ASSAY FOR MEASURING FACTOR VIIa-ANTITHROMBIN COMPLEXES

Abstract

A method for determining the concentration of factor VIIa-antithrombin complexes is disclosed which has application to estimating the level of intravascular exposure of tissue factor, assessing patient risk for hypercoagulation or other coagulopathies, and monitoring patients for factor VIIa-antithrombin complexes over time which can reveal changes in risk for hypercoagulation or other coagulopathies and/or effectiveness of anticoagulant therapy. Antibodies suitable for use in an in vitro assay for determining the concentration of factor VIIa-antithrombin complexes and methods for making the same are also disclosed.

1 . A method of measuring changes in a patient's intravascular exposure of tissue factor comprising at a first time point, mixing a quantity of said patient's plasma sample with a primary capture antibody fixed to a solid phase, said capture antibody characterized by its ability to bind to factor VIIa-antithrombin complexes which might be present in said plasma sample, and incubating said mixture under conditions to promote binding between said capture antibody and said factor VIIa-antithrombin complexes which might be present in said plasma sample to form bound factor VIIa-antithrombin complexes and an unbound mixture; removing said unbound mixture; mixing said bound factor VIIa-antithrombin complexes with a secondary antibody characterized by its ability to bind to said bound factor VIIa-antithrombin complexes to form bound factor VIIa-antithrombin-secondary antibody complexes and unbound secondary antibody; removing said unbound secondary antibody; determining the amount of bound secondary antibody; and comparing the amount of bound secondary antibody to a standards amount of bound secondary antibody determined for bound factor VIIa-antithrombin complexes of a known concentration to obtain the concentration of factor VIIa-antithrombin complexes in said patient plasma sample at said first time point; at a second time point, determining the concentration of factor VIIa-antithrombin complexes in said patient's plasma sample by repeating the method used at said first time point, and comparing the amount of factor VIIa-antithrombin complexes at each time point to see if the amount is rising, falling, or remaining the same, wherein an increase in the concentration of factor VIIa-antithrombin complexes in said patient's plasma sample at said second time point compared to the concentration of factor VIIa-antithrombin complexes in said patient's plasma sample at said first time point represents an increase in said patient's intravascular exposure of tissue factor and wherein a decrease in the concentration of factor VIIa-antithrombin complexes in said patient's plasma sample at said second time point compared to the concentration of factor VIIa-antithrombin complexes in said patient's plasma sample at said first time point represents an decrease in said patients intravascular exposure of tissue factor. 
   
   
       2 . The method of  claim 1 , wherein said bound capture antibody binds to the factor VIIa portion of a factor VIIa-antithrombin complex. 
   
   
       3 . The method of  claim 1 , wherein said bound capture antibody binds to the antithrombin portion of a factor VIIa-antithrombin complex. 
   
   
       4 . A method of measuring a patient's risk for entering a hypercoagulable state comprising measuring the amount of factor VIIa-antithrombin complexes in a plasma sample from said patient at a first time point; measuring the amount of factor VIIa-antithrombin complexes in a plasma sample from said patient at a second time point; and comparing the amount of determining the amount of factor VIIa-antithrombin complexes in a plasma sample from said patient at said second time point to the amount of factor VIIa-antithrombin complexes in a plasma sample from said patient at said first time point, wherein an increase in the amount of factor VIIa-antithrombin complexes in a plasma sample from said patient at said second time point over the amount of factor VIIa-antithrombin complexes in a plasma sample from said patient at said first time point indicates an increase in said patient's risk for entering a hypercoagulable state and wherein a decrease in the amount of factor VIIa-antithrombin complexes in a plasma sample from said patient at said second time point over the amount of factor VIIa-antithrombin complexes in a plasma sample from said patient at said first time point indicates an decrease in said patient's risk for entering a hypercoagulable state. 
   
   
       5 . A method for monitoring the effectiveness of anticoagulant therapy by testing for factor VIIa-antithrombin complexes at a first time point and a second time point, and comparing the values obtained wherein an increase in said complexes at said second time point is indicative of possible failure of said therapy or the need for adjustment of said therapy to prevent the onset of undesirable clotting events.