Two simple and generic antibody-independent kinase assays: comparison of a bioluminescent and a microfluidic assay format.
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 microl. 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.
Pommereau
Antje
A
Aventis Lead Discovery Technologies, Industriepark Hoechst, H811, D-65926 Frankfurt am Main, Germany.
Pap
Everard
E
Kannt
Aimo
A
D000255:Adenosine Triphosphate / Q000378:metabolism; D000906:Antibodies / Q000378:metabolism*; D007700:Kinetics; D008163:Luminescent Measurements; D044085:Microfluidics; D010770:Phosphotransferases / Q000378:metabolism*
D000906:Antibodies; D000255:Adenosine Triphosphate; D010770:Phosphotransferases