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Sensitive protein detection in microtiter plates by proximity ligation with rolling circle amplification (PLARCA)
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylära verktyg.
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylära verktyg.
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylära verktyg.
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylära verktyg.
Vise andre og tillknytning
(engelsk)Manuskript (preprint) (Annet vitenskapelig)
Emneord [en]
Enzyme-linked immunosorbent assay, immunoassay and rolling circle amplification
HSV kategori
Identifikatorer
URN: urn:nbn:se:uu:diva-320376OAI: oai:DiVA.org:uu-320376DiVA, id: diva2:1089360
Tilgjengelig fra: 2017-04-19 Laget: 2017-04-19 Sist oppdatert: 2017-05-02
Inngår i avhandling
1. Development of Enhanced Molecular Diagnostic Tools for Protein Detection and Analysis
Åpne denne publikasjonen i ny fane eller vindu >>Development of Enhanced Molecular Diagnostic Tools for Protein Detection and Analysis
2017 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Uppsala: Acta Universitatis Upsaliensis, 2017. s. 83
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1336
Emneord
protein detection, proximity ligation assays, proximity extension assay, rolling circle amplification, ELISA, flow cytometry, fluorescence microscopy
HSV kategori
Identifikatorer
urn:nbn:se:uu:diva-320380 (URN)978-91-554-9930-3 (ISBN)
Disputas
2017-06-14, B/B42, BMC, Husargatan 3, Uppsala, 13:00 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2017-05-22 Laget: 2017-04-25 Sist oppdatert: 2017-06-08

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