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Molecular & Cellular Proteomics 5:1681-1696, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

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Technology

Optimal Design of Microarray Immunoassays to Compensate for Kinetic Limitations

Theory and Experiment ^*,S

Wlad Kusnezow^,, Yana V. Syagailo, Sven Rüffer, Nina Baudenstiel^¶, Christoph Gauer^||, Jörg D. Hoheisel, David Wild^** and Igor Goychuk

From the Divisions of Functional Genome Analysis and ^¶ Biophysics of Macromolecules, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany, ^|| Advalytix AG, Eugen-Sänger-Ring 4, Gewerbegebiet Brunnthal Nord, D-85649 Brunnthal, Germany, ^** ConvaTec, Deeside Industrial Park, Deeside, Flintshire CH5 2NU, United Kingdom, and Institute of Physics, University of Augsburg, Universitätsstr. 1, D-86135 Augsburg, Germany

In this report we examine the limitations of existing microarray immunoassays and investigate how best to optimize them using theoretical and experimental approaches. Derived from DNA technology, microarray immunoassays present a major technological challenge with much greater physicochemical complexity. A key physicochemical limitation of the current generation of microarray immunoassays is a strong dependence of antibody microspot kinetics on the mass flux to the spot as was reported by us previously. In this report we analyze, theoretically and experimentally, the effects of microarray design parameters (incubation vessel geometry, incubation time, stirring, spot size, antibody-binding site density, etc.) on microspot reaction kinetics and sensitivity. Using a two-compartment model, the quantitative descriptors of the microspot reaction were determined for different incubation and microarray design conditions. This analysis revealed profound mass transport limitations in the observed kinetics, which may be slowed down as much as hundreds of times compared with the solution kinetics. The data obtained were considered with relevance to microspot assay diffusional and adsorptive processes, enabling us to validate some of the underlying principles of the antibody microspot reaction mechanism and provide guidelines for optimal microspot immunoassay design. For an assay optimized to maximize the reaction velocity on a spot, we demonstrate sensitivities in the am and low fm ranges for a system containing a representative sample of antigen-antibody pairs. In addition, a separate panel of low abundance cytokines in blood plasma was detected with remarkably high signal-to-noise ratios.

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