US4290774A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Purification of lipoprotein cholesterol for use as a cholesterol reference material
Assignee: EMD Millipore Corp
•
Filing: 1980-01-07
•
Granted: 1981-09-22
•
Expires: 2000-01-07
Abstract
Abstract A method for the purification of lipoprotein cholesterol. The method involves the steps of adsorbing lipoprotein material on a silica adsorbant, eluting the adsorbed lipoprotein with water at a pH of about 10 to 11.5, adjusting the salt concentration to about less than 0.05M, heating the lipoprotein to about 50 DEG to 100 DEG C. for a period of about 5 minutes to 24 hours, adding an alkaline carbonate and alkaline earth salt to form a precipitate and removing the precipitate, adjusting the pH from about 6.5 to 9.0 and recovering purified lipoprotein cholesterol. Also a method for using the cholesterol as a cholesterol reference material.
Inventors
- 👤 Makram M. Girgis
- 👤 David E. Jackson
- 👤 Frank J. Mannuzza
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-01 16:23
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/92; G01N33/96; Y; Y10; Y10T; Y10T436/00; Y10T436/10; Y10T436/104165
Claims Analysis
Total Claims: 15
Claim 1
Independent Claim
136 words
In a method for purifying lipoprotein cholesterol which involves the steps of adsorbing a solution of a lipoprotein-containing substance onto a silica adsorbant, separating the adsorbed lipoprotein from any excess solution present, freezing and thawing the adsorbed lipoprotein, eluting the adsorbed lipoprotein, concentrating the lipoprotein to a desired concentration and recovering purified lipoprotein cholesterol therefrom, the improvement comprising the steps of eluting the adsorbed lipoprotein at a pH from about 10 to 11.5, adjusting the salt concentration to about less than 0.05 M, heating the eluted lipoprotein to a temperature of from about 50° to 100° C. for a period of time sufficient to increase the storage stability of the purified lipoprotein cholesterol, adding an alkaline carbonate and an alkaline earth salt; adjusting the pH from about 6.5 to 9.0; and recovering therefrom said lipoprotein cholesterol.
Claim 2
Dependent Claim
136 words
In a method for purifying lipoprotein cholesterol which involves the steps of adsorbing a solution of a lipoprotein-containing substance onto a silica adsorbant, separating the adsorbed lipoprotein from any excess solution present, freezing and thawing the adsorbed lipoprotein, eluting the adsorbed lipoprotein, concentrating the lipoprotein to a desired concentration and recovering purified lipoprotein cholesterol therefrom, the improvement comprising the steps of eluting the adsorbed lipoprotein at a pH from about 10 to 11.5, adjusting the salt concentration to about less than 0.05 M, heating the eluted lipoprotein to a temperature of from about 50° to 100° C. for a period of time sufficient to increase the storage stability of the purified lipoprotein cholesterol, adding an alkaline carbonate and an alkaline earth salt; adjusting the pH from about 6.5 to 9.0; and recovering therefrom said lipoprotein cholesterol.
Claim 3
Dependent Claim
14 words
A method as claimed in claim 1 wherein the lipoprotein-containing substance is bovine lipoprotein.
Claim 4
Dependent Claim
21 words
A method as claimed in claim 2 wherein the pH is maintained from about 10.4 to 10.6 during the eluting step.
Claim 5
Dependent Claim
23 words
A method as claimed in claim 2 wherein the temperature is maintained at about 80° C. for a period of about 8 hours.
Claim 6
Dependent Claim
24 words
A method as claimed in claim 2 which additionally includes the steps of diluting the cholesterol with a protein-containing diluent and lyophilizing the cholesterol.
Claim 7
Dependent Claim
27 words
A method as claimed in claim 2 wherein from 0.1 to 1.0 M alkaline carbonate and from 1 mM to 100 mM alkaline earth salt are added.
Claim 8
Independent Claim
143 words
A method for purifying lipoprotein cholesterol which comprises the steps of: (a) adsorbing a solution of lipoprotein-containing substance onto a silica adsorbant; (b) separating the adsorbed lipoprotein from any excess solution present; (c) freezing and thawing the adsorbed lipoprotein; (d) eluting the adsorbed lipoprotein at a pH of from about 10 to 11.5; (e) concentrating the lipoprotein obtained from step (d) to a desired concentration of cholesterol; (f) adjusting the pH of the lipoprotein from about 7.0 to 10.0; (g) adjusting the salt concentration to about less than 0.05 M; (h) heating the lipoprotein to a temperature of from about 50° to 100° C. for a period of about 5 minutes to 24 hours; (i) adding an alkaline carbonate and an alkaline earth salt; (j) adjusting the pH of the lipoprotein from about 6.5 to 9.0; and (k) recovering therefrom purified lipoprotein cholesterol.
Claim 9
Independent Claim
143 words
A method for purifying lipoprotein cholesterol which comprises the steps of: (a) adsorbing a solution of lipoprotein-containing substance onto a silica adsorbant; (b) separating the adsorbed lipoprotein from any excess solution present; (c) freezing and thawing the adsorbed lipoprotein; (d) eluting the adsorbed lipoprotein at a pH of from about 10 to 11.5; (e) concentrating the lipoprotein obtained from step (d) to a desired concentration of cholesterol; (f) adjusting the pH of the lipoprotein from about 7.0 to 10.0; (g) adjusting the salt concentration to about less than 0.05 M; (h) heating the lipoprotein to a temperature of from about 50° to 100° C. for a period of about 5 minutes to 24 hours; (i) adding an alkaline carbonate and an alkaline earth salt; (j) adjusting the pH of the lipoprotein from about 6.5 to 9.0; and (k) recovering therefrom purified lipoprotein cholesterol.
Claim 10
Dependent Claim
14 words
A method as claimed in claim 7 wherein the lipoprotein-containing substance is bovine lipoprotein.
Claim 11
Dependent Claim
24 words
A method as claimed in claim 7 which additionally includes the steps of diluting the cholesterol with a protein-containing diluent and lyophilizing the cholesterol.
Claim 12
Dependent Claim
141 words
A purified lipoprotein cholesterol prepared by the steps of: (a) adsorbing a solution of lipoprotein-containing substance onto a silica adsorbant; (b) separating the adsorbed lipoprotein from any excess solution present; (c) freezing and thawing the adsorbed lipoprotein; (d) eluting the adsorbed lipoprotein at a pH of from about 10 to 11.5; (e) concentrating the lipoprotein obtained from step (d) to a desired concentration of cholesterol; (f) adjusting the pH of the cholesterol from about 7.0 to 10.0; (g) adjusting the salt concentration to about less than 0.05 M; (h) heating the cholesterol to a temperature of from about 50° to 100° C. for a period of about 5 minutes to 24 hours; (i) adding an alkaline carbonate and an alkaline earth salt; (j) adjusting the pH of the cholesterol from about 6.5 to 9.0; and (k) recovering therefrom purified lipoprotein cholesterol.
Claim 13
Dependent Claim
141 words
A purified lipoprotein cholesterol prepared by the steps of: (a) adsorbing a solution of lipoprotein-containing substance onto a silica adsorbant; (b) separating the adsorbed lipoprotein from any excess solution present; (c) freezing and thawing the adsorbed lipoprotein; (d) eluting the adsorbed lipoprotein at a pH of from about 10 to 11.5; (e) concentrating the lipoprotein obtained from step (d) to a desired concentration of cholesterol; (f) adjusting the pH of the cholesterol from about 7.0 to 10.0; (g) adjusting the salt concentration to about less than 0.05 M; (h) heating the cholesterol to a temperature of from about 50° to 100° C. for a period of about 5 minutes to 24 hours; (i) adding an alkaline carbonate and an alkaline earth salt; (j) adjusting the pH of the cholesterol from about 6.5 to 9.0; and (k) recovering therefrom purified lipoprotein cholesterol.
Claim 14
Independent Claim
175 words
A method of determining the cholesterol content of a test sample which includes the step of using as a cholesterol reference material a composition consisting essentially of purified lipoprotein cholesterol prepared by the steps of: (a) adsorbing a solution of lipoprotein-containing substance onto a silica adsorbant; (b) separating the adsorbed lipoprotein from any excess solution present; (c) freezing and thawing the adsorbed lipoprotein; (d) eluting the adsorbed lipoprotein at a pH of from about 10 to 11.5; (e) concentrating the lipoprotein obtained from step (d) to a desired concentration of cholesterol; (f) adjusting the pH of the cholesterol from about 7.0 to 10.0; (g) adjusting the salt concentration to about less than 0.05 M; (h) heating the cholesterol to a temperature of from about 50° to 100° C. for a period of about 5 minutes to 24 hours; (i) adding an alkaline carbonate and an alkaline earth salt to form a precipitate and removing the precipitate; (j) adjusting the pH of the cholesterol from about 6.5 to 9.0; and (k) recovering therefrom purified lipoprotein cholesterol.
Claim 15
Independent Claim
175 words
A method of determining the cholesterol content of a test sample which includes the step of using as a cholesterol reference material a composition consisting essentially of purified lipoprotein cholesterol prepared by the steps of: (a) adsorbing a solution of lipoprotein-containing substance onto a silica adsorbant; (b) separating the adsorbed lipoprotein from any excess solution present; (c) freezing and thawing the adsorbed lipoprotein; (d) eluting the adsorbed lipoprotein at a pH of from about 10 to 11.5; (e) concentrating the lipoprotein obtained from step (d) to a desired concentration of cholesterol; (f) adjusting the pH of the cholesterol from about 7.0 to 10.0; (g) adjusting the salt concentration to about less than 0.05 M; (h) heating the cholesterol to a temperature of from about 50° to 100° C. for a period of about 5 minutes to 24 hours; (i) adding an alkaline carbonate and an alkaline earth salt to form a precipitate and removing the precipitate; (j) adjusting the pH of the cholesterol from about 6.5 to 9.0; and (k) recovering therefrom purified lipoprotein cholesterol.
References Cited (Backward Citations)
US4096136A
Cited
US4110077A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1984-09-21 | 1985-03-23 | 1985-09-22 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1988-09-21 | 1989-03-23 | 1989-09-22 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1992-09-21 | 1993-03-23 | 1993-09-22 | $7700.00 | Window Closed / Lapsed |