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 reactive complexes to facilitate the detection of the enzyme or enzyme inhibitor. The reactive complexes include a substrate joined (e.g., covalently bonded, physically adsorbed, etc.) to a reporter and specific binding member. In one embodiment, for example, a peptide, protein, or glycoprotein substrate is joined to a reporter (e.g., dyed latex particle) and specific binding member (e.g., biotinylated compound). In this embodiment, the substrate provides a cleavage target for a proteolytic enzyme. Specifically, upon contacting the reactive complexes, the proteolytic enzyme cleaves the substrate and releases the reporter and/or specific binding member. The signal exhibited by the released 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 within a test sample, the method comprising:
contacting a test sample with a plurality of reactive complexes to form an incubation mixture, the reactive complexes each comprising a substrate joined to a reporter and specific binding member, wherein the substrate is cleaved by the enzyme to release the reporter and the specific binding member;
applying the incubation mixture to a chromatographic medium so that the released specific binding member flows through the chromatographic medium to a first detection zone, the chromatographic medium comprising the first detection zone within which a first receptive material is immobilized, the first receptive material having an affinity for the specific binding member, wherein the chromatographic medium further comprises a second detection zone within which a second receptive material is immobilized, the second receptive material comprising a biological material and having an affinity for the reporter or complexes thereof, the reporter being capable of directly or indirectly generating a first detection signal within the first detection zone and a second detection signal within the second detection zone; and
determining the presence or intensity of the first detection signal within the first detection zone and the second detection signal within the second detection zone, wherein the amount of an enzyme within the test sample is inversely proportional to the intensity of the first detection signal and directly proportional to the intensity of the second detection signal.
2. The method of claim 1 , wherein the amount of the enzyme within the test sample is directly proportional to the ratio of the intensity of the second detection signal to the intensity of the first detection signal.
3. The method of claim 1 , wherein the enzyme is a hydrolase.
4. The method of claim 3 , wherein the hydrolase is a protease or peptidase.
5. The method of claim 3 , wherein the hydrolase is a serine protease, aspartic protease, thiol protease, metalloprotease, acid protease, or alkaline protease.
6. The method of claim 1 , wherein the substrate is a protein, glycoprotein, peptide, nucleic acid, carbohydrate, lipid, ester, or derivative thereof.
7. The method of claim 1 , wherein the substrate is casein, albumin, hemoglobin, myoglobin, keratin, gelatin, insulin, proteoglycan, fibronectin, laminin, collagen, elastin, or a derivative thereof.
8. The method of claim 1 , wherein the reporter comprises a detectable substance that is capable of directly generating the first detection signal.
9. The method of claim 1 , wherein the reporter comprises a specific binding member.
10. The method of claim 9 , further comprising contacting the incubation mixture with probes conjugated with a specific binding member, the probes further comprising a detectable substance that is capable of directly generating the first detection signal.
11. The method of claim 10 , wherein the specific binding member of the probes has specific binding affinity for the specific binding member of the reporter.
12. The method of claim 10 , wherein the specific binding member of the probes is the same as or an analog of the specific binding member of the reporter.
13. The method of claim 1 , wherein the specific binding member is selected from the group consisting of antibodies, antigens, haptens, biotin, avidin, streptavidin, neutravidin, captavidin, protein A, protein G, carbohydrates, lectins, nucleotide sequences, peptide sequences, effector and receptor molecules, hormone and hormone binding protein, enzyme cofactors and enzymes, enzyme inhibitors, and derivatives thereof.
14. The method of claim 1 , wherein the total quantity of the first receptive material is less than otherwise needed to bind to the total quantity of the reactive complexes.
15. The method of claim 1 , wherein the total quantity of the first receptive material is the same or more than otherwise needed to bind to the total quantity of the reactive complexes.
16. The method of claim 1 , wherein the first receptive material is selected from the group consisting of antibodies, antigens, haptens, biotin, avidin, streptavidin, neutravidin, captavidin, protein A, protein G, carbohydrates, lectins, nucleotide sequences, peptide sequences, effector and receptor molecules, hormone and hormone binding protein, enzyme cofactors and enzymes, enzyme inhibitors, and derivatives thereof.
17. The method of claim 1 , wherein the specific binding member is biotin.
18. The method of claim 17 , wherein the first receptive material is avidin, streptavidin, neutravidin, captavidin, or a derivative thereof.
19. A method for detecting an enzyme, within a test sample, the method comprising:
contacting a test sample with a plurality of reactive complexes to form an incubation mixture, the reactive complexes each comprising a substrate joined to a reporter and specific binding member, wherein the substrate is cleaved by the enzyme to release the reporter and the specific binding member;
applying the incubation mixture to a chromatographic medium so that the released specific binding member flows through the chromatographic medium to a first detection zone, the chromatographic medium comprising the first detection zone within which a first receptive material is immobilized, the first receptive material having an affinity for the specific binding member, wherein the chromatographic medium further comprises a second detection zone within which a second receptive material is immobilized, the second receptive material comprising a polyelectrolyte and having an affinity for the reporter or complexes thereof, the reporter being capable of directly or indirectly generating a first detection signal within the first detection zone and a second detection signal within the second detection zone; and
determining the presence or intensity of the first detection signal within the first detection zone and the second detection signal within the second detection zone, wherein the amount of an enzyme within the test sample is inversely proportional to the intensity of the first detection signal and directly proportional to the intensity of the second detection signal.
20. The method of claim 19 , wherein the amount of the enzyme within the test sample is directly proportional to the ratio of the intensity of the second detection signal to the intensity of the first detection signal.
21. The method of claim 19 , wherein the enzyme is a hydrolase.
22. The method of claim 21 , wherein the hydrolase is a protease or peptidase.
23. The method of claim 21 , wherein the hydrolase is a serine protease, aspartic protease, thiol protease, metalloprotease, acid protease, or alkaline protease.
24. The method of claim 19 , wherein the substrate is a protein, glycoprotein, peptide, nucleic acid, carbohydrate, lipid, ester, or derivative thereof.
25. The method of claim 19 , wherein the substrate is casein, albumin, hemoglobin, myoglobin, keratin, gelatin, insulin, proteoglycan, fibronectin, laminin, collagen, elastin, or a derivative thereof.
26. The method of claim 19 , wherein the reporter comprises a detectable substance that is capable of directly generating the first detection signal.
27. The method of claim 19 , wherein the reporter comprises a specific binding member.
28. The method of claim 27 , further comprising contacting the incubation mixture with probes conjugated with a specific binding member, the probes further comprising a detectable substance that is capable of directly generating the first detection signal.
29. The method of claim 28 , wherein the specific binding member of the probes has specific binding affinity for the specific binding member of the reporter.
30. The method of claim 28 , wherein the specific binding member of the probes is the same as or an analog of the specific binding member of the reporter.
31. The method of claim 19 , wherein the specific binding member is selected from the group consisting of antibodies, antigens, haptens, biotin, avidin, streptavidin, neutravidin, captavidin, protein A, protein G, carbohydrates, lectins, nucleotide sequences, peptide sequences, effector and receptor molecules, hormone and hormone binding protein, enzyme cofactors and enzymes, enzyme inhibitors, and derivatives thereof.
32. The method of claim 19 , wherein the total quantity of the first receptive material is less than otherwise needed to bind to the total quantity of the reactive complexes.
33. The method of claim 19 , wherein the total quantity of the first receptive material is the same or more than otherwise needed to bind to the total quantity of the reactive complexes.
34. The method of claim 19 , wherein the first receptive material is selected from the group consisting of antibodies, antigens, haptens, biotin, avidin, streptavidin, neutravidin, captavidin, protein A, protein G, carbohydrates, lectins, nucleotide sequences, peptide sequences, effector and receptor molecules, hormone and hormone binding protein, enzyme cofactors and enzymes, enzyme inhibitors, and derivatives thereof.
35. The method of claim 19 , wherein the specific binding member is biotin.
36. The method of claim 35 , wherein the first receptive material is avidin, streptavidin, neutravidin, captavidin, or a derivative thereof.