Development of an assay for label-free high-throughput screening of thrombin inhibitors by use of reflectometric interference spectroscopy.
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.
Birkert
Oliver
O
Institute of Physical Chemistry, University of Tübingen, Germany. oliver.birkert@ipc.uni-tuebingen.de
Gauglitz
Günter
G
D000818:Animals; D002417:Cattle; D002645:Chickens; D020128:Inhibitory Concentration 50; D015842:Serine Proteinase Inhibitors / Q000032:analysis*; D012709:Serum Albumin / Q000032:analysis; D012710:Serum Albumin, Bovine / Q000032:analysis; D013057:Spectrum Analysis / Q000295:instrumentation / Q000379:methods; D013917:Thrombin / Q000037:antagonists & inhibitors*
D015842:Serine Proteinase Inhibitors; D012709:Serum Albumin; D012710:Serum Albumin, Bovine; D013917:Thrombin