Method for measuring the activity of cathepsin B after de-inhibition

A method for measuring activity of cathepsin B in a liquid biological sample being selected from the group consisting of blood plasma and blood serum and containing at least cathepsin B and at least one enzyme inhibitor corresponding to cathepsin B, wherein the enzyme inhibitor blocks enzyme activity of cathepsin B by binding with cathepsin B to form an enzyme-inhibitor complex, the method comprising:

• exposing an inhibitor-binding substance to the biological sample to remove the at least one enzyme inhibitor from the enzyme-inhibitor complex to provide a first sample containing active enzyme cathepsin B, the inhibitor binding substance associated with a de-inhibition device, the de-inhibition device being a rigid carrier configured for immersion into the liquid biological sample and removal therefrom;
• mixing an enzyme specific substrate with the first sample to form a second sample, wherein the enzyme specific substrate includes a di- or oligopeptide with the fluorogenic group 7-Amino-4-trifluoromethylcoumarin (AFC) bound to the C-terminum of the di- or oligopeptide and with a protecting group bound to the N-terminus of the di- or oligopeptide, the fluorogenic group being cleavable during enzymatic reaction with the active enzyme cathepsin B;
• incubating the second sample to effect the enzymatic reaction thereby forming a cleavage product containing the cleaved fluorogenic group;
• and
• measuring amount of cleavage product produced during the enzymatic reaction using fluorescence as a measuring parameter;
• wherein the fluorescence measuring parameter for the enzyme activity is at a wavelength of at least about 500 nm;
• and
• wherein the inhibitor-binding substance effects the removal of the at least one enzyme inhibitor from the liquid biological sample with the removal of the de-inhibition device and associated binding substance from the biological sample.