Method of Determining Enzymatic Activity in Biological Media

Abstract

A reliable, fast and convenient method is provided to determine the change in concentration of enzyme in time in a biological medium in the presence of a signal substrate that corrects for substrate consumption, color-dependency of the signal and non-linearity between the concentration of the leaving group of the signal substrate and the amount of signal. The method comprises the measurement of a calibrator curve in a suitable medium, such as a buffer, to determine the characteristics of the measured curve. These characteristics will then allow to obtain this whole curve or a sufficient part thereof again through a mathematical procedure based on a single-point measurement in a sample of a medium in which the enzyme generation takes place. This has the great advantage that there is no need to measure the whole calibrator curve in each individual medium, since a single-point measurement is adequate.

1 . A kit suitable for determining the course of thrombin activity comprising:
 a reagent with a known concentration of thrombin;   a trigger reagent to start thrombin generation;   an additive for facilitating a diagnostic value of a test;   a reagent containing a signal substrate or a signal substrate to which a signal-producing leaving group is added;   a software program directly loadable into the memory of a computer, wherein the software program is capable of calculating enzyme activity by correcting curves, wherein said calculating comprises:
 (i) generating a first curve that shows the time course of signal development in a reaction mixture, and determining an initial slope of the first curve, an end-level of the first curve, and a curvature of the first curve wherein said first curve is created based on a measurement of a detectable signal that relates to an amount of conversion product generated after adding a signal substrate to a reaction mixture that contains a predetermined amount of thrombin activity; 
 (ii) generating a second curve that shows a time course of signal development in a reaction mixture, wherein said second curve is created based on a measurement of detectable signal that relates to an amount of conversion product generated after adding a signal substrate to a plasma sample in which thrombin generation takes place, wherein the detectable signal is a measurement of fluorescence in time; 
 (iii) correcting for non-linearity an intensity of fluorescence in time signal described in (ii) that relates to a conversion product formed upon reaction with thrombin; 
 (iv) subtracting an intensity of fluorescence in time signal that relates to a conversion product formed upon reaction with alpha2-Macroglobulin-thrombin; 
 (v) calculating a concentration in time of thrombin from an intensity of fluorescence in time signal described in (ii) after said correcting described in (iii) and said subtracting described in (iv) using said initial slope described in (i) thereby calculating enzyme activity; and 
 (vi) correcting the concentration in time of thrombin calculated in (v) for color and/or turbidity of said sample of plasma using a measurement of 
   a calibrated amount of fluorophore that can be used inside or outside the medium in which thrombin generation takes place; or   a fluorescent source that can be used inside or outside the medium in which thrombin generation takes place.   
     
     
         2 . The kit according to  claim 1  further comprising freeze-dried reagents. 
     
     
         3 . The kit according to  claim 1 , wherein said trigger reagent comprises elagic acid, kaolin, tissue factor or phospholipids, or a combination thereof. 
     
     
         4 . The kit according to  claim 1 , wherein the additive comprises a prothrombotic or antithrombotic agent. 
     
     
         5 . The kit according to  claim 4 , wherein the additive comprises thrombomodulin, heparin, direct thrombin inhibitors, antithrombin or pentasaccharides.