Procedure for the determination of the activity of the protease which activates factor VII from protein solutions

Abstract

A procedure for the determination of the activity of the protease which activates blood clotting factor VII from protein solutions is describes, in which the protein solution comprising the protease and/or its proenzyme is incubated with a solid phase to which an antibody which binds the protease and/or its proenzyme has been coupled beforehand, and after washing the solid phase, the protease and/or its proenzyme fixed thereto, are incubated with reagents which allow for the determination of their activity.

We claim:  
     
         1 . A method for determining the activity of the protease that activates blood clotting factor VII in a protein solution, comprising: 
 1) coupling an antibody that binds the protease and its proenzyme to a solid phase;    2) binding the protease and its proenzyme to the antibody by incubating a protein solution containing the protease and its proenzyme with the solid phase;    3) removing unbound protease and proenzyme by washing the solid phase;    4) incubating the solid phase with a reagent to produce a signal;    5) detecting the signal produced by the reagent; and    6) comparing the level of the signal with a reference standard to determine the amount of activity of the protease and its proenzyme.    
     
     
         2 . A method for determining the activity of the protease that activates blood clotting factor VII in a protein solution, comprising 
 1) coupling an antibody that binds the protease or its proenzyme to a solid phase;    2) binding the protease or its proenzyme to the antibody by incubating a protein solution containing the protease or its proenzyme with the solid phase;    3) removing unbound protease or proenzyme by washing the solid phase;    4) incubating the solid phase with a reagent to produce a signal;    5) detecting the signal produced by the reagent; and    6) comparing the level of the signal with a reference standard to determine the amount of activity of the protease or its proenzyme.    
     
     
         3 . The method as claimed in  claim 1  or  2 , wherein the reagent comprises a chromogenic substrate, and wherein the activity is measured through photometric extinction of the signal produced by the chromogenic substrate.  
     
     
         4 . The method as claimed in  claim 1  or  2 , wherein the activity of the protease is measured by determining: 
 1) inactivation of blood clotting factors VIII/VIIIa or V/Va;  
 2) reduction of the time of blood clotting as measured in a global clotting test;  
 3) activation of plasminogen activators; or  
 4) activation of FVII.  
 
     
     
         5 . The method as claimed in  claim 1  or  2 , wherein the antibody directed against the protease and its proenzyme is a polyclonal antibody, a monoclonal antibody, or an F(ab) or F(ab) 2  antibody fragment.  
     
     
         6 . A kit for determining the activity of the protease that activates factor VII comprising a solid phase, an antibody that binds the protease and its proenzyme, and a reagent that allows determination of the activity of the protease and its proenzyme, wherein the kit provides components for determining the amount of protease activating blood clotting factor VII.  
     
     
         7 . A kit for determining the activity of the protease that activates factor VII comprising a solid phase, an antibody that binds the protease or its proenzyme, and a reagent that allows determination of the activity of the protease or its proenzyme, wherein the kit provides components for determining the amount of protease activating blood clotting factor VII.  
     
     
         8 . The method of  claim 1  or  2 , wherein early detection of an infarct, or stable or unstable angina pectoris can be made by comparing the level of protease activity in the protein solution to the level of protease activity in protein solutions taken from patients suffering from an infarct or stable or unstable angina pectoris.  
     
     
         9 . The method of  claim 1  or  2 , wherein an assessment of the effect of administering a prophylactic to a patient suffering from angina pectoris can be made can be made by comparing the level of protease activity in the protein solution to the level of protease activity in protein solutions taken from patients suffering from angina pectoris who have not been administered a prophylactic.  
     
     
         10 . The method of  claim 1  or  2 , wherein an assessment of the effect of administering therapy to a patient suffering from angina pectoris can be made by comparing the level of protease activity in the protein solution to the level of protease activity in protein solutions taken from patients suffering from angina pectoris who have not been administered therapy.  
     
     
         11 . The method of  claim 1  or  2 , wherein diagnosis of the severity of an infarct can be made by comparing the level of protease activity in the protein solution to the level of protease activity in protein solutions taken from patients who have suffered an infarct.  
     
     
         12 . The method of  claim 1  or  2 , wherein the method enables monitoring of the level of protease activity in protein solutions taken from pregnant women, and wherein an increasing level of protease activity indicates a reduced risk of thromboembolic complications.  
     
     
         13 . The method of  claim 1  or  2 , wherein early detection of the risk of thrombosis can be made by comparing the level of protease activity in the protein solution to the level of protease activity in protein solutions taken from patients with thrombosis.