US4668638A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Liposome composition for lupus assay
Assignee: Elan Pharmaceuticals LLC
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Filing: 1983-09-26
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Granted: 1987-05-26
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Expires: 2003-09-26
Abstract
Abstract An immunoassay utilizing a novel liposome composition in which there is incorporated a stabilizing or destabilizing component and an antigen. The presence of cognate antibodies in test samples is detected by the alteration of the supramolecular structure of the liposomes resulting in changes in stabilization. Destabilization can be detected, and in certain cases, caused by, the addition of magnesium or calcium ions. Increased stabilization can be detected by the polymerization of bilayer components in response to ultraviolet light. Antibody-antigen interactions at the liposome surface mediate the stabilization/destabilization response.
Inventors
- 👤 Andrew S. Janoff
- 👤 Marc J. Ostro
- 👤 Alan L. Weiner
- 👤 Gerald Weissmann
- 👤 James R. Seibold
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-28 16:02
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/543; G01N33/554; G01N33/564; G01N2800/00; G01N2800/10; G01N2800/101; G01N2800/104; Y; Y10; Y10S; Y10S436/00; Y10S436/829; Y10T; Y10T428/00; Y10T428/29; Y10T428/2982; Y10T428/2984
Claims Analysis
Total Claims: 62
Claim 1
Independent Claim
121 words
A method for detecting antibodies in serum, comprising: (a) contacting serum, for a period of time at least sufficient to permit equilibration of binding between an antigen and its cognate antibody, with liposomes having a multicomponent bilayer comprising a flexible double tailed phospholipid and a destabilization component, wherein at least one of the liposome membrane constituents is an antigen; (b) exposing the liposomes to conditions which cause destabilization of the liposomes in the absence of cognate antibodies directed against the antigen and which do not cause destabilization of the liposomes in the presence of the cognate antibodies; (c) detecting any destabilization of the liposome, the method being performed under conditions non-inhibiting to the binding between the antigen and its cognate antibody.
Claim 2
Independent Claim
121 words
A method for detecting antibodies in serum, comprising: (a) contacting serum, for a period of time at least sufficient to permit equilibration of binding between an antigen and its cognate antibody, with liposomes having a multicomponent bilayer comprising a flexible double tailed phospholipid and a destabilization component, wherein at least one of the liposome membrane constituents is an antigen; (b) exposing the liposomes to conditions which cause destabilization of the liposomes in the absence of cognate antibodies directed against the antigen and which do not cause destabilization of the liposomes in the presence of the cognate antibodies; (c) detecting any destabilization of the liposome, the method being performed under conditions non-inhibiting to the binding between the antigen and its cognate antibody.
Claim 3
Dependent Claim
13 words
The method according to claim 1 wherein the destabilizing agent is a cardiolipin.
Claim 4
Dependent Claim
14 words
The method according to claim 1 wherein the destabilizing agent is a phosphatidic acid.
Claim 5
Independent Claim
121 words
A method for detecting antibodies in serum, comprising: (a) contacting serum, for a period of time at least sufficient to permit equilibration of binding between an antigen and its cognate antibody, with liposomes having a multicomponent bilayer comprising a flexible double tailed phospholipid and a destabilization component, wherein at least one of the liposome membrane constituents is an antigen; (b) exposing the liposomes to conditions which do not cause destabilization of the liposomes in the absence of cognate antibodies directed against the antigen and which cause destabilization of the liposomes in the presence of the cognate antibodies; (c) detecting any destabilization of the liposome, the method being performed under conditions non-inhibiting to the binding between the antigen and its cognate antibody.
Claim 6
Independent Claim
121 words
A method for detecting antibodies in serum, comprising: (a) contacting serum, for a period of time at least sufficient to permit equilibration of binding between an antigen and its cognate antibody, with liposomes having a multicomponent bilayer comprising a flexible double tailed phospholipid and a destabilization component, wherein at least one of the liposome membrane constituents is an antigen; (b) exposing the liposomes to conditions which do not cause destabilization of the liposomes in the absence of cognate antibodies directed against the antigen and which cause destabilization of the liposomes in the presence of the cognate antibodies; (c) detecting any destabilization of the liposome, the method being performed under conditions non-inhibiting to the binding between the antigen and its cognate antibody.
Claim 7
Dependent Claim
13 words
The method according to claim 4 wherein the destabilizing agent is a cardiolipin.
Claim 8
Dependent Claim
14 words
The method according to claim 4 wherein the destabilizing agent is a phosphatidic acid.
Claim 9
Dependent Claim
33 words
The method according to claim 2 for detecting SLE antibodies in serum wherein the phospholipid phosphatidylcholine and the antigen is cardiolipin in a molar ratio of from about 1:7 to about 7:1 respectively.
Claim 10
Dependent Claim
16 words
The method according to claim 7, wherein said ratio is from about 2:5 to about 4:3.
Claim 11
Dependent Claim
42 words
The method according to claim 7, wherein said liposomes contain entrapped therein a divalent cation responsive colorimetric indicator, and said conditions causing destabilization comprise contacting said liposomes with divalent cations which results in leakage and a change in color of the indicator.
Claim 12
Dependent Claim
15 words
The method according to claim 9, wherein the divalent cations are magnesium or calcium cations.
Claim 13
Dependent Claim
66 words
The method according to claim 3 for detecting SLE antibodies in serum wherein the phospholipid is phosphatidylcholine and the antigen is phosphatidic acid in molar ratios of from about 9:1 to about 3:7, respectively, said liposomes being unstable in the presence of calcium ions unless SLE antibodies are present and wherein the condition which cause destabilization of the liposomes comprise exposing said liposomes to calcium ions.
Claim 14
Dependent Claim
36 words
The method according to claim 11, wherein said phosphatidic acid is dipalmitoylphosphatidic acid, and said liposomes have a multicomponent liposome bilayer comprising egg phosphatidylcholine, dipalmitoylphosphatidic acid, cholesterol and dicetylphosphate in molar ratios of about 7:1:1:1, respectively.
Claim 15
Dependent Claim
41 words
The method according to claim 11, wherein said liposomes contain entrapped therein a divalent cation responsive colorimetric indicator, and said destablization results in an influx of divalent cations into the liposomes which results in a change in color of the indicator.
Claim 16
Dependent Claim
58 words
A method according to claim 3 for detecting SLE antibodies in serum wherein the phospholipid is phosphatidylcholine and the antigen is phosphatidic acid in a molar ratio of from about 1:7 to about 7:1, respectively, said liposomes being stable in the presence of serum containing SLE antibodies and unstable in the presence of serum not containing SLE antibodies.
Claim 17
Dependent Claim
16 words
The method according to claim 14, wherein said ratio is from about 2:5 to about 4:3.
Claim 18
Dependent Claim
13 words
The method according to claim 14, wherein said phosphatidic acid is 1-palmitoyl-2-oleoylphosphatidic acid.
Claim 19
Dependent Claim
39 words
The method according to claim 14, wherein said liposomes contain entrapped therein a colorimetric indicator, and said instability is detected by a color change in said indicator which is released from the liposomes in the absence of SLE antibodies.
Claim 20
Dependent Claim
26 words
The method according to claim 17, wherein said indicator is responsive to divalent metal ions and is exposed to divalent cations upon release from the liposomes.
Claim 21
Dependent Claim
14 words
The method according to claim 18, wherein the divalent metal ions are magnesium ions.
Claim 22
Dependent Claim
22 words
The method according to claim 7, 11 or 14, wherein anionic lipid components of the multicomponent bilayer are cesium or potassium salts.
Claim 23
Dependent Claim
14 words
The method according to claim 7, 11 or 14, wherein said phosphatidylcholine is 1-palmitoyl-2-oleoyl-phosphatidylcholine.
Claim 24
Dependent Claim
22 words
The method according to claim 7, 11 or 14, wherein said liposome bilayer contains a stabilizing amount of a rigid steroid stabilizer.
Claim 25
Dependent Claim
11 words
The method according to claim 22, wherein said steroid is cholesterol.
Claim 26
Dependent Claim
32 words
The method according to claim 23, wherein the stabilizing amount is from about 5 to about 30 molar percent, relative to the total amount of said phosphatidylcholine and phosphatidic acid or cardiolipin.
Claim 27
Dependent Claim
41 words
The method according to claim 7 or 14, wherein said liposome bilayer contains from about 10 to about 20 molar percent, relative to the total amount of said phosphatidylcholine and phosphatidic acid or cardiolipin, of a liposome compatible negatively charged compound.
Claim 28
Dependent Claim
15 words
The method according to claim 25, wherein said liposome compatible negatively charged compound is phosphatidylglycerol.
Claim 29
Dependent Claim
11 words
The method according to claim 26, wherein said phosphatidylglycerol is dipalmitoyl-phosphatidylglycerol.
Claim 30
Dependent Claim
31 words
The method according to claim 11, wherein said liposome bilayer contains from about 10 to about 20 molar percent, relative to the total amount of phosphatidylcholine and phosphatidic acid, of dicetylphosphate.
Claim 31
Dependent Claim
20 words
The method according to claim 7, 11 or 14, wherein said liposome bilayer contains a stabilizing amount of an antioxidant.
Claim 32
Dependent Claim
36 words
The method according to claim 29, wherein said antioxidant is alpha-tocopherol in an amount corresponding to from about 1 to about 5 molar percent of the total amount of said phosphatidylcholine and phosphatidic acid or cardiolipin.
Claim 33
Dependent Claim
25 words
The method according to claim 7, wherein said bilayer comprises 1-palmitoyl-2-oleoyl-phosphatidylcholine, cardiolipin, cholesterol, alpha-tocopherol and dipalmitoyl phosphatidylglycerol in a molar ratio of about 3:4:1.9:0.1:1, respectively.
Claim 34
Dependent Claim
26 words
The method according to claim 14, wherein said bilayer comprises 1-palmitoyl-2-oleoyl-phosphatidylcholine, 1-palmitoyl-2-oleoyl-phosphatidic acid, cholesterol, alpha-tocopherol and dipalmitoyl phosphatidylglycerol in a molar ratio of about 3:4:1.9:0.1:1, respectively.
Claim 35
Dependent Claim
15 words
The method according to claim 9, 13 or 17, wherein said indicator is arsenazo III.
Claim 36
Dependent Claim
48 words
A liposome composition comprising liposome vesicles, said vesicles having (i) a multicomponent liposome bilayer comprising a flexible double tailed phosphatidylcholine and a lipid antigen for SLE antibodies in a molar ratio of from about 1:7 to about 9:1, respectively, and (ii) a colorimetric indicator entrapped within said liposome.
Claim 37
Dependent Claim
48 words
A liposome composition comprising liposome vesicles, said vesicles having (i) a multicomponent liposome bilayer comprising a flexible double tailed phosphatidylcholine and a lipid antigen for SLE antibodies in a molar ratio of from about 1:7 to about 9:1, respectively, and (ii) a colorimetric indicator entrapped within said liposome.
Claim 38
Dependent Claim
16 words
A composition according to claim 34, wherein said indicator is a divalent cation responsive colorimetric indicator.
Claim 39
Dependent Claim
16 words
A composition according to claim 34, wherein said ratio is from about 2:5 to about 4:3.
Claim 40
Dependent Claim
16 words
A composition according to claim 34, wherein said lipid antigen is a phosphatidic acid or cardiolipin.
Claim 41
Dependent Claim
11 words
A composition according to claim 34, wherein said phosphatidylcholine is 1-palimitoyl-2-oleoyl-phosphatidylcholine.
Claim 42
Dependent Claim
28 words
A composition of claim 34, wherein said vesicles have a multicomponent liposome bilayer comprising egg phosphatidylcholine, dipalmitoylphosphatidic acid, cholesterol and dicetylphosphate in molar ratios of about 1:1:1, respectively.
Claim 43
Dependent Claim
19 words
A composition according to claim 34, wherein said liposome bilayer contains a stabilizing amount of a rigid steroid stabilizer.
Claim 44
Dependent Claim
11 words
A composition according to claim 40, wherein said steroid is cholesterol.
Claim 45
Dependent Claim
32 words
A composition according to claim 40, wherein the stabilizing amount is from about 5 to about 30 molar percent, relative to the total of amount of said phosphatidylcholine and said lipid antigen.
Claim 46
Dependent Claim
37 words
A composition according to claim 34, wherein said liposome bilayer contains from about 10 to about 20 molar percent, relative to the total of said phosphatidylcholine and said lipid antigen, of a liposome compatible negatively charged compound.
Claim 47
Dependent Claim
16 words
A composition according to claim 43, wherein said liposome compatible negatively charged compound is a phosphatidylglycerol.
Claim 48
Dependent Claim
11 words
A composition according to claim 44, wherein said phosphatidylglycerol is dipalmitoyl-phosphatidylglycerol.
Claim 49
Dependent Claim
17 words
A composition according to claim 34, wherein said liposome bilayer contains a stabilizing amount of an antioxidant.
Claim 50
Dependent Claim
35 words
A composition according to claim 46, wherein said antioxidant is alpha-tocopherol in an amount corresponding to from about 1 to about 5 molar percent of the total amount of said phosphatidylcholine and said lipid antigen.
Claim 51
Dependent Claim
26 words
A composition according to claim 34, wherein said liposome bilayer comprises 1-palmitoyl-2-oleoyl-phosphatidylcholine, cardiolipin, cholesterol, alpha-tocopherol and dipalmitoyl phosphatidylglycerol in a molar ratio of about 3:4:1.9:0.1:1, respectively.
Claim 52
Dependent Claim
26 words
A composition according to claim 34, wherein said liposome bilayer comprises 1-palmitoyl-2-oleoyl-phosphatidylcholine, 1-palmitoyl-2-oleoyl-phosphatidic acid, cholesterol, alpha-tocopherol and dipalmitoylphosphatidylglycerol in a molar ratio of about 3:4:1.9:0.1:1, respectively.
Claim 53
Dependent Claim
14 words
A composition according to claim 34 or 48, wherein said indicator is arsenazo III.
Claim 54
Dependent Claim
16 words
A composition according to claim 34, wherein said lipid antigen is a cesium or potassium salt.
Claim 55
Dependent Claim
28 words
A composition according to claim 34, wherein said lipid antigen is the cesium salt of cardiolipin and wherein said liposome bilayer further comprises the potassium salt of dipalmitoylphosphatidylglycerol.
Claim 56
Independent Claim
101 words
A method for preparing liposomes having a compound entrapped therein, comprising: (a) forming a dispersion of at least one amphipathic lipid in an organic solvent; (b) forming an aqueous mixture containing a compound to be entrapped in the liposomes; (c) injecting the dispersion into the aqueous mixture heated to a temperature above the boiling point of the organic solvent to form, as the organic solvent escapes, a suspension of liposomes having said compounds entrapped therein; and (d) exposing the suspension to a vacuum produced by a water aspirator for from about one to five hours to remove excess remaining organic solvent.
Claim 57
Independent Claim
101 words
A method for preparing liposomes having a compound entrapped therein, comprising: (a) forming a dispersion of at least one amphipathic lipid in an organic solvent; (b) forming an aqueous mixture containing a compound to be entrapped in the liposomes; (c) injecting the dispersion into the aqueous mixture heated to a temperature above the boiling point of the organic solvent to form, as the organic solvent escapes, a suspension of liposomes having said compounds entrapped therein; and (d) exposing the suspension to a vacuum produced by a water aspirator for from about one to five hours to remove excess remaining organic solvent.
Claim 58
Independent Claim
77 words
A method for detecting antibodies in serum, comprising: (a) contacting serum, for a period of time at least sufficient to permit equilibration of binding between an antigen and its cognate antibody, with liposomes having a multicomponent bilayer comprising a diacetylenic phospholipid and an antigen; (b) exposing the liposomes to ultraviolet light; and (b) detecting any polymerization of the liposome membrane, the method being performed under conditions non-inhibiting to the binding between the antigen and its cognate antibody.
Claim 59
Independent Claim
77 words
A method for detecting antibodies in serum, comprising: (a) contacting serum, for a period of time at least sufficient to permit equilibration of binding between an antigen and its cognate antibody, with liposomes having a multicomponent bilayer comprising a diacetylenic phospholipid and an antigen; (b) exposing the liposomes to ultraviolet light; and (b) detecting any polymerization of the liposome membrane, the method being performed under conditions non-inhibiting to the binding between the antigen and its cognate antibody.
Claim 60
Dependent Claim
21 words
The method according to claim 54 wherein the antigen is a diacetylenic phospholipid and the other bilayer components are non-diacetylenic phospholipids.
Claim 61
Dependent Claim
70 words
The method according to claim 54 wherein the diacetylenic phospholipid has a charged polar head group and further comprising the step prior to exposure to ultraviolet light, of exposing the liposomes to conditions which cause phase separation of the polar head group in the absence of cognate antibodies directed against the antigen and which do not cause phase separation of the phosphatidic acid in the presence of the cognate antibodies.
Claim 62
Dependent Claim
21 words
The method according to claim 56 wherein the antigen is a diacetylenic phospholipid and the other bilayer components are non-diacetylenic phospholipids.
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) | 1990-05-25 | 1990-11-24 | 1991-05-26 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1994-05-25 | 1994-11-24 | 1995-05-26 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1998-05-25 | 1998-11-24 | 1999-05-26 | $7700.00 | Window Closed / Lapsed |