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Classification of protein profiles from antibody microarrays using heat and detergent treatment.
KTH, School of Biotechnology (BIO), Proteomics.ORCID iD: 0000-0002-0056-1313
KTH, School of Biotechnology (BIO), Proteomics.ORCID iD: 0000-0002-1855-703X
KTH, School of Biotechnology (BIO), Proteomics.
KTH, School of Biotechnology (BIO), Proteomics.
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2011 (English)In: New Biotechnology, ISSN 1871-6784, Vol. 29, no 5, 564-570 p.Article in journal (Refereed) Published
Abstract [en]

Antibody microarrays offer new opportunities for exploring the proteome and to identify biomarker candidates in human serum and plasma. Here, we have investigated the effect of heat and detergents on an antibody-based suspension bead array (SBA) assay using polyclonal antibodies and biotinylated plasma samples. With protein profiles from more than 2300 antibodies generated in 384-plex antibody SBAs, three major classes of heat and detergent susceptibility could be described. The results show that washing of the beads with SDS (rather than Tween) after target binding lowered intensity levels of basically all profiles and that about 50% of the profiles appeared to be lowered to a similar extent by heating of the sample. About 33% of the profiles appeared to be insensitive to heat treatment while another 17% showed a positive influence of heat to yield elevated profiles. The results suggest that the classification of antibodies is driven by the molecular properties of the antibody-antigen interaction and can generally not be predicted based on protein class or Western blot data. The experimental scheme presented here can be used to systematically categorize antibodies and thereby combine antibodies with similar properties into targeted arrays for analysis of plasma and serum.

Place, publisher, year, edition, pages
2011. Vol. 29, no 5, 564-570 p.
National Category
Medical Biotechnology
URN: urn:nbn:se:kth:diva-52450DOI: 10.1016/j.nbt.2011.10.005ISI: 000305606500008PubMedID: 22023822ScopusID: 2-s2.0-84862011672OAI: diva2:466666
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
QC 20111216Available from: 2011-12-16 Created: 2011-12-16 Last updated: 2013-02-08Bibliographically approved
In thesis
1. Bead based protein profiling in blood
Open this publication in new window or tab >>Bead based protein profiling in blood
2013 (Swedish)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis is about protein profiling in blood-derived samples using suspension bead ar- rays built with protein affinity reagents, and the evaluation of binding characteristics and potential disease relation of such profiles.

A central aim of the presented work was to discover and verify disease associated protein profiles in blood-derived samples such as serum or plasma. This was based on immobiliz- ing antigens or antibodies on color-coded beads for a multiplexed analysis. This concept generally allow for a dual multiplexing because hundreds of samples can be screened for hundreds of proteins in a miniaturized and parallelized fashion. At first, protein antigens were used to study humoral immune responses in cattle suffering from a mycoplasma infec- tion (Paper I). Here, the most immunogenic of the applied antigens were identified based on reactivity profiles from the infected cattle, and were combined into an antigen cocktail to serve as a diagnostic assay in a standard ELISA set-up. Next, antibodies and their em- ployment in assays with directly labeled human samples was initiated. This procedure was applied in a study of kidney disorders where screening of plasma resulted in the discovery of a biomarker candidate, fibulin-1 (Paper II). In parallel to the disease related applica- tions, systematic evaluations of the protein profiles were conducted. Protein profiles from 2,300 antibodies were classified on the bases of binding properties in relation to sample heating and stringent washing (Paper III). With a particular focus on heat dependent de- tectability, a method was developed to visualize those proteins that were captured to the beads in an immunoassay by using Western blotting (Paper IV). In conclusion, this thesis presents examples of the possibilities of comparative plasma profiling enabled by protein bead arrays. 

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2013. 116 p.
Trita-BIO-Report, ISSN 1654-2312 ; 2013:4
Affinity proteomics, protein array, suspension bead array, antigen, antibody, biomarker discovery, serology, selectivity, sensitivity, serum, plasma
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
urn:nbn:se:kth:diva-117960 (URN)978-91-7501-629-0 (ISBN)
Public defence
2013-03-01, Gardaulan, Smittskyddsinstitutet, Nobels väg 18, Solna, 10:00 (English)

QC 20130208

Available from: 2013-02-08 Created: 2013-02-07 Last updated: 2013-02-08Bibliographically approved

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