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Towards a human proteome atlas: High-throughput generation of mono-specific antibodies for tissue profiling
KTH, School of Biotechnology (BIO), Proteomics.ORCID iD: 0000-0002-4657-8532
KTH, School of Biotechnology (BIO).
KTH, School of Biotechnology (BIO), Proteomics.
KTH, School of Biotechnology (BIO), Proteomics.ORCID iD: 0000-0001-8993-048X
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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.

Place, publisher, year, edition, pages
2005. Vol. 5, 4327-4337 p.
Keyword [en]
antibody generation; protein microarray; proteome atlas; tissue microarray; tissue profiling
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-11428DOI: 10.1002/pmic.200500072ISI: 000233686100002Scopus ID: 2-s2.0-28444433078OAI: oai:DiVA.org:kth-11428DiVA: diva2:276021
Note
QC 20100727Available from: 2009-11-10 Created: 2009-11-10 Last updated: 2017-12-12Bibliographically 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. Protein microarrays for validation of affinity binders
Open this publication in new window or tab >>Protein microarrays for validation of affinity binders
2011 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Is specificity an important issue regarding affinity reagents? What about the validation of affinity reagents today, is it good enough? This depends on the application and the producer of the reagent. Validation should be the most important marketing argument that can be found.Today there is a continuous growth of both the number of affinity reagents that are produced and the different types of affinity reagents that are developed. In proteomics they become more and more important in exploring the human proteome. Therefore, validated affinity reagents should be on top of every proteomic researcher’s list. How should this be accomplished?Better international agreements on how affinity reagents should be tested to be regarded as functional reagents are needed. One of the most important issues is the specificity of the affinity reagent. An international standard for which specific validation that is needed for different kinds of applications would be very useful.In this thesis, it is shown that the protein microarray platform that was established within the HPA project at KTH is a very good tool to determine the specificity of different affinity binders.In the first study, the production of mono-specific antibodies for tissue profiling in the Human Protein Atlas (HPA) project is presented. The section describing the use of protein microarrays for validation of the antibodies is relevant for this thesis. The implementation of protein microarrays in the HPA workflow was an important addition, because a deeper insight of the specificity of all the antibodies produced were now available.In a second study, bead based arrays were compared to planar protein microarrays used in the HPA project. In this study, 100 different bead identities were coupled with 100 different antigens and mixed together to generate an array. The correlation between the two types of assays was very high and the conclusion was that the methods can be used as backup to each other.A third study was a part of an international initiative to produce renewable affinity binders against proteins containing SH2 domain. Here, the HPA protein microarrays were modified to analyze different types of reagents produced at six laboratories around the world. Monoclonal antibodies, single chain fragment and fibronectin scaffolds were tested as well as mono-specific antibodies. It was shown to be possible to adapt protein microarrays used in the HPA project to validate other kinds of affinity reagents.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2011. 31 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2011:23
Keyword
Microarray, protein, antibody, antigen, affinity, validation
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-48256 (URN)978-91-7501-149-3 (ISBN)
Presentation
2011-12-09, FB42, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 10:00 (Swedish)
Opponent
Supervisors
Projects
Development and applications of protein microarraysThe Swedish Human Proteome Resource (HPR) program
Note
QC 20111117Available from: 2011-11-17 Created: 2011-11-16 Last updated: 2011-11-17Bibliographically approved

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Uhlén, MathiasHober, SophiaAl-Khalili Szigyarto, Cristina

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