Cholesterol assay

• US 3,907,645 A
• Filed: 01/23/1974
• Issued: 09/23/1975
• Est. Priority Date: 09/22/1971
• Status: Expired due to Term

• Current Assignee: British Technology Group Ltd.
• Original Assignee: William Richmond
• Inventors: Richmond, William
• Primary Examiner(s): Tanenholtz, Alvin E.
• Application Number:
  US05/435,892
• Time in Patent Office: 608 Days
• Field of Search: 195/103.5
• US Class Current: 435/11
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: C12Q 1/60 involving cholesterol
• CPC Class Codes: Y10S 210/922 Oil spill cleanup, e.g. bacterial
• CPC Class Codes: Y10S 435/815 by sorption
• CPC Class Codes: Y10S 435/866 Mycobacterium smegmatis
• CPC Class Codes: Y10S 435/872 Nocardia
• CPC Class Codes: Y10S 435/911 using fungi
• CPC Class Codes: Y10S 435/913 Aspergillus
• CPC Class Codes: Y10S 435/923 Candida lipolytica
• CPC Class Codes: Y10S 435/924 Candida tropicalis
• CPC Class Codes: Y10S 435/942 Saccharomyces cerevisiae

Abstract

Enzyme preparations which will convert cholesterol to Delta 4cholestenone and hydrogen peroxide are obtained from certain Nocardia species belonging to the Mycobacterium rhodocrous group. The preparations have a cholesterol oxidase specific activity of at least 1 unit per 5 mg of protein nitrogen and, when in liquid form, a potency of at least 10 2 units/ml. They are prepared by growing the organism and recovering the enzyme preparation, preferably by extracting the harvested cells with a surface active agent such as Triton X-100. The enzyme preparations are used to assay for cholesterol by measuring the amount in which one of the products of the cholesterol oxidase reaction, preferably hydrogen peroxide, is formed or the quantity in which oxygen is used.

Claims

1. A METHOD OF ASSAYING FOR CHOLESTEROL IN A LIQUID WHICH 2 COMPRISES INCUBATING SAID LIQUID WITH AN ENZYME PREPARATION CAPABLE OF OXIDISING CHOLESTEROL INTO $4-CHOLESTENONE AND HYDROGEN PEROXIDE AND DETERMINING THE AMOUNT OF CHOLESTEROL PRESENT IN SAID LIQUID BY MEASURING THE AMOUNT OF HYDROGEN PEROXIDE PRODUCT.

2. A method according to claim 1 in which said liquid is serum or another biological fluid.

3. A method of assaying for cholesterol in a liquid which comprises incubating said liquid with an enzyme preparation derived from Norcadia species NCIB 10554 (NRRL 5635) or NCIB 10555 (NRRL 5636), said enzyme preparation having cholesterol oxidase activity capable of oxidizing cholesterol to Delta 4 cholestenone and hydrogen peroxide and determining the amount of cholesterol present in said liquid by measuring the amount in which a product of the cholesterol oxidase reaction is formed or the amount in which oxygen is used in the cholesterol oxidase reaction.

4. A method according to claim 1 and wherein said enzyme preparation is derived from a Nocardia species.

5. A method according to claim 4 in which said enzyme preparation has a cholesterol oxidase specific activity of at least 1 unit per 5 mg of protein nitrogen and a potency of at least 10 2 units ml when in liquid form and the amount of hydrogen peroxide produced is measured by a fluorimetric method.

6. A method according to claim 4 in which said enzyme preparation has a cholesterol oxidase specific-activity of at least 1 unit per 5 mg of protein nitrogen and a potency of at least 10 1 units/ml when in liquid form and the amount of hydrogen peroxide produced is measured colorimetrically.

7. A method according to claim 4 in which the amount of hydrogen peroxide produced is measured by a system which comprises a chromogenic reagent or reagents capable of undergoing a colour change in the presence of hydrogen peroxide, said amount of hydrogen peroxide being measured by colorimetrically measuring the colour change of the chromogenic reagent or reagents.

8. A method according to claim 7 in which said liquid is serum or another biological fluid.

9. A method according to claim 8 in which saponified serum or other biological fluid is incubated with said enzyme preparation to measure total cholesterol.

10. A method according to claim 3 in which said enzyme preparation has a cholesterol oxidase specific activity of at least 1 unit per 50 Mu g of protein nitrogen and a potency of at least 0.5 unit ml when in liquid form.

11. A method according to claim 3 carried out as an individual test on a single sample.

12. A method according to claim 3 in which a plurality of samples are tested on an automated basis.

13. A kit for use in assaying for cholesterol in a liquid comprising in association a. an enzyme preparation which is derived from Nocardia species NCIB 10554 (NRRL 5635) or NCIB 10555 (NRRL 5636), which has a cholesterol oxidase specific activity of at least 1 unit per 5 mg of protein nitrogen and is capable of oxidizing cholesterol into 66 4-cholestenone and hydrogen peroxide;

and b. at least one reagent which is capable of being used in the determination of the amount in which hydrogen peroxide is formed in the cholesterol oxidase reaction.

14. A kit according to claim 13 in which component (b) comprises at least one reagent capable of taking part in a reaction by means of which hydrogen peroxide can be determined fluorimetrically.

15. A kit according to claim 13 which includes in addition at least one standard cholesterol solution.

16. A kit for use in assaying for cholesterol in a liquid comprising in association (a) an enzyme preparation capable of oxidizing cholesterol to Delta 4-cholestenone and hydrogen peroxide;

and (b) at least one reagent which is capable of being used in the determination of the amount of hydrogen peroxide produced.

17. A Kit according to claim 16 wherein said enzyme preparation is derived from a Nocardia species.

18. A kit according to claim 17 in which enzyme preparation (a) is in liquid form and has a potency of at least 10 2 units/ml.

19. A kit according to claim 17 in which component (b) comprises at least one reagent capable of taking part in a reaction by means of which hydrogen peroxide can be determined colorimetrically.

20. A kit according to claim 17 in which said enzyme preparation (a) has a cholesterol oxidase specific activity of at least 1 unit per 50 Mu g of protein nitrogen and when in liquid form, has a potency oF at least 0.5 unit/ml.

21. A kit according to claim 17 in which component (a) is a freeze-dried enzyme preparation which upon addition of an aqueous solvent and a suitable buffer forms an aqueous preparation of the required potency.

22. A kit according to claim 17 which contains each component in an amount required for a defined number of tests.

23. A kit according to claim 22 which contains a single amount of each component, the amount of each component required for an individual test to be extracted from this single amount.

24. A kit according to claim 22 which contains a separate amount of enzyme component (a) for each test.

25. A kit according to claim 24, in which separate amounts of enzyme component (a) which correspond to that amount required for a single test are provided in freeze dried or concentrated form in individual vials to be reconstituted with buffer before each test.

26. A kit according to claim 13 for the assay of total cholesterol in serum which contains in addition to components (a) and (b) at least one acid reagent capable of neutralising saponified serum.

27. A method of assaying for cholesterol in a liquid which comprises incubating said liquid with a cholesterol oxidase capable of oxidizing cholesterol to Delta 4 cholestenone and hydrogen peroxide and having a potency of at least 10 1 units/ml and determining the amount of cholesterol by measuring the amount in which Delta 4-cholestenone is formed or oxygen is consumed in the cholesterol oxidase reaction.

28. A kit for use in assaying for cholesterol in a liquid comprising in association a. a cholesterol oxidase capable of oxidizing cholesterol to Delta 4 cholestenone and hydrogen peroxide and having a potency of at least 10 1 units/ml;

and b. at least one reagent which is capable of being used in the determination of the amount in which Delta 4-cholestenone is formed or oxygen is consumed in the cholesterol oxidase reaction.

29. A method according to claim 27 wherein the cholesterol oxidase has a specific activity of at least 1 unit per 50 Mu g of protein nitrogen.

30. A kit according to claim 28 wherein the cholesterol oxidase has a specific activity of at least 1 unit per 50 Mu g of protein nitrogen.

31. A method according to claim 1 wherein the enzyme preparation has a catalase activity of less than 10% of the cholesterol oxidase activity.

32. A kit according to claim 16 wherein the enzyme preparation has a catalase activity of less than 10% of the cholesterol oxidase activity.

33. A method according to claim 1 wherein the enzyme preparation has a catalase activity of less than 1% of the cholesterol oxidase activity.

34. A kit according to claim 16 wherein the enzyme preparation has a catalase activity of less than 1% of the cholesterol oxidase activity.

35. A method according to claim 4 wherein the enzyme preparation has a cholesterol oxidase specific activity of at least 1 unit per 50 Mu g of protein nitrogen and a potency of at least 0.5 units/ml.

36. A kit according to claim 17 wherein the enzyme preparation has a cholesterol oxidase specific activity of at least 1 unit per 50 Mu g of protein nitrogen and a potency of at least 0.5 units/ml.

37. A method according to claim 4 wherein the enzyme preparation has a cholesterol oxidase specific activity of at least 1 unit per 28 Mu g of protein nitrogen, a potency of 5 units/ml and contains 3% v/v Triton X-100.

38. A kit according to claim 17 wherein the enzyme preparation has a cholesterol oxidase specific activity of 1 unit per 28 Mu g of protein nitrogen, a potency of 5 units/ml and contains 3% v/v Triton X-100.

39. A method according to claim 2 in which saponified serum or other biological fluid is incubated with said enzyme preparation to measure total cholesterol.

40. A method according to claim 1 in which said enzyme preparation has a cholesterol oxidase specific activity of at least 1 unit per 5 mg of protein nitrogen and a potency of at least 10 2 units/ml when in liquid form and the amount of hydrogen peroxide produced is measured by a fluorimetric method.

41. A method according to claim 1 in which said enzyme preparation has a cholesterol oxidase specific activity of at least 1 unit per 5 mg of protein nitrogen and a potency of at least 10 1 units/ml when in liquid form and the amount of hydrogen peroxide produced is measured colorimetrically.

42. A kit according to claim 16 in which enzyme preparation (a) is in liquid form and has a potency of at least 10 2 units/ml.

43. A kit according to claim 16 in which the at least one reagent of component (b) is capable of taking part in a reaction by means of which hydrogen peroxide can be determined colorimetrically.

44. A kit according to claim 16 in which component (a) is a freeze-dried enzyme preparation which upon addition of an aqueous solvent and a suitable buffer forms an aqueous preparation of the required potency.

45. A kit according to claim 28 wherein said cholesterol oxidase is derived from a Nocardia species.

46. A kit according to claim 28 wherein said cholesterol oxidase is derived from Nocardia species NCIB 10554 (NRRL 5635) or NCIB 10555 (NRRL 5636).