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Antibody based plasma protein profiling
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
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 [en]
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: urn:nbn:se:kth:diva-126270ISBN: 978-91-7501-829-4 (print)OAI: oai:DiVA.org:kth-126270DiVA: diva2:642014
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
List of papers
1. Comparative protein profiling of serum and plasma using an antibody suspension bead array approach
Open this publication in new window or tab >>Comparative protein profiling of serum and plasma using an antibody suspension bead array approach
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2010 (English)In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 10, no 3, 532-540 p.Article in journal (Refereed) Published
Abstract [en]

In the pursuit towards a systematic analysis of human diseases, array-based approaches within antibody proteomics offer high-throughput strategies to discover protein biomarkers in serum and plasma. To investigate the influence of sample preparation on such discovery attempts, we report on a systematic effort to compare serum and plasma protein profiles determined with an antibody suspension bead array. The intensity levels were used to define protein profiles and no significant differences between serum and plasma were observed for 79% of the 174 antibodies (targeting 156 proteins). By excluding 36 antibodies giving rise to differential intensity levels, cluster analysis revealed donor-specific rather than preparation-dependent grouping. With a cohort from a clinically relevant medical condition, the metabolic syndrome, the influence of the sample type on a multiplexed biomarker discovery approach was further investigated. Independent comparisons of protein profiles in serum and plasma revealed an antibody targeting ADAMTSL-4, a protein that would qualify to be studied further in association with the condition. In general, the preparation type had an impact on the results of the applied antibody suspension bead array, and while the technical variability was equal, plasma offered a greater biological variability and allowed to give rise to more discoveries than serum.

Keyword
Metabolic syndrome, Plasma, Protein arrays, Protein profiling, Serum, Suspension bead array, mass-spectrometry, extracellular-matrix, proteomics, identification, generation, affinity, disease, tissues, atlas
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-19229 (URN)10.1002/pmic.200900657 (DOI)000274707400015 ()2-s2.0-76649124562 (Scopus ID)
Funder
Knut and Alice Wallenberg Foundation
Note
QC 20100525Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2017-12-12Bibliographically approved
2. Profiling post-centrifugation delay of serum and plasma with antibody bead arrays
Open this publication in new window or tab >>Profiling post-centrifugation delay of serum and plasma with antibody bead arrays
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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.

Keyword
protein profiling, plasma, antibody, affinity proteomics, biomark- ers, multiplex, assay development
National Category
Medical Biotechnology
Identifiers
urn:nbn:se:kth:diva-126285 (URN)10.1016/j.jprot.2013.04.020 (DOI)000332495700005 ()23631827 (PubMedID)2-s2.0-84888297693 (Scopus ID)
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
3. Toward Next Generation Plasma Profiling via Heat-induced Epitope Retrieval and Array-based Assays
Open this publication in new window or tab >>Toward Next Generation Plasma Profiling via Heat-induced Epitope Retrieval and Array-based Assays
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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.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-30996 (URN)10.1074/mcp.M110.001560 (DOI)000285055900013 ()2-s2.0-78149323414 (Scopus ID)
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Note
QC 20110318Available from: 2011-03-18 Created: 2011-03-07 Last updated: 2017-12-11Bibliographically approved
4. Generation of monospecific antibodies based on affinity capture of polyclonal antibodies
Open this publication in new window or tab >>Generation of monospecific antibodies based on affinity capture of polyclonal antibodies
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2011 (English)In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 20, no 11, 1824-1835 p.Article in journal (Refereed) Published
Abstract [en]

A method is described to generate and validate antibodies based on mapping the linear epitopes of a polyclonal antibody followed by sequential epitope-specific capture using synthetic peptides. Polyclonal antibodies directed towards four proteins RBM3, SATB2, ANLN, and CNDP1, potentially involved in human cancers, were selected and antibodies to several non-overlapping epitopes were generated and subsequently validated by Western blot, immunohistochemistry, and immunofluorescence. For all four proteins, a dramatic difference in functionality could be observed for these monospecific antibodies directed to the different epitopes. In each case, at least one antibody was obtained with full functionality across all applications, while other epitope-specific fractions showed no or little functionality. These results present a path forward to use the mapped binding sites of polyclonal antibodies to generate epitope-specific antibodies, providing an attractive approach for large-scale efforts to characterize the human proteome by antibodies.

Keyword
epitope mapping, monospecific antibody, affinity chromatography
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-47969 (URN)10.1002/pro.716 (DOI)000296273700009 ()2-s2.0-80054717087 (Scopus ID)
Funder
Knut and Alice Wallenberg FoundationScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note
QC 20111117Available from: 2011-11-17 Created: 2011-11-15 Last updated: 2017-12-08Bibliographically approved
5. Plasma levels of carnosine dipeptidase 1 decrease in prostate cancer patients with lymph node metastasis
Open this publication in new window or tab >>Plasma levels of carnosine dipeptidase 1 decrease in prostate cancer patients with lymph node metastasis
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

There is a need for a better differentiation of aggressive tumors in prostate cancer to design a tailored treatment for each patient, preferably by a minimally invasive analysis of blood samples. In a previous study, we discovered a decrease of plasma levels of carnosine dipeptidase 1 (CNDP1) in association with aggressive prostate cancer. Now this relation has been investigated and characterized further by generating several new antibodies for extended analysis of CNDP1 in plasma. Multi-antibody sandwich assays were developed and applied to 1,214 samples from two Swedish cohorts that confirmed decreased levels of CNDP1 in plasma for patients with advanced disease. Therein, CNDP1 assays revealed superior differentiation for tumor N stages than clinical tPSA. Further investigations can now elucidate mechanisms behind decreasing levels of CNDP1 in plasma and primary in regards to lymph node metastasis.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-126386 (URN)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceVinnovaKnut and Alice Wallenberg FoundationSwedish Research Council
Note

QS 2013

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

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