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Last Updated: March 29, 2024

Claims for Patent: 7,026,111


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Summary for Patent: 7,026,111
Title:Methods and reagents for improved cell-based assays
Abstract: The ability to efficiently determine the state of enzyme expression in cells has long been desired as material to the diagnosis of disease. This invention relates to cytoenzymology, and more particularly to improved reagents for use in cell-based assays, especially those using fluorogenic substrates.
Inventor(s): Clausell; Adrian (San Diego, CA), Gu; Jirong (Irvine, CA), Reddy; M. Parameswara (Brea, CA)
Assignee: Beckman Coulter, Inc. (Fullerton, CA)
Application Number:09/978,498
Patent Claims:1. A method for assaying a metabolically active whole cell for the presence or activity of an enzyme, comprising the steps: (a) providing a sample comprising an intact metabolically active whole cell in medium; and (b) providing a mixture composition comprising a substrate of said enzyme or an analyte compound and an agent that enhances/uptake of said substrate or analyte compound; and (c) adding said mixture composition to said sample, wherein said mixture composition subsides within said medium of said sample to form a layer of said mixture composition over said intact metabolically active whole cell, wherein said layer is not homogeneously mixed with said medium of said sample; and (d) assaying said sample for any change in concentration of said substrate or analyte compound or of a product formed via action of said enzyme on said substrate or analyte, wherein said cell remains intact at the conclusion of said assay, wherein a change in said concentration is indicative of the presence or activity of said enzyme in said metabolically active whole cell; and wherein said agent that enhances uptake of said substrate or analyte is selected from the group consisting of glycerol, dimethyl sulfoxide (DMSO), trehalose, glutamate, betaine, ethylene glycol, threitol, ribose, and trimethylamine N-oxide, with the proviso that when said agent is dimethyl sulfoxide (DMSO), said agent will be present at a concentration of between about 20% and about 60% (v/v).

2. The method of claim 1, wherein said enzyme is selected from the group consisting of a 5' nucleotidase, acetylcholinesterase, an acid phosphatase, an acidic esterase, an acidic esterase I, an acidic esterase II, an acidic non-specific esterase, an adenosine deaminase, an adenosine monophosphate deaminase, an alkaline phosphatase, an aminopeptidase A, an aminopeptidase B, an aminopeptidase M, an aminopentidase N, an angiotensin converting enzyme, a caspase, a cathepsin B, a cathepsin B1, a cathepsin C, a cathepsin D, a cathepsin H, a cathepsin L, a cholinesterase, a chymotrypsin, a collagenase, a cytosine deaminase, a DPP I, a DPP II, a DPP IV, an elastase, an endopeptidase I, an endopeptidase II, an ester proteinase, a galactopyranosidase, a glucoronidase, a glycopyranosidase, a guanine deaminase, an HIV Protease, a lipase, a membrane associated endopeptidase I, a membrane associated endopeptidase II, a neutral endopeptidase, a neutral esterase, a neutral esterase I, a neutral esterase II, a neutral non-specific esterase, a nucleosidase, a pancreatin, a phospholipase A, a phospholipase C, a phospholipase D, a plasmin, a serine phosphatase, a tartrate resistant phosphatase, a tartrate resistant phosphatase, a threonine phosphatase, a thymidine deaminase, a tripeptidyl peptidase, a trypsin, a tyrosine phosphatase, a urokinase, and a .gamma.-glutamyl transpeptidase (.gamma.-GT).

3. The method of claim 2, wherein said enzyme is a caspase.

4. The method of claim 3, wherein said caspase is caspase 1, caspase 3, caspase 6, caspase 8 or caspase 9.

5. The method of claim 1, wherein multiple enzymes are simultaneously assayed.

6. The method of claim 1, wherein multiple enzymes are sequentially assayed.

7. The method of claim 1, wherein said substrate or analyte compound comprises an indicator group and one or more leaving groups, each of said leaving groups being selected for cleavage by said enzyme, said indicator group being in a first state when bonded to a leaving group, and being in a second state when said leaving group is cleaved from said indicator group by said enzyme; and wherein said step (b) comprises sensing whether said second state of said indicator group is produced; wherein the production of said second state of said indicator group is indicative of the presence or activity of said enzyme in said metabolically active whole cell.

8. The method of claim 7, wherein said indicator group is a fluorescent, colorimetric, bioluminescent or chemiluminescent indicator group.

9. The method of claim 7, wherein said uptake-enhancing agent is glycerol.

10. The method of claim 9, wherein said glycerol concentration is between about 5% and about 60% (v/v).

11. The method of claim 10, wherein said glycerol concentration is between about 20% and about 60% (v/v).

12. The method of claim 11, wherein said glycerol concentration is between about 25% and about 40% (v/v).

13. The method of claim 1, wherein said uptake-enhancing agent is dimethyl sulfoxide (DMSO).

14. The method of claim 1, wherein said uptake-enhancing agent is glutamate.

15. The method of claim 14, wherein said glutamate concentration is between about 0.25 M and about 2 M.

16. The method of claim 15, wherein said glutamate concentration is between about 1 M and about 2 M.

17. The method of claim 1, wherein said uptake-enhancing agent is betaine.

18. The method of claim 17, wherein said betaine concentration is about 0.3 M or greater.

19. The method of claim 1, wherein said uptake-enhancing agent is trehalose.

20. The method of claim 19, wherein said trehalose concentration is between about 0.1 M and about 1.5 M.

21. The method of claim 1, wherein said uptake-enhancing agent is ethylene glycol.

22. The method of claim 21, wherein said ethylene glycol concentration is between about 2 M and about 7 M.

23. The method of claim 1, wherein said uptake-enhancing agent is threitol.

24. The method of claim 23, wherein said threitol concentration is between about 1 M and about 5 M.

25. The method of claim 1, wherein said uptake-enhancing agent is ribose.

26. The method of claim 25, wherein said ribose concentration is between about 0.4 M and about 4 M.

27. The method of claim 1, wherein said uptake-enhancing agent is triethylamine N-oxide.

28. The method of claim 27, wherein said triethylamine N-oxide concentration is between about 0.4 M and about 4 M.

29. The method of claim 7, wherein said enzyme is selected from the group consisting of a 5' nucleotidase, acetylcholinesterase, an acid phosphatase, an acidic esterase, an acidic esterase I, an acidic esterase II, an acidic non-specific esterase, an adenosine deaminase, an adenosine monophosphate deaminase, an alkaline phosphatase, an aminopeptidase A, an aminopeptidase B, an aminopeptidase M, an aminopentidase N, an angiotensin converting enzyme, a caspase, a cathepsin B, a cathepsin B1, a cathepsin C, a cathepsin D, a cathepsin H, a cathepsin L, a cholinesterase, a chymotrypsin, a collagenase, a cytosine deaminase, a DPP I, a DPP II, a DPP IV, an elastase, an endopeptidase I, an endopeptidase II, an ester proteinase, a galactopyranosidase, a glucoronidase, a glycopyranosidase, a guanine deaminase, an HIV Protease, a lipase, a membrane associated endopeptidase I, a membrane associated endopeptidase II, a neutral endopeptidase, a neutral esterase, a neutral esterase I, a neutral esterase II, a neutral non-specific esterase, a nucleosidase, a pancreatin, a phospholipase A, a phospholipase C, a phospholipase D, a plasmin, a serine phosphatase, a tartrate resistant phosphatase, a tartrate resistant phosphatase, a threonine phosphatase, a thymidine deaminase, a tripeptidyl peptidase, a trypsin, a tyrosine phosphatase, a urokinase, and a .gamma.-glutamyl transpeptidase (.gamma.-GT).

30. The method of claim 29, wherein said enzyme is a caspase.

31. The method of claim 30, wherein said caspase is caspase 1, caspase 3,caspase 6, caspase 8, or caspase 9.

32. The method of claim 7, wherein multiple enzymes are simultaneously assayed.

33. The method of claim 7, wherein multiple enzymes are sequentially assayed.

34. The method of claim 7, wherein said step (b) includes measuring an intensity of said second state against time.

35. The method of claim 7, wherein said step (b) includes measuring a magnitude of said second state at a point of time.

36. The method of claim 7, wherein said substrate or analyte compound comprises more than one leaving group, and wherein each of said substrate's leaving groups is cleaved sequentially by said enzyme.

37. The method of claim 7, wherein said indicator group is selected from the group consisting of rhodamine 110, rhodol, fluorescein, coumarin, and derivatives thereof.

38. The method of claim 37, wherein said derivatives of rhodamine 110, rhodol, fluorescein and coumarin are selected from the group consisting of 4'(5')thiofluorescein, 4'(5')-aminofluorescein, 4'(5')-carboxyfluorescein, 4'(5')-chlorofluorescein, 4'(5')-methylfluorescein, 4'(5')-sulfofluorescein, 4'(5')-aminorhodol, 4'(5')-carboxyrhodol, 4'(5')-chlororhodol, 4'(5')-methylrhodol, 4'(5')-sulforhodol; 4'(5')-aminorhodamine 110, 4'(5')-sulforhodamine 110, 4'(5')thiorhodamine 110, 7-aminocoumarin, and sulfonated coumarin.

39. The method of claim 7, wherein said assay detects the presence or absence of an abnormality in the activity of said enzyme by comparing the production of said second state of said indicator group by said test cell to the production of said second state of said indicator group by a reference normal cell.

40. The method of claim 39, wherein said abnormality is a morphological or disease state.

41. The method of claim 40, wherein said morphological state is an apoptotic state.

42. The method of claim 40, wherein said disease state is a tumorigenic state.

43. The method of claim 7, wherein said substrate or analyte compound contains a blocking group.

44. The method of claim 43, wherein said blocking group is a Cbz blocking group.

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