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US4780409A
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Thermally induced phase separation immunoassay
Assignee: Genetic Systems Corp
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Filing: 1986-04-28
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Granted: 1988-10-25
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Expires: 2006-04-28
Abstract
Abstract An immunoassay in which a thermally induced phase separation is used to effect the separation of specifically bound reactants from free reactants is disclosed. A first reactant is conjugated to a temperature-sensitive polymer to form a polymer/reactant conjugate, and a second reactant is conjugated to a reporter to form a reporter/reactant conjugate. The polymer/reactant, reporter/reactant, and biological fluid samples suspected of containing the analyte are admixed in solution at a temperature other than that at which the polymer will precipitate. Specific binding is allowed to occur, thereby forming a ternary complex. The salt concentration of the adjusted solution is then adjusted to a concentration sufficient to cause the complex to precipitate from the solution, the amount of reporter activity in the precipitated complex or in the solution measured and the presence and/or concentration of the analyte therefrom determined. Alternatively, the first reactant may be conjugated to a monomer and subsequently copolymerized with additional monomers to yield a temperature-sensitive copolymer. Multiple analyses may also be performed on a single sample by choosing a variety of polymers, each polymer having a different specific binding partner conjugated thereto and a different critical solution temperature. By altering the temperature and/or the salt concentration of the solution incrementally, the reporter associated with each of the complexes precipitated with each temperature or concentration increment may be measured, and the presence and/or concentration of each of the analytes determined.
Inventors
- 👤 Nobuo Monji
- 👤 Allan S. Hoffman
- 👤 John H. Priest
- 👤 Raymond L. Houghton
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-24 13:48
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/543; G01N33/544; G01N33/545; G01N33/531; G01N33/536; G01N33/537; G01N33/5375; G01N33/569; G01N33/56911; G01N33/56927; Y; Y10; Y10S; Y10S435/00; Y10S435/971; Y10S435/973; Y10S436/00; Y10S436/824; Y10S436/827
Claims Analysis
Total Claims: 119
Claim 1
Independent Claim
167 words
A method for determining the presence and/or concentration of an analyte in a biological fluid sample, comprising: conjugating a first reactant which is capable of specifically binding with said analyte to a temperature-sensitive polymer to form a polymer/reactant conjugate; conjugating a second reactant which is capable of specifically binding with said analyte to a reporter to form a reporter/reactant conjugate; admixing in solution said polymer/reactant, reporter/reactant and said biological fluid sample suspected of containing said analyte at a temperature other than that at which said polymer will precipitate such that specific binding occurs between said first and second reactants and said analyte, thereby forming a ternary complex; adjusting the salt concentration of the admixed polymer/reactant, reporter/reactant, and analyte containing sample solution to a concentration sufficient to alter the critical solution temperature of the polymer, such that said complex precipitates from the solution; and measuring the amount of reporter activity in the precipitated complex or in the solution and therefrom determining the presence and/or concentration of said analyte.
Claim 2
Independent Claim
167 words
A method for determining the presence and/or concentration of an analyte in a biological fluid sample, comprising: conjugating a first reactant which is capable of specifically binding with said analyte to a temperature-sensitive polymer to form a polymer/reactant conjugate; conjugating a second reactant which is capable of specifically binding with said analyte to a reporter to form a reporter/reactant conjugate; admixing in solution said polymer/reactant, reporter/reactant and said biological fluid sample suspected of containing said analyte at a temperature other than that at which said polymer will precipitate such that specific binding occurs between said first and second reactants and said analyte, thereby forming a ternary complex; adjusting the salt concentration of the admixed polymer/reactant, reporter/reactant, and analyte containing sample solution to a concentration sufficient to alter the critical solution temperature of the polymer, such that said complex precipitates from the solution; and measuring the amount of reporter activity in the precipitated complex or in the solution and therefrom determining the presence and/or concentration of said analyte.
Claim 3
Dependent Claim
18 words
The method of claim 1 including, after the step of adjusting, separating the precipitated complex from the solution.
Claim 4
Dependent Claim
21 words
The method of claim 1 including, after the step of conjugating said first reactant to said polymer, purifying said polymer/reactant conjugate.
Claim 5
Dependent Claim
16 words
The method of claim 1 wherein said polymer is characterized by a lower critical solution temperature.
Claim 6
Dependent Claim
31 words
The method of claim 2 including, after the step of separating said precipitated complex from said solution, redissolving the precipitated complex in a substantially smaller volume than the original solution volume.
Claim 7
Dependent Claim
37 words
The method of claim 1 wherein said analyte is capable of simultaneously reacting with said first and second reactants and is one selected from the group consisting of drugs, vitamins, hormones, proteins, metabolites, cells, viruses, and microorganisms.
Claim 8
Dependent Claim
28 words
The method of claim 1 wherein said first and second reactants are selected from the group consisting of antibodies, antigens, lectins, receptors, transport proteins and non-immunoglobulin antibody-binding proteins.
Claim 9
Dependent Claim
26 words
The method of claim 1 wherein the salt is selected from the group consisting of ammonium sulfate, sodium sulfate, sodium carbonate, potassium carbonate, and sodium phosphate.
Claim 10
Dependent Claim
11 words
The method of claim 1 wherein the salt is ammonium sulfate.
Claim 11
Dependent Claim
23 words
The method of claim 1 wherein said reporter is one selected from the group consisting of enzymes, fluorophores, radioisotopes, luminescers and dye particles.
Claim 12
Dependent Claim
10 words
The method of claim 1 wherein said polymer is poly-N-isopropylacrylamide.
Claim 13
Dependent Claim
22 words
The method of claim 1 wherein said polymer is a copolymer formed from monomers selected to achieve a desired critical solution temperature.
Claim 14
Dependent Claim
17 words
The method of claim 12 wherein said copolymer is formed from N-isopropylacrylamide monomers copolymerized with acrylamide monomers.
Claim 15
Dependent Claim
34 words
The method of claim 12 wherein said copolymer is formed from N-isopropylacrylamide monomers copolymerized with monomers selected from the group consisting of acrylamide monomers, N-ethyl acrylamide monomers, N-methyl acrylamide monomers, and N-isopropyl acrylamide monomers.
Claim 16
Dependent Claim
25 words
The method of claim 1 wherein said first reactant is conjugated to a monomer and subsequently copolymerized with additional monomers to yield a temperature-sensitive copolymer.
Claim 17
Dependent Claim
45 words
The method of claim 15 wherein said first reactant is conjugated to the N-hydroxysuccinimide ester of acrylic acid and the resultant monomerized reactant copolymerized with a monomer selected from the group consisting of acrylamide monomers, N-isopropyl acrylamide monomers, N-ethyl acrylamide monomers, and N-methyl acrylamide monomers.
Claim 18
Dependent Claim
45 words
The method of claim 15 wherein said first reactant is conjugated to the N-hydroxysuccinimide ester of vinyl benzoate and the resultant monomerized reactant copolymerized with a monomer selected from the group consisting of acrylamide monomers, N-isopropyl acrylamide monomers, N-ethyl acrylamide monomers and N-methyl acrylamide monomers.
Claim 19
Independent Claim
233 words
A method for conducting multiple analyses on a single biological fluid sample suspected of containing one or more analytes, comprising: conjugating a plurality of selected first reactants, each of said first reactants being capable of specifically binding with one of said analytes, to a plurality of temperature-sensitive polymers, each of said first reactants being conjugated to a polymer having a critical solution temperature, thereby forming multiple polymer/reactant conjugates; conjugating a plurality of selected second reactants, each of said second reactants being capable of specifically binding with one of said analytes, to a reporter to form multiple reporter/reactant conjugates; admixing in solution said multiple polymer/reactant, multiple reporter/reactant, and said biological fluid sample suspected of containing one or more analytes at a temperature other than that at which any of said polymers will precipitate such that specific binding occurs between said reactants and said analytes, thereby forming a plurality of ternary complexes; adjusting the salt concentration of the admixed polymer/reactant, reporter/reactant, and analyte sample containing solution incrementally to alter the critical solution temperature of the polymers such that the complex precipitated with each concentration increment can be separated from said solution prior to the precipitation of a complex with a different critical solution temperature; and measuring the amount of the respective reporter activity in each of the precipitated complexes or in the solution and therefrom determining the presence and/or concentration of each of said analytes.
Claim 20
Independent Claim
233 words
A method for conducting multiple analyses on a single biological fluid sample suspected of containing one or more analytes, comprising: conjugating a plurality of selected first reactants, each of said first reactants being capable of specifically binding with one of said analytes, to a plurality of temperature-sensitive polymers, each of said first reactants being conjugated to a polymer having a critical solution temperature, thereby forming multiple polymer/reactant conjugates; conjugating a plurality of selected second reactants, each of said second reactants being capable of specifically binding with one of said analytes, to a reporter to form multiple reporter/reactant conjugates; admixing in solution said multiple polymer/reactant, multiple reporter/reactant, and said biological fluid sample suspected of containing one or more analytes at a temperature other than that at which any of said polymers will precipitate such that specific binding occurs between said reactants and said analytes, thereby forming a plurality of ternary complexes; adjusting the salt concentration of the admixed polymer/reactant, reporter/reactant, and analyte sample containing solution incrementally to alter the critical solution temperature of the polymers such that the complex precipitated with each concentration increment can be separated from said solution prior to the precipitation of a complex with a different critical solution temperature; and measuring the amount of the respective reporter activity in each of the precipitated complexes or in the solution and therefrom determining the presence and/or concentration of each of said analytes.
Claim 21
Dependent Claim
31 words
The method of claim 18 including, after the step of adjusting the salt concentration of the solution, separating each of said complexes from the solution as the concentration is altered incrementally.
Claim 22
Dependent Claim
21 words
The method of claim 18 including, after the step of conjugating said first reactants to said polymers, purifying said polymer/reactant conjugates.
Claim 23
Dependent Claim
16 words
The method of claim 18 wherein said polymer is characterized by a lower critical solution temperature.
Claim 24
Dependent Claim
31 words
The method of claim 19 including, after the step of separating said precipitated complexes from said solution, redissolving the precipitated complexes in a substantially smaller volume than the original solution volume.
Claim 25
Dependent Claim
26 words
The method of claim 18 wherein the salt is selected from the group consisting of ammonium sulfate, sodium sulfate, sodium carbonate, potassium carbonate, and sodium phosphate.
Claim 26
Dependent Claim
11 words
The method of claim 18 wherein the salt is ammonium sulfate.
Claim 27
Dependent Claim
37 words
The method of claim 18 wherein said analyte is capable of simultaneously reacting with said first and second reactants and is one selected from the group consisting of drugs, vitamins, hormones, proteins, metabolites, cells, viruses, and microorganisms.
Claim 28
Dependent Claim
28 words
The method of claim 18 wherein said first and second reactants are selected from the group consisting of antibodies, antigens, lectins, receptors, transport proteins, and non-immunoglobulin antibody-binding proteins.
Claim 29
Dependent Claim
23 words
The method of claim 18 wherein said reporter is one selected from the group consisting of enzymes, fluorophores, radioisotopes, luminescers and dye particles.
Claim 30
Dependent Claim
10 words
The method of claim 18 wherein said polymer is poly-N-isopropylacrylamide.
Claim 31
Dependent Claim
22 words
The method of claim 18 wherein said polymer is a copolymer formed from monomers selected to achieve a desired critical solution temperature.
Claim 32
Dependent Claim
17 words
The method of claim 29 wherein said copolymer is formed from N-isopropylacrylamide monomers copolymerized with acrylamide monomers.
Claim 33
Dependent Claim
34 words
The method of claim 29 wherein said copolymer is formed from N-isopropylacrylamide monomers copolymerized with monomers selected from the group consisting of acrylamide monomers, N-ethyl acrylamide monomers, N-methyl acrylamide monomers, and N-isopropyl acrylamide monomers.
Claim 34
Dependent Claim
25 words
The method of claim 18 wherein said first reactant is conjugated to a monomer and subsequently copolymerized with additional monomers to yield a temperature-sensitive copolymer.
Claim 35
Dependent Claim
45 words
The method of claim 32 wherein said first reactant is conjugated to the N-hydroxysuccinimide ester of acrylic acid and the resultant monomerized reactant copolymerized with a monomer selected from the group consisting of acrylamide monomers, N-isopropyl acrylamide monomers, N-ethyl acrylamide monomers, and N-methyl acrylamide monomers.
Claim 36
Dependent Claim
44 words
The method of claim 32 wherein said first reactant is conjugated to the N-hydroxysuccinimide ester of vinyl benzoate and the resultant monomerized reactant copolymerized with a monomer selected from the group consisting of acrylamide monomers, N-isopropylacrylamide monomers, N-ethyl acrylamide monomers, and N-methyl acrylamide monomers.
Claim 37
Independent Claim
179 words
A method for determining the presence and/or concentration of an analyte in a biological fluid sample, comprising: conjugating a first reactant which is capable of specifically binding with said analyte to a monomer to form a monomer/reactant conjugate; copolymerizing said monomer/reactant conjugate with additional monomers to yield a temperature-sensitive copolymer/reactant; conjugating a second reactant which is capable of specifically binding with said analyte to a reporter to form a reporter/reactant conjugate; admixing in solution said copolymer/reactant, reporter/reactant, and said biological fluid sample suspected of containing said analyte at a temperature other than that at which said copolymer will precipitate such that specific binding occurs between said first and second reactants and said analyte, thereby forming a ternary complex; adjusting the salt concentration of the admixed copolymer/reactant, reporter/reactant, and analyte containing sample solution to a salt concentration sufficient to alter the critical solution temperature of the copolymer, such that said complex precipitates from the solution; and measuring the amount of reporter activity in the precipitated complex or in the solution and therefrom determining the presence and/or concentration of said analyte.
Claim 38
Independent Claim
179 words
A method for determining the presence and/or concentration of an analyte in a biological fluid sample, comprising: conjugating a first reactant which is capable of specifically binding with said analyte to a monomer to form a monomer/reactant conjugate; copolymerizing said monomer/reactant conjugate with additional monomers to yield a temperature-sensitive copolymer/reactant; conjugating a second reactant which is capable of specifically binding with said analyte to a reporter to form a reporter/reactant conjugate; admixing in solution said copolymer/reactant, reporter/reactant, and said biological fluid sample suspected of containing said analyte at a temperature other than that at which said copolymer will precipitate such that specific binding occurs between said first and second reactants and said analyte, thereby forming a ternary complex; adjusting the salt concentration of the admixed copolymer/reactant, reporter/reactant, and analyte containing sample solution to a salt concentration sufficient to alter the critical solution temperature of the copolymer, such that said complex precipitates from the solution; and measuring the amount of reporter activity in the precipitated complex or in the solution and therefrom determining the presence and/or concentration of said analyte.
Claim 39
Dependent Claim
18 words
The method of claim 35 including, after the step of adjusting, separating the precipitated complex from the solution.
Claim 40
Dependent Claim
31 words
The method of claim 36 including, after the step of separating said precipitated complex from said solution, redissolving the precipitated complex in a substantially smaller volume that the original solution volume.
Claim 41
Dependent Claim
37 words
The method of claim 35 wherein said analyte is capable of simultaneously reacting with said first and second reactants and is one selected from the group consisting of drugs, vitamins, hormones, proteins, metabolites, cells, viruses, and microorganisms.
Claim 42
Dependent Claim
28 words
The method of claim 35 wherein said first and second reactants are selected from the group consisting of antibodies, antigens, lectins, receptors, transport proteins and non-immunoglobulin antibody-binding proteins.
Claim 43
Dependent Claim
23 words
The method of claim 35 wherein said reporter is one selected from the group consisting of enzymes, fluorophores, radioisotopes, luminescers and dye particles.
Claim 44
Dependent Claim
26 words
The method of claim 35 wherein the salt is selected from the group consisting of ammonium sulfate, sodium sulfate, sodium carbonate, potassium carbonate, and sodium phosphate.
Claim 45
Dependent Claim
11 words
The method of claim 35 wherein the salt is ammonium sulfate.
Claim 46
Dependent Claim
17 words
The method of claim 35 wherein said polymer is one characterized by a lower critical solution temperature.
Claim 47
Dependent Claim
44 words
The method of claim 35 wherein said first reactant is conjugated to the N-hydroxysuccinimide ester of acrylic acid and the resultant monomerized reactant copolymerized with a monomer selected from the group consisting of acrylamide monomers, N-isopropylacrylamide monomers, N-ethyl acrylamide monomers, and N-methyl acrylamide monomers.
Claim 48
Dependent Claim
44 words
The method of claim 35 wherein said first reactant is conjugated to the N-hydroxysuccinimie ester of vinyl benzoate and the resultant monomerized reactant copolymerized with a monomer selected from the group consisting of acrylamide monomers, N-isopropylacrylamide monomers, N-ethyl acrylamide monomers, and N-methyl acrylamide monomers.
Claim 49
Independent Claim
225 words
A method for conducting multiple analyses on a single biological fluid sample suspected of containing one or more analytes, comprising: conjugating a plurality of selected first reactants, each of said first reactants being capable of specifically binding with one of said analytes to a plurality of temperature-sensitive polymers, each of said first reactants being conjugated to a polymer having a critical solution temperature, thereby forming multiple polymer/reactant conjugates; conjugating a plurality of selected second reactants, each of said second reactants being capable of specifically binding with one of said analytes, to one or more reporters to form multiple reporter/reactant conjugates; admixing in solution said multiple polymer/reactant, multiple reporter/reactant, and said biological fluid sample suspected of containing one or more analytes at a temperature other than that at which any of said polymers will precipitate such that specific binding occurs between said reactants and said analytes, thereby forming a plurality of ternary complexes; altering the temperature of the admixed polymer/reactant, reporter/reactant, and analyte sample containing solution incrementally such that the complex precipitated with each temperature increment can be separated from said solution prior to the precipitation of a complex with a different critical solution temperature; and measuring the amount of the respective reporter activity in each of the precipitated complexes or in the solution and therefrom determining the presence and/or concentration of each of said analytes.
Claim 50
Independent Claim
225 words
A method for conducting multiple analyses on a single biological fluid sample suspected of containing one or more analytes, comprising: conjugating a plurality of selected first reactants, each of said first reactants being capable of specifically binding with one of said analytes to a plurality of temperature-sensitive polymers, each of said first reactants being conjugated to a polymer having a critical solution temperature, thereby forming multiple polymer/reactant conjugates; conjugating a plurality of selected second reactants, each of said second reactants being capable of specifically binding with one of said analytes, to one or more reporters to form multiple reporter/reactant conjugates; admixing in solution said multiple polymer/reactant, multiple reporter/reactant, and said biological fluid sample suspected of containing one or more analytes at a temperature other than that at which any of said polymers will precipitate such that specific binding occurs between said reactants and said analytes, thereby forming a plurality of ternary complexes; altering the temperature of the admixed polymer/reactant, reporter/reactant, and analyte sample containing solution incrementally such that the complex precipitated with each temperature increment can be separated from said solution prior to the precipitation of a complex with a different critical solution temperature; and measuring the amount of the respective reporter activity in each of the precipitated complexes or in the solution and therefrom determining the presence and/or concentration of each of said analytes.
Claim 51
Dependent Claim
30 words
The method of claim 46 including, after the step of altering the temperature of the solution, separating each of said complexes from the solution as the temperature is altered incrementally.
Claim 52
Dependent Claim
21 words
The method of claim 46 including, after the step of conjugating said first reactants to said polymers, purifying said polymer/reactant conjugates.
Claim 53
Dependent Claim
31 words
The method of claim 47 including, after the step of separating said precipitated complexes from said solution, redissolving the precipitated complexes in a substantially smaller volume than the original solution volume.
Claim 54
Dependent Claim
37 words
The method of claim 46 wherein said analyte is capable of simultaneously reacting with said first and second reactants and is one selected from the group consisting of drugs, vitamins, hormones, proteins, metabolites, cells, viruses, and microorganisms.
Claim 55
Dependent Claim
28 words
The method of claim 46 wherein said first and second reactants are selected from the group consisting of antibodies, antigens, lectins, receptors, transport proteins, and non-immunoglobulin antibody-binding proteins.
Claim 56
Dependent Claim
23 words
The method of claim 46 wherein said reporter is one selected from the group consisting of enzymes, fluorophores, radioisotopes, luminescers and dye particles.
Claim 57
Dependent Claim
26 words
The method of claim 46 wherein said polymer is one characterized by a lower critical solution temperature, thereby precipitating upon heating said solution above said temperature.
Claim 58
Dependent Claim
28 words
The method of claim 53 wherein said polymer is one selected from the group consisting of polyvinyl methyether, polyvinylmethyl oxazolidone, polymethacrylic acid, poly-N-isopropylacrylamide, hydroxypropyl cellulose and methyl cellulose.
Claim 59
Dependent Claim
10 words
The method of claim 53 wherein said polymer is poly-N-isopropylacrylamide.
Claim 60
Dependent Claim
26 words
The method of claim 46 wherein said polymer is one characterized by an upper critical solution temperature, thereby precipitating upon cooling said solution below said temperature.
Claim 61
Dependent Claim
24 words
The method of claim 56 wherein said polymer is one selected from the group consisting of polyethylene glycol, polyacrylic acid, polymethacrylamide and polyvinyl alcohol.
Claim 62
Dependent Claim
24 words
The method of claim 53 or 56 wherein said polymer is a copolymer formed from monomers selected to achieve a desired critical solution temperature.
Claim 63
Dependent Claim
17 words
The method of claim 58 wherein said copolymer is formed from N-isopropylacrylamide monomers copolymerized with acrylamide monomers.
Claim 64
Dependent Claim
34 words
The method of claim 58 wherein said copolymer is formed from N-isopropylacrylamide monomers copolymerized with monomers selected from the group consisting of acrylamide monomers, N-ethyl acrylamide monomers, N-methyl acrylamide monomers, and N-isopropyl acrylamide monomers.
Claim 65
Dependent Claim
25 words
The method of claim 46 wherein said first reactant is conjugated to a monomer and subsequently copolymerized with additional monomers to yield a temperature-sensitive copolymer.
Claim 66
Dependent Claim
45 words
The method of claim 61 wherein said first reactant is conjugated to the N-hydroxysuccinimide ester of acrylic acid and the resultant monomerized reactant copolymerized with a monomer selected from the group consisting of acrylamide monomers, N-isopropyl acrylamide monomers, N-ethyl acrylamide monomers, and N-methyl acrylamide monomers.
Claim 67
Dependent Claim
45 words
The method of claim 61 wherein said first reactant is conjugated to the N-hydroxysuccinimide ester of vinyl benzoate and the resultant monomerized reactant copolymerized a monomer selected from the group consisting of with acrylamide monomers, N-isopropyl acrylamide monomers, N-ethyl acrylamide monomers, and N-methyl acrylamide monomers.
Claim 68
Independent Claim
167 words
A method for determining the presence and/or concentration of a member of the genus Chlamydia in a biological fluid sample, comprising: providing a temperature-sensitive polymer characterized by a critical solution temperature that is capable of binding to the elementary bodies of a member of the genus Chlamydia; conjugating a first reactant which is capable of specifically binding with said member to a reporter to form a reporter/reactant conjugate; admixing in solution said polymer, reporter/reactant and said biological fluid sample suspected of containing said member at a temperature other than that at which said polymer will precipitate such that binding occurs between said polymer and said first reactant and said member, thereby forming a ternary complex; incubating the admixed polymer, reporter/reactant, and member containing sample solution at a temperature sufficient to cause said complex to precipitate from the solution; and measuring the amount of reporter activity in the precipitated complex or in the solution and therefrom determining the presence and/or concentration of said member of the genus Chlamydia.
Claim 69
Independent Claim
167 words
A method for determining the presence and/or concentration of a member of the genus Chlamydia in a biological fluid sample, comprising: providing a temperature-sensitive polymer characterized by a critical solution temperature that is capable of binding to the elementary bodies of a member of the genus Chlamydia; conjugating a first reactant which is capable of specifically binding with said member to a reporter to form a reporter/reactant conjugate; admixing in solution said polymer, reporter/reactant and said biological fluid sample suspected of containing said member at a temperature other than that at which said polymer will precipitate such that binding occurs between said polymer and said first reactant and said member, thereby forming a ternary complex; incubating the admixed polymer, reporter/reactant, and member containing sample solution at a temperature sufficient to cause said complex to precipitate from the solution; and measuring the amount of reporter activity in the precipitated complex or in the solution and therefrom determining the presence and/or concentration of said member of the genus Chlamydia.
Claim 70
Dependent Claim
15 words
The method of claim 64 wherein said member of the genus Chlamydia is Chlamydia trachomatis.
Claim 71
Dependent Claim
10 words
The method of claim 64 wherein said polymer is poly-N-isopropylacrylamide.
Claim 72
Dependent Claim
18 words
The method of claim 64 including, after the step of incubating, separating the precipitated complex from the solution.
Claim 73
Dependent Claim
22 words
The method of claim 64 wherein said reporter is selected from the group consisting of enzymes, fluorophores, radioisotopes, luminescers, and dye particles.
Claim 74
Dependent Claim
12 words
The method of claim 64 wherein said first reactant is an antibody.
Claim 75
Independent Claim
253 words
A method for conducting multiple analyses on a single biological fluid sample suspected of containing one or more analytes, comprising: conjugating a plurality of selected first reactants, each of said first reactants being capable of specifically binding with one of said analytes, to a plurality of temperature-sensitive polymers, each of said first reactants being conjugated to a polymer having a critical solution temperature, thereby forming multiple polymer/reactant conjugates; conjugating a plurality of selected second reactants, each of said second reactants being capable of specifically binding with one of said analytes, to one or more reporters to form multiple reporter/reactant conjugates; admixing in solution said multiple polymer/reactant, multiple reporter/reactant, and said biological fluid sample suspected of containing one or more analytes at a temperature other than that at which any of said polymers will precipitate such that specific binding occurs between said reactants and said analytes, thereby forming a plurality of ternary complexes; selectively modifying the conditions of the admixed polymer/reactant, reporter/reactant, and analyte sample containing solution by incubating the admixed solution and adjusting the salt concentration of the admixed solution at least once to alter the critical solution temperature of the polymers such that the complex precipitated with each temperature or concentration increment can be separated from said solution prior to the precipitation of a complex with a different critical solution temperature; and measuring the amount of the respective reporter activity in each of the precipitated complexes or in the solution and therefrom determining the presence and/or concentration of each of said analytes.
Claim 76
Independent Claim
253 words
A method for conducting multiple analyses on a single biological fluid sample suspected of containing one or more analytes, comprising: conjugating a plurality of selected first reactants, each of said first reactants being capable of specifically binding with one of said analytes, to a plurality of temperature-sensitive polymers, each of said first reactants being conjugated to a polymer having a critical solution temperature, thereby forming multiple polymer/reactant conjugates; conjugating a plurality of selected second reactants, each of said second reactants being capable of specifically binding with one of said analytes, to one or more reporters to form multiple reporter/reactant conjugates; admixing in solution said multiple polymer/reactant, multiple reporter/reactant, and said biological fluid sample suspected of containing one or more analytes at a temperature other than that at which any of said polymers will precipitate such that specific binding occurs between said reactants and said analytes, thereby forming a plurality of ternary complexes; selectively modifying the conditions of the admixed polymer/reactant, reporter/reactant, and analyte sample containing solution by incubating the admixed solution and adjusting the salt concentration of the admixed solution at least once to alter the critical solution temperature of the polymers such that the complex precipitated with each temperature or concentration increment can be separated from said solution prior to the precipitation of a complex with a different critical solution temperature; and measuring the amount of the respective reporter activity in each of the precipitated complexes or in the solution and therefrom determining the presence and/or concentration of each of said analytes.
Claim 77
Dependent Claim
30 words
The method of claim 70 including, after the step of selectively modifying the conditions, separating each of said complexes from the solution as the temperature or concentration is altered incrementally.
Claim 78
Dependent Claim
21 words
The method of claim 70 including, after the step of conjugating said first reactants to said polymers, purifying said polymer/reactant conjugates.
Claim 79
Dependent Claim
17 words
The method of claim 70 wherein said polymer is one characterized by a lower critical solution temperature.
Claim 80
Dependent Claim
31 words
The method of claim 71 including, after the step of separating said precipitated complexes from said solution, redissolving the precipitated complexes in a substantially smaller volume than the original solution volume.
Claim 81
Dependent Claim
26 words
The method of claim 70 wherein the salt is selected from the group consisting of ammonium sulfate, sodium sulfate, sodium carbonate, potassium carbonate, and sodium phosphate.
Claim 82
Dependent Claim
11 words
The method of claim 70 wherein the salt is ammonium sulfate.
Claim 83
Dependent Claim
37 words
The method of claim 70 wherein said analyte is capable of simultaneously reacting with said first and second reactants and is one selected from the group consisting of drugs, vitamins, hormones, proteins, metabolites, cells, viruses, and microorganisms.
Claim 84
Dependent Claim
28 words
The method of claim 70 wherein said first and second reactants are selected from the group consisting of antibodies, antigens, lectins, receptors, transport proteins, and non-immunoglobulin antibody-binding proteins.
Claim 85
Dependent Claim
23 words
The method of claim 70 wherein said reporter is one selected from the group consisting of enzymes, fluorophores, radioisotopes, luminescers and dye particles.
Claim 86
Dependent Claim
10 words
The method of claim 70 wherein said polymer is poly-N-isopropylacrylamide.
Claim 87
Dependent Claim
22 words
The method of claim 70 wherein said polymer is a copolymer formed from monomers selected to achieve a desired critical solution temperature.
Claim 88
Dependent Claim
25 words
The method of claim 70 wherein said first reactant is conjugated to a monomer and subsequently copolymerized with additional monomers to yield a temperature-sensitive copolymer.
Claim 89
Independent Claim
161 words
A method for determining the presence and/or concentration of an analyte in a biological fluid sample, comprising: conjugating a first reactant which is capable of specifically binding with said analyte to a temperature-sensitive polymer characterized by a critical solution temperature to form a polymer/reactant conjugate; conjugating a second reactant which is capable of specifically binding with said analyte to a reporter to form a reporter/reactant conjugate; admixing in solution said polymer/reactant, reporter/reactant and said biological fluid sample suspected of containing said analyte at a temperature other than that at which said polymer will precipitate such that specific binding occurs between said first and second reactants and said analyte, thereby forming a ternary complex; incubating the admixed polymer/reactant, reporter/reactant, and analyte containing sample solution at a temperature sufficient to cause said complex to precipitate from the solution; and measuring the amount of reporter activity in the precipitated complex or in the solution and therefrom determining the presence and/or concentration of said analyte.
Claim 90
Independent Claim
161 words
A method for determining the presence and/or concentration of an analyte in a biological fluid sample, comprising: conjugating a first reactant which is capable of specifically binding with said analyte to a temperature-sensitive polymer characterized by a critical solution temperature to form a polymer/reactant conjugate; conjugating a second reactant which is capable of specifically binding with said analyte to a reporter to form a reporter/reactant conjugate; admixing in solution said polymer/reactant, reporter/reactant and said biological fluid sample suspected of containing said analyte at a temperature other than that at which said polymer will precipitate such that specific binding occurs between said first and second reactants and said analyte, thereby forming a ternary complex; incubating the admixed polymer/reactant, reporter/reactant, and analyte containing sample solution at a temperature sufficient to cause said complex to precipitate from the solution; and measuring the amount of reporter activity in the precipitated complex or in the solution and therefrom determining the presence and/or concentration of said analyte.
Claim 91
Dependent Claim
18 words
The method of claim 83 including, after the step of incubating, separating the precipitated complex from the solution.
Claim 92
Dependent Claim
21 words
The method of claim 83 including, after the step of conjugating said first reactant to said polymer, purifying said polymer/reactant conjugate.
Claim 93
Dependent Claim
17 words
The method of claim 85 wherein said polymer/reactant conjugate is purified by gel-permeation chromatography or serial precipitation.
Claim 94
Dependent Claim
31 words
The method of claim 84 including, after the step of separating said precipitated complex from said solution, redissolving the precipitated complex in a substantially smaller volume than the original solution volume.
Claim 95
Dependent Claim
37 words
The method of claim 83 wherein said analyte is capable of simultaneously reacting with said first and second reactants and is one selected from the group consisting of drugs, vitamins, hormones, proteins, metabolites, cells, viruses, and microorganisms.
Claim 96
Dependent Claim
28 words
The method of claim 83 wherein said first and second reactants are selected from the group consisting of antibodies, antigens, lectins, receptors, transport proteins and non-immunoglobulin antibody-binding proteins.
Claim 97
Dependent Claim
13 words
The method of claim 89 wherein said non-immunoglobulin antibody-binding protein is protein A.
Claim 98
Dependent Claim
23 words
The method of claim 83 wherein said reporter is one selected from the group consisting of enzymes, fluorophores, radioisotopes, luminescers and dye particles.
Claim 99
Dependent Claim
24 words
The method of claim 83 wherein said reporter is a fluorophore selected from the group consisting of fluorescein, rhodamine, phycoerythrin, phycocyanin, and Nile blue.
Claim 100
Dependent Claim
23 words
The method of claim 83 wherein said reporter is an enzyme selected from the group consisting of horseradish peroxidase, β-galactosidase and alkaline phosphatase.
Claim 101
Dependent Claim
28 words
The method of claim 83 wherein said polymer is one selected from the group consisting of polyvinyl methylether, polyvinylmethyl oxazolidone, polymethacrylic acid, poly-N-isopropylacrylamide, hydroxypropyl cellulose and methyl cellulose.
Claim 102
Dependent Claim
10 words
The method of claim 83 wherein said polymer is poly-N-isopropylacrylamide.
Claim 103
Dependent Claim
22 words
The method of claim 83 wherein said polymer is a copolymer formed from monomers selected to achieve a desired critical solution temperature.
Claim 104
Dependent Claim
17 words
The method of claim 96 wherein said copolymer is formed from N-isopropylacrylamide monomers copolymerized with acrylamide monomers.
Claim 105
Dependent Claim
32 words
The method of claim 83 wherein said first reactant is conjugated to a monomer and subsequently copolymerized with additional monomers to yield a temperature-sensitive copolymer characterized by a lower critical solution temperature.
Claim 106
Dependent Claim
26 words
The method of claim 98 wherein said first reactant is conjugated to the N-hydroxysuccinimide ester of acrylic acid and the resultant monomerized reactant copolymerized with N-isopropylacrylamide.
Claim 107
Dependent Claim
26 words
The method of claim 98 wherein said first reactant is conjugated to the N-hydroxysuccinimide ester of vinyl benzoate and the resultant monomerized reactant copolymerized with N-isopropylacrylamide.
Claim 108
Dependent Claim
24 words
The method of claim 83 wherein said polymer is one selected from the group consisting of polyethylene glycol, polyacrylic acid, polymethacrylamide and polyvinyl alcohol.
Claim 109
Dependent Claim
22 words
The method of claim 83 wherein said polymer is a copolymer formed from monomers selected to achieve a desired critical solution temperature.
Claim 110
Independent Claim
166 words
A method for determining the presence and/or concentration of an analyte in a biological fluid sample, comprising: conjugating a first reactant which is capable of specifically binding with said analyte to a monomer to form a monomer/reactant conjugate; copolymerizing said monomer/reactant conjugate with additional monomers to yield a temperature-sensitive copolymer/reactant; conjugating a second reactant which is capable of specifically binding with said analyte to a reporter to form a reporter/reactant conjugate; admixing in solution said copolymer/reactant, reporter/reactant, and said biological fluid sample suspected of containing said analyte at a temperature other than that at which said copolymer will precipitate such that specific binding occurs between said first and second reactants and said analyte, thereby forming a ternary complex; incubating the admixed copolymer/reactant, reporter/reactant, and analyte containing sample solution at a temperature sufficient to cause said complex to precipitate from the solution; and measuring the amount of reporter activity in the precipitated complex or in the solution and therefrom determining the presence and/or concentration of said analyte.
Claim 111
Independent Claim
166 words
A method for determining the presence and/or concentration of an analyte in a biological fluid sample, comprising: conjugating a first reactant which is capable of specifically binding with said analyte to a monomer to form a monomer/reactant conjugate; copolymerizing said monomer/reactant conjugate with additional monomers to yield a temperature-sensitive copolymer/reactant; conjugating a second reactant which is capable of specifically binding with said analyte to a reporter to form a reporter/reactant conjugate; admixing in solution said copolymer/reactant, reporter/reactant, and said biological fluid sample suspected of containing said analyte at a temperature other than that at which said copolymer will precipitate such that specific binding occurs between said first and second reactants and said analyte, thereby forming a ternary complex; incubating the admixed copolymer/reactant, reporter/reactant, and analyte containing sample solution at a temperature sufficient to cause said complex to precipitate from the solution; and measuring the amount of reporter activity in the precipitated complex or in the solution and therefrom determining the presence and/or concentration of said analyte.
Claim 112
Dependent Claim
26 words
The method of claim 103 wherein said polymer is one characterized by a lower critical solution temperature, thereby precipitating upon heating said solution above said temperature.
Claim 113
Dependent Claim
26 words
The method of claim 103 wherein said polymer is one characterized by an upper critical solution temperature, thereby precipitating upon cooling said solution below said temperature.
Claim 114
Dependent Claim
26 words
The method of claim 103 wherein said first reactant is conjugated to the N-hydroxysuccinimide ester of acrylic acid and the resultant monomerized reactant copolymerized with N-isopropylacrylamide.
Claim 115
Dependent Claim
26 words
The method of claim 103 wherein said first reactant is conjugated to the N-hydroxysuccinimide ester of vinyl benzoate and the resultant monomerized reactant copolymerized with N-isopropylacrylamide.
Claim 116
Independent Claim
248 words
A method for determining the presence and/or concentration of an analyte in a biological fluid sample, comprising: conjugating a first reactant which is capable of specifically binding with said analyte to a temperature-sensitive polymer to form a polymer/reactant conjugate; conjugating a second reactant which is capable of specifically binding with said analyte to a reporter to form a reporter/reactant conjugate; admixing in solution said polymer/reactant and said biological fluid sample suspected of containing said analyte at a temperature other than that at which said polymer will precipitate such that specific binding occurs between said first reactant and said analyte, thereby forming a binary complex; adjusting the salt concentration of the admixed polymer/reactant and analyte containing sample solution to a concentration sufficient to alter the critical solution temperature of the polymer, such that said complex precipitates from the solution; separating said precipitated binary complex from the solution; resolubilizing the precipitated binary complex; admixing in solution said binary complex and said reporter/reactant conjugate at a temperature other than that at which said polymer will precipitate, such that specific binding occurs between said second reactant and said analyte, thereby forming a ternary complex; adjusting the salt concentration of the ternary complex containing solution to a concentration sufficient to alter the critical solution temperature of the polymer, such that said ternary complex precipitates from the solution; and measuring the amount of reporter activity in the precipitated ternary complex or in the solution and therefrom determining the presence and/or concentration of said analyte.
Claim 117
Independent Claim
248 words
A method for determining the presence and/or concentration of an analyte in a biological fluid sample, comprising: conjugating a first reactant which is capable of specifically binding with said analyte to a temperature-sensitive polymer to form a polymer/reactant conjugate; conjugating a second reactant which is capable of specifically binding with said analyte to a reporter to form a reporter/reactant conjugate; admixing in solution said polymer/reactant and said biological fluid sample suspected of containing said analyte at a temperature other than that at which said polymer will precipitate such that specific binding occurs between said first reactant and said analyte, thereby forming a binary complex; adjusting the salt concentration of the admixed polymer/reactant and analyte containing sample solution to a concentration sufficient to alter the critical solution temperature of the polymer, such that said complex precipitates from the solution; separating said precipitated binary complex from the solution; resolubilizing the precipitated binary complex; admixing in solution said binary complex and said reporter/reactant conjugate at a temperature other than that at which said polymer will precipitate, such that specific binding occurs between said second reactant and said analyte, thereby forming a ternary complex; adjusting the salt concentration of the ternary complex containing solution to a concentration sufficient to alter the critical solution temperature of the polymer, such that said ternary complex precipitates from the solution; and measuring the amount of reporter activity in the precipitated ternary complex or in the solution and therefrom determining the presence and/or concentration of said analyte.
Claim 118
Independent Claim
230 words
A method for determing the presence and/or concentration of an analyte in a biological fluid sample, comprising: conjugating a first reactant which is capable of specifically binding with said analyte to a temperature-sensitive polymer characterized by a critical solution temperature to form a polymer/reactant conjugate; conjugating a second reactant which is capable of specifically binding with said analyte to a reporter to form a reporter/reactant conjugate; admixing in solution said polymer/reactant and said biological fluid sample suspected of containing said analyte at a temperature other than that at which said polymer will precipitate such that specific binding occurs between said first reactant and said analyte, thereby forming a binary complex; incubating the admixed polymer/reactant and analyte containing sample solution at a temperature sufficient to cause said complex to precipitate from the solution; separating said precipitated binary complex from the solution; resolubilizing the precipitated binary complex; admixing in solution said binary complex and said reporter/reactant conjugate at a temperature other than that at which said polymer will precipitate, such that specific binding occurs between said second reactant and said analyte, thereby forming a ternary complex; incubating the ternary complex containing solution at a temperature sufficient to cause said ternary complex to precipitate from the solution; and measuring the amount of reporter activity in the precipitated ternary complex or in the solution and therefrom determining the presence and/or concentration of said analyte.
Claim 119
Independent Claim
230 words
A method for determing the presence and/or concentration of an analyte in a biological fluid sample, comprising: conjugating a first reactant which is capable of specifically binding with said analyte to a temperature-sensitive polymer characterized by a critical solution temperature to form a polymer/reactant conjugate; conjugating a second reactant which is capable of specifically binding with said analyte to a reporter to form a reporter/reactant conjugate; admixing in solution said polymer/reactant and said biological fluid sample suspected of containing said analyte at a temperature other than that at which said polymer will precipitate such that specific binding occurs between said first reactant and said analyte, thereby forming a binary complex; incubating the admixed polymer/reactant and analyte containing sample solution at a temperature sufficient to cause said complex to precipitate from the solution; separating said precipitated binary complex from the solution; resolubilizing the precipitated binary complex; admixing in solution said binary complex and said reporter/reactant conjugate at a temperature other than that at which said polymer will precipitate, such that specific binding occurs between said second reactant and said analyte, thereby forming a ternary complex; incubating the ternary complex containing solution at a temperature sufficient to cause said ternary complex to precipitate from the solution; and measuring the amount of reporter activity in the precipitated ternary complex or in the solution and therefrom determining the presence and/or concentration of said analyte.
References Cited (Backward Citations)
US3817837A
Cited
US4065310A
Cited
US4088538A
Cited
US4231999A
Cited
US4493793A
Cited
US4371515A
Cited
US4281061A
Cited
US4469796A
Cited
US4522922A
Cited
US4530900A
Cited
EP0124050A1
Cited
US4511478A
Cited
US4609707A
Cited
US4711840A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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| 3.5 Years (Year 4) | 1991-10-25 | 1992-04-25 | 1992-10-25 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1995-10-25 | 1996-04-25 | 1996-10-25 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1999-10-25 | 2000-04-25 | 2000-10-25 | $7700.00 | Window Closed / Lapsed |