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Validating the selectivity of antibodies used in multiplexed serum profiling via parallel immunocapture analysis
KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0002-1855-703X
KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova. KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).ORCID iD: 0000-0003-4214-6991
KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).ORCID iD: 0000-0001-8993-048X
KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0002-4657-8532
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The increasing availability of antibodies towards human proteins drives the generation of new and exploratory data, which can be generated by profiling e.g. plasma samples using multiplexed arrays. However, antibody assays can lead to erroneous results due to cross-­‐reactivity to off-­‐targets. Here, we describe an approach in which an antibody-­‐based suspension bead array is combined with subsequent validation of on-­‐ target binding using a coupled affinity purification assay. Based on differential heat treatment of samples, antibody performance was investigated and the results for antibodies directed towards several complement factors provide insights on the detection of proteins in sera from patients with complement deficiency. In conclusion, the combined parallel flow and blot based immunocapture analysis serves an important, first line tool for resolving differently detected serum or plasma protein profiles proposed by antibody arrays. 

Keyword [en]
Suspension bead array, protein profiling, antibody, immunocapture, Western blot
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:kth:diva-117959OAI: oai:DiVA.org:kth-117959DiVA: diva2:604021
Note

QS 2013

Available from: 2013-02-07 Created: 2013-02-07 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.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2013:4
Keyword
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)
Identifiers
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)
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Supervisors
Note

QC 20130208

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

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Neiman, MajaNygren, Per-ÅkeUhlén, MathiasSchwenk, Jochen M.

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Neiman, MajaNygren, Per-ÅkeUhlén, MathiasNilsson, PeterSchwenk, Jochen M.
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Proteomics (closed 20130101)Science for Life Laboratory, SciLifeLabAlbanova VinnExcellence Center for Protein Technology, ProNovaMolecular Biotechnology (closed 20130101)
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)

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