Evaluating the utility of the HTRF Transcreener ADP assay technology: a comparison with the standard HTRF assay technology.
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Hong, Lin, Quinn, Christopher and Jia, Yong (2009) Evaluating the utility of the HTRF Transcreener ADP assay technology: a comparison with the standard HTRF assay technology. Analytical Biochemistry, 391 (1). pp. 31-38. ISSN 1096-0309
Abstract
The HTRF (homogeneous time-resolved fluorescence) Transcreener ADP assay is a new kinase assay technology marketed by Cis-Bio International (Bagnols-Cèze, France). It measures kinase activity by detecting the formation of ADP using a monoclonal antibody and HTRF detection principles. In this article, we compare this technology with a standard HTRF kinase assay using EGFR [L858R/T790M] mutant enzyme as a case study. We demonstrate that the HTRF Transcreener ADP assay generated similar kinetic constants and inhibitor potency compared with the standard HTRF assay. However, the smaller dynamic window and lower Z' factor of the HTRF Transcreener ADP assay make this format less preferable for high-throughput screening. Based on the assay principle, the HTRF Transcreener ADP assay can detect both kinase and ATPase activities simultaneously. The ability to probe ATPase activity opens up new avenues for assaying kinases with intrinsic ATPase activity without the need to identify substrates, and this can speed up the drug discovery process. However, caution must be exercised because any contaminating ATPase activity will result in an invalid assay. The inability to tolerate high concentrations of ATP in the assay will also limit the application of this technology, especially in compound mechanistic studies such as ATP competition. Overall, the HTRF Transcreener ADP assay provides a new alternative tool to complement existing assay technologies for drug discovery.
| Item Type: | Article |
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| Additional Information: | author can archive post-print (ie final draft post-refereeing); Publisher's version/PDF cannot be used |
| Keywords: | HTRF; Transcreener; ADP; Kinase assay; ATPase; Inhibitor |
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| Date Deposited: | 14 Dec 2009 13:49 |
| Last Modified: | 31 Jan 2013 00:57 |
| URI: | https://oak.novartis.com/id/eprint/1205 |