Enzyme detection technique

• US 7,504,235 B2
• Filed: 08/31/2005
• Issued: 03/17/2009
• Est. Priority Date: 08/31/2005
• Status: Expired due to Fees

• Current Assignee: Kimberly-Clark Worldwide Incorporated (Kimberly-Clark Corporation)
• Original Assignee: Kimberly-Clark Worldwide Incorporated (Kimberly-Clark Corporation)
• Inventors: Song, Xuedong
• Primary Examiner(s): Weber; Jon P
• Assistant Examiner(s): Shen; Bin
• Application Number:
  US11/217,097
• Publication Number:
  US 20070048815A1
• Time in Patent Office: 1,294 Days
• Field of Search: None
• US Class Current: 435/19
• CPC Class Codes: C12Q 1/44 involving esterase
• CPC Class Codes: G01N 33/52 Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72
• CPC Class Codes: G01N 33/558 using diffusion or migration of antigen or antibody immunochromatographic test strips G01N33/54387
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes

Abstract

A diagnostic test kit for detecting the presence or absence of an enzyme (e.g., leukocyte esterase) within a test sample is provided. The test kit comprises a substrate that is capable of being cleaved in the presence of the enzyme to release a nucleophilic aromatic compound. The kit also comprises a lateral flow device that comprises a chromatographic medium. The chromatographic medium defines a detection zone within which is contained a first reagent (e.g., diazonium ion) that is capable of reacting with the nucleophilic aromatic compound to form a second reagent (e.g., aromatic azo compound). The second reagent exhibits a color that is different than the color of the first reagent. The lateral flow device also includes an absorbent material located adjacent to the chromatographic medium, the absorbent material receiving the test sample after flowing through the chromatographic medium.

Claims

1. A diagnostic test kit for detecting the presence or absence of an enzyme within a test sample, the test kit comprising:

a substrate that is capable of being cleaved in the presence of the enzyme to release a nucleophilic aromatic compound; and

a lateral flow device that comprises;

a chromatographic medium defining a discrete detection zone within which is contained a first reagent, wherein the nucleophilic aromatic compound is capable of flowing through the chromatographic medium and reacting with the first reagent within the detection zone to form a second reagent, the second reagent exhibiting a color that is different than the color of the first reagent; and

an absorbent material positioned adjacent to the chromatographic medium and located downstream from the detection zone, wherein the absorbent material receives the test sample after flowing through the chromatographic medium.

2. The diagnostic test kit of claim 1, wherein the enzyme is an esterase.

3. The diagnostic test kit of claim 2, wherein the esterase is leukocyte esterase.

4. The diagnostic test kit of claim 2, wherein the substrate is an aromatic ester.

5. The diagnostic test kit of claim 4, wherein the substrate is an indoxyl ester, lactate ester, thiazole ester, pyrrole ester, thiophene ester, naphthyl ester, phenoxyl ester, or quinolinyl ester.

6. The diagnostic test kit of claim 1, wherein the nucleophilic aromatic compound is a benzopyrrole.

7. The diagnostic test kit of claim 1, wherein the first reagent is a diazonium ion.

8. The diagnostic test kit of claim 7, wherein the diazonium ion is provided by a salt selected from the group consisting of diazonium chlorides, diazonium acid sulphates, diazonium alkyl sulphates, diazonium fluoborates, diazonium benzenesulphonates, and diazonium acid 1,5-naphthalenedisulphonates.

9. The diagnostic test kit of claim 7, wherein the second reagent is an aromatic azo compound.

10. The diagnostic test kit of claim 1, wherein the chromatographic medium is a porous membrane.

11. The diagnostic test kit of claim 1, wherein the substrate is disposed on the lateral flow assay device.

12. The diagnostic test kit of claim 11, wherein the lateral flow assay device further comprises a reagent zone within which is contained the substrate.

13. The diagnostic test kit of claim 12, further comprising a sample application zone that is located upstream from the reagent zone.

14. The diagnostic test kit of claim 1, wherein the lateral flow device comprises a quenching zone located upstream from the detection zone.

15. The diagnostic test kit of claim 14, wherein the quenching zone contains an agent capable of reacting with a contaminant in the test sample.

16. The diagnostic test kit of claim 1, wherein the lateral flow device comprises a control zone that is capable of signaling the presence of the test sample.

17. The diagnostic test kit of claim 16, further comprising a control reagent that is capable being detected within the control zone.

18. The diagnostic test kit of claim 1, wherein the test kit further comprises an accelerator for the substrate.

19. The diagnostic test kit of claim 18, wherein the accelerator is contained within an accelerator zone located upstream from the detection zone.

20. The diagnostic test kit of claim 1, wherein the test sample is urine.

31. The diagnostic test kit of claim 1, wherein the absorbent material includes nitrocellulose, a cellulosic material, a porous polyethylene pad, glass fiber filter paper, or a combination thereof.

33. The diagnostic test kit of claim 1, wherein the lateral flow device further comprises a reagent pad on which the substrate is disposed.

34. The diagnostic test kit of claim 33, wherein the lateral flow device further comprises a sample pad configured to receive the test sample.

35. The diagnostic test kit of claim 34, wherein the sample pad also defines a quenching zone that contains an agent capable of reacting with a contaminant in the test sample.

21. A diagnostic test kit for detecting the presence or absence of leukocyte esterase within urine, the test kit comprising:

an aromatic ester substrate that is capable of being hydrolyzed in the presence of leukocyte esterase to release a nucleophilic aromatic compound; and

a lateral flow device that comprises;

a porous membrane defining a discrete detection zone within which is contained a diazonium ion, wherein the nucleophilic aromatic compound is capable of flowing through the chromatographic medium and reacting with the diazonium ion within the detection zone to form an aromatic azo compound, the aromatic azo compound exhibiting a color that is different than the color of the diazonium ion; and

an absorbent material positioned adjacent to the porous membrane and located downstream from the detection zone, wherein the absorbent material receives the urine after flowing through the porous membrane.

22. The diagnostic test kit of claim 21, wherein the substrate is an indoxyl ester, lactate ester, thiazole ester, pyrrole ester, thiophene ester, naphthyl ester, phenoxyl ester, or quinolinyl ester.

23. The diagnostic test kit of claim 21, wherein the nucleophilic aromatic compound is a benzopyrrole.

24. The diagnostic test kit of claim 21, wherein the diazonium ion is provided by a salt selected from the group consisting of diazonium chlorides, diazonium acid sulphates, diazonium alkyl sulphates, diazonium fluoborates, diazonium benzenesulphonates, and diazonium acid 1,5-naphthalenedisulphonates.

25. The diagnostic test kit of claim 21, wherein the substrate is disposed on the lateral flow assay device.

26. The diagnostic test kit of claim 25, wherein the lateral flow assay device further comprises a reagent zone within which the substrate is disposed.

27. The diagnostic test kit of claim 26, further comprising a sample application zone that is positioned upstream from the reagent zone.

28. The diagnostic test kit of claim 21, wherein the lateral flow device comprises a quenching zone located upstream from the detection zone.

29. The diagnostic test kit of claim 21, wherein the lateral flow device comprises a control zone that is capable of signaling the presence of the urine.

30. The diagnostic test kit of claim 21, wherein the test kit further comprises an accelerator for the substrate, the accelerator being contained within an accelerator zone located upstream from the detection zone.

32. The diagnostic test kit of claim 21, wherein the absorbent material includes nitrocellulose, a cellulosic material, a porous polyethylene pad, glass fiber filter paper, or a combination thereof.

36. The diagnostic test kit of claim 21, wherein the lateral flow device further comprises a reagent pad on which the substrate is disposed.

37. The diagnostic test kit of claim 36, wherein the lateral flow device further comprises a sample pad configured to receive the test sample.

38. The diagnostic test kit of claim 37, wherein the sample pad also defines a quenching zone that contains an agent capable of reacting with a contaminant in the test sample.