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Profiling post-centrifugation delay of serum and plasma with antibody bead arrays
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0001-8603-8293
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2013 (English)In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 95, no SI, 46-54 p.Article in journal (Refereed) Published
Abstract [en]

Several biobanking initiatives have emerged to create extensive collections of specimen for biomedical studies and various analytical platforms. An affinity proteomic analysis with antibody suspension bead arrays was conducted to investigate the influence of the pre-analytical time and temperature conditions on blood derived samples. Serum and EDTA plasma prepared from 16 individuals was centrifuged and aliquots were kept either at 4. °C or in ambient temperature for 1. h and up to 36. h prior to first storage. Multiplexed protein profiles of post-centrifugation delay were generated in 384 biotinylated samples using 373 antibodies that targeted 343 unique proteins. Very few profiles were observed as significantly altered by the studied temperature and time intervals. Single binder and sandwich assays revealed decreasing levels of caldesmon 1 (CALD1) related to EDTA standard tubes and prolonged post-centrifugation delay of 36. h. Indications from changes in CALD1 levels require further confirmation in independent material, but the current data suggests that samples should preferentially be frozen during the day of collection when to be profiled with antibody arrays selected for this study. Biological significance: Affinity-based profiling of serum and plasma by microarray assays can provide unique opportunities for the discovery of biomarkers. It is though often not known how differences in sample handling after collection influence the downstream analysis. By profiling three types of blood preparations for alterations in protein profiles with respect to time and temperature post centrifugation, we addressed an important component in the analysis and of such specimen. We believe that this analysis adds valuable information to be considered when biobanking blood derived samples.This article is part of a Special Issue entitled: Standardization and Quality Control in Proteomics.

Place, publisher, year, edition, pages
2013. Vol. 95, no SI, 46-54 p.
Keyword [en]
protein profiling, plasma, antibody, affinity proteomics, biomark- ers, multiplex, assay development
National Category
Medical Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-126285DOI: 10.1016/j.jprot.2013.04.020ISI: 000332495700005PubMedID: 23631827Scopus ID: 2-s2.0-84888297693OAI: oai:DiVA.org:kth-126285DiVA: diva2:642220
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceVinnovaKnut and Alice Wallenberg Foundation
Note

QC 20140414

Available from: 2013-08-21 Created: 2013-08-21 Last updated: 2017-12-06Bibliographically approved
In thesis
1. Antibody based plasma protein profiling
Open this publication in new window or tab >>Antibody based plasma protein profiling
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis is about protein profiling in serum and plasma using antibody suspension bead arrays for the analysis of biobanked samples and in the context of prostate cancer biomarker discovery. The influence of sample preparation methods on antibody based protein profiles were investigated (Papers I-III) and a prostate cancer candidate biomarker identified and verified (Papers III-V). Furthermore, a perspective on the research area affinity proteomics and its’ employment in biomarker discovery, for improved understanding and potentially improved disease diagnosis, is provided.

Paper I presents the results of a comparative plasma and serum protein profiling study, with a targeted biomarker discovery approach in the context of metabolic syndrome. The study yielded a higher number of significant findings and a low experimental variability in blood samples prepared as plasma. Paper II investigated the effects from post-centrifugation delays at different temperatures prior sample storage of serum and plasma samples. Minor effects were found on the detected levels of more than 300 predicted or known plasma proteins. In Paper III, the detectability of proteins in plasma was explored by exposing samples to different pre-analytical heat treatments, prior target capture. Heat induced epitope retrieval was observed for approximately half of the targeted proteins, and resulted in the discovery of different candidate markers for prostate cancer. Several antibodies towards the prostate cancer candidate biomarker CNDP1 were generated, epitope mapped and evaluated in a bead based sandwich immunoassay, as presented in Papers IV and V. Furthermore, the developed sandwich immunoassay targeting multiple distinct CNDP1 epitopes in more than 1000 samples, confirmed the association of CNDP1 levels to aggres- sive prostate cancer and more specifically to prostate cancer patients with regional lymph node metastasis (Paper V).

As an outcome of the present investigations and in parallel to studies within the Biobank profiling research group, valuable lessons from study design and multiplex antibody analysis of plasma within biomarker discovery to experimental, technical and biological verifications have been collected.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2013. viii, 68 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2013:12
Keyword
protein profiling, plasma, antibody, affinity proteomics, biomark- ers, multiplex, assay development
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-126270 (URN)978-91-7501-829-4 (ISBN)
Public defence
2013-09-06, Gamma house lecture hall, SciLifeLab, Tomtebodavägen 23a, Solna, 10:00 (English)
Opponent
Supervisors
Note

QC 20130821

Available from: 2013-08-21 Created: 2013-08-20 Last updated: 2013-08-21Bibliographically approved

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Hong, Mun-GwanUhlén, MathiasSchwenk, Jochen M.

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