US3884764A
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US
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Method and composition for blood serum cholesterol analysis
Assignee: Eastman Kodak Co
•
Filing: 1974-03-25
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Granted: 1975-05-20
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Expires: 1994-03-25
Abstract
Abstract A totally enzymatic method for the quantitative analysis of cholesterol in a complex mixture containing free cholesterol and cholesterol esters comprising the steps of: 1. CONTACTING A SAMPLE TO BE TESTED WITH A NOVEL BUFFERED AQUEOUS TEST SOLUTION COMPRISING: A. A LIPASE HAVING CHOLESTEROL ESTERASE ACTIVITY; B. A PROTEASE; AND C. CHOLESTEROL OXIDASE; AND 2. MEASURING THE QUANTITY OF ONE OF THE PRODUCTS OF THE OXIDATION OF CHOLESTEROL TO DETERMINE THE TOTAL AMOUNT OF CHOLESTEROL IN THE SAMPLE UNDER EVALUATION. Optionally, an indicator system for quantitatively detecting one or more of the cholesterol oxidation products, for example, peroxidase and a color indicator system which undergoes a color change in the presence of H2O2 and peroxidase may be included in the aqueous test solution. Quantification is preferably achieved using fluorometric or spectrophotometric techniques. According to a preferred embodiment the buffers aqueous test solution includes a surfactant. A dry mixture of lyophilized enzymes which need only be dissolved in an accurate amount of water to provide an assay solution and element comprising a porous substrate into which the solution has been embibed and dried are also described.
Inventors
- 👤 Charles T Goodhue
- 👤 Hugh A Risley
- 👤 Roy E Snoke
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 14:25
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/60; Y; Y10; Y10S; Y10S435/00; Y10S435/8215; Y10S435/911; Y10S435/921
Claims Analysis
Total Claims: 93
Claim 1
Independent Claim
45 words
AN AQUEOUS TEST SOLUTION USEFUL IN THE QUANTITATIVE ASSAY OF A SAMPLE FOR TOTAL CHOLESTEROL CONTENT COMPRISING: A. A LIPASE HAVING CHOLESTEROL ESTERASE ACTIVITY; B. A PROTEASE; AND C. CHOLESTEROL OXIDASE SAID TEST SOLUTION BEING BUFFERED TO A PH OF BETWEEN ABOUT 5.5 AND 8.5.
Claim 2
Dependent Claim
45 words
AN AQUEOUS TEST SOLUTION USEFUL IN THE QUANTITATIVE ASSAY OF A SAMPLE FOR TOTAL CHOLESTEROL CONTENT COMPRISING: A. A LIPASE HAVING CHOLESTEROL ESTERASE ACTIVITY; B. A PROTEASE; AND C. CHOLESTEROL OXIDASE SAID TEST SOLUTION BEING BUFFERED TO A PH OF BETWEEN ABOUT 5.5 AND 8.5.
Claim 3
Dependent Claim
43 words
An aqueous test solution as described in claim 1 wherein said choesterol oxidase is selected from the group consisting of cholesterol oxidase enzymes produced by a bacterial strain selected from the group consisting of NRRL 5767, NRRL 5768, NRRL 5635 and NRRL 5636.
Claim 4
Dependent Claim
30 words
measuring the quantity of at least one of the products of the cholesterol induced decomposition of cholesterol to determine the total amount of free and esterified cholesterol in the sample.
Claim 5
Dependent Claim
30 words
measuring the quantity of at least one of the products of the cholesterol induced decomposition of cholesterol to determine the total amount of free and esterified cholesterol in the sample.
Claim 6
Dependent Claim
39 words
An aqueous test solution as described in claim 1 wherein said cholesterol oxidase is selected from the group consisting of cholesterol oxidase enzymes produced by a bacterial strain selected from the group consisting of NRRL 5635 and NRRL 5636.
Claim 7
Dependent Claim
14 words
An aqueous test solution as described in claim 1 which also includes a surfactant.
Claim 8
Dependent Claim
14 words
An aqueous test solution as described in claim 3 which also includes a surfactant.
Claim 9
Dependent Claim
29 words
An aqueous test solution as described in claim 4 wherein said surfactant is selected from the group consisting of deoxycholate, octyl phenoxy polyethoxy ethanols, (p-nonylphenoxy) glycerols and polyethylene glycols.
Claim 10
Dependent Claim
14 words
An aqueous test solution as described in claim 4 wherein said surfactant is nonionic.
Claim 11
Dependent Claim
14 words
An aqueous test solution as described in claim 5 wherein said surfactant is nonionic.
Claim 12
Dependent Claim
24 words
An aqueous test solution as described in claim 4 wherein the concentration of said surfactant ranges from about 0.05 to about 0.5% by weight.
Claim 13
Dependent Claim
24 words
An aqueous test solution as described in claim 5 wherein the concentration of said surfactant ranges from about 0.05 to about 0.5% by weight.
Claim 14
Dependent Claim
75 words
An aqueous test solution as described in claim 5 wherein said lipase is from a preparation 50 mg of which hydrolyzes at least about 25 mg% of the cholesteryl linoleate present in 5 ml of a standard composition comprising 200 mg of cholesteryl linoleate, 5 ml ethyl ether, 100 ml water, and 430 mg sodium cholate buffered to a pH of 7.0 with 0.1 M phosphate during incubation for 2 hours at 37*C under nitrogen.
Claim 15
Dependent Claim
16 words
An aqueous test solution as described in claim 4 wherein said lipase is a microbial lipase.
Claim 16
Dependent Claim
16 words
An aqueous test solution as described in claim 12 wherein said lipase is from Candida cylindracca.
Claim 17
Dependent Claim
16 words
An aqueous test solution as described in claim 5 wherein said lipase is from Candida cylindracca.
Claim 18
Dependent Claim
30 words
An aqueous test solution as described in claim 4 wherein said lipase is selected from the group consisting of wheat germ lipase, pancreatic lipase and the lipase from Candida cylindracca.
Claim 19
Dependent Claim
27 words
An aqueous test solution as described in claim 15 wherein the concentration of said lipase ranges from about 10 to about 50 units per ml of solution.
Claim 20
Dependent Claim
28 words
An aqueous test solution as described in claim 4 wherein the concentration of said cholesterol oxidase ranges from about 0.002 to about 0.05 units per ml of solution.
Claim 21
Dependent Claim
33 words
An aqueous test solution as described in claim 4 wherein said protease is selected from the group consisting of chymotrypsin, papain, bromelain, Streptomyces griseus protease, elastase, Bacillus subtilis protease and Aspergillus oryzae protease.
Claim 22
Dependent Claim
33 words
An aqueous test solution as described in claim 8 wherein said protease is selected from the group consisting of chymotrypsin, papain, bromelain, Streptomyces griseus protease, elastase, Bacillus subtilis protease and Aspergillus oryzae protease.
Claim 23
Dependent Claim
33 words
An aqueous test solution as described in claim 15 wherein said protease is selected from the group consisting of chymotrypsin, papain, bromelain, Streptomyces griseus protease, elastase, Bacillus subtilis protease and Aspergillus oryzae protease.
Claim 24
Dependent Claim
33 words
An aqueous test solution as described in claim 13 wherein said protease is selected from the group consisting of chymotrypsin, papain, bromelain, Streptomyces griseus protease, elastase, Bacillus subtilis protease and Aspergillus oryzae protease.
Claim 25
Dependent Claim
25 words
An aqueous test solution as described in claim 4 wherein the concentration of said protease ranges from about 0.5 to about 2.0 units per ml.
Claim 26
Dependent Claim
27 words
An aqueous test solution as described in claim 4 wherein potassium phosphate is used to buffer the solution to a pH of between about 6.5 and 7.0.
Claim 27
Dependent Claim
25 words
An aqueous test soluTion as described in claim 4 further comprising an indicator system for quantitatively detecting at least one of the cholesterol oxidation products.
Claim 28
Dependent Claim
33 words
An aqueous test solution as described in claim 8 wherein said indicator system comprises peroxidase and a color indicator system which undergoes a color change in the presence of peroxidase and hydrogen peroxide.
Claim 29
Dependent Claim
34 words
An aqueous test solution as described in claim 25 wherein said color indicator system comprises a leuco dye which undergoes a detectable color change upon oxidation under the influence of peroxidase and hydrogen peroxide.
Claim 30
Dependent Claim
55 words
An aqueous test solution as described in claim 25 wherein said color indicator system comprises at least one substance which undergoes no substantial color change upon oxidation in the presence of hydrogen peroxide and peroxidase, but which in its oxidized form reacts with a coupler eto cause a quantifable energy absorption shift or color change.
Claim 31
Dependent Claim
25 words
An aqueous test solution as described in claim 12 wherein said substance which undergoes no substantial color change is 4-amino-antipyrine and said coupler is 1,7-dihydroxynaphthalene.
Claim 32
Dependent Claim
34 words
An aqueous test solution as described in claim 27 wherein said substance which undergoes no substantial color change upon oxidation and said coupler are a single compound which undergoes autocoupling in its oxidized state.
Claim 33
Dependent Claim
15 words
An aqueous test solution as described in claim 14 wherein said single compound is 4-methoxy-1-naphthalene.
Claim 34
Dependent Claim
25 words
An aqueous test solution as described in claim 24 which is buffered to a pH of between about 6.5 and 7.0 with a phosphate buffer.
Claim 35
Dependent Claim
21 words
An aqueous test solution as described in claim 25 comprising from about 3 to about 6 units of peroxidase per ml.
Claim 36
Dependent Claim
89 words
An aqueous test solution useful in the quantitative assay of a sample for total cholesterol content comprising: a. from about 0.002 to about 0.5 units of a cholesterol oxidase per ml; b. from about 10 to about 50 units of a lipase having cholesterol esterase activity per ml; c. from about 0.5 to about 2.0 units of a protease per ml; and d. from about .05 to about 0.5 percent by weight of a surfactant, said test solution being buffered to a pH of between about 5.5 and 8.5.
Claim 37
Dependent Claim
89 words
An aqueous test solution useful in the quantitative assay of a sample for total cholesterol content comprising: a. from about 0.002 to about 0.5 units of a cholesterol oxidase per ml; b. from about 10 to about 50 units of a lipase having cholesterol esterase activity per ml; c. from about 0.5 to about 2.0 units of a protease per ml; and d. from about .05 to about 0.5 percent by weight of a surfactant, said test solution being buffered to a pH of between about 5.5 and 8.5.
Claim 38
Dependent Claim
39 words
An aqueous test solution as described in claim 33 wherein said cholesterol oxidase is selected from the group consisting of cholesterol oxidase enzymes produced by a bacterial strain selected from the group consisting of NRRL 5635 and NRRL 5636.
Claim 39
Dependent Claim
29 words
An aqueous test solution as described in claim 33 wherein said surfactant is selected from the group consisting of deoxycholate, octyl phenoxy polyethoxy ethanols, (p-nonylphenoxy) glycerols and polyethylene glycols.
Claim 40
Dependent Claim
24 words
An aqueous test solution as described in claim 33 wherein the concentration of said surfactant ranges from about 0.05 to about 0.5% by weight.
Claim 41
Dependent Claim
16 words
An aqueous test solution as described in claim 33 wherein said lipase is from Candida cylindracca.
Claim 42
Dependent Claim
16 words
An aqueous test solution as described in claim 37 wherein said lipase is from Candida cylindracca.
Claim 43
Dependent Claim
33 words
An aqueous test solution as described in claim 33 wherein said protease is selected from the group consisting of chymotrypsin, papain, bromelain, Streptomyces griseus protease, elastase, Bacillus subtilis protease and Aspergillus oryzae protease.
Claim 44
Dependent Claim
16 words
An aqueous test solution as described in claim 38 wherein said protease is from Bacillus subtilis.
Claim 45
Dependent Claim
27 words
An aqueous test solution as described in claim 33 wherein potassium phosphate is used to buffer the solution to a pH of between about 6.5 and 7.0.
Claim 46
Dependent Claim
25 words
An aqueous test solution as described in claim 33 further comprising an indicator system for quantitatively detecting at least one of the cholesterol oxidation products.
Claim 47
Dependent Claim
33 words
An aqueous test solution as described in claim 42 wherein said indicator system compriSes peroxidase and a color indicator system which undergoes a color change in the presence of peroxidase and hydrogen peroxide.
Claim 48
Dependent Claim
55 words
An aqueous test solution as described in claim 43 wherein said color indicator system comprises at least one substance which undergoes no substantial color change upon oxidation in the presence of hydrogen peroxide and peroxidase, but which in its oxidized form reacts with a coupler to cause a quantifable energy absorption shift or color change.
Claim 49
Dependent Claim
25 words
An aqueous test solution as described in claim 44 wherein said substance which undergoes no substantial color change is 4-aminoantipyrine and said coupler is 1,7-dihydroxynaphthalene.
Claim 50
Dependent Claim
34 words
An aqueous test solution as described in claim 45 wherein said substance which undergoes no substantial color change upon oxidation and said coupler are a single compound which undergoes autocoupling in its oxidized state.
Claim 51
Dependent Claim
15 words
An aqueous test solution as described in claim 46 wherein said single compound is 4-methoxy-1-naphthalene.
Claim 52
Dependent Claim
25 words
An aqueous test solution as described in claim 47 which is buffered to a pH of between about 6.5 and 7.0 with a phosphate buffer.
Claim 53
Dependent Claim
21 words
An aqueous test solution as described in claim 48 comprising from about 3 to about 6 units of peroxidase per ml.
Claim 54
Dependent Claim
23 words
A totally enzymatic method for the quantitative analysis of cholesterol in a complex mixture of cholesterol and cholesterol esters comprising the steps of:
Claim 55
Dependent Claim
23 words
A totally enzymatic method for the quantitative analysis of cholesterol in a complex mixture of cholesterol and cholesterol esters comprising the steps of:
Claim 56
Dependent Claim
35 words
A method as described in claim 50 wherein said sample is contacted with said test solution for a period of at least about 10 minutes at a temperature of between about 25*C and about 50*C.
Claim 57
Dependent Claim
39 words
An aqueous test solution as described in claim 51 wherein said cholesterol oxidase is selected from the group consisting of cholesterol oxidase enzymes produced by a bacterial strain selected from the group consisting of NRRL 5635 and NRRL 5636.
Claim 58
Dependent Claim
29 words
An aqueous test solution as described in claim 52 wherein said surfactant is selected from the group consisting of deoxycholate, octyl phenoxy polyethoxy ethanols, (p-nonylphenoxy) glycerols and polyethylene glycols.
Claim 59
Dependent Claim
24 words
An aqueous test solution as described in claim 52 wherein the concentration of said surfactant ranges from about 0.05 to about 0.5% by weight.
Claim 60
Dependent Claim
16 words
An aqueous test solution as described in claim 52 wherein said lipase is from Candida cylindracca.
Claim 61
Dependent Claim
16 words
An aqueous test solution as described in claim 52 wherein said lipase is from Candida cylindracca.
Claim 62
Dependent Claim
33 words
An aqueous test solution as described in claim 52 wherein said protease is selected from the group consisting of chymotrypsin, papain, bromelain, Streptomyces griseus protease, elastase, Bacillus subtilis protease and Aspergillus oryzae protease.
Claim 63
Dependent Claim
16 words
An aqueous test solution as described in claim 52 wherein said protease is from Bacillus subtilis.
Claim 64
Dependent Claim
27 words
An aqueous test solution as described in claim 52 wherein potassium phosphate is used to buffer the solution to a PH of between about 6.5 and 7.0.
Claim 65
Dependent Claim
25 words
An aqueous test solution as described in claim 52 further comprising an indicator system for quantitatively detecting at least one of the cholesterol oxidation products.
Claim 66
Dependent Claim
33 words
An aqueous test solution as described in claim 52 wherein said indicator system comprises peroxidase and a color indicator system which undergoes a color change in the presence of peroxidase and hydrogen peroxide.
Claim 67
Dependent Claim
55 words
An aqueous test solution as described in claim 52 wherein said color indicator system comprises at least one substance which undergoes no substantial color change upon oxidation in the presence of hydrogen peroxide and peroxidase, but which in its oxidized form reacts with a coupler to cause a quantizable energy absorption shift or color change.
Claim 68
Dependent Claim
25 words
An aqueous test solution as described in claim 62 wherein said substance which undergoes no substantial color change is 4-aminoantipyrine and said coupler is 1,7-hydroxy-naphthalene.
Claim 69
Dependent Claim
34 words
An aqueous test solution as described in claim 62 wherein said substance which undergoes no substantial color change upon oxidation and said coupler are a single compound which undergoes autocoupling in its oxidized state.
Claim 70
Dependent Claim
15 words
An aqueous test solution as described in claim 62 wherein said single compound is 4-methoxy-1-naphthalene.
Claim 71
Dependent Claim
25 words
An aqueous test solution as described in claim 52 which is buffered to a pH of between about 6.5 and 7.0 with a phosphate buffer.
Claim 72
Dependent Claim
21 words
An aqueous test solution as described in claim 52 comprising from about 3 to about 6 units of peroxidase per ml.
Claim 73
Dependent Claim
109 words
A composition of matter comprising in dry form sufficient cholesterol oxidase, lipase and protease to provide when dissolved in water an aqueous test solution useful in the quantitative assay of a sample for total cholesterol content comprising: a. from about 0.002 to about 0.5 units of a cholesterol oxidase per ml; b. from about 10 to about 50 units of a lipase having cholesterol esterase activity per ml; c. from about 0.5 to about 2.0 units of a protease per ml; and d. from about .05 to about 0.5 percent by weight of a surfactant, said test solution being buffered to a pH of between about 5.5 and 8.5.
Claim 74
Dependent Claim
109 words
A composition of matter comprising in dry form sufficient cholesterol oxidase, lipase and protease to provide when dissolved in water an aqueous test solution useful in the quantitative assay of a sample for total cholesterol content comprising: a. from about 0.002 to about 0.5 units of a cholesterol oxidase per ml; b. from about 10 to about 50 units of a lipase having cholesterol esterase activity per ml; c. from about 0.5 to about 2.0 units of a protease per ml; and d. from about .05 to about 0.5 percent by weight of a surfactant, said test solution being buffered to a pH of between about 5.5 and 8.5.
Claim 75
Dependent Claim
24 words
The composition of claim 68 further comprising sufficient buffer to buffer said aqueous test solution to a pH of between about 5.5 and 8.5.
Claim 76
Dependent Claim
37 words
A composition as described in claim 68 wherein said cholesterol oxidase is selected from the group consisting of cholesterol oxidase enzymes produced by a bacterial strain selected from the group consisting of NRRL 5635 and NRRL 5636.
Claim 77
Dependent Claim
27 words
A composition as described in claim 68 wherein said surfactant is selected from the group consisting of deoxycholate, octyl phenoxy polyethoxy ethanols, (p-nonylphenoxy) glycerols and polyethylene glycols.
Claim 78
Dependent Claim
22 words
A composition as described in claim 68 wherein the concentration of said surfactant ranges from about 0.05 to about 0.5% by weight.
Claim 79
Dependent Claim
14 words
A composition as described in claim 68 wherein said lipase is from Candida cylindracca.
Claim 80
Dependent Claim
14 words
A composition as described in claim 73 wherein said lipase is from Candida cylindracca.
Claim 81
Dependent Claim
31 words
A composition as described in claim 68 wherein said protease is selected from the group consisting of chymotrypsin, papain, bromelain, Streptomyces griseus protease, elastase, Bacillus subtilis protease and Aspergillus oryzae protease.
Claim 82
Dependent Claim
14 words
A composition as described in claim 75 wherein said protease is from Bacillus subtilis.
Claim 83
Dependent Claim
25 words
A composition as described in claim 68 wherein potassium phosphate is used to buffer the solution to a pH of between about 6.5 and 7.0.
Claim 84
Dependent Claim
23 words
A composition as described in claim 68 further comprising an indicator system for quantitatively detecting at least one of the cholesterol oxidation products.
Claim 85
Dependent Claim
31 words
A composition as described in claim 78 whereiN said indicator system comprises peroxidase and a color indicator system which undergoes a color change in the presence of peroxidase and hydrogen peroxide.
Claim 86
Dependent Claim
53 words
A composition as described in claim 79 wherein said color indicator system comprises at least one substance which undergoes no substantial color change upon oxidation in the presence of hydrogen peroxide and peroxidase, but which in its oxidized form reacts with a coupler to cause a quantizable energy absorption shift or color change.
Claim 87
Dependent Claim
22 words
A composition as described in claim 80 wherein said substance which undergoes no substantial color change is 4-aminoantipyrine and said coupler 1,7-dihydroxy-naphthalene.
Claim 88
Dependent Claim
32 words
A composition as described in claim 81 wherein said substance which undergoes no substantial color change upon oxidation and said coupler are a single compound which undergoes autocoupling in its oxidized state.
Claim 89
Dependent Claim
13 words
A composition as described in claim 82 wherein said single compound is 4-methoxy-1-naphthalene.
Claim 90
Dependent Claim
23 words
A composition as described in claim 83 which is buffered to a pH of between about 6.5 and 7.0 with a phosphate buffer.
Claim 91
Dependent Claim
19 words
A composition as described in claim 84 comprising from about 3 to about 6 units of peroxidase per ml.
Claim 92
Dependent Claim
116 words
An analytical element useful in the quantitative analysis of complex solutions for cholesterol content comprising a bibulous substate impregnated with the dried residue of a solution comprising an aqueous test solution useful in the quantitative assay of a sample for total cholesterol content comprising: a. from about 0.002 to about 0.5 units of a cholesterol oxidase per ml; b. from about 10 to about 50 units of a lipase having cholesterol esterase activity per ml; c. from about 0.5 to about 2.0 units of a protease per ml; and d. from about .05 to about 0.5 percent by weight of a surfactant, said test solution being buffered to a pH of between about 5.5 and 8.5.
Claim 93
Dependent Claim
116 words
An analytical element useful in the quantitative analysis of complex solutions for cholesterol content comprising a bibulous substate impregnated with the dried residue of a solution comprising an aqueous test solution useful in the quantitative assay of a sample for total cholesterol content comprising: a. from about 0.002 to about 0.5 units of a cholesterol oxidase per ml; b. from about 10 to about 50 units of a lipase having cholesterol esterase activity per ml; c. from about 0.5 to about 2.0 units of a protease per ml; and d. from about .05 to about 0.5 percent by weight of a surfactant, said test solution being buffered to a pH of between about 5.5 and 8.5.
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) | 1978-05-19 | 1978-11-18 | 1979-05-20 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1982-05-19 | 1982-11-18 | 1983-05-20 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1986-05-19 | 1986-11-18 | 1987-05-20 | $7700.00 | Window Closed / Lapsed |