US4745074A
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US
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Blood-fluid composition for cell lysis system
Assignee: LIPSOME TECHNOLOGY Inc
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Filing: 1984-02-23
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Granted: 1988-05-17
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Expires: 2004-02-23
Abstract
Abstract A blood fluid composition for use in a complement-mediated cell lysis system. The composition includes a blood fluid, which may be either a serum source of complement, analyte-containing serum, or both, and lipid vesicles capable of reducing the extent of non-specific cell lysis produced when the blood fluid is added to lysable target cells in the system. The vesicles are present in an amount which increases the ratio of ligand-specific to non-specific cell lysis in the system at least 2-fold and preferably 4-fold or more over that achievable in the system in the absence of the vesicles.
Inventors
- 👤 Hans Schreier
- 👤 Francis J. Martin
- 👤 Viola T. Kung
- 👤 Francis C. Szoka
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-31 22:17
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/5306; Y; Y10; Y10S; Y10S436/00; Y10S436/821; Y10S436/825; Y10S436/829
Claims Analysis
Total Claims: 16
Claim 1
Independent Claim
119 words
A homogeneous liposome immunoassay system comprising liposomes having surface-bonded ligand molecules and encapsulated reporter molecules which are detactable upon cell lysis, serum complement, anti-ligand molecules capable of reacting specifically and with high affinity with said ligand molecules in the presence of said complement, to induce ligand-specific cell lysis and release of the encapsulated reporter molecules, and lipid vesicles effective to increase the ratio of ligand-specific lysis to nonspecific lysis produced in the absence of said anti-ligand, where said vesicles are present in an amount which increases said specific to nonspecific ratio more than 2-fold over that achievable in the system in the absence of said vesicles, where both the liposomes and lipid vesicles contain phosphatidylcholine as a major component.
Claim 2
Dependent Claim
119 words
A homogeneous liposome immunoassay system comprising liposomes having surface-bonded ligand molecules and encapsulated reporter molecules which are detactable upon cell lysis, serum complement, anti-ligand molecules capable of reacting specifically and with high affinity with said ligand molecules in the presence of said complement, to induce ligand-specific cell lysis and release of the encapsulated reporter molecules, and lipid vesicles effective to increase the ratio of ligand-specific lysis to nonspecific lysis produced in the absence of said anti-ligand, where said vesicles are present in an amount which increases said specific to nonspecific ratio more than 2-fold over that achievable in the system in the absence of said vesicles, where both the liposomes and lipid vesicles contain phosphatidylcholine as a major component.
Claim 3
Dependent Claim
35 words
The system of claim 1, for use in detecting an anti-ligand analyte in a fluid sample, wherein the anti-ligand molecules are supplied by addition of the fluid sample to the liposomes and the serum complement.
Claim 4
Dependent Claim
36 words
The system of claim 1, for use in detecting an analyte which is effective in competing with the surface-bound molecules for binding to the anti-ligand, wherein the system further includes a fluid sample containing the analyte.
Claim 5
Dependent Claim
138 words
In a homogeneous liposome immunoassay method in which a suspension of liposomes containing surface-bound ligand molecules and encapsulated reporter molecules is reacted with serum complement and anti-ligand molecules, producing both ligand-specific complement-mediated liposome lysis which is related to the concentration of anti-ligand molecules in the reaction mixture, and nonspecific liposome lysis which is independent of the concentration of anti-ligand molecules, wherein the improvement comprises enhancing the sensitivity and reproducibility of the immunoassay by adding to the serum complement, an amount of lipid vesicles which, when the vesicle-containing complement is reacted with said liposomes and anti-ligand molecules, is effective to increase the ratio of specific to nonspecific complement-mediate liposome lysis at least about 2-fold over that achievable in the absence of the added vesicles, where both the liposomes and lipid vesicles contain phosphatidyl choline as a major lipid component.
Claim 6
Dependent Claim
138 words
In a homogeneous liposome immunoassay method in which a suspension of liposomes containing surface-bound ligand molecules and encapsulated reporter molecules is reacted with serum complement and anti-ligand molecules, producing both ligand-specific complement-mediated liposome lysis which is related to the concentration of anti-ligand molecules in the reaction mixture, and nonspecific liposome lysis which is independent of the concentration of anti-ligand molecules, wherein the improvement comprises enhancing the sensitivity and reproducibility of the immunoassay by adding to the serum complement, an amount of lipid vesicles which, when the vesicle-containing complement is reacted with said liposomes and anti-ligand molecules, is effective to increase the ratio of specific to nonspecific complement-mediate liposome lysis at least about 2-fold over that achievable in the absence of the added vesicles, where both the liposomes and lipid vesicles contain phosphatidyl choline as a major lipid component.
Claim 7
Dependent Claim
119 words
A homogeneous liposomes immunoassay system comprising liposomes having surface-bound ligand molecules and encapsulated reporter molecules which are detectable upon cell lysis, serum complement, anti-ligand molecules capable of reacting specifically and with high affinity with said ligand molecules in the presence of said complement, to induce ligand-specific cell lysis and release of the encapsulated reporter molecules, and lipid vesicles effective to increase the ratio of ligand-specific lysis to nonspecific lysis produced in the absence of said anti-ligand, where said vesicles are present in an amount which increases said specific to nonspecific ratio more than 2-fold over that achievable in the system in the absence of said vesicles, where both the liposomes and lipid vesicles contain phosphatidylethanolamine or a phosphatidylethanolamine derivative.
Claim 8
Dependent Claim
119 words
A homogeneous liposomes immunoassay system comprising liposomes having surface-bound ligand molecules and encapsulated reporter molecules which are detectable upon cell lysis, serum complement, anti-ligand molecules capable of reacting specifically and with high affinity with said ligand molecules in the presence of said complement, to induce ligand-specific cell lysis and release of the encapsulated reporter molecules, and lipid vesicles effective to increase the ratio of ligand-specific lysis to nonspecific lysis produced in the absence of said anti-ligand, where said vesicles are present in an amount which increases said specific to nonspecific ratio more than 2-fold over that achievable in the system in the absence of said vesicles, where both the liposomes and lipid vesicles contain phosphatidylethanolamine or a phosphatidylethanolamine derivative.
Claim 9
Dependent Claim
45 words
The system of claim 5, wherein the phosphatidylethanolamine or phosphatidylethanolamine derivative has the form: ##STR1## where R 1 and R 2 are saturated or unsaturated hydrocarbon chains, and X=H, CO 2 H, N-4-(p-moleimidophenyl) butyryl or a hapten against which said anti-ligand molecules are immunospecifically reactive.
Claim 10
Dependent Claim
12 words
The system of claim 6, wherein the lipid includes N-4(P-malimidophenyl) butyryl phosphatidylethanolamine.
Claim 11
Dependent Claim
136 words
In a homogeneous liposome immunoassay method in which a suspension of liposomes containing surface-bound ligand molecules and encapsulated reporter molecules is reacted with serum complement and anti-ligand molecules, producing both ligand-specific complement-mediated liposome lysis which is related to the concentration of anti-ligand molecules in the reaction mixture, and nonspecific liposome lysis which is independent of the concentration of anti-ligand molecules, wherein the improvement comprises enhancing the sensitivity and reproducibility of the immunoassay by adding to the serum complement, an amount of lipid vesicles which, when the vesicle-containing complement is reacted with said liposomes and anti-ligand molecules, is effective to increase the ratio of specific to nonspecific complement-mediated liposome lysis at least about 2-fold over the achievable in the absence of the added vesicles, where both the liposomes and lipid vesicles contain phosphatidylethanolamine or a derivative thereof.
Claim 12
Dependent Claim
136 words
In a homogeneous liposome immunoassay method in which a suspension of liposomes containing surface-bound ligand molecules and encapsulated reporter molecules is reacted with serum complement and anti-ligand molecules, producing both ligand-specific complement-mediated liposome lysis which is related to the concentration of anti-ligand molecules in the reaction mixture, and nonspecific liposome lysis which is independent of the concentration of anti-ligand molecules, wherein the improvement comprises enhancing the sensitivity and reproducibility of the immunoassay by adding to the serum complement, an amount of lipid vesicles which, when the vesicle-containing complement is reacted with said liposomes and anti-ligand molecules, is effective to increase the ratio of specific to nonspecific complement-mediated liposome lysis at least about 2-fold over the achievable in the absence of the added vesicles, where both the liposomes and lipid vesicles contain phosphatidylethanolamine or a derivative thereof.
Claim 13
Dependent Claim
45 words
The method of claim 8, wherein the phosphatidyl ethanolamine or derivative includes lipids of the form: ##STR2## where R 1 and R 2 are saturated or unsaturated hydrocarbon chains, and X=H, C 2 H N-4-(p-maleimidophenyl) butyryl or a hapten against which such-anti-ligand is immunospecifically reactive.
Claim 14
Dependent Claim
15 words
The method of claim 9, wherein the lipid vesicles include an N-4-(p-maleimidophenyl) butyryl phosphatidyl ethanolamine.
Claim 15
Dependent Claim
141 words
In a homogeneous liposomes immunoassay method in which a suspension of liposomes containing surface-bound ligand molecules and encapsulated reporter molecules is reacted with serum complement and anti-ligand molecules, producing both ligand-specific complement-mediated liposome lysis which is related to the concentration of anti-ligand molecules in the reaction mixture, and nonspecific liposome lysis which is independent of the concentration of anti-ligand molecules, wherein the improvement comprises enhancing the sensitivity and reproducibility of the immunoassay, where the anti-ligand molecules are analyte molecules contained in a serum or plasma sample, by adding to the serum complement, an amount of lipid vesicles which, when the vesicle-containing complement is reacted with said liposomes and said sample containing the anti-ligand molecules, is effective to increase the ratio of specific to nonspecific complement-mediated liposomes lysis at least about 2-fold over the achievable in the absence of the added vesicles.
Claim 16
Dependent Claim
141 words
In a homogeneous liposomes immunoassay method in which a suspension of liposomes containing surface-bound ligand molecules and encapsulated reporter molecules is reacted with serum complement and anti-ligand molecules, producing both ligand-specific complement-mediated liposome lysis which is related to the concentration of anti-ligand molecules in the reaction mixture, and nonspecific liposome lysis which is independent of the concentration of anti-ligand molecules, wherein the improvement comprises enhancing the sensitivity and reproducibility of the immunoassay, where the anti-ligand molecules are analyte molecules contained in a serum or plasma sample, by adding to the serum complement, an amount of lipid vesicles which, when the vesicle-containing complement is reacted with said liposomes and said sample containing the anti-ligand molecules, is effective to increase the ratio of specific to nonspecific complement-mediated liposomes lysis at least about 2-fold over the achievable in the absence of the added vesicles.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1991-05-17 | 1991-11-16 | 1992-05-17 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1995-05-17 | 1995-11-16 | 1996-05-17 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1999-05-17 | 1999-11-16 | 2000-05-17 | $7700.00 | Window Closed / Lapsed |