Assay methods for detecting serum proteases, particularly activated protein C

A method for determining the amount of a protease in a body fluid sample comprising the steps of:

• a) contacting an immobilized antibody molecule composition, comprising antibody molecules affixed to a solid support, with an irreversible protease inhibitor in an amount of inhibitor sufficient to inhibit any protease associated with the composition to form a first admixture, said antibody molecules immunoreactive with said protease to form an immunoreaction complex having protease activity and the ability to bind a reversible protease inhibitor;
• b) maintaining the first admixture for a time period sufficient for said irreversible protease inhibitor to inhibit said protease and form a protease activity-free immobilized antibody composition;
• c) removing excess irreversible protease inhibitor from said protease activity-free immobilized antibody composition formed in step (b);
• d) admixing a body fluid sample with a coagulation inhibiting buffer containing a protease inhibiting amount of said reversible protease inhibitor, to form a second admixture;
• e) admixing said second admixture with the protease activity-free antibody composition formed in step (c) to form an immunoreaction admixture having a liquid phase and a solid phase;
• f) maintaining said immunoreaction admixture under immunoreaction conditions for a time period sufficient for said protease present in said body sample to immunoreact with the antibody molecules present on the solid support and form a first solid-phase immunoreaction product;
• g) removing the reversible protease inhibitor from said first solid phase immunoreaction product to form an inhibitor-free solid-phase immunoreaction product;
• and
• h) determining the amount of said protease present in the inhibitor-free solid-phase immunoreaction product formed in step (g) and thereby the amount of protease in the body sample.