One-step enzymatic and amine detection technique

• US 20060003336A1
• Filed: 06/30/2004
• Published: 01/05/2006
• Est. Priority Date: 06/30/2004
• Status: Active Grant

• Current Assignee: Kimberly-Clark Worldwide Incorporated (Kimberly-Clark Corporation)
• Original Assignee: Kimberly-Clark Worldwide Incorporated (Kimberly-Clark Corporation)
• Inventors: Song, Xuedong, Chidebelu-Eze, Chibueze Obi, Boga, RameshBabu
• Granted Patent: US 7,521,226 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: G01N 33/54388 based on lateral flow
• CPC Class Codes: G01N 33/558 using diffusion or migration of antigen or antibody immunochromatographic test strips G01N33/54387

Abstract

A technique for detecting the presence or quantity of an enzyme (or enzyme inhibitor) and/or an amine within a test sample is provided. For example, in one embodiment, a diagnostic test kit is employed that utilizes reactive complexes that each includes a substrate joined (e.g., covalently bonded, physically adsorbed, etc.) to a reporter and a separation species. Upon contacting the reactive complexes, enzymes may cleave the substrate and release the reporter. Moreover, the test kit may also employ a chemichromic dye, i.e., a dye that exhibits a detectable color change upon chemical reaction with one or more functional groups, such as amino groups. The signal generated (directly or indirectly) by the reporter and chemichromic dye may then be used to indicate the presence or quantity of an enzyme (or enzyme inhibitor) and amine, respectively, within the test sample.

Claims

1. A diagnostic kit for detecting an amine, enzyme, or an enzyme inhibitor within a test sample, the kit comprising:

a plurality of reactive complexes that each comprises a substrate joined to a reporter and a separation species, said substrate being cleavable by an enzyme to release said reporter; and

a chromatographic medium that defines a first enzyme detection zone within which an enzyme detection signal is capable of being generated, wherein the presence or quantity of an enzyme, or an inhibitor thereof, is determinable from said enzyme detection signal, said chromatographic medium further defining an amine detection zone within which is contained a chemichromic dye, said chemichromic dye being capable of undergoing a color change in the presence of an amine, wherein the presence or quantity of an amine is determinable from said color change.

2. A diagnostic test kit as defined in claim 1, wherein the enzyme is a protease or peptidase.

3. A diagnostic test kit as defined in claim 1, wherein said substrate is a protein, glycoprotein, peptide, nucleic acid, carbohydrate, lipid, ester, or derivative thereof.

4. A diagnostic test kit as defined in claim 1, wherein said substrate is casein, albumin, hemoglobin, myoglobin, keratin, gelatin, insulin, proteoglycan, fibronectin, laminin, collagen, elastin, or a derivative thereof.

5. A diagnostic test kit as defined in claim 1, wherein said reporter comprises a detectable substance that is capable of directly generating said enzyme detection signal.

6. A diagnostic test kit as defined in claim 1, wherein said reporter comprises a specific binding member.

7. A diagnostic test kit as defined in claim 6, further comprising probes conjugated with a specific binding member, said probes comprising a detectable substance that is capable of directly generating said enzyme detection signal.

8. A diagnostic test kit as defined in claim 1, wherein said separation species is a specific binding member.

9. A diagnostic test kit as defined in claim 8, wherein a receptive material is immobilized within said first enzyme detection zone that has an affinity for said specific binding member.

10. A diagnostic test kit as defined in claim 1, wherein said separation species is a magnetic particle.

11. A diagnostic test kit as defined in claim 10, further comprising a magnetic device positioned adjacent to said chromatographic medium to immobilize said magnetic particle within a separation zone.

12. A diagnostic test kit as defined in claim 1, wherein said chromatographic medium further comprises a second enzyme detection zone within which a second enzyme detection signal is capable of being generated.

13. A diagnostic test kit as defined in claim 12, wherein a second receptive material is immobilized within said second detection zone that is capable of binding to said reporter or complexes thereof to generate said second enzyme detection signal.

14. A diagnostic test kit as defined in claim 12, wherein a second receptive material is immobilized within said second detection zone that is capable of binding to probes or complexes thereof to generate said second enzyme detection signal.

15. A diagnostic test kit as defined in claim 1, wherein said chemichromic dye is an arylmethane.

16. A diagnostic test kit as defined in claim 1, wherein said chemichromic dye is a triarylmethane having the following general structure:

17. A diagnostic test kit as defined in claim 1, wherein said chemichromic dye is a diarylmethane.

18. A diagnostic test kit as defined in claim 1, wherein said amine detection zone is positioned downstream from said first enzyme detection zone.

19. A diagnostic kit for detecting an amine or a hydrolytic enzyme within a test sample, the kit comprising:

a plurality of reactive complexes that each comprises a substrate joined to a reporter and a specific binding member, said substrate being cleavable by a hydrolytic enzyme to release said reporter; and

a chromatographic medium that defines a first enzyme detection zone within which an enzyme detection signal is capable of being generated, wherein the presence or quantity of a hydrolytic enzyme is determinable from said enzyme detection signal, said chromatographic medium further defining an amine detection zone positioned downstream from said first enzyme detection zone, wherein a chemichromic dye is contained within said amine detection zone, said chemichromic dye being capable of undergoing a color change in the presence of an amine, wherein the presence or quantity of an amine is determinable from said color change.

20. A diagnostic test kit as defined in claim 19, wherein said reporter comprises a detectable substance that is capable of directly generating said enzyme detection signal.

21. A diagnostic test kit as defined in claim 19, wherein said reporter comprises a specific binding member.

22. A diagnostic test kit as defined in claim 21, further comprising probes conjugated with a specific binding member, said probes comprising a detectable substance that is capable of directly generating said enzyme detection signal.

23. A diagnostic test kit as defined in claim 19, wherein said chromatographic medium further comprises a second enzyme detection zone within which a second enzyme detection signal is capable of being generated.

24. A diagnostic test kit as defined in claim 23, wherein said second detection zone is capable of capturing said reporter or complexes thereof to generate said second enzyme detection signal.

25. A diagnostic test kit as defined in claim 19, wherein said chemichromic dye is a triarylmethane having the following general structure:

26. A diagnostic test kit as defined in claim 19, wherein said chemichromic dye is a diarylmethane.

27. A method for detecting an amine, enzyme, or enzyme inhibitor within a test sample, the method comprising:

i) contacting the test sample with a chromatographic medium, said chromatographic medium defining an enzyme detection zone and an amine detection zone, wherein an enzyme detection signal is capable of being generated within said enzyme detection zone and an amine detection signal is capable of being generated within said amine detection zone;

ii) determining the presence or quantity of an enzyme or enzyme inhibitor from said enzyme detection signal; and

iii) determining the presence or quantity of an amine from said amine detection signal.

28. A method as defined in claim 27, wherein the quantity of an enzyme within the test sample is inversely proportional to the intensity of said enzyme detection signal.

29. A method as defined in claim 27, wherein the quantity of an enzyme within the test sample is directly proportional to the intensity of said enzyme detection signal.

30. A method as defined in claim 27, wherein said chromatographic medium further comprises a second enzyme detection zone within which a second enzyme detection signal is capable of being generated.

31. A method as defined in claim 30, wherein the quantity of an enzyme within the test sample is directly proportional to the intensity of said second enzyme detection signal.

32. A method as defined in claim 27, further comprising selectively controlling the pH level of the test sample to optimize the activity of an enzyme.

33. A method as defined in claim 27, wherein the test sample is obtained from vaginal fluid.