| 摘要 |
The present method utilizes the capture of the functionally active form of an enzyme that covalently binds or binds with a dissociation constant of 1 x 10<-9>M or less to an enzyme inhibitor or mutant thereof. The present invention is directed to a method for detecting a functionally active form of an enzyme in a biological sample, comprising contacting an enzyme inhibitor or mutant thereof immobilized on a solid substrate with the biological sample, and measuring the binding of the enzyme inhibitor or mutant thereof to the active form of the enzyme by a detectable label, wherein the enzyme inhibitor specifically forms a covalent bond or binds with a dissociation constant of 1 x 10<-9>M or less with the active form of the enzyme. Further, the present invention is directed to an analytical element useful for carrying out the detection of a functionally active form of an enzyme in biological sample, that includes an enzyme inhibitor or mutant thereof immobilized on a solid substrate, wherein the enzyme inhibitor specifically binds or binds with a dissociation constant of 1 x 10<-9>M or less to the active form the enzyme. This analytical element is included in a kit with an analytical reagent conjugated to a detectable label or conjugated to a reactive molecule that generates a detectable label, wherein the reagent specifically binds to the active form of the enzyme that binds to the enzyme inhibitor. Specific enzyme inhibitors or mutants thereof are designed to covalently bind to specific clinically important enzymes. These enzyme inhibitors contain modifications that facilitate binding to a solid support and optionally modifications that affect the binding to a target enzyme or affect the stability of the inhibitor. The method is particularly useful in measuring the presence of enzymes, such as tPA, elastase, cathepsin G and prostate specific antigen. Also disclosed is a method of immobilizing enzyme inhibitors or mutants thereof on a solid substrate yet retaining the property of covantly binding or binding with a dissociation constant of 1 x 10<-9>M or less to a funcitonally active enzyme. |
