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Expanding assay dynamics: A combined competitive and direct assay system for the quantification of proteins in multiplexed Immunoassays
NMI–Natural and Medical Sciences Institute at the University of Tübingen.
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2008 (English)In: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 54, no 6, 956-963 p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: The concurrent detection and quantification of analytes that vary widely in concentration present a principal problem in multiplexed assay systems. Combining competitive and sandwich immunoassays permits coverage of a wide concentration range, and both highly abundant molecules and analytes present in low concentration can be quantified within the same assay. METHODS: The use of different fluorescence readout channels allows the parallel use of a competitive system and a sandwich configuration. The 2 generated assay signals are combined and used to calculate the amount of analyte. The measurement range can be adjusted by varying the competitor concentration, and an extension of the assay system's dynamic range is possible. RESULTS: We implemented the method in a planar protein microarray-based autoimmune assay to detect autoantibodies against 13 autoantigens and to measure the concentration of a highly abundant protein, total human IgG, in one assay. Our results for autoantibody detection and IgG quantification agreed with results obtained with commercially available assays. The use of 2 readout channels in the protein microarray-based system reduced spot-to-spot variation and intraassay variation. CONCLUSIONS: By combining a direct immunoassay with a competitive system, analytes present in widely varying concentrations can be quantified within a single multiplex assay. Introducing a second readout channel for analyte quantification is an effective tool for spot-to-spot normalization and helps to lower intraassay variation.

Place, publisher, year, edition, pages
2008. Vol. 54, no 6, 956-963 p.
National Category
Analytical Chemistry
Identifiers
URN: urn:nbn:se:kth:diva-17572DOI: 10.1373/clinchem.2007.099812ISI: 000256325800005Scopus ID: 2-s2.0-44849128262OAI: oai:DiVA.org:kth-17572DiVA: diva2:335616
Note
QC 20100525Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2017-12-12Bibliographically approved
In thesis
1. 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)
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Note
QC 20101112Available from: 2010-11-12 Created: 2010-11-12 Last updated: 2010-11-15Bibliographically approved

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