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Generation and characterization of antibodies for proteomics research
KTH, School of Biotechnology (BIO), Proteomics. (Proteomics)
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 [en]
antibody, antibody validation, immunohistochemistry, immunofluorescence, tissue microarray, Western blot, epitope mapping, antigen design
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-11425ISBN: 978-91-7415-439-9 (print)OAI: oai:DiVA.org:kth-11425DiVA: diva2:275849
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
List of papers
1. Towards a human proteome atlas: High-throughput generation of mono-specific antibodies for tissue profiling
Open this publication in new window or tab >>Towards a human proteome atlas: High-throughput generation of mono-specific antibodies for tissue profiling
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2005 (English)In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 5, 4327-4337 p.Article in journal (Refereed) Published
Abstract [en]

A great need exists for the systematic generation of specific antibodies to explore the human proteome. Here, we show that antibodies specific to human proteins can be generated in a high-throughput manner involving stringent affinity purification using recombinant protein epitope signature tags (PrESTs) as immunogens and affinity-ligands. The specificity of the generated affinity reagents, here called mono-specific antibodies (msAb), were validated with a novel protein microarray assay. The success rate for 464 antibodies generated towards human proteins was more than 90% as judged by the protein array assay. The antibodies were used for parallel profiling of patient biopsies using tissue microarrays generated from 48 human tissues. Comparative analysis with well-characterized monoclonal antibodies showed identical or similar specificity and expression patterns. The results suggest that a comprehensive atlas containing extensive protein expression and subcellular localization data of the human proteome can be generated in an efficient manner with mono-specific antibodies.

Keyword
antibody generation; protein microarray; proteome atlas; tissue microarray; tissue profiling
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-11428 (URN)10.1002/pmic.200500072 (DOI)000233686100002 ()2-s2.0-28444433078 (Scopus ID)
Note
QC 20100727Available from: 2009-11-10 Created: 2009-11-10 Last updated: 2017-12-12Bibliographically approved
2. Multiplexed PrEST immunization for high-throughput affinity proteomics
Open this publication in new window or tab >>Multiplexed PrEST immunization for high-throughput affinity proteomics
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2006 (English)In: JIM - Journal of Immunological Methods, ISSN 0022-1759, E-ISSN 1872-7905, Vol. 315, 110-120 p.Article in journal (Refereed) Published
Abstract [en]

Monospecific antibodies dfdfdfdf (msAbs) generated through antigen specific purification of polyclonal antisera are valuable tools in proteome analyses. However, proteome wide generation of msAbs would require extensive immunization programs. Therefore, it would be desirable to develop efficient immunization and purification methods to reduce the number of animals needed for such antibody-based research. Here we describe a multiplex immunization strategy for generation of msAbs towards recombinantly produced human protein fragments, denoted PrESTs. Antisera from rabbits immunized with a mixture of two, three, five and up to ten different PrESTs have been purified by a two-step immunoaffinity-based protocol and the efficiency of the purification method was analyzed using a two-color protein array concept. The obtained results showed that almost 80% of the animals immunized with antigens composed of two or three different PrESTs yielded antibodies recognizing all the included PrESTs. Furthermore, the modified two-step purification method effectively eliminated all background binding and produced pure antibody pools against individual PrESTs. This indicates that the multiplexed PrEST immunization strategy described here could become useful for high-throughput antibody-based proteomics initiatives, thus significantly reducing the number of animals needed in addition to providing a more cost-efficient method for production of msAbs.

Keyword
Affinity purification; Monospecific antibody; Multiplex antigens; PrEST; Proteomics
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-11429 (URN)10.1016/j.jim.2006.07.014 (DOI)000241291500013 ()2-s2.0-33748764953 (Scopus ID)
Note

QC 20100727

Available from: 2009-11-10 Created: 2009-11-10 Last updated: 2017-12-12Bibliographically approved
3. Characterization of PrEST-based antibodies towards human Cytokeratin-17
Open this publication in new window or tab >>Characterization of PrEST-based antibodies towards human Cytokeratin-17
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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.

Keyword
Cytokeratin-17; PrEST; Epitope mapping; Antibody
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-11430 (URN)10.1016/j.jim.2008.11.013 (DOI)000264685300003 ()
Note
QC 20100712Available from: 2009-11-10 Created: 2009-11-10 Last updated: 2017-12-12Bibliographically approved
4. Novel antigen design for the generation of G-protein-coupled receptorantibodies.
Open this publication in new window or tab >>Novel antigen design for the generation of G-protein-coupled receptorantibodies.
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(English)Manuscript (preprint) (Other academic)
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-14226 (URN)
Note
QC 20100727Available from: 2010-07-27 Created: 2010-07-27 Last updated: 2010-07-27Bibliographically approved

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