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Solid-phase Proximity Ligation Assays: High-performance and multiplex protein analyses
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. (Molecular Medicine)
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Protein biomarkers circulating in blood hold the promise of improved diagnosis, prognosis and follow-up of treatment of disease via minimally invasive procedures. For the discovery and validation of such biomarkers, methods are needed that can facilitate parallel, highly specific and in-depth analysis of the blood proteome. The work presented in this thesis intends to develop and apply such assays, building on the concept of the proximity ligation assay (PLA).

In paper I, I present an easy and non-expensive alternative for the conjugation of oligonucleotides to antibodies via biotin-streptavidin-biotin interaction. This approach can be used when large sets of antibodies and/or oligos need to be validated for their performance as probes in PLA reactions.

In paper II, a solid-phase variant of PLA (SP-PLA) for the detection and quantification of proteins in blood is presented. SP-PLA exhibited an improved limit of detection compared to commercial ELISA assays by two orders of magnitude. In addition SP-PLA exhibited a broader dynamic range by at least one order of magnitude and required only 5 μl of sample, rendering the method very well suited for analyses of precious bio-banked material. Last but not least, SP-PLA was used to validate the diagnostic potential of GDF-15 as a biomarker for cardiovascular disease in a set of cardiovascular disease patients and healthy controls.

Paper III discusses the development of a multiplex SP-PLA (MultiPLAy) for the simultaneous detection of 36 proteins in just 5 μl of sample. MultiPLAy exhibited an improved LOD when compared to state-of-the-art bead-based sandwich assays. Most importantly, we observed only a minimal tendency to increased background with multiplexing, compared to a sandwich assay, suggesting that much higher levels of multiplexing will be possible. The assay was used to identify putative biomarkers in sample cohorts of colorectal cancer (CRC) and cardiovascular disease (CVD). Subsequent multivariate analysis revealed previously known diagnostic biomarkers. Furthermore, we successfully applied next-generation sequencing as a readout for the protein assays, allowing for the first time digital recording of protein profiles in blood.

In paper IV, we investigated the suitability of prostasomes as blood biomarkers in patients with prostate cancer using a newly developed PLA assay (4PLA) that utilizes five binders for the detection of complex target molecules. The assay successfully detected significantly elevated levels of prostasomes in blood samples from prostate cancer patients prior to radical prostatectomy, compared to controls and men with benign biopsy results.

 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2011. , 43 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 640
Keyword [en]
proximity ligation assay, multiplexed, biomarkers, solid-phase, microparticles, cancer, sequencing
National Category
Biochemistry and Molecular Biology
Research subject
Biology with specialization in Molecular Biotechnology
Identifiers
URN: urn:nbn:se:uu:diva-144093ISBN: 978-91-554-7999-2OAI: oai:DiVA.org:uu-144093DiVA: diva2:395519
Public defence
2011-03-25, Rudbeckhall, Rudbeck Laboratory, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2011-03-04 Created: 2011-01-27 Last updated: 2011-05-04
List of papers
1. Self-assembly of proximity probes for flexible and modular proximity ligation assays
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2007 (English)In: BioTechniques, ISSN 0736-6205, Vol. 43, no 4, 443-450 p.Article in journal (Refereed) Published
Abstract [en]

Proximity ligation assay (PLA) is a recently developed strategy for protein analysis in which antibody-based detection of a target protein via a DNA ligation reaction of oligonucleotides linked to the antibodies results in the formation of an amplifiable DNA strand suitable for analysis. Here we describe a faster and more cost-effective strategy to construct the antibody-based proximity ligation probes used in PLA that is based on the noncovalent interaction of biotinylated oligonucleotides with streptavidin followed by the interaction of this complex with biotinylated antibodies.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-95759 (URN)10.2144/000112551 (DOI)000205886000019 ()180119334 (PubMedID)
Available from: 2007-04-23 Created: 2007-04-23 Last updated: 2011-05-04Bibliographically approved
2. Sensitive plasma protein analysis by microparticle-based proximity ligation assays
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2010 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 9, no 2, 327-335 p.Article in journal (Refereed) Published
Abstract [en]

Detection of proteins released in the bloodstream from tissues damaged by disease can promote early detection of pathological conditions, differential diagnostics, and follow-up of therapy. Despite these prospects and a plethora of candidate biomarkers, efforts in recent years to establish new protein diagnostic assays have met with limited success. One important limiting factor has been the challenge of detecting proteins present at trace levels in complex bodily fluids. To achieve robust, sensitive, and specific detection, we have developed a microparticle-based solid-phase proximity ligation assay, dependent on simultaneous recognition of target proteins by three antibody molecules for added specificity. After capture on a microparticle, solid-phase pairs of proximity probes are added followed by washes, enabling detection and identification of rare protein molecules in blood while consuming small amounts of sample. We demonstrate that single polyclonal antibody preparations raised against target proteins of interest can be readily used to establish assays where detection depends on target recognition by three individual antibody molecules, recognizing separate epitopes. The assay was compared with state-of-the-art sandwich ELISAs for detection of vascular endothelial growth factor, interleukin-8 and interleukin-6, and it was found to be superior both with regard to dynamic range and minimal numbers of molecules detected. Furthermore, the assays exhibited excellent performance in undiluted plasma and serum as well as in whole blood, producing comparable results for nine different antigens. We thus show that solid-phase proximity ligation assay is suitable for validation of a variety of protein biomarkers over broad dynamic ranges in clinical samples.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-123198 (URN)10.1074/mcp.M900248-MCP200 (DOI)000275506200010 ()19955079 (PubMedID)
Available from: 2010-04-26 Created: 2010-04-26 Last updated: 2014-01-08Bibliographically approved
3. Multiplexed solid-phase proximity ligation assays: Highly specific and parallel protein measurements with DNA sequencing readout
Open this publication in new window or tab >>Multiplexed solid-phase proximity ligation assays: Highly specific and parallel protein measurements with DNA sequencing readout
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Identification and validation of protein biomarkers is a very important step towards the understanding of the underlying mechanisms of disease, early diagnosis and efficient patient treatment. To carry out this task, methods are needed that would allow us to mine the proteome with sufficient sensitivity and specificity in large sets of samples. We present herein the development of a Multiplexed Proximity Ligation Assay (MultiPLAy), to facilitate efficient protein profiling in a parallel, sensitive and specific manner. We showed that for the simultaneous analysis of 35 proteins MultiPLAy exhibited an improved sensitivity over conventional sandwich assays as well as a smaller susceptibility to background signal increase in the transition from singleplex to multiplex. We used MultiPLAy to identify putative biomarkers in two separate sample cohorts of colorectal cancer (CRC) and cardiovascular disease (CVD) and with the use a novel multivariate analysis approach were able to identify new, as well as already known diagnostic biomarkers. Furthermore we were able to combine MultiPLAy with the use of next-generation sequencing allowing for the first time digital recording of protein profiles in blood. We demonstrated good reproducibility of MultiPLAy coupled to next-generation sequencing, as well as a satisfactory correlation to standard real-time PCR readout. We conclude that MultiPLAy has great potential as a basis for highly multiplexed protein detection assays that can be utilized for the identification of large numbers of proteins or protein variants. This will allow extensive validation of protein expression patterns in biobanked samples and in prospective studies, and can provide a much-needed platform for efficient validation of diagnostic markers for clinical use.

 

Keyword
multiplex, proximity ligation assay, sequencing, colorectal cancer, cardiovascular disease, biomarkers
Identifiers
urn:nbn:se:uu:diva-145007 (URN)
Funder
Knut and Alice Wallenberg FoundationEU, FP7, Seventh Framework ProgrammeSwedish Research Council
Available from: 2011-02-04 Created: 2011-02-04 Last updated: 2011-05-04
4. Multiple recognition assay reveals prostasomes as promising plasma biomarkers for prostate cancer
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2011 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 108, no 21, 8809-8814 p.Article in journal (Refereed) Published
Abstract [en]

Prostasomes are microvesicles (mean diameter, 150 nm) that are produced and secreted by normal and malignant prostate acinar cells. It has been hypothesized that invasive growth of malignant prostate cells may cause these microvesicles, normally released into seminal fluid, to appear in interstitial space and therewith into peripheral circulation. The suitability of prostasomes as blood biomarkers in patients with prostate cancer was tested by using an expanded variant of the proximity ligation assay (PLA). We developed an extremely sensitive and specific assay (4PLA) for detection of complex target structures such as microvesicles in which the target is first captured via an immobilized antibody and subsequently detected by using four other antibodies with attached DNA strands. The requirement for coincident binding by five antibodies to generate an amplifiable reporter results in both increased specificity and sensitivity. The assay successfully detected significantly elevated levels of prostasomes in blood samples from patients with prostate cancer before radical prostatectomy, compared with controls and men with benign biopsy results. The medians for prostasome levels in blood plasma of patients with prostate cancer were 2.5 to sevenfold higher compared with control samples in two independent studies, and the assay also distinguished patients with high and medium prostatectomy Gleason scores (8/9 and 7, respectively) from those with low score (<= 6), thus reflecting disease aggressiveness. This approach that enables detection of prostasomes in peripheral blood may be useful for early diagnosis and assessment of prognosis in organ-confined prostate cancer.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-144092 (URN)10.1073/pnas.1019330108 (DOI)000290908000058 ()21555566 (PubMedID)
Funder
EU, FP7, Seventh Framework ProgrammeKnut and Alice Wallenberg FoundationSwedish Research Council
Available from: 2011-01-27 Created: 2011-01-27 Last updated: 2012-06-12Bibliographically approved

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