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Multiplexed PrEST immunization for high-throughput affinity proteomics
KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
KTH, School of Biotechnology (BIO).
KTH, School of Biotechnology (BIO).ORCID iD: 0000-0001-8993-048X
KTH, School of Biotechnology (BIO).ORCID iD: 0000-0003-0605-8417
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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.

Place, publisher, year, edition, pages
2006. Vol. 315, 110-120 p.
Keyword [en]
Affinity purification; Monospecific antibody; Multiplex antigens; PrEST; Proteomics
National Category
Industrial Biotechnology
URN: urn:nbn:se:kth:diva-11429DOI: 10.1016/j.jim.2006.07.014ISI: 000241291500013ScopusID: 2-s2.0-33748764953OAI: diva2:276022

QC 20100727

Available from: 2009-11-10 Created: 2009-11-10 Last updated: 2016-09-27Bibliographically 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.
Trita-BFE, ISSN 1104-4101 ; 2009:21
antibody, antibody validation, immunohistochemistry, immunofluorescence, tissue microarray, Western blot, epitope mapping, antigen design
National Category
Industrial Biotechnology
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)
QC 20100727Available from: 2009-11-10 Created: 2009-11-09 Last updated: 2011-11-23Bibliographically approved

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