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Clinical Chemistry 54: 956-963, 2008. First published April 24, 2008; 10.1373/clinchem.2007.099812
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Right arrow Automation and Analytical Techniques
(Clinical Chemistry. 2008;54:956-963.)
© 2008 American Association for Clinical Chemistry, Inc.

Automation and Analytical Techniques

Expanding Assay Dynamics: A Combined Competitive and Direct Assay System for the Quantification of Proteins in Multiplexed Immunoassays

Michael Hartmann1, Monika Schrenk1, Anette Döttinger1, Sarah Nagel1, Johan Roeraade2, Thomas O. Joos1 and Markus F. Templin1,a

1 NMI–Natural and Medical Sciences Institute at the University of Tübingen, Reutlingen, Germany; 2 Department of Analytical Chemistry, Royal Institute of Technology, Stockholm, Sweden.

aAddress correspondence to this author at: NMI–Natural and Medical Sciences Institute at the University of Tübingen, Markwiesenstr. 55, 72770 Reutlingen, Germany. Fax 49 (0)71321 51530 816; e-mail templin{at}nmi.de

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.




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 Clin. Chem.Home page
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Clin. Chem., June 1, 2008; 54(6): 937 - 939.
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