Assay Method Information
Assay Name:  In Vitro Inhibitory Effect of the Compounds of the Present Invention on the Enzymatic Activity of Blood-Coagulation Factor XIa
Description:  The following method was used to test the in vitro inhibitory effect of the compounds of the present invention on the activity of human blood-coagulation factor XIa, expressed by the Inhibition constant Ki.Solution preparation: reaction buffer: 0.03M HEPES acid, 0.145M NaCl, 0.005 M KCl, 0.1% PEG-8000, pH=7.5;HEPES 8.499 g, NaCl 8.47 g, KCl 0.3725, PEG 8000 1 g, plus ddH2O 800 ml; the pH was adjusted to 7.4 with HCl, and the volume was metered to 1 L.S2366 substrate stock solution (2 mM): a volume of substrate (25 ml) was dissolved in 23 ml sterile deionized water, aliquoted, and stored at 4° C. in darkness.S2366 substrate working solution: the stock solution was diluted by 4 folds with the reaction buffer before use.FXIa working solution: 1 μl FXIa stock solution was added to 10 ml reaction buffer and thoroughly mixed before use.Method: 15 μl test sample working solution (15 μl DMSO for the control group) and 75 μl FXIa working solution were added to a 96-well plate, and incubated at room temperature for 15 min. Then 60 μl S2366 substrate working solution was added to initiate the reaction. The absorbance at 405 nm for the test compounds was continually assayed once every 3 minutes, and a ΔA-time curve was plotted to calculate the slope as the reaction rate. In accordance with the following equation, IC50 of each test sample when the substrate concentration was 200 μM was calculated with spss 16.0. Inhibition (I %) and Ki of the test samples were calculated in accordance with the follow equations, and the results are shown in Table 1. I %=(V0−Vi)/V0×100 where V0 is the reaction rate in the control wells, and Vi is the reaction rate in the test sample wells; IC50=Ki(1+[S]/Km) where Km=0.2 mM, and [S] is the substrate concentration=200 μM.
Affinity data for this assay
 
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