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Autoimmunity detection via proximity assays
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
Department of Medicine, Solna (MedS), K2, Karolinska Institute, Stockholm, Sweden .
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
Department of Clinical Sciences, Skåne University Hospital SUS, Lund University, Malmö, Sweden.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Since autoantibodies are recognized as valuable biomarkers for clinical diagnostics and prognostics in autoimmune diseases such as Stiff Person Syndrome (SPS) and Type 1 diabetes, detection of such markers at improved sensitivity and specificity could be of significant interest. In addition, as proximity assays have been shown to offer highly sensitive and specific detection of multiple proteins, the technique could be expanded to applications for autoimmunity detection. In the present study, we have applied the newly developed proximity ligation assay with rolling circle amplification (PLARCA), and proximity extension assay (PEA) for the detection of GADA, autoantibodies specific for glutamic acid decarboxylase 65 (GAD65). Through the use of oligonucleotide conjugated autoantigen GAD65 and anti-human antibodies, as proximity probes, we were able to apply these proximity assays to detect GADA in a set of SPS patient samples. In summary, we have applied and established both PLARCA and PEA, as a proof of concept, for the use of the specific and sensitive autoimmune detection.

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:uu:diva-334535OAI: oai:DiVA.org:uu-334535DiVA, id: diva2:1159781
Available from: 2017-11-23 Created: 2017-11-23 Last updated: 2017-11-24
In thesis
1. Development and Application of Proximity Assays for Proteome Analysis in Medicine
Open this publication in new window or tab >>Development and Application of Proximity Assays for Proteome Analysis in Medicine
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Along with proteins, a myriad of different molecular biomarkers, such as post-translational modifications and autoantibodies, could be used in an attempt to improve disease detection and progression. In this thesis, I build on several iterations of the proximity ligation assay to develop and apply new adaptable methods to facilitate detection of proteins, autoantibodies and post-translational modifications.

In paper I, we present an adaptation of the solid-phase proximity ligation assay (SP-PLA) for the detection of post-translational modification of proteins (PTMs). The assay was adapted for the detection of two of the most commons PTMs present in proteins, glycosylation and phosphorylation, offering the encouraging prospect of using detection of PTMs in a diagnostic or prognostic capacity. 

In paper II, we developed a variant of the proximity ligation assay using micro titer plate for detection and quantification of protein using optical density as readout in the fluorometer, termed PLARCA. With a detection limit considerably lower than ELISA, PLARCA detected femtomolar levels of these proteins in patient samples.

In paper III, we aim to compare detection values of samples collected from earlobe capillary, venous plasma, as well as capillary plasma stored in dried plasma spots (DPS) assessed with a 92-plex inflammation panel using multiplex proximity extension assay (PEA). Despite the high variability in protein measurements between the three sample sources, we were able to conclude that earlobe capillary sampling is a suitable less invasive alternative, to venipuncture.

In paper IV, we describe the application of PLARCA and proximity extension assay (PEA) for the detection of GAD65 autoantibodies (GADA). Thus, offering highly sensitive and specific autoimmunity detection.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2018. p. 60
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1400
Keyword
Solid-phase proximity ligation assay, post-translational modifications, glycosylation, phosphorylation, Enzyme-linked immunosorbent assay, immunoassay and rolling circle amplification, Proximity Extension Assay; inflammation protein biomarkers, autoantibodies; autoimmune disease
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-334536 (URN)978-91-513-0164-8 (ISBN)
Public defence
2018-01-18, B:41, BMC, Husargatan 3, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2017-12-15 Created: 2017-11-23 Last updated: 2018-03-08

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