Diagnostic test for determining the concentration of transient proteolytic activity in composite biological media

A method for determining in real time the course of thrombin activity in a biological sample as it appears in and disappears from the sample comprising the following steps:

• a) adding a protease activator to a first portion of said sample to generate thrombin activity to produce an activated sample;
• b) adding a signal substrate to the activated sample of step a), such that a detectable signal related to the amount of conversion product is formed upon reaction by the generated thrombin activity;
• c) adding a known stable proteolytic activity selected from the group consisting of α2-macroglobulin-thrombin complex and staphylocoagulase-prothrombin complex that acts on the same signal substrate as defined in step b) to a second parallel portion of said sample to produce a combined second portion, wherein said proteolytic activity is otherwise inert and said proteolytic activity is not triggered;
• d) adding the same signal substrate as defined in to step b) to the combined second portion of said sample of step c), said signal substrate causing a detectable signal upon reaction by the means with known stable proteolytic activity;
• e) determining the time course of signal development in reaction of step b) and step d) to provide a signal curve from each reaction;
• and
• f) comparing said curves to derive the course of thrombin activity in time in the sample.