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Protein microarrays for validation of affinity binders
KTH, School of Biotechnology (BIO), Proteomics.
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 [en]
Microarray, protein, antibody, antigen, affinity, validation
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-48256ISBN: 978-91-7501-149-3 (print)OAI: oai:DiVA.org:kth-48256DiVA: diva2:457131
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
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. Determination of binding specificities in highly multiplexed bead-based assays for antibody proteomics
Open this publication in new window or tab >>Determination of binding specificities in highly multiplexed bead-based assays for antibody proteomics
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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.

Keyword
protein microarrays, atlas
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-16314 (URN)10.1074/mcp.T600035-MCP200 (DOI)000243312000011 ()2-s2.0-33846544538 (Scopus ID)
Note

QC 20100525

Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2017-12-12Bibliographically approved
3. Validation of affinity reagents using antigen microarrays
Open this publication in new window or tab >>Validation of affinity reagents using antigen microarrays
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2011 (English)In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 29, no 5, 555-563 p.Article in journal (Refereed) Published
Abstract [en]

There is a need for standardised validation of affinity reagents to determine their binding selectivity and specificity. This is of particular importance for systematic efforts that aim to cover the human proteome with different types of binding reagents. One such international program is the SH2-consortium, which was formed to generate a complete set of renewable affinity reagents to the SH2-domain containing human proteins. Here, we describe a microarray strategy to validate various affinity reagents, such as recombinant single-chain antibodies, mouse monoclonal antibodies and antigen-purified polyclonal antibodies using a highly multiplexed approach. An SH2-specific antigen microarray was designed and generated, containing more than 6000 spots displayed by 14 identical subarrays each with 406 antigens, where 105 of them represented SH2-domain containing proteins. Approximately 400 different affinity reagents of various types were analysed on these antigen microarrays carrying antigens of different types. The microarrays revealed not only very detailed specificity profiles for all the binders, but also showed that overlapping target sequences of spotted antigens were detected by off-target interactions. The presented study illustrates the feasibility of using antigen microarrays for integrative, high-throughput validation of various types of binders and antigens.

Keyword
protein microarray, antibody validation, affinity reagent, antigen, specificity, SH2
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-48322 (URN)10.1016/j.nbt.2011.11.009 (DOI)000305606500007 ()2-s2.0-84862014285 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceKnut and Alice Wallenberg Foundation
Available from: 2011-11-17 Created: 2011-11-17 Last updated: 2017-12-08Bibliographically approved

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