Detection of Cleavage Activity Of An Enzyme

A method for detecting the presence or absence in a test sample of cleavage activity of an enzyme capable of cleaving a substrate, the method comprising:

• (i) bringing an indicator molecule into contact with the test sample, said indicator molecule comprising:
• (a) a cleavage region comprising at least one cleavage site, which can be cleaved by said enzyme if said enzyme cleavage activity is present;
• and
• (b) a capture site which remains intact following cleavage of the indicator molecule;
• wherein cleavage of the at least one cleavage site produces a novel binding site;
• (ii) adding to the test sample binding molecules capable of binding to the novel binding site, wherein the binding molecules are incapable of binding to the indicator molecule unless and until cleavage has occurred;
• (iii) capturing the part of the indicator molecule containing the novel binding site at a capture zone through binding of capture molecules in the capture zone to the capture site, said capture molecules being able to bind to the capture site irrespective of whether or not the indicator molecule has been cleaved;
• and
• (iv) detecting cleavage of the at least one cleavage site by determining binding of the binding molecules to the novel binding site of the indicator molecule captured in the capture zone.

A method for diagnosing a respiratory condition in a test sample by detecting cleavage activity of an enzyme capable of cleaving a substrate, the method comprising:

• (i) bringing an indicator molecule into contact with the test sample, said indicator molecule comprising:
• (a) a cleavage region comprising at least one cleavage site, which can be cleaved by said enzyme if said enzyme cleavage activity is present;
• and
• (b) a capture site which remains intact following cleavage of the indicator molecule;
• wherein cleavage of the at least one cleavage site produces a novel binding site;
• (ii) adding to the test sample binding molecules capable of binding to the novel binding site, wherein the binding molecules are incapable of binding to the indicator molecule unless and until cleavage has occurred;
• (iii) capturing the part of the indicator molecule containing the novel binding site at a capture zone through binding of capture molecules in the capture zone to the capture site, said capture molecules being able to bind to the capture site irrespective of whether or not the indicator molecule has been cleaved;
• and
• (iv) detecting cleavage of the at least one cleavage site by determining binding of the binding molecules to the novel binding site of the indicator molecule captured in the capture zone wherein an increased level of cleavage compared to a control diagnoses the respiratory condition.

An enzyme detection kit for detecting the presence in a test sample of cleavage activity of an enzyme capable of cleaving a substrate, the kit comprising:

• (i) an indicator molecule for adding to the test sample, said indicator molecule comprising
• (a) a cleavage region comprising at least one cleavage site, which can be cleaved by said enzyme if said enzyme cleavage activity is present;
• and
• (b) a capture site which remains intact following cleavage of the indicator molecule;
• wherein cleavage of the at least one cleavage site produces a novel binding site;
• (ii) capture molecules capable of binding to the capture site of the indicator molecule, irrespective of whether or not the indicator molecule has been cleaved;
• (iii) a solid support to which the capture molecules can be attached (i.e. are attachable or attached) to form a capture zone to receive the test sample;
• and
• (iv) binding molecules capable of binding to the novel binding site, wherein the binding molecules are incapable of binding to the indicator molecule unless and until cleavage has occurred.

An indicator molecule for use in detecting the presence in a test sample of cleavage activity of an enzyme capable of cleaving a substrate, the indicator molecule comprising:

• (a) a cleavage region comprising at least one cleavage site, which can be cleaved by said enzyme if said enzyme cleavage activity is present;
• (b) a capture site which remains intact following cleavage of the indicator molecule;
• and
• (c) a scaffold molecule which acts to connect at least two parts of the indicator molecule outside of the at least one cleavage site, optionally outside of the cleavage region;
• wherein the scaffold further acts to structurally constrain the indicator molecule in a manner such that cleavage of the at least one cleavage site produces a novel binding site to which binding molecules bind, but wherein the binding molecules are incapable of binding to the indicator molecule unless and until cleavage has occurred.