US4215993A
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US
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Precipitating reagent and method for isolation and determination of high density lipoproteins in human serum
Assignee: Data Medical Associates Inc
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Filing: 1978-12-04
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Granted: 1980-08-05
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Expires: 1998-12-04
Abstract
Abstract A precipitating reagent and method is provided for isolating high density lipoproteins from low density lipoproteins in human serum together with a quantitative determination of high density lipoprotein cholesterol. Precipitation of low density lipoproteins is accomplished by the precipitating reagent without the addition of metal ions into the sample. The precipitating reagent lowers the pH of the human serum approximately to the isoelectric point of the low density lipoproteins through the use of an organic buffer. The precipitating reagent also contains a polyanion and neutral polymer. The preferred composition of the precipitating reagent contains about 0.4% phosphotungstic acid by weight thereof, about 2.5% of polyethylene glycol by weight thereof and 2-(N-morpholino) ethane sulfonic acid as the buffer present in a concentration of from about 0.2 molar to about 0.5 molar. According to the method provided, the precipitating reagent is added to the human serum sample thereby causing the low density lipoproteins to form a precipitate, leaving the high density lipoproteins in the resulting supernatant liquid. The supernatant is separated from the precipitate and a cholesterol assay reagent is added to the supernatant. The cholesterol assay reagent reacts with the high density lipoprotein to produce a compound that absorbs radiation at a specific wavelength. The amount of high density lipoprotein cholesterol present in the human serum sample is then determined by comparing the absorbance of a sample with the absorbance of a known standard.
Inventors
- 👤 James L. Sanders
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-02 21:17
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/60; G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/92; G01N2800/00; G01N2800/04; G01N2800/044
Claims Analysis
Total Claims: 30
Claim 1
Independent Claim
72 words
A precipitating reagent for selectively precipitating low density lipoproteins from human serum which comprises phosphotungstic acid, an organic buffer having a pH buffering range of from about 4.8-6.8 and a concentration of from about 0.2 molar to about 1.2 molar and a neutral polymer selected from the group consisting of polyvinylpyrollidone, polyethylene glycol and poly-N-vinyl-5-methyl-2-oxazolidinone, said phosphotungstic acid and said neutral polymer present in effective amounts for selectively precipitating low density lipoproteins.
Claim 2
Dependent Claim
72 words
A precipitating reagent for selectively precipitating low density lipoproteins from human serum which comprises phosphotungstic acid, an organic buffer having a pH buffering range of from about 4.8-6.8 and a concentration of from about 0.2 molar to about 1.2 molar and a neutral polymer selected from the group consisting of polyvinylpyrollidone, polyethylene glycol and poly-N-vinyl-5-methyl-2-oxazolidinone, said phosphotungstic acid and said neutral polymer present in effective amounts for selectively precipitating low density lipoproteins.
Claim 3
Dependent Claim
55 words
The precipitating reagent as recited in claim 1 wherein the polyvinylpyrollidone has a molecular weight in the range of from about 30,000 grams per mole to about 50,000 grams per mole and the polyethylene glycol has a molecular weight in the range of from about 5,000 grams per mole to about 6,500 grams per mole.
Claim 4
Dependent Claim
19 words
The precipitating reagent as recited in claim 1 wherein at least about 0.2% by weight thereof is phosphotungstic acid.
Claim 5
Dependent Claim
16 words
The precipitating reagent as recited in claim 3 wherein the buffer is 2-(N-morpholino) ethane sulfonic acid.
Claim 6
Dependent Claim
110 words
A precipitating reagent for selectively precipitating low density lipoproteins from human serum which comprises: (a) from about 0.2% to about 3.0% phosphotungstic acid by weight thereof; (b) a zwitterionic buffer having a useful pH buffering range of from about 5.2 to about 6.8 and a pKa of about 6.15 at 20° C. present in a concentration of from about 0.2 molar to about 1.2 molar; and (c) from about 2.0% to about 18.0% by weight thereof is a neutral polymer selected from the group consisting of polyvinylpyrollidone having a molecular weight of approximately 40,000 grams per mole and polyethylene glycol having a molecular weight of approximately 6,000 grams per mole.
Claim 7
Dependent Claim
110 words
A precipitating reagent for selectively precipitating low density lipoproteins from human serum which comprises: (a) from about 0.2% to about 3.0% phosphotungstic acid by weight thereof; (b) a zwitterionic buffer having a useful pH buffering range of from about 5.2 to about 6.8 and a pKa of about 6.15 at 20° C. present in a concentration of from about 0.2 molar to about 1.2 molar; and (c) from about 2.0% to about 18.0% by weight thereof is a neutral polymer selected from the group consisting of polyvinylpyrollidone having a molecular weight of approximately 40,000 grams per mole and polyethylene glycol having a molecular weight of approximately 6,000 grams per mole.
Claim 8
Dependent Claim
44 words
The precipitating reagent as recited in claim 5, wherein: (a) about 0.4% by weight thereof is phosphotungstic acid; (b) the zwitterionic buffer is 2-(N-morpholino) ethane sulfonic acid; and (c) the neutral polymer is polyvinylpyrollidone present in a concentration of about 6% by weight thereof.
Claim 9
Dependent Claim
45 words
The precipitating reagent as recited in claim 5, wherein: (a) about 0.4% by weight thereof is phosphotungstic acid; (b) the zwitterionic buffer is 2-(N-morpholino) ethane sulfonic acid; and (c) the neutral polymer is polyethylene glycol present in a concentration of about 2.5% by weight thereof.
Claim 10
Dependent Claim
12 words
The precipitating reagent as recited in claim 5 further comprising ethylenediaminetetraacetic acid.
Claim 11
Dependent Claim
53 words
The precipitating reagent as recited in claim 5 wherein: (a) about 2.4% by weight thereof is phosphotungstic acid; (b) the zwitterionic buffer is 2-(N-morpholino) ethane sulfonic acid present in a concentration of about 1.0 molar; and (c) the neutral polymer is polyethylene glycol present in a concentration of about 2.5% by weight thereof.
Claim 12
Dependent Claim
25 words
The precipitating reagent as recited in claim 9 further comprising ethylenediaminetetraacetic acid present in the range of from about 10 millimolar to about 60 millimolar.
Claim 13
Independent Claim
108 words
A method for precipitating low density lipoproteins and determining the amount of high density lipoprotein cholesterol in human serum without the addition of metal ions which comprises adjusting the pH of said serum approximately to the low density lipoprotein isoelectric point with an organic buffer, introducing phosphotungstic acid into said serum and introducing a neutral polymer into said serum, said phosphotungstic acid and said neutral polymer being introduced in said serum in effective amounts for precipitating low density lipoproteins thereby forming precipitate and supernatant fractions, said neutral polymer selected from the group consisting of polyvinylpyrollidone, polyethylene glycol and poly-N-vinyl-5-methyl-2-oxazolidinone, and analyzing the supernatant for high density lipoprotein cholesterol.
Claim 14
Independent Claim
108 words
A method for precipitating low density lipoproteins and determining the amount of high density lipoprotein cholesterol in human serum without the addition of metal ions which comprises adjusting the pH of said serum approximately to the low density lipoprotein isoelectric point with an organic buffer, introducing phosphotungstic acid into said serum and introducing a neutral polymer into said serum, said phosphotungstic acid and said neutral polymer being introduced in said serum in effective amounts for precipitating low density lipoproteins thereby forming precipitate and supernatant fractions, said neutral polymer selected from the group consisting of polyvinylpyrollidone, polyethylene glycol and poly-N-vinyl-5-methyl-2-oxazolidinone, and analyzing the supernatant for high density lipoprotein cholesterol.
Claim 15
Dependent Claim
96 words
The method as recited in claim 11 wherein: (a) the concentration of the organic buffer in the serum is from about 0.1 molar to about 0.25 molar; (b) the concentration of phosphotungstic acid in the serum is from about 0.1% to about 0.5% by weight thereof; (c) the concentration of neutral polymer in the serum is from about 1.0% to about 4.0% by weight thereof; and (d) said concentrations of organic buffer, phosphotungstic acid and neutral polymer being the concentrations in the serum after addition of said organic buffer, said phosphotungstic acid and said neutral polymer.
Claim 16
Dependent Claim
115 words
The method as recited in claim 11 wherein said analyzing comprises: (a) separating said supernatant fraction from said precipitate fraction; (b) adding to said supernatant fraction a cholesterol assay reagent containing cholesterol esterase, cholesterol oxidase, peroxidase, 4-aminoantipyrine and phenol to form a resulting mixture; (c) incubating said resulting mixture; (d) determining the absorbance of said incubated resulting mixture at about 500 nm; (e) determining the absorbance at about 500 nm at a calibrated reference having a known concentration of cholesterol; (f) calculating the high density lipoprotein cholesterol concentration of the resulting mixture by multiplying the absorbance of the resulting mixture over the absorbance of the calibrated reference times the cholesterol concentration of the calibrated reference.
Claim 17
Dependent Claim
20 words
The method as recited in claim 13 wherein the separation of said supernatant fraction from said precipitate fraction includes centrifugation.
Claim 18
Dependent Claim
38 words
The method as recited in claim 13 wherein said cholesterol assay reagent added to said supernatant fraction contains about 0.36 U/cholesterol esterase, about 0.4 U/cholesterol oxidase, about 100 U/oxidase, about 0.4 milligrams 4-aminoantipyrine and about 6.0 milligrams phenol.
Claim 19
Dependent Claim
21 words
The method as recited in claim 13 wherein said resulting mixture is incubated at about 37° C. for about 15 minutes.
Claim 20
Independent Claim
111 words
A method for the determination of high density lipoprotein components in human serum selected from the group consisting of triglycerides, phospholipids, apo-proteins A, B and C and cholesterol which comprises: (a) adjusting the pH of said serum approximately to the low density lipoprotein isoelectric point with an organic buffer; (b) introducing phosphotungstic acid into said serum; (c) introducing a neutral polymer into said serum selected from the group consisting of polyvinylpyrollidone, polyethylene glycol and poly-N-vinyl-5-methyl-2-oxazolidinone, said phosphotungstic acid and said neutral polymer present in effective amounts for precipitating low density lipoproteins and forming precipitate and supernatant fractions; and (d) assaying the supernatant fraction for the specific high density lipoprotein component desired.
Claim 21
Independent Claim
111 words
A method for the determination of high density lipoprotein components in human serum selected from the group consisting of triglycerides, phospholipids, apo-proteins A, B and C and cholesterol which comprises: (a) adjusting the pH of said serum approximately to the low density lipoprotein isoelectric point with an organic buffer; (b) introducing phosphotungstic acid into said serum; (c) introducing a neutral polymer into said serum selected from the group consisting of polyvinylpyrollidone, polyethylene glycol and poly-N-vinyl-5-methyl-2-oxazolidinone, said phosphotungstic acid and said neutral polymer present in effective amounts for precipitating low density lipoproteins and forming precipitate and supernatant fractions; and (d) assaying the supernatant fraction for the specific high density lipoprotein component desired.
Claim 22
Dependent Claim
97 words
The method as recited in claim 17 wherein: (a) the concentration of the organic buffer in the serum is from about 0.1 molar to about 0.25 molar; (b) the concentration of phosphotungstic acid in the serum is from about 0.1% to about 0.5% by weight thereof; (c) the concentration of neutral polymer in the serum is from about 1% to about 4% by weight thereof; and (d) said concentrations of organic buffer, phosphotungstic acid and a neutral polymer being the concentrations in the serum after addition of said organic buffer, said phosphotungstic acid and said neutral polymer.
Claim 23
Independent Claim
196 words
A method for precipitating low density lipoproteins without the addition of metal ions which comprises introducing a reagent into said human serum, said reagent comprising: (a) from about 0.2% to about 3.0% phosphotungstic acid by weight of said reagent; (b) a zwitterionic buffer having a useful pH buffering range of from about 5.2 to about 6.8, pKa of about 6.15 at 20° C. present in a concentration in said reagent of from about 0.2 molar to about 1.2 molar; (c) from about 2.0% to about 18.0% by weight of said reagent of a neutral polymer selected from the group consisting of polyvinylpyrollidone having a molecular weight of about 40,000 and polyethylene glycol having a molecular weight of about 6,000; and (d) optionally from about 3.2 millimolar to about 60 millimolar ethylenediaminetetraacetic acid, said reagent being introduced into said human serum so that concentration of the reagent components in the combined reagent and human serum is from about 0.1% to about 0.5% by weight phosphotungstic acid; from about 1.0% to about 4.0% by weight neutral polymer; from about 0.1 molar to about 0.25 molar organic buffer; and from about 1.6 millimolar to about 10 millimolar ethylenediaminetetraacetic acid.
Claim 24
Independent Claim
196 words
A method for precipitating low density lipoproteins without the addition of metal ions which comprises introducing a reagent into said human serum, said reagent comprising: (a) from about 0.2% to about 3.0% phosphotungstic acid by weight of said reagent; (b) a zwitterionic buffer having a useful pH buffering range of from about 5.2 to about 6.8, pKa of about 6.15 at 20° C. present in a concentration in said reagent of from about 0.2 molar to about 1.2 molar; (c) from about 2.0% to about 18.0% by weight of said reagent of a neutral polymer selected from the group consisting of polyvinylpyrollidone having a molecular weight of about 40,000 and polyethylene glycol having a molecular weight of about 6,000; and (d) optionally from about 3.2 millimolar to about 60 millimolar ethylenediaminetetraacetic acid, said reagent being introduced into said human serum so that concentration of the reagent components in the combined reagent and human serum is from about 0.1% to about 0.5% by weight phosphotungstic acid; from about 1.0% to about 4.0% by weight neutral polymer; from about 0.1 molar to about 0.25 molar organic buffer; and from about 1.6 millimolar to about 10 millimolar ethylenediaminetetraacetic acid.
Claim 25
Dependent Claim
60 words
The method as recited in claim 19 wherein: (a) the precipitating reagent contains about 0.4% phosphotungstic acid by weight thereof; (b) the zwitterionic buffer is 2-N-morpholino ethane sulfonic acid; and (c) the neutral polymer present in the precipitating reagent is polyethylene glycol having a molecular weight of about 6,000 and present in a concentration of about 2.5% by weight thereof.
Claim 26
Dependent Claim
32 words
The method as recited in claim 19 wherein the neutral polymer is polyvinylpyrollidone having a molecular weight of about 40,000 and is present in a concentration of about 6% by weight thereof.
Claim 27
Dependent Claim
20 words
The method as recited in claim 19 wherein equal volumes of said human serum and said precipitating reagents are utilized.
Claim 28
Independent Claim
154 words
A method for the determination of high density lipoprotein components in human serum selected from the group consisting of triglycerides, phospholipids, apoproteins A, B and C and cholesterol which comprises: introducing a reagent into said human serum, which reagent comprises from about 0.2% to about 3.0% phosphotungstic acid by weight of said reagent, a zwitterionic buffer having a useful pH buffering range of from about 5.2 to about 6.8, a pKa of about 6.15 at 20° C. and present in a concentration of from about 0.2 molar to about 1.2 molar and from about 2.0% to about 18.0% by weight of said reagent of a neutral polymer selected from the group consisting of polyvinylpyrollidone having a molecular weight of about 40,000 and polyethylene glycol having a molecular weight of about 6,000, said reagent being introduced into said serum so that the pH of said serum is approximately at the low density lipoprotein isoelectric point.
Claim 29
Independent Claim
154 words
A method for the determination of high density lipoprotein components in human serum selected from the group consisting of triglycerides, phospholipids, apoproteins A, B and C and cholesterol which comprises: introducing a reagent into said human serum, which reagent comprises from about 0.2% to about 3.0% phosphotungstic acid by weight of said reagent, a zwitterionic buffer having a useful pH buffering range of from about 5.2 to about 6.8, a pKa of about 6.15 at 20° C. and present in a concentration of from about 0.2 molar to about 1.2 molar and from about 2.0% to about 18.0% by weight of said reagent of a neutral polymer selected from the group consisting of polyvinylpyrollidone having a molecular weight of about 40,000 and polyethylene glycol having a molecular weight of about 6,000, said reagent being introduced into said serum so that the pH of said serum is approximately at the low density lipoprotein isoelectric point.
Claim 30
Dependent Claim
42 words
The method as recited in claim 23 further comprising the addition to the serum of ethylenediaminetetraacetic acid resulting in a concentration in the combined serum and reagent between about 1.6 millimolar and 10 millimolar, said ethylenediaminetetraacedic acid being contained in said reagent.
References Cited (Backward Citations)
US3770631A
Cited
DE2265121A1
Cited
US3955925A
Cited
US4086218A
Cited
US4110077A
Cited
US4126416A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1983-08-05 | 1984-02-04 | 1984-08-05 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1987-08-05 | 1988-02-04 | 1988-08-05 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1991-08-05 | 1992-02-04 | 1992-08-05 | $7700.00 | Window Closed / Lapsed |