Customer Application (system support)
Use of Statistical Design and Automation in Optimizing a Protein Tyrosine Phosphatase-1B - Activity Assay
Maureen Beresini1, Sherry Yeh2, and Saloumeh Kadkhodayan1
BioAnalytical Research and Development1 and Assay and Automation
Technology2, Genentech, Inc. South San Francisco, CA 94080
Genentech, Inc. - www.genentech.com
Protein tyrosine phosphatase-1B is a potential therapeutic target for the treatment of type 2 diabetes. Antagonists of this enzyme may inhibit dephosphorylation of the insulin receptor, and consequently, maintain it in an activated state leading to increased insulin sensitivity. To evaluate the activity of potential antagonists of this enzyme, a fluorescein diphosphatebased activity assay was developed. Performance of this assay was variable; furthermore, both enzyme and antagonist activity varied with buffer conditions. Therefore, the assay was re-optimized using the SAGIAN™ Automated Assay Optimization (AAO) system on a Beckman Biomek® 2000. Ten buffer factors were evaluated in an iterative fashion for their effects on assay performance as assessed in terms of total activity, background activity, inhibition by antagonist, and signal-to-noise ratio. Enzyme and substrate concentrations were then optimized in the refined buffer system. Statistical design proved to be a powerful tool for efficient optimization of this assay, and automation greatly facilitated its application.
Features
- Statistical design is a powerful tool for assay optimization.
- Together statistical design and automation facilitate the coverage of a greater amount of experimental space than can be covered by traditional methods.
- The AAO system was successfully used for optimizing the PTP-1B activity assay through an iterative process of screening and optimization followed by verification.
* All trademarks are the property of their respective owners. Where applicable, the PCR process is covered by patents owned by Roche Molecular Systems, Inc., and F. Hoffman-LaRoche, Ltd.
