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Two Simple and Generic Antibody-Independent Kinase Assays: Comparison of a Bioluminescent and a Microfluidic Assay Format

Aventis Lead Discovery Technologies, Industriepark Hoechst, H811, D-65926 Frankfurt am Main, Germany

Everard Pap

Aventis Lead Discovery Technologies, Industriepark Hoechst, H811, D-65926 Frankfurt am Main, Germany

Aimo Kannt

Aventis Lead Discovery Technologies, Industriepark Hoechst, H811, D-65926 Frankfurt am Main, Germany

In this study, the authors have compared the performance of 2 high-throughput screening assays for a serin/threonine kinase: a microplate-based, bioluminescent assay that uses the luciferin/luciferase system to monitor ATP consumption, and a microfluidic assay that measures the change in mobility in an electric field of a fluorescently labeled peptide upon phosphorylation. Both assays are homogeneous, nonradioactive, antibody independent and could be miniaturized to a reaction volume of 4 µl. The robustness of both formats was demonstrated by Z' values > 0.8. Screening of a small library (2133 compounds) showed that the results obtained with both technologies correlate very well. Although the threshold for hits was set to a comparably low value—22.2% and 13.7% inhibition for the ATP consumption and microfluidic assay, respectively, corresponding to mean plus 3 standard deviations—the overlap of active compounds identified with the 2 assay formats was greater than 94%. Thus, both assays allow the identification of even low potency inhibitors with a high level of confidence.

Key Words: HTS • serine/threonine kinase assay • bioluminescence • luciferase • microfluidics • caliper

Journal of Biomolecular Screening, Vol. 9, No. 5, 409-416 (2004)
DOI: 10.1177/1087057104264175


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