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Toward Next Generation Plasma Profiling via Heat-induced Epitope Retrieval and Array-based Assays
KTH, School of Biotechnology (BIO), Proteomics.ORCID iD: 0000-0001-8141-8449
KTH, School of Biotechnology (BIO), Proteomics.
KTH, School of Biotechnology (BIO), Proteomics.ORCID iD: 0000-0002-1855-703X
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2010 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 9, no 11, 2497-2507 p.Article in journal (Refereed) Published
Abstract [en]

There is a need for high throughput methods for screening patient samples in the quest for potential biomarkers for diagnostics and patient care. Here, we used a combination of undirected target selection, antibody suspension bead arrays, and heat-induced epitope retrieval to allow for protein profiling of human plasma in a novel and systematic manner. Several antibodies were found to reveal altered protein profiles upon epitope retrieval at elevated temperatures with limits of detection improving into lower ng/ml ranges. In a study based on prostate cancer patients, several proteins with differential profiles were discovered and subsequently validated in an independent cohort. For one of the potential biomarkers, the human carnosine dipeptidase 1 protein (CNDP1), the differences were determined to be related to the glycosylation status of the targeted protein. The study shows a path of pursuit for large scale screening of biobank repositories in a flexible and proteome-wide fashion by utilizing heat-induced epitope retrieval and using an antibody suspension bead array format. Molecular & Cellular Proteomics 9:2497-2507, 2010.

Place, publisher, year, edition, pages
2010. Vol. 9, no 11, 2497-2507 p.
National Category
Biochemistry and Molecular Biology
URN: urn:nbn:se:kth:diva-30996DOI: 10.1074/mcp.M110.001560ISI: 000285055900013ScopusID: 2-s2.0-78149323414OAI: diva2:404803
Swedish Research CouncilKnut and Alice Wallenberg Foundation
QC 20110318Available from: 2011-03-18 Created: 2011-03-07 Last updated: 2013-08-21Bibliographically 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.
Trita-BIO-Report, ISSN 1654-2312 ; 2013:12
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)
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)

QC 20130821

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

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