| Assay Method Information | |
| | Enzymatic Activity Assay |
| Description: | Determination of the Selectivity To demonstrate the selectivity of the substances with respect to thrombin and factor Xa inhibition, the test substances are examined for their inhibition of other human serine proteases, such as factor factor XIa, trypsin, plasmin, tissue plasminogen activator (TPA), and plasma kallikrein. The determinations are carried out in microtitre plates. To determine the enzymatic activity of factor XIa (0.15 nmol/l from Kordia), trypsin (42 mU/ml from Sigma), plasmin (0.1 μg/ml from Kordia), TPA (1 nmol/l from Kordia) and plasma kallikrein (0.2 nmol/l from Loxo), these enzymes are dissolved (50 mmol/l of Tris buffer [C,C,C-tris(hydroxymethyl)aminomethane], 100 mmol/l of sodium chloride, 0.1% BSA [bovine serum albumin], 5 mmol/l of calcium chloride, pH 7.4) and incubated for 15 min with test substance in various concentrations in dimethyl sulphoxide and also with dimethyl sulphoxide without test substance. The enzymatic reaction is then started by addition of the appropriate substrates (5 μmol/l of Boc-Glu(OBzl)-Ala-Arg-AMC from Bachem for factor XIa, 5 μmol/l of Boc-Ile-Glu-Gly-Arg-AMC from Bachem for Trypsin, 50 μmol/l of MeOSuc-Ala-Phe-Lys-AMC from Bachem for plasmin, 5 μmol/l of CH3SO2-D-Phe-Gly-Arg-AMC from Pentapharm for TPA and 5 μmol/l of H-Pro-Phe-Arg-AMC from Bachem for plasma kallikrein). After an incubation time of 30 min at 22° C., fluorescence is measured (excitation: 360 nm, emission: 460 nm). |
| Affinity data for this assay | |
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