Detection of cleavage activity of an enzyme
The present invention relates to detecting cleavage activity of an enzyme. The various aspects of the invention include an enzyme detection device, kit, method and use for detecting or measuring the presence in a test sample of the activity of an enzyme capable of cleaving a substrate. The invention also relates to indicator and binding molecules useful for carrying out the invention. The enzyme substrate contains a hidden binding site which is only revealed upon cleavage by the enzyme.
1. An enzyme detection kit for detecting the presence of cleavage activity of an enzyme, the kit comprising:
(i) an intact indicator molecule, the intact indicator molecule comprising
(a) a cleavage region comprising at least one cleavage site that is specific for the enzyme, the cleavage region defining an end of a detectable fragment or cleavage part of the indicator molecule when the intact indicator molecule is cleaved at the cleavage site, the detectable fragment or cleavage part of the indicator molecule comprising a binding site, wherein the binding site of the detectable fragment or cleavage part of the indicator molecule is a conformational epitope that is not formed in the intact indicator molecule; and
(b) a capture site which is present in the intact indicator molecule and is present in the detectable fragment or cleavage part of the indicator molecule;
(ii) capture molecules which are (a) capable of binding to the capture site of the intact indicator molecule and (b) capable of binding to the capture site of the detectable fragment or cleavage part of the indicator molecule;
(iii) a solid support to which the capture molecules are attachable or attached to form a capture zone; and
(iv) binding molecules which are (a) capable of binding to the binding site of the detectable fragment or cleavage part of the indicator molecule and (b) incapable of binding to the intact indicator molecule.