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Self-assembly of proximity probes for flexible and modular proximity ligation assays
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
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2007 (English)In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 43, no 4, 443-450 p.Article in journal (Refereed) Published
Abstract [en]

Proximity ligation assay (PLA) is a recently developed strategy for protein analysis in which antibody-based detection of a target protein via a DNA ligation reaction of oligonucleotides linked to the antibodies results in the formation of an amplifiable DNA strand suitable for analysis. Here we describe a faster and more cost-effective strategy to construct the antibody-based proximity ligation probes used in PLA that is based on the noncovalent interaction of biotinylated oligonucleotides with streptavidin followed by the interaction of this complex with biotinylated antibodies.

Place, publisher, year, edition, pages
2007. Vol. 43, no 4, 443-450 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-95759DOI: 10.2144/000112551ISI: 000205886000019PubMedID: 180119334OAI: oai:DiVA.org:uu-95759DiVA: diva2:170091
Available from: 2007-04-23 Created: 2007-04-23 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Development and Application of Triple Specific Proximity Ligation Assays (3PLA)
Open this publication in new window or tab >>Development and Application of Triple Specific Proximity Ligation Assays (3PLA)
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

After the completion of the human genome project the human genome was annotated with the surprisingly small amount of 24 000 (www.ensemble.com) genes. This has focused research on the contribution of splice variants, posttranslational modifications and interactions of proteins at the proteome level and other regulatory elements in the cell to fully understand the complexity of functions in a higher organism. Proteomic oriented projects are currently aiming to investigate all the splice variants and posttranslational modifications of all the proteins present in an organism or cell type and annotate their function and interaction partners. Projects on this scale are at the moment difficult to achieve and new methodologies are needed.

Proximity ligation assays (PLAs) are based on a novel protein detection strategy that converts the presence of a target molecule in a unique DNA tag through ligation reactions. PLA detection of proteins requires several independent recognition events by affinity reagents that have been converted into proximity probes. Different formats of the proximity ligation strategy have been developed in both heterogeneous and homogeneous format[1-4]. This thesis presents the development of an antibody based proximity ligation approach and the development of a novel proximity ligation based detection strategy named triple specific proximity ligation (3PLA). To extend the range of target molecules we adapted the proximity ligation assay for the use with antibodies by converting matched monoclonal antibody pairs and polyclonal antibody batches into proximity probes and used them for the detection of several cytokines in complex biological fluids. The novel 3PLA requires the simultaneous detection by three independent affinity binders to create one specific DNA based signal. This requirement for triple recognition extends the biological specificity of immunoassays and allows a proximity ligation design with reduced background signal and thus higher sensitivity. We have established proof of principle detection of the biomarkers troponin I and prostate specific antigen (PSA) alone and in complex with 1-alpha-antichymotrypsin (ACT) and detected as little as 100 molecules of vascular endothelial growth factor (VEGF). To further explore the extended biological specificity of 3PLA we adapted the assay for detection of protein complexes formed during NFκB signaling and used this system to profile the mode of action of three small molecular weight inhibitors of the IκB Kinase (IKK). The development of new protein detection methods hold promises for the investigation of complex interactions and mechanism on the proteome level which are not accessible with current technologies. We have developed tools and protocols useful for the development of new proximity ligation strategies and designs. These protocols allow the rapid and low cost custom set up of PLAs without the need for extensive conjugation protocols or purification procedures.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2007. 36 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 252
Keyword
Molecular medicine, proxmity ligation, method development, cell siganling, Molekylärmedicin
Identifiers
urn:nbn:se:uu:diva-7819 (URN)978-91-554-6865-1 (ISBN)
Public defence
2007-05-15, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarsjköldsväg 20, Uppsala, 13:15
Opponent
Supervisors
Available from: 2007-04-23 Created: 2007-04-23Bibliographically approved
2. Solid-phase Proximity Ligation Assays: High-performance and multiplex protein analyses
Open this publication in new window or tab >>Solid-phase Proximity Ligation Assays: High-performance and multiplex protein analyses
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Protein biomarkers circulating in blood hold the promise of improved diagnosis, prognosis and follow-up of treatment of disease via minimally invasive procedures. For the discovery and validation of such biomarkers, methods are needed that can facilitate parallel, highly specific and in-depth analysis of the blood proteome. The work presented in this thesis intends to develop and apply such assays, building on the concept of the proximity ligation assay (PLA).

In paper I, I present an easy and non-expensive alternative for the conjugation of oligonucleotides to antibodies via biotin-streptavidin-biotin interaction. This approach can be used when large sets of antibodies and/or oligos need to be validated for their performance as probes in PLA reactions.

In paper II, a solid-phase variant of PLA (SP-PLA) for the detection and quantification of proteins in blood is presented. SP-PLA exhibited an improved limit of detection compared to commercial ELISA assays by two orders of magnitude. In addition SP-PLA exhibited a broader dynamic range by at least one order of magnitude and required only 5 μl of sample, rendering the method very well suited for analyses of precious bio-banked material. Last but not least, SP-PLA was used to validate the diagnostic potential of GDF-15 as a biomarker for cardiovascular disease in a set of cardiovascular disease patients and healthy controls.

Paper III discusses the development of a multiplex SP-PLA (MultiPLAy) for the simultaneous detection of 36 proteins in just 5 μl of sample. MultiPLAy exhibited an improved LOD when compared to state-of-the-art bead-based sandwich assays. Most importantly, we observed only a minimal tendency to increased background with multiplexing, compared to a sandwich assay, suggesting that much higher levels of multiplexing will be possible. The assay was used to identify putative biomarkers in sample cohorts of colorectal cancer (CRC) and cardiovascular disease (CVD). Subsequent multivariate analysis revealed previously known diagnostic biomarkers. Furthermore, we successfully applied next-generation sequencing as a readout for the protein assays, allowing for the first time digital recording of protein profiles in blood.

In paper IV, we investigated the suitability of prostasomes as blood biomarkers in patients with prostate cancer using a newly developed PLA assay (4PLA) that utilizes five binders for the detection of complex target molecules. The assay successfully detected significantly elevated levels of prostasomes in blood samples from prostate cancer patients prior to radical prostatectomy, compared to controls and men with benign biopsy results.

 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 43 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 640
Keyword
proximity ligation assay, multiplexed, biomarkers, solid-phase, microparticles, cancer, sequencing
National Category
Biochemistry and Molecular Biology
Research subject
Biology with specialization in Molecular Biotechnology
Identifiers
urn:nbn:se:uu:diva-144093 (URN)978-91-554-7999-2 (ISBN)
Public defence
2011-03-25, Rudbeckhall, Rudbeck Laboratory, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2011-03-04 Created: 2011-01-27 Last updated: 2011-05-04

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