uu.seUppsala University Publications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Validation of affinity reagents using antigen microarrays
KTH, Proteomik.
KTH, Proteomik.
KTH, Skolan för bioteknologi (BIO).
KTH, Skolan för bioteknologi (BIO).
Show others and affiliations
2011 (English)In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 29, no 5, 555-563 p.Article in journal (Other academic) 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.

Place, publisher, year, edition, pages
2011. Vol. 29, no 5, 555-563 p.
Keyword [en]
protein microarray, antibody validation, affinity reagent, antigen, specificity, SH2
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:uu:diva-208949DOI: 10.1016/j.nbt.2011.11.009ISI: 000305606500007OAI: oai:DiVA.org:uu-208949DiVA: diva2:655455
Available from: 2011-11-17 Created: 2013-10-11 Last updated: 2017-12-06
In thesis
1. Mass Spectrometry and Affinity Based Methods for Analysis of Proteins and Proteomes
Open this publication in new window or tab >>Mass Spectrometry and Affinity Based Methods for Analysis of Proteins and Proteomes
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteomics is a fast growing field and there has been a tremendous increase of knowledge the last two decades. Mass spectrometry is the most used method for analysis of complex protein samples. It can be used both in large scale discovery studies as well as in targeted quantitative studies. In parallel with the fast improvements of mass spectrometry-based proteomics there has been a fast growth of affinity-based methods. A common challenge is the large dynamic range of protein concentrations in biological samples. No method can today cover the whole dynamic range. If affinity and mass spectrometry-based proteomics could be used in better combination, this would be partly solved. The challenge for affinity-based proteomics is the poor specificity that has been seen for many of the commercially available antibodies. In mass spectrometry, the challenges are sensitivity and sample throughput. In this thesis, large scale approaches for validation of antibodies and other binders are presented. Protein microarrays were used in four validation studies and one was based on mass spectrometry. It is shown that protein microarrays can be valuable tools to check the specificity of antibodies produced in a large scale production. Mass spectrometry was shown to give similar results as Western blot and Immunohistochemistry regarding specificity, but did also provide useful information about which other proteins that were bound to the antibody.

Mass spectrometry has many applications and in this thesis two methods contributing with new knowledge in animal proteomics are presented. A combination of high affinity depletion, SDS PAGE and mass spectrometry revealed 983 proteins in dog cerebrospinal fluid, of which 801 were marked as uncharacterized in UniProt. A targeted quantitative study of cat serum based on parallel reaction monitoring showed that mass spectrometry can be an applicable method instead of ELISA in animal proteomic studies. Mass spectrometry is a generic method and has the advantage of shorter and less expensive development costs for specific assays that are not hampered by cross-reactivity.

Mass spectrometry supported by affinity based applications will be an attractive tool for further improvements in the proteomic field.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. 82 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1272
Keyword
Mass spectrometry, proteomics, microarray, protein, antibody, antigen, affinity, validation
National Category
Analytical Chemistry
Research subject
Chemistry with specialization in Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-259623 (URN)978-91-554-9300-4 (ISBN)
Public defence
2015-09-25, C4:305, BMC, Husargatan 3, Uppsala, 10:15 (Swedish)
Opponent
Supervisors
Available from: 2015-09-03 Created: 2015-08-10 Last updated: 2015-10-01

Open Access in DiVA

No full text

Other links

Publisher's full text

Authority records BETA

Sjöberg, RonaldSundberg, MårtenGundberg, AnnaSivertsson, ÅsaSchwenk, Jochen M.Uhlén, MathiasNilsson, Peter

Search in DiVA

By author/editor
Sjöberg, RonaldSundberg, MårtenGundberg, AnnaSivertsson, ÅsaSchwenk, Jochen M.Uhlén, MathiasNilsson, Peter
In the same journal
New Biotechnology
Industrial Biotechnology

Search outside of DiVA

GoogleGoogle Scholar

doi
urn-nbn

Altmetric score

doi
urn-nbn
Total: 355 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf