Patent US3907645A successfully refreshed from Google Patents.
US3907645A
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Cholesterol assay
Assignee: BTG International Ltd
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Filing: 1974-01-23
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Granted: 1975-09-23
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Expires: 1994-01-23
Abstract
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.
Inventors
- 👤 William Richmond
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 18:08
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/0004; C12N9/0006; C12Q; C12Q1/00; C12Q1/26; C12Q1/60; Y; Y10; Y10S; Y10S210/00; Y10S210/918; Y10S210/922; Y10S435/00; Y10S435/814; Y10S435/815; Y10S435/8215; Y10S435/822; Y10S435/863; Y10S435/866; Y10S435/872; Y10S435/911; Y10S435/913; Y10S435/921; Y10S435/923; Y10S435/924; Y10S435/94; Y10S435/942
Claims Analysis
Total Claims: 52
Claim 1
Independent Claim
46 words
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.
Claim 2
Independent Claim
46 words
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.
Claim 3
Dependent Claim
16 words
A method according to claim 1 in which said liquid is serum or another biological fluid.
Claim 4
Independent Claim
87 words
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.
Claim 5
Independent Claim
87 words
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.
Claim 6
Dependent Claim
17 words
A method according to claim 1 and wherein said enzyme preparation is derived from a Nocardia species.
Claim 7
Dependent Claim
55 words
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.
Claim 8
Dependent Claim
50 words
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.
Claim 9
Dependent Claim
57 words
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.
Claim 10
Dependent Claim
16 words
A method according to claim 7 in which said liquid is serum or another biological fluid.
Claim 11
Dependent Claim
24 words
A method according to claim 8 in which saponified serum or other biological fluid is incubated with said enzyme preparation to measure total cholesterol.
Claim 12
Dependent Claim
42 words
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.
Claim 13
Dependent Claim
16 words
A method according to claim 3 carried out as an individual test on a single sample.
Claim 14
Dependent Claim
18 words
A method according to claim 3 in which a plurality of samples are tested on an automated basis.
Claim 15
Dependent Claim
92 words
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.
Claim 16
Dependent Claim
92 words
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.
Claim 17
Dependent Claim
32 words
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.
Claim 18
Dependent Claim
16 words
A kit according to claim 13 which includes in addition at least one standard cholesterol solution.
Claim 19
Dependent Claim
50 words
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.
Claim 20
Dependent Claim
50 words
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.
Claim 21
Dependent Claim
16 words
A Kit according to claim 16 wherein said enzyme preparation is derived from a Nocardia species.
Claim 22
Dependent Claim
25 words
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.
Claim 23
Dependent Claim
32 words
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.
Claim 24
Dependent Claim
43 words
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.
Claim 25
Dependent Claim
34 words
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.
Claim 26
Dependent Claim
20 words
A kit according to claim 17 which contains each component in an amount required for a defined number of tests.
Claim 27
Dependent Claim
31 words
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.
Claim 28
Dependent Claim
18 words
A kit according to claim 22 which contains a separate amount of enzyme component (a) for each test.
Claim 29
Dependent Claim
43 words
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.
Claim 30
Dependent Claim
33 words
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.
Claim 31
Independent Claim
64 words
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.
Claim 32
Independent Claim
64 words
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.
Claim 33
Dependent Claim
72 words
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.
Claim 34
Dependent Claim
72 words
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.
Claim 35
Dependent Claim
26 words
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.
Claim 36
Dependent Claim
26 words
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.
Claim 37
Dependent Claim
23 words
A method according to claim 1 wherein the enzyme preparation has a catalase activity of less than 10% of the cholesterol oxidase activity.
Claim 38
Dependent Claim
23 words
A kit according to claim 16 wherein the enzyme preparation has a catalase activity of less than 10% of the cholesterol oxidase activity.
Claim 39
Dependent Claim
23 words
A method according to claim 1 wherein the enzyme preparation has a catalase activity of less than 1% of the cholesterol oxidase activity.
Claim 40
Dependent Claim
23 words
A kit according to claim 16 wherein the enzyme preparation has a catalase activity of less than 1% of the cholesterol oxidase activity.
Claim 41
Dependent Claim
36 words
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.
Claim 42
Dependent Claim
36 words
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.
Claim 43
Dependent Claim
39 words
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.
Claim 44
Dependent Claim
37 words
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.
Claim 45
Dependent Claim
24 words
A method according to claim 2 in which saponified serum or other biological fluid is incubated with said enzyme preparation to measure total cholesterol.
Claim 46
Dependent Claim
54 words
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.
Claim 47
Dependent Claim
51 words
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.
Claim 48
Dependent Claim
25 words
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.
Claim 49
Dependent Claim
34 words
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.
Claim 50
Dependent Claim
34 words
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.
Claim 51
Dependent Claim
16 words
A kit according to claim 28 wherein said cholesterol oxidase is derived from a Nocardia species.
Claim 52
Dependent Claim
24 words
A kit according to claim 28 wherein said cholesterol oxidase is derived from Nocardia species NCIB 10554 (NRRL 5635) or NCIB 10555 (NRRL 5636).
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1978-09-22 | 1979-03-24 | 1979-09-23 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1982-09-22 | 1983-03-24 | 1983-09-23 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1986-09-22 | 1987-03-24 | 1987-09-23 | $7700.00 | Window Closed / Lapsed |