Patent ID: 7256011

Claim:
A method for determining target protease activity in a sample, employing a protein reagent comprising first and second hindering entities, the hindering entities linked together by an enzyme donor (“ED”) fragment and an amino acid sequence comprising a covalent bond susceptible to cleavage by said target protease activity, wherein said ED in the absence of at least one hindering entity is able to form an active indicator enzyme with an enzyme acceptor (“EA”) fragment, wherein cleavage of the cleavable sequence by the target protease produces a protein reagent fragment active with said EA to produce an active indicator enzyme, wherein indicator enzyme activity is related to said target protease activity, and wherein said protein reagent fragment is at least 5 times as active as said protein reagent in complexing with said EA in forming active indicator enzyme, said method comprising: combining said protein reagent, said sample, enzyme acceptor and indicator enzyme substrate for a time sufficient for said target protease activity to cleave said covalent bond; and measuring the indicator enzyme activity as an indication of the protease activity.