Patent US4311665A successfully refreshed from Google Patents.
US4311665A
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Separation and isolation of conjugated and unconjugated bilirubin
Assignee: Clinical Diagnostic Systems Inc
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Filing: 1979-12-10
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Granted: 1982-01-19
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Expires: 1999-12-10
Abstract
Abstract A method is described for the separation and isolation of conjugated and unconjugated bilirubin from an aqueous liquid containing one or both of the bilirubin components. The aqueous liquid is contacted together with an interactive mordant for bilirubin to mordant bilirubin, the mordanted bilirubin is separated from the aqueous liquid, and the mordanted bilirubin is treated in an aqueous medium with a chaotropic agent at least slightly soluble in the medium to release the conjugated and/or unconjugated bilirubin from the mordant. A preferred embodiment also provides for selectively separating conjugated bilirubin from unconjugated bilirubin by selectively solubilizing either the conjugated or unconjugated bilirubin released from the mordant. Isolated conjugated bilirubin is thereby obtained having a purity in excess of 75%. A reference composition comprising the isolated conjugated bilirubin is also disclosed.
Inventors
- 👤 Tai-Wing Wu
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-30 11:19
CPC Classifications
C; C07; C07D; C07D207/00; C07D207/02; C07D207/44; C07D207/444; C07H; C07H7/00; C07H7/02; C07H7/033; C09; C09B; C09B23/00; C09B23/02; C09B23/04; C09B67/00; C09B67/0096; G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/72; G01N33/728; Y; Y10; Y10T; Y10T436/00; Y10T436/14; Y10T436/145555; Y10T436/146666; Y10T436/25; Y10T436/25375
Claims Analysis
Total Claims: 27
Claim 1
Independent Claim
145 words
A method for the separation and isolation of conjugated bilirubin (B c ) and unconjugated bilirubin (B u ) from an aqueous liquid containing B c , B u , or a mixture of B c and B u , said method comprising: (a) contacting together said liquid and an interactive mordant having binding sites for bilirubin to mordant said B c , B u , or mixtures of B c and B u , said mordant comprising at least one moiety having a hydrophobic organic matrix containing a charge-bearing cationic group; (b) separating the mordanted B c and/or B u of step (a) from said liquid; and (c) treating the mordanted B c and/or B u of step (b) in an aqueous medium with a chaotropic agent at least slightly soluble in said medium to release B c and/or B u from said mordant.
Claim 2
Independent Claim
145 words
A method for the separation and isolation of conjugated bilirubin (B c ) and unconjugated bilirubin (B u ) from an aqueous liquid containing B c , B u , or a mixture of B c and B u , said method comprising: (a) contacting together said liquid and an interactive mordant having binding sites for bilirubin to mordant said B c , B u , or mixtures of B c and B u , said mordant comprising at least one moiety having a hydrophobic organic matrix containing a charge-bearing cationic group; (b) separating the mordanted B c and/or B u of step (a) from said liquid; and (c) treating the mordanted B c and/or B u of step (b) in an aqueous medium with a chaotropic agent at least slightly soluble in said medium to release B c and/or B u from said mordant.
Claim 3
Dependent Claim
33 words
A method as defined in claim 1 wherein said chaotropic agent in step (c) is selected from the group consisting of ionizable salts, non-ionic surfactants, xanthine and alkylated xanthines, urea, and mixtures thereof.
Claim 4
Dependent Claim
70 words
A method as defined in claim 1 or 2 wherein said interactive mordant is a polymeric mordant having monomeric units in the polymer chain of the following formula ##STR10## wherein A represents an organo group constituting a portion of the polymer backbone; Q represents a chemical bond(s) or a chemical group linking M.sup.⊕ to A; M.sup.⊕ represents a hydrophobic organic moiety containing a cation; and X.sup.⊖ represents an acid anion.
Claim 5
Independent Claim
154 words
A method for the selective separation and isolation of conjugated bilirubin (B c ) and unconjugated bilirubin (B u ) from an aqueous liquid containing a mixture of B c and B u , said method comprising: (a) contacting together said liquid and an interactive mordant having binding sites for bilirubin to mordant said B c and B u in said liquid, said mordant comprising at least one moiety having a hydrophobic organic matrix containing a charge-bearing cationic group; (b) separating the mordanted B c and B u of step (a) from said liquid; and (c) treating the mordanted B c and B u of step (b) in an aqueous medium with a chaotropic agent at least slightly soluble in said medium to release B c and B u from the mordant; and (d) selectively solubilizing the released B c and B u of step (c) to separate B c from B u .
Claim 6
Independent Claim
154 words
A method for the selective separation and isolation of conjugated bilirubin (B c ) and unconjugated bilirubin (B u ) from an aqueous liquid containing a mixture of B c and B u , said method comprising: (a) contacting together said liquid and an interactive mordant having binding sites for bilirubin to mordant said B c and B u in said liquid, said mordant comprising at least one moiety having a hydrophobic organic matrix containing a charge-bearing cationic group; (b) separating the mordanted B c and B u of step (a) from said liquid; and (c) treating the mordanted B c and B u of step (b) in an aqueous medium with a chaotropic agent at least slightly soluble in said medium to release B c and B u from the mordant; and (d) selectively solubilizing the released B c and B u of step (c) to separate B c from B u .
Claim 7
Dependent Claim
50 words
A method of claim 4 wherein step (d) is carried out during or after step (c) by adjusting the pH of said aqueous medium to a value effective to solubilize the released B c in said medium while the released B u forms an insoluble solid phase in said medium.
Claim 8
Dependent Claim
27 words
A method of claim 1, 4 or 5 wherein a volatile water-miscible organic solvent is added to said mordanted B c and B u following step (b).
Claim 9
Dependent Claim
25 words
A method of claim 4 wherein step (d) is carried out by extraction in a water-immiscible organic solvent in which B u is selectively solubilized.
Claim 10
Dependent Claim
25 words
A method of claim 4 wherein step (d) is carried out by extraction in chloroform, dichloromethane, or mixtures thereof to selectively solubilize B u therein.
Claim 11
Dependent Claim
26 words
A method of claim 1 or 4 wherein said method is carried out at a temperature in the range of from about 0° to 10° C.
Claim 12
Dependent Claim
17 words
A method of claim 1 or 4 wherein said method is carried out in a non-oxidizing atmosphere.
Claim 13
Dependent Claim
18 words
A method of claim 1 or 4 wherein step (a) is carried out in a dry test element.
Claim 14
Dependent Claim
26 words
A method of claim 1 or 4 wherein step (a) is carried out by contacting together in an aqueous medium said interactive mordant and said liquid.
Claim 15
Independent Claim
215 words
A method for the selective separation and isolation of conjugated bilirubin (B c ) and unconjugated bilirubin (B u ) from an aqueous liquid containing a mixture of B c and B u , said method comprising: (a) contacting together said liquid and an interactive mordant having binding sites for bilirubin to mordant said B c and B u , said interactive mordant representing a polymeric mordant having monomeric units in the polymer chain of the following formula ##STR11## wherein A represents an organo group constituting a portion of the polymer backbone, Q represents a chemical bond(s) or a chemical group linking M.sup.⊕ to A, M.sup.⊕ represents a hydrophobic organic moiety containing a cation, and X.sup.⊖ represents an acid anion; (b) separating the mordanted B c and B u of step (a) from said liquid; (c) treating the mordanted B c and B u of step (b) in an aqueous medium with a chaotropic agent at least slightly soluble in said medium to release B c and B u from the mordant, said chaotropic agent selected from the group consisting of ionizable salts, non-ionic surfactants, xanthine and alkylated xanthines, urea, and mixtures thereof; and (d) selectively solubilizing the released B c or B u of step (c) to separate B c from B u .
Claim 16
Independent Claim
215 words
A method for the selective separation and isolation of conjugated bilirubin (B c ) and unconjugated bilirubin (B u ) from an aqueous liquid containing a mixture of B c and B u , said method comprising: (a) contacting together said liquid and an interactive mordant having binding sites for bilirubin to mordant said B c and B u , said interactive mordant representing a polymeric mordant having monomeric units in the polymer chain of the following formula ##STR11## wherein A represents an organo group constituting a portion of the polymer backbone, Q represents a chemical bond(s) or a chemical group linking M.sup.⊕ to A, M.sup.⊕ represents a hydrophobic organic moiety containing a cation, and X.sup.⊖ represents an acid anion; (b) separating the mordanted B c and B u of step (a) from said liquid; (c) treating the mordanted B c and B u of step (b) in an aqueous medium with a chaotropic agent at least slightly soluble in said medium to release B c and B u from the mordant, said chaotropic agent selected from the group consisting of ionizable salts, non-ionic surfactants, xanthine and alkylated xanthines, urea, and mixtures thereof; and (d) selectively solubilizing the released B c or B u of step (c) to separate B c from B u .
Claim 17
Dependent Claim
45 words
A method of claim 13 wherein said interactive mordant is a copolymer containing about 10 to 90 wt% of repeating units having the formula specified in step (a) of claim 13 and up to about 75 wt% of repeating units of a noninterfering hydrophobic monomer.
Claim 18
Dependent Claim
49 words
A method of claim 13 wherein step (d) is carried out during step (c) by adjusting the pH of said aqueous medium to a value less than 7.0 whereby released B c is solubilized in said medium and released B u forms an insoluble solid phase in said medium.
Claim 19
Dependent Claim
38 words
A method of claim 13 or 15 wherein said chaotropic agent comprises a salt of a strong acid and a strong base and wherein a volatile, water-miscible organic solvent is added to said aqueous medium in step (c).
Claim 20
Dependent Claim
34 words
A method of claim 13 or 15 wherein said chaotropic agent comprises a salt of a strong acid and a strong base and wherein propanol is added to said aqueous medium in step (c).
Claim 21
Dependent Claim
28 words
A method of claim 13 wherein said method is carried out under a non-oxidizing atmosphere at a temperature within the range of from about 0° to 60° C.
Claim 22
Dependent Claim
20 words
Isolated conjugated bilirubin having a purity in excess of 75% as determined by quantitative nuclear magnetic resonance and gravimetric analysis.
Claim 23
Dependent Claim
20 words
Isolated conjugated bilirubin having a purity in excess of 75% as determined by quantitative nuclear magnetic resonance and gravimetric analysis.
Claim 24
Dependent Claim
21 words
Isolated conjugated bilirubin having a purity of about 85% or higher as determined by quantitative nuclear magnetic resonance and gravimetric analysis.
Claim 25
Dependent Claim
21 words
Isolated conjugated bilirubin having a purity of about 85% or higher as determined by quantitative nuclear magnetic resonance and gravimetric analysis.
Claim 26
Dependent Claim
43 words
An article of manufacture for the assay of bilirubin contained in an aqueous liquid, said article comprising a wet or dry matrix, and isolated conjugated bilirubin having a purity of 75% or higher as determined by quantitative nuclear magnetic resonance and gravimetric analysis.
Claim 27
Dependent Claim
43 words
An article of manufacture for the assay of bilirubin contained in an aqueous liquid, said article comprising a wet or dry matrix, and isolated conjugated bilirubin having a purity of 75% or higher as determined by quantitative nuclear magnetic resonance and gravimetric analysis.
References Cited (Backward Citations)
US4069017A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1985-01-18 | 1985-07-20 | 1986-01-19 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1989-01-18 | 1989-07-20 | 1990-01-19 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1993-01-18 | 1993-07-20 | 1994-01-19 | $7700.00 | Window Closed / Lapsed |