Fisher D K, Higgins T J
Department of Immunopharmacology, Sterling Winthrop Pharmaceuticals Research Division, Collegeville, Pennsylvania 19426-0900.
Pharm Res. 1994 May;11(5):759-63. doi: 10.1023/a:1018996817529.
Protein phosphatases are intimately involved in a variety of cellular processes, many of which are of interest to the pharmaceutical industry. Phosphatase assays generally employ radioisotopes, making them tedious to perform, costly, and hazardous, while other procedures require antibodies and/or are unsuitable for mass screening efforts. To facilitate screening for inhibitors of the CD45 protein tyrosine phosphatase (PTPase), we have developed a sensitive colorimetric assay, using small volumes in 96-well microtiter plates and read on a standard ELISA plate reader. The assay was sensitive down to 0.5 nmol of released phosphate and can be easily run by robotics to assay thousands of compounds in a day. The assay is sparing of reagents and has been successfully used with all classes of phosphatases. The reagents are nonradioactive, readily obtainable, and minimal in cost. This assay should facilitate the search for specific inhibitors of phosphatases.
蛋白磷酸酶密切参与多种细胞过程，其中许多过程是制药行业所关注的。磷酸酶检测通常使用放射性同位素，这使得检测操作繁琐、成本高昂且具有危险性，而其他方法需要抗体且/或不适用于大规模筛选工作。为便于筛选CD45蛋白酪氨酸磷酸酶（PTPase）的抑制剂，我们开发了一种灵敏的比色测定法，该方法在96孔微量滴定板中使用小体积样本，并在标准酶标仪上读取结果。该检测方法对释放的磷酸盐的检测灵敏度低至0.5 nmol，并且可以通过机器人轻松操作，每天检测数千种化合物。该检测方法试剂用量少，已成功应用于各类磷酸酶的检测。试剂无放射性，易于获得且成本极低。这种检测方法应有助于寻找磷酸酶的特异性抑制剂。
