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

Abstract

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.