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Development of Enhanced Molecular Diagnostic Tools for Protein Detection and Analysis
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Improved diagnosis, prognosis and disease follow-up is a fundamental procedure and a constant challenge in medicine.  Among the different molecular biomarkers, proteins are the essential regulatory component in blood; hence, by developing enhanced specific and sensitive molecular tools will gives great insight into the different processes in disease treatment.  In this thesis, we build on the proximity ligation assay to develop and apply new adaptable methods to facilitate protein detection.

In paper I, I present a variant of the proximity ligation assay (we call PLARCA) using micro titer plate for detection and quantification of protein using optical density as readout in the fluorometer. PLARCA detected femtomolar levels of these proteins in patient samples, which was considerably below the detection threshold for ELISA.

In paper II, we developed and adapted a new method into the in situ PLA methods for detection and identification of extracellular vesicles (EVs) using flow cytometry as readout (a method we call ExoPLA).  We identified five target proteins on the surface of the Evs and using three colors, we identified the EV using flow cytometer.

In paper III, we aim to improve the efficiency of in situ PLA by creating and developing new designs and versions of the assay we called Unfold probes Through comparison of detection of protein using in situ PLA versus Unfold probes, we observed considerable decrease in non-specific signals, and also a lower detection threshold.

In paper IV, we describe the development of a solid phase proximity extension (sp-PEA) assay for protein detection and quantification. We compared detection of IL-8, TNF-alpha, IL-10 and IL-6 using spPEA and PEA; spPEA demonstrations over 2 orders of magnitudes in the lower detection concentrations by decreased in background noise.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. , 83 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1336
Keyword [en]
protein detection, proximity ligation assays, proximity extension assay, rolling circle amplification, ELISA, flow cytometry, fluorescence microscopy
National Category
Medical Biotechnology
Identifiers
URN: urn:nbn:se:uu:diva-320380ISBN: 978-91-554-9930-3 (print)OAI: oai:DiVA.org:uu-320380DiVA: diva2:1090627
Public defence
2017-06-14, B/B42, BMC, Husargatan 3, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2017-05-22 Created: 2017-04-25 Last updated: 2017-06-08
List of papers
1. Sensitive protein detection in microtiter plates by proximity ligation with rolling circle amplification (PLARCA)
Open this publication in new window or tab >>Sensitive protein detection in microtiter plates by proximity ligation with rolling circle amplification (PLARCA)
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(English)Manuscript (preprint) (Other academic)
Keyword
Enzyme-linked immunosorbent assay, immunoassay and rolling circle amplification
National Category
Medical Biotechnology
Identifiers
urn:nbn:se:uu:diva-320376 (URN)
Available from: 2017-04-19 Created: 2017-04-19 Last updated: 2017-05-02
2. Detecting individual extracellular vesicles using a multicolor in situ proximity ligation assay with flow cytometric readout
Open this publication in new window or tab >>Detecting individual extracellular vesicles using a multicolor in situ proximity ligation assay with flow cytometric readout
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2016 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, 34358Article in journal (Refereed) Published
Abstract [en]

Flow cytometry is a powerful method for quantitative and qualitative analysis of individual cells. However, flow cytometric analysis of extracellular vesicles (EVs), and the proteins present on their surfaces has been hampered by the small size of the EVs - in particular for the smallest EVs, which can be as little as 40 nm in diameter, the limited number of antigens present, and their low refractive index. We addressed these limitations for detection and characterization of EV by flow cytometry through the use of multiplex and multicolor in situ proximity ligation assays (in situ PLA), allowing each detected EV to be easily recorded over background noise using a conventional flow cytometer. By targeting sets of proteins on the surface that are specific for distinct classes of EVs, the method allows for selective recognition of populations of EVs in samples containing more than one type of EVs. The method presented herein opens up for analyses of EVs using flow cytometry for their characterization and quantification.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-306259 (URN)10.1038/srep34358 (DOI)000384185900001 ()27681459 (PubMedID)
Funder
EU, European Research Council, 259796; 294409Swedish Research Council
Available from: 2016-10-26 Created: 2016-10-26 Last updated: 2017-04-25Bibliographically approved
3. Enhanced in situ proximity ligation assays via Unfolding PLA probes
Open this publication in new window or tab >>Enhanced in situ proximity ligation assays via Unfolding PLA probes
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(English)Manuscript (preprint) (Other academic)
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-248874 (URN)
Funder
Swedish Foundation for Strategic Research EU, FP7, Seventh Framework Programme, 278568EU, FP7, Seventh Framework Programme, 264737
Available from: 2015-04-08 Created: 2015-04-08 Last updated: 2017-05-04
4. Protein detection by sensitive magnetic bead-based proximity extension assays
Open this publication in new window or tab >>Protein detection by sensitive magnetic bead-based proximity extension assays
(English)Manuscript (preprint) (Other academic)
Keyword
protein detection, magnetic bead, sensitivity and detection limits
National Category
Medical Biotechnology
Identifiers
urn:nbn:se:uu:diva-320377 (URN)
Available from: 2017-04-19 Created: 2017-04-19 Last updated: 2017-05-04

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