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DNA-Assisted Immunoassays for High-Performance Protein Analyses
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab. (Molecular tools)
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins play important roles in most cellular functions, such as, replication, transcription regulation, signal transduction, for catalyzing chemical reaction, etc. Technologies developed to identify proteins rely either on observing their own properties such as charge, size, mass to charge ratio or sequence composition; or on using affinity reagents that recognize specific protein targets. Immunoassays utilizing functionalized affinity reagents are powerful for targeted proteomics. Among them, DNA-assisted immunoassays in which affinity reagents are labeled with DNA molecules, offer some unique advantages.

In this thesis, I will present works to improve current DNA-assisted immunoassays such as proximity ligation assays (PLA), as well as to take advantage of DNA reactions to adress other problems. In paper I, a new solid support (MBC-Ts) was functionalized with antibodies and used in the solid-phase PLA for detection of VEGF. The assay using MBC-Ts was compared among the commercially available solid supports in different matrices and it was shown to exhibit enhanced limit of detection in complex matrices. In paper II, a two-step protocol was described to prepare high-quality probes used in homogeneous and in situ PLA by purifying DNA-labeled affinity reagents from unconjugated affinity reagents and excess oligonucleotides. In paper III, PLA was applied on a capillary western blotting instrument so that both the sensitivity and specificity of the original assay were improved. In paper IV, a new method was introduced to profile protein components in individual protein complexes by DNA-barcoded antibodies. This method has been used to profile protein complexes such as surface proteins on individual secreted vesicles.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. , 53 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1055
Keyword [en]
proximity ligation assay, magnetic beads, conjugation, phosphorylation, affinity binder, protein complexes, recombinant binder, sensitivity, specificity
National Category
Biochemistry and Molecular Biology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biology with specialization in Molecular Biotechnology
Identifiers
URN: urn:nbn:se:uu:diva-236591ISBN: 978-91-554-9114-7 (print)OAI: oai:DiVA.org:uu-236591DiVA: diva2:765757
Public defence
2015-01-16, B41, Biomedicalcenter, Husargatan 3, 75018, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2014-12-19 Created: 2014-11-20 Last updated: 2015-02-03
List of papers
1. A tosyl-activated magnetic bead cellulose as solid support for sensitive protein detection
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2013 (English)In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 167, no 3, 235-240 p.Article in journal (Refereed) Published
Abstract [en]

Magnetic bead cellulose (MBC) was prepared using sol-gel transition of viscose in the presence of maghemite (gamma-Fe2O3) nanoparticles. The MBC particles were then activated with p-toluenesulfonyl chloride to yield tosyl-activated magnetic bead cellulose (MBC-Ts). The microspheres were characterized by light and electron microscopy, elemental analysis and atomic absorption spectroscopy to determine morphology, size, polydispersity and content of iron and tosyl groups. The functionality of the MBC-Ts microspheres was demonstrated using proximity ligation assay (PLA) to detect vascular endothelial growth factor in femtomolar concentration range. The MBC-Ts microspheres performed equally well as commercially available microparticles that are routinely used as solid support in solid phase PLA.

Keyword
Bead cellulose, Magnetic, Protein detection, Proximity ligation assay
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-208643 (URN)10.1016/j.jbiotec.2013.06.010 (DOI)000324040300005 ()
Available from: 2013-10-07 Created: 2013-10-07 Last updated: 2017-12-06Bibliographically approved
2. A Universal Approach to Prepare Reagents for DNA-Assisted Protein Analysis
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2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 9, e108061- p.Article in journal (Refereed) Published
Abstract [en]

The quality of DNA-labeled affinity probes is critical in DNA-assisted protein analyses, such as proximity ligation and extension assays, immuno-PCR, and immuno-rolling circle amplification reactions. Efficient, high-performance methods are therefore required for isolation of pure conjugates from reactions where DNA strands have been coupled to antibodies or recombinant affinity reagents. Here we describe a universal, scalable approach for preparing high-quality oligonucleotide-protein conjugates by sequentially removing any unconjugated affinity reagents and remaining free oligonucleotides from conjugation reactions. We applied the approach to generate high-quality probes using either antibodies or recombinant affinity reagents. The purified high-grade probes were used in proximity ligation assays in solution and in situ, demonstrating both augmented assay sensitivity and improved signal-to-noise ratios.

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-236454 (URN)10.1371/journal.pone.0108061 (DOI)000342921200076 ()25233463 (PubMedID)
Available from: 2014-11-19 Created: 2014-11-19 Last updated: 2017-12-05Bibliographically approved
3. Highly sensitive and specific protein detection via proximity ligation in capillary westerns
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Detection and quantification of proteins and their post-translational modifications are crucial todecipher functions of complex networks in cell biology and medicine. Affinity-based protein analyseshave an important role in proteomic research, and western blotting is one of the most-widely usedtechniques. The NanoPro 1000 system developed by ProteinSimple is an instrument that can resolveand identify proteins and their isoforms through capillary isoelectric focusing with antibody baseddetection directly in the capillaries, in analogy to conventional western blotting. The in situ proximityligation assay (PLA) serves to detect the location of proteins using pairs of oligonucleotide-conjugatedantibodies that mediate formation of rolling circle amplification (RCA) products upon recognition ofthe target protein, or single or dual primary antibodies binding the target. PLA can offer improvedspecificity of detection, and increased sensitivity via, RCA initiated by oligonucleotides attached tothe antibody pairs. Here we have used in situ PLA to detect single or pairs of primary antibodiesbinding proteins separated in the NanoPro 1000 system for highly sensitive and specific detection ofproteins and their isoforms. We achieved at least 10-fold improved detection limits for Erk protein inHDMEC cell lysates, compared with the standard NanoPro 1000 assays, and we demonstrated greatlyenhanced specificity of detection by combining pairs of antibodies, each of which exhibited crossreactivesignals when used on their own.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-236588 (URN)
Available from: 2014-11-20 Created: 2014-11-20 Last updated: 2015-02-03
4. Profiling individual protein complexes by proximity-dependent barcoding
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(English)Manuscript (preprint) (Other academic)
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Molecular Biotechnology
Identifiers
urn:nbn:se:uu:diva-220093 (URN)
Available from: 2014-03-11 Created: 2014-03-10 Last updated: 2015-02-03

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