Enzymatic detection techniques
View Patent ↗A diagnostic test kit for detecting the presence or quantity of an enzyme or enzyme inhibitor is provided. The diagnostic kit utilizes substrate conjugates to facilitate the detection of the enzyme or enzyme inhibitor via direct detection of the substrate and/or a product formed in an enzyme-catalyzed reaction of the substrate. The substrate conjugates include a substrate joined (e.g., covalently bonded, physically adsorbed, etc.) to a reporter. In one embodiment, for example, a peptide, protein, or glycoprotein substrate is joined to a reporter (e.g., dyed latex particle). In this embodiment, the substrate provides a cleavage target for an enzyme. Specifically, upon contacting the substrate conjugate, the enzyme catalyzes a reaction with the substrate and forms a product conjugate that includes the product of the enzyme catalyzed reaction joined to the reporter. The signal exhibited by the reporters may then be used to indicate the presence or quantity of an enzyme or enzyme inhibitor within the test sample.
1. A method for detecting an enzyme, or an inhibitor thereof, within a test sample, the method comprising:
contacting a test sample with a plurality of substrate conjugates to form an incubation mixture that includes the substrate conjugates and the test sample, said substrate conjugates each comprising a substrate joined to a reporter, wherein an enzyme-catalyzed reaction of said substrate conjugate forms a product conjugate, said product conjugate comprising a product of the enzyme-catalyzed reaction joined to the reporter, said reporter being capable of directly or indirectly generating an optically detectable detection signal;
applying said incubation mixture to a chromatographic medium, said chromatographic medium comprising a first detection zone within which said product conjugate preferentially binds, said chromatographic medium further comprising a second detection zone within which said substrate conjugate preferentially binds, said incubation mixture migrating through said chromatographic medium to reach said first detection zone and said second detection zone, wherein the binding of the product conjugate generates a first detection signal within said first detection zone, and wherein the binding of the substrate conjugate generates a second detection signal within said second detection zone; and
optically determining the presence or intensity of said first detection signal within said first detection zone and said second detection signal within said second detection zone.
2. The method of claim 1 , wherein the amount of an enzyme within said test sample is inversely proportional to the intensity of said second detection signal in said second detection zone.
3. The method of claim 1 , wherein the amount of an enzyme within said test sample is directly proportional to the intensity of said first detection signal in said first detection zone.
4. The method of claim 1 , wherein the enzyme is a hydrolase.
5. The method of claim 1 , wherein the enzyme is a transferase, a ligase, or a polymerase.
6. The method of claim 5 , wherein the transferase is a kinase or a methylase.
7. The method of claim 1 , wherein said substrate is a protein, glycoprotein, peptide, nucleic acid, carbohydrate, lipid, ester, antibody, antigen, or a derivative thereof.
8. The method of claim 7 , wherein said substrate is casein, albumin, hemoglobin, myoglobin, keratin, gelatin, insulin, proteoglycan, fibronectin, laminin, collagen, elastin, or a derivative thereof.
9. The method of claim 1 , wherein product conjugate and unreacted substrate conjugate are capable of reaching said first detection zone.