Development of an assay for label-free high-throughput screening of thrombin inhibitors by use of reflectometric interference spectroscopy
Development of an assay for label-free high-throughput screening of thrombin inhibitors by use of reflectometric interference spectroscopy
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DOI：
10.1007/s00216-001-1196-4
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发表时间：
2002-01-01
影响因子：
4.3
通讯作者：
Gauglitz, G
中科院分区：
文献类型：
--
作者：
Birkert, O;Gauglitz, G
我们描述了凝血酶抑制剂的平行亲和测定法的发展和分析条件，该方法适用于第一个无标记的光学筛选HTS检测装置，该装置完全可集成到筛选平台中。为了实现与药物库的兼容性，通过将96孔和384孔塑料微孔板的无底支架粘合到换能器载玻片上来实现HTS换能器。传感器涂有葡聚糖，以确保生物相容性和功能性，和一个已知的凝血酶抑制剂共价连接到it.By适应反射干涉光谱的同时阅读的整个传感器板，我们能够检测凝血酶的结合在所有的威尔斯的微孔板在线，并行，和时间分辨。通过使用抑制试验，筛选凝血酶活性的384种物质可以在小于15分钟的测定时间内进行。我们还表明，数据质量足够高，平行定量库物质的IC 50值。
We describe the development of, and analytical conditions used for, parallel affinity assay for thrombin inhibitors adapted to the first label-free optical screening HTS detection set-up fully integrable into a screening platform. To achieve compatibility with pharmaceutical libraries, an HTS-transducer was realized by gluing the bottomless scaffolds of 96- and 384-well plastic microplates on to transducer slides. The transducer are coated with a dextran, to ensure biocompatibility and functionality, and a known thrombin inhibitor was attached covalently to it. By adapting reflectometric interference spectroscopy for simultaneous reading of the whole transducer plate we were able to detect the binding of thrombin in all the wells of the microplates on-line, in parallel, and time resolved. By using an inhibition assay, the screening of 384 substances for thrombin activity can be performed within an assay time of less than 15 min. We also show that the data quality is high enough for parallel quantification of the IC 50 values of the library substances.