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Sensitive plasma protein analysis by microparticle-based proximity ligation assays
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
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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.

Place, publisher, year, edition, pages
2010. Vol. 9, no 2, 327-335 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-123198DOI: 10.1074/mcp.M900248-MCP200ISI: 000275506200010PubMedID: 19955079OAI: oai:DiVA.org:uu-123198DiVA: diva2:313158
Available from: 2010-04-26 Created: 2010-04-26 Last updated: 2014-01-08Bibliographically approved
In thesis
1. Solid-phase Proximity Ligation Assays: High-performance and multiplex protein analyses
Open this publication in new window or tab >>Solid-phase Proximity Ligation Assays: High-performance and multiplex protein analyses
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
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:nbn:se:uu:diva-144093 (URN)978-91-554-7999-2 (ISBN)
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
2. Proximity Ligation Assays for Disease Biomarkers Analysis
Open this publication in new window or tab >>Proximity Ligation Assays for Disease Biomarkers Analysis
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

One of the pressing needs in the field of disease biomarker discovery is new technologies that could allow high performance protein analysis in different types of clinical material, such as blood and solid tissues. This thesis includes four approaches that address important limitations of current technologies, thus enabling highly sensitive, specific and parallel protein measurements.

Paper I describes a method for sensitive singleplex protein detection in complex biological samples, namely solid phase proximity ligation assay (SP-PLA). SP-PLA exhibited improved sensitivity compared to conventional sandwich immunoassays. We applied SP-PLA to validate the potential of GDF-15 as a biomarker for cardiovascular disease.  

Paper II describes ProteinSeq, a multiplexed immunoassay based on the principle of SP-PLA, for parallel detection of 36 proteins using next-generation sequencing as readout. ProteinSeq exhibited improved sensitivity compared to multiplexed sandwich immunoassays, and the potential to achieve even higher levels of multiplexing while preserving a high sensitivity and specificity. We applied ProteinSeq to analyze 36 proteins, including one internal control, in 5 μl of plasma samples in a cohort of patients with cardiovascular disease and healthy controls.

Paper III describes PLA-DTM, a strategy for recording all possible interactions between sets of proteins in clinical samples. Individual proteins and their interactions are first encoded to dual barcoded DNA by PLA, and the barcodes are interrogated by a method named dual tag microarray (DTM). We applied the method for studying interactions among protein members of the NFκB signaling pathway.

Paper IV describes a novel probing strategy for analyzing individual biomolecules in solution or in situ. The technique employs a new class of probes for unfolding proximity ligation assays - uPLA probes. The probes are designed so that each probe set is sufficient in forming and replicating circular DNA reporter, without interactions among themselves when incubated with the sample. The uPLA probing strategy provides ease in the design of multiple probe sets in parallelized assays while enhancing the specificity of detection. We used the uPLA probes to detect various targets, including synthetic DNA and cancer-related transcripts in situ.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 42 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 703
Keyword
proximity ligation assay, blood biomarkers, protein interactions, pathway analysis, single molecule, next-generation sequencing
National Category
Biomedical Laboratory Science/Technology
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-158634 (URN)978-91-554-8158-2 (ISBN)
Public defence
2011-10-28, Rudbecksalen, Rudbecklaboratory, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Supervisors
Available from: 2011-10-07 Created: 2011-09-12 Last updated: 2015-08-10Bibliographically approved
3. DNA-Mediated Detection and Profiling of Protein Complexes
Open this publication in new window or tab >>DNA-Mediated Detection and Profiling of Protein Complexes
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins are the effector molecules of life. They are encoded in DNA that is inherited from generation to generation, but most cellular functions are executed by proteins. Proteins rarely act on their own – most actions are carried out through an interplay of tens of proteins and other biomolecules.

Here I describe how synthetic DNA can be used to study proteins and protein complexes. Variants of proximity ligation assays (PLA) are used to generate DNA reporter molecules upon proximal binding by pairs of DNA oligonucleotide-modified affinity reagents. In Paper I, a robust protocol was set up for PLA on paramagnetic microparticles, and we demonstrated that this solid phase PLA had superior performance for detecting nine candidate cancer biomarkers compared to other immunoassays. Based on the protocol described in Paper I I then developed further variants of PLA that allows detection of protein aggregates and protein interactions. I sensitively detected aggregated amyloid protofibrils of prion proteins in paper II, and in paper III I studied binary interactions between several proteins of the NFκB family. For all immunoassays the selection of high quality affinity binders represents a major challenge. I have therefore established a protocol where a large set of protein binders can be simultaneously validated to identify optimal pairs for dual recognition immunoassays (Paper IV).  

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. 43 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 923
Keyword
Proximity ligation assay, Protein complexes, Protein interactions, Biomarkers, Prions, Antibodies
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Medical Science
Identifiers
urn:nbn:se:uu:diva-204861 (URN)978-91-554-8718-8 (ISBN)
Public defence
2013-09-27, Rudbecksalen, Rudbeck Laboratory, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2013-09-06 Created: 2013-08-12 Last updated: 2014-01-08

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Siegbahn, AgnetaLandegren, UlfKamali-Moghaddam, Masood

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