US4517303A
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Specific binding assays utilizing analyte-cytolysin conjugates
Assignee: Dade Behring Inc
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Filing: 1982-10-20
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Granted: 1985-05-14
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Expires: 2002-10-20
Abstract
Abstract A novel analyte-cytolysin conjugate and its use in a lipid vesicle mediated measurement process is described for a wide variety of analytes present at very low concentration. The method involves forming a reaction system consisting of analyte, analyte specific binding agent, analyte-cytolysin conjugate, and vesicles containing detectable marker material in such proportions that uncombined conjugate alters the permeability of the vesicles resulting in the release and quantitative detection of marker material which can be correlated with the amount of analyte initially present.
Inventors
- 👤 J. William Freytag
- 👤 William J. Litchfield
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-01 14:12
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/543; G01N33/554; G01N33/58; G01N33/585; G01N33/586; G01N33/82; G01N33/94; G01N33/9453; Y; Y10; Y10S; Y10S435/00; Y10S435/966; Y10S435/972; Y10S436/00; Y10S436/803; Y10S436/811; Y10S436/813; Y10S436/815; Y10S436/817; Y10S436/827; Y10S436/828; Y10S436/829; Y10S930/00; Y10S930/01; Y10S930/28
Claims Analysis
Total Claims: 46
Claim 1
Independent Claim
39 words
A conjugate for the detection of an analyte in a test sample, comprising in combination: (1) cytolysin means for altering the permeability of a membrane of a vesicle, said means being conjugated to (2) at least one analyte molecule.
Claim 2
Dependent Claim
39 words
A conjugate for the detection of an analyte in a test sample, comprising in combination: (1) cytolysin means for altering the permeability of a membrane of a vesicle, said means being conjugated to (2) at least one analyte molecule.
Claim 3
Dependent Claim
49 words
The conjugate of claim 1 wherein the cytolysin is selected from the group consisting of aerolysin, amphotericin B, alamethicin, A-23187 (calcium ionophore), ATP translocase, cereolysin, diptheria toxin, filipin, gramicidin, melittin, nigericin, nystatin, polymyxin B, Staphylococcus aureus toxin α, β, γ, or δ, Streptolysin O, Streptolysin S, tubulin and valinomycin.
Claim 4
Dependent Claim
10 words
The conjugate of claim 1 wherein the cytolysin is melittin.
Claim 5
Dependent Claim
31 words
The conjugate of claim 3 wherein the attachment of analyte derivative is through an amino acid residue selected from the group consisting of lysine, serine, threonine, N-terminal glycine, and C-terminal glutamine.
Claim 6
Dependent Claim
35 words
The conjugate of claim 1 wherein the analyte derivative is a drug, metabolite, hormone, steroid, pesticide, environmental pollutant, food toxin, vitamin, protein, microbial surface marker, cancer cell marker, fungus, protozoan, virus, cell or tissue antigen.
Claim 7
Dependent Claim
12 words
The conjugate of claim 5 wherein the analyte derivative is a drug.
Claim 8
Dependent Claim
10 words
The conjugate of claim 6 wherein the drug is ouabain.
Claim 9
Dependent Claim
23 words
The conjugate of claim 4 wherein at least one ouabain molecule is attached to melittin through an ε-amino group of a lysine residue.
Claim 10
Dependent Claim
12 words
The conjugate of claim 5 wherein the analyte derivative is a vitamin.
Claim 11
Dependent Claim
10 words
The conjugate of claim 9 wherein the vitamin is biotin.
Claim 12
Dependent Claim
24 words
The conjugate of claim 4 wherein at least one biotin derivative molecule is attached to melittin through an ε-amino group of a lysine residue.
Claim 13
Dependent Claim
147 words
An analyte-cytolysin conjugate comprising a substituted melittin molecule of the formula: ##STR1## wherein at least one of R 1 through R 8 is an analyte derivative molecule selected from the group consisting of drug, vitamin, hormone, metabolite, steroid, pesticide, environmental pollutant, food toxin, protein, microbial surface marker, cancer cell marker, fungus, protozoan, cell or tissue antigen, provided that when at least one of R 2 , R 6 , or R 7 is said analyte derivative, attachment is through an ε-amino group of the amino acid side chain; when at least one of R 3 , R 4 , or R 5 is said analyte derivative, attachment is through a hydroxyl group of said amino acid side chain; when R 1 is said analyte derivative, attachment is through the N-terminal amino group; and when R 8 is said analyte derivative, attachment is through the C-terminal carboxyl group.
Claim 14
Dependent Claim
147 words
An analyte-cytolysin conjugate comprising a substituted melittin molecule of the formula: ##STR1## wherein at least one of R 1 through R 8 is an analyte derivative molecule selected from the group consisting of drug, vitamin, hormone, metabolite, steroid, pesticide, environmental pollutant, food toxin, protein, microbial surface marker, cancer cell marker, fungus, protozoan, cell or tissue antigen, provided that when at least one of R 2 , R 6 , or R 7 is said analyte derivative, attachment is through an ε-amino group of the amino acid side chain; when at least one of R 3 , R 4 , or R 5 is said analyte derivative, attachment is through a hydroxyl group of said amino acid side chain; when R 1 is said analyte derivative, attachment is through the N-terminal amino group; and when R 8 is said analyte derivative, attachment is through the C-terminal carboxyl group.
Claim 15
Dependent Claim
23 words
The conjugate of claim 12 wherein at least one of R 2 , R 6 , or R 7 is a ouabain derivative.
Claim 16
Dependent Claim
23 words
The conjugate of claim 12 wherein at least one of R 2 , R 6 , or R 7 is a biotin derivative.
Claim 17
Independent Claim
89 words
A method for determining the amount of an analyte in liquid medium comprising the steps of: (A) forming a reaction system by contacting said liquid medium with (1) analyte specific binding agent; (2) analyte-cytolysin conjugate; and (3) vesicles containing marker material sequestered within the vesicle in proportions such that the interaction of unbound analyte-cytolysin conjugate with said vesicles results in the release of said marker material; and (B) measuring said marker material thereby released which is related to the amount of said analyte initially present in said liquid medium.
Claim 18
Independent Claim
89 words
A method for determining the amount of an analyte in liquid medium comprising the steps of: (A) forming a reaction system by contacting said liquid medium with (1) analyte specific binding agent; (2) analyte-cytolysin conjugate; and (3) vesicles containing marker material sequestered within the vesicle in proportions such that the interaction of unbound analyte-cytolysin conjugate with said vesicles results in the release of said marker material; and (B) measuring said marker material thereby released which is related to the amount of said analyte initially present in said liquid medium.
Claim 19
Dependent Claim
34 words
The method of claim 15 wherein the analyte is a drug, metabolite, hormone, steroid, pesticide, environmental pollutant, food toxin, vitamin, protein, microbial surface marker, cancer cell marker, fungus, protozoan, virus, cell or tissue antigen.
Claim 20
Dependent Claim
11 words
The method of claim 15 wherein the analyte is a drug.
Claim 21
Dependent Claim
10 words
The method of claim 17 wherein the drug is digoxin.
Claim 22
Dependent Claim
11 words
The method of claim 15 wherein the analyte is a vitamin.
Claim 23
Dependent Claim
10 words
The method of claim 19 wherein the vitamin is biotin.
Claim 24
Dependent Claim
24 words
The method of claim 15 wherein the analyte binding agent is selected from the group consisting of antibody, hormone-receptor, lectin, and specific binding protein.
Claim 25
Dependent Claim
12 words
The method of claim 21 wherein the binding agent is an antibody.
Claim 26
Dependent Claim
12 words
The method of claim 22 wherein the antibody is specific for digoxin.
Claim 27
Dependent Claim
14 words
The method of claim 21 wherein the binding agent is a specific binding protein.
Claim 28
Dependent Claim
12 words
The method of claim 24 wherein the specific binding protein is avidin.
Claim 29
Dependent Claim
11 words
The method of claim 15 wherein the analyte-cytolysin conjugate is ouabain-melittin.
Claim 30
Dependent Claim
11 words
The method of claim 15 wherein the analyte-cytolysin conjugate is biotin-melittin.
Claim 31
Dependent Claim
30 words
The method of claim 15 wherein the vesicle utilized to sequester marker material is selected from the group consisting of lipid vesicle, red blood cell, and red blood cell ghost.
Claim 32
Dependent Claim
12 words
The method of claim 28 wherein the vesicle is a lipid vesicle.
Claim 33
Dependent Claim
26 words
The method of claim 15 wherein the marker material is a substance selected from the group consisting of enzyme, cofactor, chromophore, fluorophore, spin label, and ion.
Claim 34
Dependent Claim
12 words
The method of claim 29 wherein the marker material is an enzyme.
Claim 35
Dependent Claim
11 words
The method of claim 30 wherein the enzyme is alkaline phosphatase.
Claim 36
Dependent Claim
10 words
The method of claim 30 wherein the enzyme is β-galactosidase.
Claim 37
Dependent Claim
10 words
The method of claim 30 wherein the fluorophore is 6-carboxyfluorescein.
Claim 38
Independent Claim
94 words
A method for determining the amount of digoxin in liquid medium comprising the steps of: (A) forming a reaction system by contacting said liquid medium with (1) digoxin specific antibody; (2) ouabain-melittin conjugate; (3) lipid vesicles containing sequestered alkaline phosphatase; and (4) p-nitrophenyl phosphate in proportions such that the interaction of unbound ouabain-melittin conjugate and said lipid vesicles results in the conversion of said p-nitrophenyl phosphate by said alkaline phosphatase; and (B) measuring the concentration of p-nitrophenlate anion produced which is related to the amount of said digoxin initially present in said liquid medium.
Claim 39
Independent Claim
94 words
A method for determining the amount of digoxin in liquid medium comprising the steps of: (A) forming a reaction system by contacting said liquid medium with (1) digoxin specific antibody; (2) ouabain-melittin conjugate; (3) lipid vesicles containing sequestered alkaline phosphatase; and (4) p-nitrophenyl phosphate in proportions such that the interaction of unbound ouabain-melittin conjugate and said lipid vesicles results in the conversion of said p-nitrophenyl phosphate by said alkaline phosphatase; and (B) measuring the concentration of p-nitrophenlate anion produced which is related to the amount of said digoxin initially present in said liquid medium.
Claim 40
Dependent Claim
43 words
A conjugate for the detection of an analyte in a test sample, comprising in combination: (1) cytolysin means for altering the permeability of a membrane of a vesicle, said means being conjugaed to (2) an antibody molecule capable of binding to the analyte.
Claim 41
Dependent Claim
43 words
A conjugate for the detection of an analyte in a test sample, comprising in combination: (1) cytolysin means for altering the permeability of a membrane of a vesicle, said means being conjugaed to (2) an antibody molecule capable of binding to the analyte.
Claim 42
Dependent Claim
24 words
The conjugate of claim 36 wherein the antibody molecule is a monovalent antibody molecule selected from the group consisting of Fab, Fab', and half-molecules.
Claim 43
Dependent Claim
49 words
The conjugate of claim 36 wherein the cytolysin is selected from the group consisting of aerolysin, amphotericin B, alamethicin, A-23187 (calcium ionophore), ATP translocase, cerolysin, diptheria toxin, filipin, gramicidin, melittin, nigericin, nystatin, polymyxin B, Staphylococcus aureus toxin α, β, γ, or δ, Streptolysin O, Streptolysin S, tubulin and valinomycin.
Claim 44
Dependent Claim
10 words
The conjugate of claim 8 wherein the cytolysin is mellitin.
Claim 45
Dependent Claim
132 words
A heterogeneous immunoassay for detecting an analyte in a liquid sample, comprising: (1) forming a reaction mixture by contacting the sample with a cytolysin/anti-analyte antibody conjugate in immunochemical excess over the analyte, whereby a fraction of said conjugate binds to analyte to form a complex and a fraction of said conjugate remains free, (2) contacting the reaction mixture with a solid phase having analyte or analyte-analog bound thereto to separate the complex from the free conjugate, and (3) contacting either the complex or the free conjugate with vesicles having marker material sequestered therein, whereby the permeability of the vesicle increases, thereby releasing marker material, (4) measuring the amount of released marker material, and (5) relating the amount of released marker material to the amount of analyte initially present in the test sample.
Claim 46
Dependent Claim
132 words
A heterogeneous immunoassay for detecting an analyte in a liquid sample, comprising: (1) forming a reaction mixture by contacting the sample with a cytolysin/anti-analyte antibody conjugate in immunochemical excess over the analyte, whereby a fraction of said conjugate binds to analyte to form a complex and a fraction of said conjugate remains free, (2) contacting the reaction mixture with a solid phase having analyte or analyte-analog bound thereto to separate the complex from the free conjugate, and (3) contacting either the complex or the free conjugate with vesicles having marker material sequestered therein, whereby the permeability of the vesicle increases, thereby releasing marker material, (4) measuring the amount of released marker material, and (5) relating the amount of released marker material to the amount of analyte initially present in the test sample.
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) | 1988-05-13 | 1988-11-12 | 1989-05-14 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1992-05-13 | 1992-11-12 | 1993-05-14 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1996-05-13 | 1996-11-12 | 1997-05-14 | $7700.00 | Window Closed / Lapsed |