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Determination of binding specificities in highly multiplexed bead-based assays for antibody proteomics
KTH, School of Biotechnology (BIO).ORCID iD: 0000-0001-8141-8449
KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).ORCID iD: 0000-0001-8993-048X
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2007 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 6, no 1, 125-132 p.Article in journal (Refereed) Published
Abstract [en]

One of the major challenges of antibody-based proteomics is the quality assurance of the generated antibodies to ensure specificity to the target protein. Here we describe a single tube multiplex approach to simultaneously analyze the binding of antibodies to a large number of different antigens. This bead-based assay utilizes the full multiplexing capacity theoretically offered by the Luminex suspension array technology. A protocol for an increased coupling throughput for the immobilization of antigens was developed and used to set up complex and stabile 100-plex bead mixtures. The possibility of using a two-dimensional multiplexing, in terms of high numbers of both analytes and samples or as in this case antigens and antibodies, enables the specificity of 96 antibodies versus 100 different antigens to be determined in 2 h. This high throughput analysis will potentially have great impact on the possibility for the utilization of different antibody proteomics approaches where the quality assessment of antibodies is of the utmost importance.

Place, publisher, year, edition, pages
2007. Vol. 6, no 1, 125-132 p.
Keyword [en]
protein microarrays, atlas
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-16314DOI: 10.1074/mcp.T600035-MCP200ISI: 000243312000011Scopus ID: 2-s2.0-33846544538OAI: oai:DiVA.org:kth-16314DiVA: diva2:334356
Note

QC 20100525

Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2017-12-12Bibliographically approved
In thesis
1. 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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Schwenk, Jochen M.Uhlén, Mathias

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