US4708933A
Expired / Lapsed
US
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Immunoliposome assay-methods and products
Assignee: BIOWHITTAKER, INC. A CORP. OF DELAWARE
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Filing: 1984-06-12
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Granted: 1987-11-24
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Expires: 2004-06-12
Abstract
Abstract This invention is directed to a new membrane lytic immunoassay. In one embodiment of this assay, an antigen is first covalently coupled with lipids and this antigen-lipid complex is mixed with a hexagonal phase forming lipid to form bilayer liposome vesicles additionally containing a self-quenching fluorescent dye. When this antigen-containing liposome is brought into contact with a solid surface coated with antibody molecules, binding occurs between the antigen and the antibody, disrupting the liposome and releasing the dye. To assay a biological fluid for free antigen the fluid is first contacted with the solid surface-antibody complex, to saturate the bound antibody. Binding by the liposomes is thereby inhibited, leading to reduced dye release. Comparing dye release against a standardized curve of dye release versus known antigen concentrations allows for rapid determination of the unknown antigen concentration in the biological fluid. Similarly, antibodies and other entities, e.g., enzymes, drugs, etc., may be determined using slightly modified versions of this assay.
Inventors
- 👤 Leaf Huang
- 👤 Rodney J. Y. Ho
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 09:05
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/531; G01N33/532; G01N33/58; G01N33/585; G01N33/586; Y; Y10; Y10S; Y10S435/00; Y10S435/966; Y10S435/967; Y10S435/975; Y10S436/00; Y10S436/815; Y10S436/829
Claims Analysis
Total Claims: 41
Claim 1
Independent Claim
138 words
An immunoassay method for detecting or quantifying an analyte of interest in a test fluid, said method comprising: (a) forming liposomes having the analyte of interest incorporated onto the surface membrane thereof and a marker compound incorporated in the interior aqueous phase thereof; (b) providing a solid phase inert support having attached thereto a receptor for the analyte of interest; (c) mixing said test fluid with said receptor-solid phase support of step (b) for sufficient time to saturate said receptor with any analyte present in said test fluid; (d) mixing said liposomes formed in step (a) with said saturated receptor-solid phase support from step (c) causing the lysis of said liposomes, without the addition of any membrane lytic molecules, ions, or active complements, and; (e) determining the presence of marker compound released by the liposomes in step (d).
Claim 2
Dependent Claim
138 words
An immunoassay method for detecting or quantifying an analyte of interest in a test fluid, said method comprising: (a) forming liposomes having the analyte of interest incorporated onto the surface membrane thereof and a marker compound incorporated in the interior aqueous phase thereof; (b) providing a solid phase inert support having attached thereto a receptor for the analyte of interest; (c) mixing said test fluid with said receptor-solid phase support of step (b) for sufficient time to saturate said receptor with any analyte present in said test fluid; (d) mixing said liposomes formed in step (a) with said saturated receptor-solid phase support from step (c) causing the lysis of said liposomes, without the addition of any membrane lytic molecules, ions, or active complements, and; (e) determining the presence of marker compound released by the liposomes in step (d).
Claim 3
Dependent Claim
28 words
The immunoassay of claim 1 wherein step (e) further comprises quantifying the amount of marker compound released and determining the amount of analyte present in the test fluid.
Claim 4
Dependent Claim
13 words
The immunoassay of claim 1 wherein said analyte of interest is an antigen.
Claim 5
Dependent Claim
13 words
The immunoassay of claim 1 wherein said analyte of interest is an enzyme.
Claim 6
Dependent Claim
13 words
The immunoassay of claim 1 wherein said analyte of interest is a drug.
Claim 7
Dependent Claim
21 words
The immunoassay of claim 1 wherein said liposomes are comprised of an H II phase forming lipid and an analyte-lipid complex.
Claim 8
Dependent Claim
73 words
An immunoassay kit, useful in the method of claim 1, said kit containing in (a) a sufficient quantity of reagents suitable for preparing a predetermined ligand-lipid complex; (b) an H II phase forming lipid; (c) a marker compound capable of being encapsulated within the interior aqueous phase of liposomes; (d) analyte standards; or (e) receptor standards; said kit requiring the use of no membrane lytic molecules, ions, or active complements for its use.
Claim 9
Dependent Claim
114 words
An immunoassay kit, useful in the method of claim 1 for detecting the presence of an analyte in a test sample, said kit containing in combination: (a) a sufficient quantity of stable liposomes having a marker compound incorporated into the interior aqueous phase thereof, and said analyte incorporated onto the surface membrane thereof, wherein said liposomes are comprised of an H II phase forming lipid and an analyte-lipid complex; (b) a solid support having bound thereto a sufficient quantity of a receptor for said analyte; and (c) a standard comprising a known amount of said analyte; (d) said kit requiring the use of no membrane lytic molecules, ions, or active complement for its operation.
Claim 10
Dependent Claim
13 words
The immunoassay of claim 1 wherein said solid phase inert support is glass.
Claim 11
Dependent Claim
15 words
The immunoassay of claim 9 wherein said glass support is in the form of beads.
Claim 12
Dependent Claim
14 words
The immunoassay of claim 1 wherein said marker compound is a self-quenching fluorescent dye.
Claim 13
Dependent Claim
11 words
The immunoassay of claim 11 wherein said self-quenching dye is calcein.
Claim 14
Dependent Claim
13 words
The immunoassay of claim 1 wherein said solid phase support is a polymer.
Claim 15
Dependent Claim
10 words
The immunoassay of claim 13 wherein said polymer is latex.
Claim 16
Dependent Claim
12 words
The immunoassay of claim 14 wherein said latex is in bead form.
Claim 17
Dependent Claim
94 words
A system for the detection of a biological analyte of interest useful in the method of claim 1, said system consisting of: (a) marker material encapsulated within the interior aqueous phase and analyte of interest incorporated onto the surface membrane of liposomes, wherein said liposomes are comprised of an H II phase forming lipid and an analyte-lipid complex; and (b) a receptor for said analyte, said receptor being bound to a solid phase support; said system requiring the use of no membrane lytic molecules, ions, or active complement for the detection of said analyte.
Claim 18
Dependent Claim
17 words
The system of claim 16, wherein said liposomes are composed of dioleyl phosphatidylethanolamine and an antigen-lipid complex.
Claim 19
Dependent Claim
13 words
The system of claim 16, wherein said analyte of interest is an antigen.
Claim 20
Dependent Claim
14 words
The system of claim 18, wherein said receptor is an antibody for said antigen.
Claim 21
Dependent Claim
12 words
The system of claim 16, wherein said solid phase support is glass.
Claim 22
Dependent Claim
15 words
The system of claim 20, wherein said glass support is in the form of beads.
Claim 23
Dependent Claim
142 words
An immunoassay for detecting or quantifying an analyte of interest in a test fluid, said immunoassay comprising: (a) forming liposomes having the receptor for the analyte of interest incorporated onto the surface membrane thereof and a marker compound incorporated in the interior aqueous phase thereof; (b) providing a solid phase inert support having attached thereto the analyte of interest; (c) mixing said test fluid with the liposomes of step (a) for sufficient time to react with the binding sites on said liposomes with any analyte of interest present in said test fluid; (d) mixing said reacted liposomes from step (c) with said solid phase bound analyte of step (b) causing the lysis of said liposomes, without the addition of any membrane lytic molecules, ions, or active complement, and; (e) determining the presence of marker compound released by the liposomes in step (d).
Claim 24
Dependent Claim
142 words
An immunoassay for detecting or quantifying an analyte of interest in a test fluid, said immunoassay comprising: (a) forming liposomes having the receptor for the analyte of interest incorporated onto the surface membrane thereof and a marker compound incorporated in the interior aqueous phase thereof; (b) providing a solid phase inert support having attached thereto the analyte of interest; (c) mixing said test fluid with the liposomes of step (a) for sufficient time to react with the binding sites on said liposomes with any analyte of interest present in said test fluid; (d) mixing said reacted liposomes from step (c) with said solid phase bound analyte of step (b) causing the lysis of said liposomes, without the addition of any membrane lytic molecules, ions, or active complement, and; (e) determining the presence of marker compound released by the liposomes in step (d).
Claim 25
Dependent Claim
28 words
The immunoassay of claim 22, wherein step (e) further comprises quantifying the amount of marker compound released and determining the amount of analyte present in the test fluid.
Claim 26
Dependent Claim
27 words
The immunoassay of claim 22, wherein said liposomes are comprised of a mixture of an H II phase forming lipid and a receptor or a receptor-lipid complex.
Claim 27
Dependent Claim
12 words
The immunoassay of claim 22, wherein said solid phase support is glass.
Claim 28
Dependent Claim
13 words
The immunoassay of claim 22, wherein said solid phase support is a polymer.
Claim 29
Dependent Claim
122 words
An immunoassay kit, useful in the method of claim 22 for detecting the presence of an analyte in a test sample, said kit containing in combination: (a) a sufficient quantity of stable liposomes having a marker compound incorporated into the interior aqueous phase thereof, and a receptor for the analyte of interest incorporated onto the surface membrane thereof, wherein said liposomes are comprised of a mixture of an H II phase forming lipid and a receptor or receptor-lipid complex; (b) a solid support having a sufficient quantity of said analyte of interest bound thereto; and (c) a standard comprising a known amount of said analyte; said kit requiring the use of no membrane lytic molecules, ions, or active complement for its operation.
Claim 30
Dependent Claim
13 words
The immunoassay of claim 22, wherein said analyte of interest is an antibody.
Claim 31
Dependent Claim
11 words
The immunoassay of claim 28, wherein said receptor is an antigen.
Claim 32
Dependent Claim
14 words
The immunoassay of claim 22, wherein said marker compound is a self-quenching fluorescent dye.
Claim 33
Dependent Claim
11 words
The immunoassay of claim 30, wherein said self-quenching dye is calcein.
Claim 34
Dependent Claim
105 words
A system for the detection of an analyte of interest, using the method of claim 22, which system comprises: (a) a marker material encapsulated in the interior aqueous phase and a sufficient quantity of a receptor for the analyte of interest incorporated onto the surface membrane of liposomes, wherein said liposomes are comprised of a mixture of an H II phase forming lipid and a receptor or a receptor-lipid complex; and (b) a sufficient quantity of analyte of interest bound to a solid phase support; said system requiring the use of no membrane lytic molecules, ions, or active complement for the detection of said analyte.
Claim 35
Dependent Claim
21 words
The system of claim 32, wherein said liposomes are composed of dioleyl phosphatidylethanolamine and and a receptor or a receptor-lipid complex.
Claim 36
Dependent Claim
13 words
The system of claim 32, wherein the analyte of interest is an antibody.
Claim 37
Dependent Claim
16 words
The system of claim 34, wherein the receptor for the analyte of interest is an antigen.
Claim 38
Dependent Claim
12 words
The system of claim 32, wherein said solid phase support is glass.
Claim 39
Dependent Claim
15 words
The system of claim 36, wherein said glass support is in the form of beads.
Claim 40
Dependent Claim
18 words
The immunoassay of claim 6 or 24, wherein said H II phase forming lipid is an unsaturated phosphatidylethanolamine.
Claim 41
Dependent Claim
21 words
The immunoassay of claim 38, wherein said unsaturated phosphatidylethanolamine is selected from the group consisting of dioleyl phosphatidylethanolamine and egg phosphatidylethanolamine.
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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) | 1990-11-23 | 1991-05-25 | 1991-11-24 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1994-11-23 | 1995-05-25 | 1995-11-24 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1998-11-23 | 1999-05-25 | 1999-11-24 | $7700.00 | Window Closed / Lapsed |