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Sensitive detection of A beta protofibrils by proximity ligation: relevance for Alzheimer's disease
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics. (Molekylär geriatrik)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics. (Molekylär geriatrik)
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2010 (English)In: BMC neuroscience (Online), ISSN 1471-2202, E-ISSN 1471-2202, Vol. 11, 124- p.Article in journal (Refereed) Published
Abstract [en]

Background: Protein aggregation plays important roles in several neurodegenerative disorders. For instance, insoluble aggregates of phosphorylated tau and of A beta peptides are cornerstones in the pathology of Alzheimer's disease. Soluble protein aggregates are therefore potential diagnostic and prognostic biomarkers for their cognate disorders. Detection of the aggregated species requires sensitive tools that efficiently discriminate them from monomers of the same proteins. Here we have established a proximity ligation assay (PLA) for specific and sensitive detection of A beta protofibrils via simultaneous recognition of three identical determinants present in the aggregates. PLA is a versatile technology in which the requirement for multiple target recognitions is combined with the ability to translate signals from detected target molecules to amplifiable DNA strands, providing very high specificity and sensitivity. Results: For specific detection of A beta protofibrils we have used a monoclonal antibody, mAb158, selective for A beta protofibrils in a modified PLA, where the same monoclonal antibody was used for the three classes of affinity reagents required in the assay. These reagents were used for detection of soluble Ab aggregates in solid- phase reactions, allowing detection of just 0.1 pg/ml A beta protofibrils, and with a dynamic range greater than six orders of magnitude. Compared to a sandwich ELISA setup of the same antibody the PLA increases the sensitivity of the Ab protofibril detection by up to 25- fold. The assay was used to measure soluble Ab aggregates in brain homogenates from mice transgenic for a human allele predisposing to A beta aggregation. Conclusions: The proximity ligation assay is a versatile analytical technology for proteins, which can provide highly sensitive and specific detection of A beta aggregates - and by implication other protein aggregates of relevance in Alzheimer's disease and other neurodegenerative disorders.

Place, publisher, year, edition, pages
2010. Vol. 11, 124- p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-133826DOI: 10.1186/1471-2202-11-124ISI: 000283243900001PubMedID: 20923550OAI: oai:DiVA.org:uu-133826DiVA: diva2:371278
Available from: 2010-11-19 Created: 2010-11-16 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Proximity Ligation and Barcoding Assays: Tools for analysis of proteins and protein complexes
Open this publication in new window or tab >>Proximity Ligation and Barcoding Assays: Tools for analysis of proteins and protein complexes
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins are fundamental structural, enzymatic and regulatory components of cells. Analysis of proteins, such as by measuring their concentrations, characterizing their modifications, and detecting their interactions, provides insights in how biological systems work physiologically or pathologically at the molecular level. To perform such analysis, molecular tools with good sensitivity, specificity, high multiplexing and throughput capacity are needed.

In this thesis, four different assays were developed and applied to detect and profile proteins and protein complexes in human body fluids, and in cells or tissues. These assays are based on targeting proteins or protein complexes by oligonucleotide-conjugated antibodies, and subsequent proximity dependent enzymatic reactions involving the attached DNA reporter sequences.

In paper I, a solid-phase proximity ligation assay (SP-PLA) was applied to detect synthetic and endogenous amyloid beta protofibrils. The SP-PLA provided better sensitivity and increased dynamic range than a traditional enzyme-linked immunosorbent assay (ELISA).

In paper II, in situ PLA was applied to investigate the correlation between MARK2-dependent phosphorylation of tau and Alzheimer’s disease. Greater numbers of MARK2-tau interactions and of phosphorylated tau proteins were observed in brain tissues from Alzheimer’s patients than in healthy controls.

In paper III, a multiplex SP-PLA was applied to identify protein biomarker candidates in amyotrophic lateral sclerosis (ALS) disease and in the analgesic mechanism of spinal cord stimulation (SCS). Among 47 proteins in human cerebrospinal fluid (CSF) samples, four were found at significantly lower concentrations (p-values < 0.001) in the samples from ALS patients compared to those from healthy controls (follistatin, IL-1α, IL-1β, and KLK5). No significant changes of the analyzed proteins were found in the CSF samples of neuropathic pain patients in   the stimulated vs. non-stimulated condition using SCS.

In paper IV, a new technology termed the proximity barcoding assay (PBA) was developed to profile individual protein complexes. The performance of PBA was demonstrated on artificially assembled streptavidin-biotin oligonucleotide complexes. PBA was also proven to be capable of profiling transcriptional pre-initiation complexes from nuclear extract of a hepatic cell line.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 43 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 980
Keyword
proximity ligation assay, proximity barcoding assay, sensitive, in situ, multiplex, profiling, amyloid-beta, MARK, tau, CSF, biomarker, protein complexes, Alzheimer’s disease, amyotrophic lateral sclerosis
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-220070 (URN)978-91-554-8901-4 (ISBN)
Public defence
2014-04-25, B21, Biomedicinskt centrum, Husargatan 3, SE-75123, Uppsala, 13:00 (English)
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Available from: 2014-04-03 Created: 2014-03-10 Last updated: 2014-04-29

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Ekholm Pettersson, FridaSehlin, DagNilsson, Lars N. G.

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