Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Proximity Ligation Assays for Disease Biomarkers Analysis
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. (Ulf Landegren)
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

One of the pressing needs in the field of disease biomarker discovery is new technologies that could allow high performance protein analysis in different types of clinical material, such as blood and solid tissues. This thesis includes four approaches that address important limitations of current technologies, thus enabling highly sensitive, specific and parallel protein measurements.

Paper I describes a method for sensitive singleplex protein detection in complex biological samples, namely solid phase proximity ligation assay (SP-PLA). SP-PLA exhibited improved sensitivity compared to conventional sandwich immunoassays. We applied SP-PLA to validate the potential of GDF-15 as a biomarker for cardiovascular disease.  

Paper II describes ProteinSeq, a multiplexed immunoassay based on the principle of SP-PLA, for parallel detection of 36 proteins using next-generation sequencing as readout. ProteinSeq exhibited improved sensitivity compared to multiplexed sandwich immunoassays, and the potential to achieve even higher levels of multiplexing while preserving a high sensitivity and specificity. We applied ProteinSeq to analyze 36 proteins, including one internal control, in 5 μl of plasma samples in a cohort of patients with cardiovascular disease and healthy controls.

Paper III describes PLA-DTM, a strategy for recording all possible interactions between sets of proteins in clinical samples. Individual proteins and their interactions are first encoded to dual barcoded DNA by PLA, and the barcodes are interrogated by a method named dual tag microarray (DTM). We applied the method for studying interactions among protein members of the NFκB signaling pathway.

Paper IV describes a novel probing strategy for analyzing individual biomolecules in solution or in situ. The technique employs a new class of probes for unfolding proximity ligation assays - uPLA probes. The probes are designed so that each probe set is sufficient in forming and replicating circular DNA reporter, without interactions among themselves when incubated with the sample. The uPLA probing strategy provides ease in the design of multiple probe sets in parallelized assays while enhancing the specificity of detection. We used the uPLA probes to detect various targets, including synthetic DNA and cancer-related transcripts in situ.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2011. , 42 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 703
Keyword [en]
proximity ligation assay, blood biomarkers, protein interactions, pathway analysis, single molecule, next-generation sequencing
National Category
Biomedical Laboratory Science/Technology
Research subject
Molecular Medicine
Identifiers
URN: urn:nbn:se:uu:diva-158634ISBN: 978-91-554-8158-2 (print)OAI: oai:DiVA.org:uu-158634DiVA: diva2:441225
Public defence
2011-10-28, Rudbecksalen, Rudbecklaboratory, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Supervisors
Available from: 2011-10-07 Created: 2011-09-12 Last updated: 2015-08-10Bibliographically approved
List of papers
1. Sensitive plasma protein analysis by microparticle-based proximity ligation assays
Open this publication in new window or tab >>Sensitive plasma protein analysis by microparticle-based proximity ligation assays
Show others...
2010 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 9, no 2, 327-335 p.Article in journal (Refereed) Published
Abstract [en]

Detection of proteins released in the bloodstream from tissues damaged by disease can promote early detection of pathological conditions, differential diagnostics, and follow-up of therapy. Despite these prospects and a plethora of candidate biomarkers, efforts in recent years to establish new protein diagnostic assays have met with limited success. One important limiting factor has been the challenge of detecting proteins present at trace levels in complex bodily fluids. To achieve robust, sensitive, and specific detection, we have developed a microparticle-based solid-phase proximity ligation assay, dependent on simultaneous recognition of target proteins by three antibody molecules for added specificity. After capture on a microparticle, solid-phase pairs of proximity probes are added followed by washes, enabling detection and identification of rare protein molecules in blood while consuming small amounts of sample. We demonstrate that single polyclonal antibody preparations raised against target proteins of interest can be readily used to establish assays where detection depends on target recognition by three individual antibody molecules, recognizing separate epitopes. The assay was compared with state-of-the-art sandwich ELISAs for detection of vascular endothelial growth factor, interleukin-8 and interleukin-6, and it was found to be superior both with regard to dynamic range and minimal numbers of molecules detected. Furthermore, the assays exhibited excellent performance in undiluted plasma and serum as well as in whole blood, producing comparable results for nine different antigens. We thus show that solid-phase proximity ligation assay is suitable for validation of a variety of protein biomarkers over broad dynamic ranges in clinical samples.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-123198 (URN)10.1074/mcp.M900248-MCP200 (DOI)000275506200010 ()19955079 (PubMedID)
Available from: 2010-04-26 Created: 2010-04-26 Last updated: 2017-12-12Bibliographically approved
2. ProteinSeq: high-performance proteomic analyses by proximity ligation and next generation sequencing
Open this publication in new window or tab >>ProteinSeq: high-performance proteomic analyses by proximity ligation and next generation sequencing
Show others...
2011 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 9, e25583- p.Article in journal (Refereed) Published
Abstract [en]

Despite intense interest, methods that provide enhanced sensitivity and specificity in parallel measurements of candidate protein biomarkers in numerous samples have been lacking. We present herein a multiplex proximity ligation assay with readout via realtime PCR or DNA sequencing (ProteinSeq). We demonstrate improved sensitivity over conventional sandwich assays for simultaneous analysis of sets of 35 proteins in 5 μl of blood plasma. Importantly, we observe a minimal tendency to increased background with multiplexing, compared to a sandwich assay, suggesting that higher levels of multiplexing are possible. We used ProteinSeq to analyze proteins in plasma samples from cardiovascular disease (CVD) patient cohorts and matched controls. Three proteins, namely P-selectin, Cystatin-B and Kallikrein-6, were identified as putative diagnostic biomarkers for CVD. The latter two have not been previously reported in the literature and their potential roles must be validated in larger patient cohorts. We conclude that ProteinSeq is promising for screening large numbers of proteins and samples while the technology can provide a much-needed platform for validation of diagnostic markers in biobank samples and in clinical use. 

Keyword
proximity ligation assay, next generation sequencing, cardiovascular disease, biomarker
National Category
Biomedical Laboratory Science/Technology
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-158802 (URN)10.1371/journal.pone.0025583 (DOI)000295939600036 ()21980495 (PubMedID)
Available from: 2011-09-15 Created: 2011-09-15 Last updated: 2017-12-08Bibliographically approved
3. Profiling cellular protein complexes by proximity ligation with dual tag microarray readout
Open this publication in new window or tab >>Profiling cellular protein complexes by proximity ligation with dual tag microarray readout
Show others...
2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 7, e40405- p.Article in journal (Refereed) Published
Abstract [en]

Patterns of protein interactions provide important insights in basic biology, and their analysis plays an increasing role in drug development and diagnostics of disease. We have established a scalable technique to compare two biological samples for the levels of all pairwise interactions among a set of targeted protein molecules. The technique is a combination of the proximity ligation assay with readout via dual tag microarrays. In the proximity ligation assay protein identities are encoded as DNA sequences by attaching DNA oligonucleotides to antibodies directed against the proteins of interest. Upon binding by pairs of antibodies to proteins present in the same molecular complexes, ligation reactions give rise to reporter DNA molecules that contain the combined sequence information from the two DNA strands. The ligation reactions also serve to incorporate a sample barcode in the reporter molecules to allow for direct comparison between pairs of samples. The samples are evaluated using a dual tag microarray where information is decoded, revealing which pairs of tags that have become joined. As a proof-of-concept we demonstrate that this approach can be used to detect a set of five proteins and their pairwise interactions both in cellular lysates and in fixed tissue culture cells. This paper provides a general strategy to analyze the extent of any pairwise interactions in large sets of molecules by decoding reporter DNA strands that identify the interacting molecules.

Keyword
proximity ligation assay, protein interaction, dual tag microarray
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-177984 (URN)10.1371/journal.pone.0040405 (DOI)000306355500039 ()22808155 (PubMedID)
Funder
Swedish Research CouncilNIH (National Institute of Health), 1R21CA126727-01A1
Note

De 2 första författarna delar förstaförfattarskapet.

Correction in: PLoS ONE 10(3): e0119890. doi: 10.1371/journal.pone.0119890ISI: 000351880000047

Available from: 2012-07-23 Created: 2012-07-23 Last updated: 2017-12-07Bibliographically approved
4. Unfolding proximity ligation probes for measuring and imaging individual nucleic acid and protein molecules
Open this publication in new window or tab >>Unfolding proximity ligation probes for measuring and imaging individual nucleic acid and protein molecules
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

We present a new class of probes for molecular detection reactions - probes for unfolding proximity ligation assays. These are secondarily structured nucleic acid reagents that can be induced to unfold in order to undergo proximity ligation reactions, followed by visualization via localized single molecule amplification. The probes enable highly specific targeting of individual nucleic acid or protein molecule. We demonstrated the performance of these new probes by detecting synthetic DNA and cancer-related transcripts in situ and multiplex probing of proteins in solution. 

Keyword
unfolding, proximity ligation, single molecule, protein biomarker, cancer-related transcript
National Category
Biomedical Laboratory Science/Technology
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-158635 (URN)
Available from: 2011-09-15 Created: 2011-09-12 Last updated: 2011-11-04

Open Access in DiVA

fulltext(2207 kB)1402 downloads
File information
File name FULLTEXT01.pdfFile size 2207 kBChecksum SHA-512
0044685586e33a22807e3455aadfd6f3f2556b3eedff17e9b4d921353f14cfdb68fb2aef01b44e39ae561a7e0193e77f0d380e55c92db6e346ead5f69bda26b5
Type fulltextMimetype application/pdf
Buy this publication >>

Search in DiVA

By author/editor
Nong, Rachel Yuan
By organisation
Molecular tools
Biomedical Laboratory Science/Technology

Search outside of DiVA

GoogleGoogle Scholar
Total: 1402 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 1109 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf