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Magnetic bead-based detection of autoimmune responses using protein microarrays.
KTH, School of Biotechnology (BIO), Nano Biotechnology.
KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
KTH, School of Biotechnology (BIO), Proteomics.ORCID iD: 0000-0001-8141-8449
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2009 (English)In: New biotechnology, ISSN 1871-6784, Vol. 26, 269-276 p.Article in journal (Refereed) Published
Abstract [en]

In the present study, a magnetic bead-based detection approach for protein microarrays is described as an alternative approach to the commonly used fluorescence-based detection system. Using the bead-based detection approach with applied magnetic force, it was possible to perform the detection step more rapidly as a result of the accelerated binding between the captured analyte in the microspot and the detection antibody, which was coupled to the magnetic beads. The resulting strong opacity shift on the microspots could be recorded with an ordinary flatbed scanner. In the context of autoimmunity, a set of 24 serum samples was analyzed for the presence of antibodies against 12 autoantigens using standard fluorescence and magnetic bead-based detection methods. Dynamic range, sensitivity, and specificity were determined for both detection methods. We propose from our findings that the magnetic bead-based detection option provides a simplified and cost effective readout method for protein microarrays.

Place, publisher, year, edition, pages
2009. Vol. 26, 269-276 p.
Keyword [en]
TECHNOLOGY; DIAGNOSTICS; DISEASES; ASSAY
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-11220DOI: 10.1016/j.nbt.2009.07.011ISI: 000273519000003PubMedID: 19664732Scopus ID: 2-s2.0-77952310841OAI: oai:DiVA.org:kth-11220DiVA: diva2:242213
Note
QC 20100713Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2011-01-20Bibliographically approved
In thesis
1. Novel diagnostic microarray assay formats towards comprehensive on-site analysis
Open this publication in new window or tab >>Novel diagnostic microarray assay formats towards comprehensive on-site analysis
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Advances in molecular methods for analyzing DNA, RNA and proteins in humans as well as in other animals, plants, fungi, bacteria or viruses have greatly increased the resolution with which we can study life’s complexity and dynamics on earth. While genomic, transcriptomic and proteomic laboratory tools for molecular diagnosis of disease are rapidly becoming more comprehensive, the access to such advanced yet often expensive and centralized procedures is limited. There is a great need for rapid and comprehensive diagnostic methods in low-resource settings or contexts where a person can not or will not go to a hospital or medical laboratory, yet where a clinical analysis is urgent.

In this thesis, results from development and characterization of novel technologies for DNA and protein microarray analysis are presented. Emphasis is on methods that could provide rapid, cost-effective and portable analysis with convenient readout and retained diagnostic accuracy. The first study presents a magnetic bead-based approach for DNA microarray analysis for a rapid visual detection of single nucleotide polymorphisms. In the second work, magnetic beads were used as detection reagents for rapid differential detection of presence of pestiviral family members using a DNA oligonucleotide microarray with read-out by means of a tabletop scanner or a digital camera. In paper three, autoimmune responses from human sera were detected on a protein autoantigen microarray, again by means of magnetic bead analysis. Here, special emphasis was made in comprehensively comparing the performance of the magnetic bead detection to common fluorescence-based detection. In the fourth study, an immunochromatographic lateral flow protein microarray assay is presented for application in the classification of contagious pleuropneumonia from bovine serum samples. The analysis could be performed within 10 minutes using a table top scanner, and the performance of the assay was shown to be comparable to that of a cocktail ELISA. In the fifth paper, the lateral flow microarray framework is investigated in further detail by means of experiments and numerical simulation. It was found that downstream effects play an important role, and the results further suggest that the downstream binding profiles may find use in simple affinity evaluation.

Place, publisher, year, edition, pages
Stockholm: KTH, 2009. xii, 90 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2009:18
Keyword
magnetic microbeads, gold nanobeads, microarrays, lateral flow, on-site diagnosis, SNPs, pestivirus, autoimmunity, contagious bovine pleuropneumonia, comsol multiphysics, finite element method
National Category
Microbiology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-11221 (URN)978-91-7415-416-0 (ISBN)
Public defence
2009-10-23, FR4 Oskar Klein, AlbaNova Universitetscentrum, Roslagstullsbacken, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
QC 20100713Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2011-11-23Bibliographically approved
2. Microfluidic Methods for Protein Microarrays
Open this publication in new window or tab >>Microfluidic Methods for Protein Microarrays
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Protein microarray technology has an enormous potential for in vitro diagnostics (IVD)1. Miniaturized and parallelized immunoassays are powerful tools to measure dozens of parameters from minute amounts of sample, whilst only requiring small amounts of reagent. Protein microarrays have become well-established research tools in basic and applied research and the first diagnostic products are already released on the market. However, in order for protein microarrays to become broadly accepted tools in IVD, a number of criteria have to be fulfilled concerning robustness and automation. Robustness and automation are key demands to improve assay performance and reliability of multiplexed assays, and to minimize the time of analysis.

These key demands are addressed in this thesis and novel methods and techniques concerning assay automation, array fabrication as well as performance and detection strategies related to protein microarrays are presented and discussed. In the first paper an automated assay format, based on planar protein microarrays is described and evaluated by the detection of several auto-antibodies from human serum and by quantification of matrix metalloproteases present in plasma. Diffusion-rate limited solid phase reactions were enhanced by microagitation, using the surface acoustic wave technology, resulting in a slightly increased signal-to-noise ratio. In the second paper of the thesis, a novel multiplexed immunoassay system was developed by combining a direct immunoassay with a competitive system. This set-up allows quantification of analytes present in widely varying concentrations within a single multiplex assay. In the third paper, a new concept for sample deposition is introduced, addressing contemporary problems of contact or non-contact microarrayers in protein microarray fabrication.

In the fourth paper, a magnetic bead-based detection method for protein microarrays is described as a cost-effective alternative approach to the commonly used fluorescence-based confocal scanning systems. The magnetic bead-based detection could easily be performed by using an ordinary flatbed scanner. In addition, applying magnetic force to the magnetic bead-based detection approach enables to run the detection step more rapidly. Finally, in paper five, a microfluidic bead-based immunoassay for multiplexed detection of receptor tyrosine kinases in breast cancer tissue is presented. Since the assay is performed inside a capillary, the amounts of sample and reagent material could be reduced by a factor of 30 or more when compared with the current standard protein microarray assay.

Place, publisher, year, edition, pages
Stockholm: KTH, 2010. ix, 51 p.
Series
Trita-CHE-Report, ISSN 1654-1081 ; 2010:45
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-26083 (URN)978-91-7415-761-1 (ISBN)
Public defence
2010-11-19, F3, Lindstedtsvägen 26, KTH, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
QC 20101112Available from: 2010-11-12 Created: 2010-11-12 Last updated: 2010-11-15Bibliographically approved

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