antibody generation, a multiplex immunization strategy was developed in a further study. Antisera from rabbits immunized with mixtures of two, three, five and up to ten different PrESTs were successfully purified and analyzed for specificity using protein arrays. Almost 80% of the animals immunized with up to three PrESTs yielded antibodies towards all the PrESTs administered, and they yielded comparable immunohistochemical staining patterns (of consecutive human tissue sections) to those of antibodies obtained from traditional single PrEST immunizations.

Proteins with highly similar sequences to other proteins present a major challenge for the proteome-wide generation of antibodies. In another study, Cytokeratin-17 which displays high sequence similarity to closely related members of the intermediate filament family, was used as a model and the specificity and cross-reactivity of antibodies generated against this target were investigated using epitope mapping in combination with comparative IHC analyses. Antibodies identified by epitope mapping as binding to the most unique parts of the Cytokeratin-17 PrESTs also showed the most Cytokeratin-17-like staining pattern, thus further supporting the strategy of using sequence identity scores as the main criteria for PrEST design.

An alternative antigen design strategy was investigated for use in raising antibodies towards G-proteincoupled receptors (GPCRs). The extracellular loops and N-terminus of each of three selected GPCRs were assembled to form single antigens and the resulting antibodies were analyzed by flow cytometric and confocal microscopic analyses of cell lines over-expressing the respective receptors. The results from both flow cytometric and immunofluorescence analyses showed that the antibodies were able to bind to their targets. In addition, the antibodies were used successfully for the in situ analysis of human brain and pancreatic islet cells.

Place, publisher, year, edition, pages
Stockholm: KTH, 2009. x, 66 p.
Series
Trita-BFE, ISSN 1104-4101 ; 2009:21
Keyword
antibody, antibody validation, immunohistochemistry, immunofluorescence, tissue microarray, Western blot, epitope mapping, antigen design
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-11425 (URN)978-91-7415-439-9 (ISBN)
Public defence
2009-12-04, FD5, Svedbergs sal, AlbaNova, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
QC 20100727Available from: 2009-11-10 Created: 2009-11-09 Last updated: 2011-11-23Bibliographically approved
2. Affinity based proteomics research tools
Open this publication in new window or tab >>Affinity based proteomics research tools
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Listen to the mantra; the mapping of the genome was finished in 2001, and the sequel research challenge is the thorough survey of the corresponding human proteome. This was stated almost a decade ago, it has been repeated over and over, and is still most certainly a hard nut to crack. The workload is daunting, much because there is no protein amplification method and no binary system for the detection of proteins, and because the complexity of the proteome is larger than that of the genome as it seems. Hence, there is a need for high throughput technologies that, at sufficiently low limits of detection and with satisfying sensitivities, may investigate protein content in human samples. With this aim, the Human Proteome Resource (HPR) project was initiated in 2003.

All work presented in this thesis relate to protein interactions; binders are either utilized such as for the depletion of high abundant proteins from serum, or analyzed such as in the validation of monospecific antibody specificity, or the epitope mapping of the same. In Paper I, the Gyrolab system is utilized in a setup for the specificity analysis of monospecific antibodies towards their antigen, and the setup is compared to planar protein arrays. Gyrolab technology is used again, in Paper II, where epitope mapping of monospecific antibodies is performed in order to analyze antibody specificity. Also, mapping serves to compare the immune-responses from parallel immunizations using the same antigen, thereby assessing reproducibility in the regeneration of antibodies. Paper III describes a high throughput approach for the depletion of high abundant proteins, in serum and plasma samples, taking advantage of Affibody molecules as binders. The last two papers, IV and V, utilize monospecific antibodies for protein analysis; in Paper IV pull out experiments show that competitive elution using the PrEST antigen can be a fruitful approach to increase specificity. And finally, in Paper V, a setup for the semi-quantitative protein content analysis in fluid samples is suggested. Again, the Gyrolab technology is used, and the setup is tested on a simplified model system.

Place, publisher, year, edition, pages
Stockholm: KTH, 2009. 86 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2009:15
National Category
Immunology Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-11184 (URN)978-91-7415-422-1 (ISBN)
Public defence
2009-10-09, FR4 Oskar Klein, AlbaNova, Roslagstullsbacken 33, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
QC 20100712Available from: 2009-10-01 Created: 2009-10-01 Last updated: 2010-07-16Bibliographically approved

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Schwenk, Jochen. M.Hober, SophiaUhlén, Mattias

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