DNA-Assisted Immunoassays for High-Performance Protein Analyses
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
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.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1055
proximity ligation assay, magnetic beads, conjugation, phosphorylation, affinity binder, protein complexes, recombinant binder, sensitivity, specificity
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
IdentifiersURN: urn:nbn:se:uu:diva-236591ISBN: 978-91-554-9114-7OAI: oai:DiVA.org:uu-236591DiVA: diva2:765757
2015-01-16, B41, Biomedicalcenter, Husargatan 3, 75018, Uppsala, 09:15 (English)
Wingren, Christer, Professor
Kamali-Moghaddam, MasoodLandegren, Ulf
List of papers