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Development and Application of Proximity Assays for Proteome Analysis in Medicine
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
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
Keywords [en]
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: urn:nbn:se:uu:diva-334536ISBN: 978-91-513-0164-8 (print)OAI: oai:DiVA.org:uu-334536DiVA, id: diva2:1159797
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
List of papers
1. Detection of post-translational modification of cancer biomarkers via proximity ligation assay
Open this publication in new window or tab >>Detection of post-translational modification of cancer biomarkers via proximity ligation assay
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2016 (English)In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 26, no 12, p. 1455-1455Article in journal, Meeting abstract (Refereed) Published
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-316637 (URN)000392935600200 ()
Conference
Annual Meeting of the Society-for-Glycobiology, NOV 19-22, 2016, New Orleans, LA
Available from: 2017-03-06 Created: 2017-03-06 Last updated: 2017-11-29Bibliographically approved
2. Analytically Sensitive Protein Detection in Microtiter Plates by Proximity Ligation with Rolling Circle Amplification
Open this publication in new window or tab >>Analytically Sensitive Protein Detection in Microtiter Plates by Proximity Ligation with Rolling Circle Amplification
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2017 (English)In: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 63, no 9, p. 1497-1505Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Detecting proteins at low concentrations in plasma is crucial for early diagnosis. Current techniques in clinical routine, such as sandwich ELISA, provide sensitive protein detection because of a dependence on target recognition by pairs of antibodies, but detection of still lower protein concentrations is often called for. Proximity ligation assay with rolling circle amplification (PLARCA) is a modified proximity ligation assay (PLA) for analytically specific and sensitive protein detection via binding of target proteins by 3 antibodies, and signal amplification via rolling circle amplification (RCA) in microtiter wells, easily adapted to instrumentation in use in hospitals.

METHODS: Proteins captured by immobilized antibodies were detected using a pair of oligonucleotide-conjugated antibodies. Upon target recognition, these PLA probes guided oligonucleotide ligation, followed by amplification via RCA of circular DNA strands that formed in the reaction. The RCA products were detected by horseradish peroxidase-labeled oligonucleotides to generate colorimetric reaction products with readout in an absorbance microplate reader.

RESULTS: We compared detection of interleukin (IL)-4, IL-6, IL-8, p53, and growth differentiation factor-15 by PLARCA and conventional sandwich ELISA or immuno RCA. PLARCA detected lower concentrations of proteins and exhibited a broader dynamic range compared ELISA and iRCA using the same antibodies. IL-4 and IL-6 were detected in clinical samples at femtomolar concentrations, considerably lower than for ELISA.

CONCLUSIONS: PLARCA offers detection of lower protein levels and increased dynamic ranges compared to ELISA. The PLARCA procedure may be adapted to routine instrumentation available in hospitals and research laboratories.

National Category
Clinical Laboratory Medicine
Identifiers
urn:nbn:se:uu:diva-326672 (URN)10.1373/clinchem.2017.271833 (DOI)000408421200013 ()28667186 (PubMedID)
Funder
Swedish Research CouncilKnut and Alice Wallenberg FoundationEU, FP7, Seventh Framework Programme, 294409 264737 313010
Available from: 2017-07-21 Created: 2017-07-21 Last updated: 2018-09-17Bibliographically approved
3. Measurements ofinflammation protein biomarkers in venous plasma, earlobe capillary plasma andin capillary plasma stored on filter paper
Open this publication in new window or tab >>Measurements ofinflammation protein biomarkers in venous plasma, earlobe capillary plasma andin capillary plasma stored on filter paper
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2017 (English)In: Cytokine, ISSN 1043-4666, E-ISSN 1096-0023Article in journal (Other academic) Submitted
Abstract [en]

Multiplex panels for protein biomarkers are increasingly used to analyze blood samples in various fields of clinical research. However, they are rarely used in studies performed outside of a clinical setting. This may in part be due to the relative invasiveness of drawing blood from the vein and because of problems associated with the separation and transport of plasma. Samples collected from the earlobe would be less invasive and could be collected more conveniently. Transportation and storage of such samples could be made easier by blotting plasma on filter paper as dried plasma spots (DPS). The objective of this study was to compare values of multiple protein biomarkers for inflammation measured from three different sources (1) venous plasma, (2) plasma derived from capillary blood from the earlobe and (3) from capillary plasma stored as DPS. Samples from twelve male individuals were assessed with a panel of 92 inflammation related proteins using multiplex proximity extension assay technology. Correlations between the three sample types varied greatly between analytes. In general, a high correlation was observed between capillary plasma and DPS, with 33 analytes showing a correlation value of ρ > 0.8 in the two sample types. At this level of correlation (ρ > 0.8) 14 analytes correlated between venous and capillary plasma in contrast to only six analytes in the comparison of venous blood with DPS. We conclude that collecting samples from the earlobe is a feasible and less invasive alternative to venipuncture, but that the comparability with measures obtained from venous samples varies greatly between proteins.

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-334534 (URN)
Available from: 2017-11-23 Created: 2017-11-23 Last updated: 2017-11-24
4. Autoimmunity detection via proximity assays
Open this publication in new window or tab >>Autoimmunity detection via proximity assays
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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:nbn:se:uu:diva-334535 (URN)
Available from: 2017-11-23 Created: 2017-11-23 Last updated: 2017-11-24

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