EP0268296A2
Expired / Lapsed
EP
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Membrane affinity concentration immunoassay
Assignee: Genetic Systems Corp
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Filing: 1987-11-19
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Granted: —
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Expires: 2007-11-19
Abstract
Abstract Methods for determining the presence and/or concentration of an analyte in a biological fluid sample are disclosed. The methods generally include admixing in solution certain polymer/reactant and reporter/reactant conjugates along with the biological fluid sample suspected of containing the analyte, thereby forming ternary complexes. The separation of the complexes from the reaction mixture is achieved through the affinity of certain selected polymer compositions for various solid phases. Upon separation, the amount of reporter activity in the solution may be measured, and therefrom the presence and/or concentration of the analyte determined. Multiple analyses on a biological fluid sample suspected of containing one or more analytes may also be performed, using either a variety of different reporters or selected polymers having varied affinity for the solid phase. Cross-Reference to Related Application This application is a continuation-in-part of co- pending application Serial No. 932,656, filed November 19, 1986.
Inventors
- 👤 Nobuo Monji
- 👤 Carol-Ann Cole
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-23 23:29
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/536; G01N33/537; G01N33/538; G01N33/543; G01N33/54306; G01N33/544; G01N33/545
Claims Analysis
Total Claims: 27
Claim 1
Independent Claim
147 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 selected 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 to form a reaction mixture, such that specific binding occurs between said first and second reactants and said analyte, thereby forming a ternary complex; contacting the reaction mixture with a solid phase capable of selectively binding said ternary complex, thereby removing said complex from the reaction mixture; and measuring the amount of reporter activity in the bound complex or in solution and therefrom determining the presence and/or concentration of said analyte.
Claim 2
Independent Claim
147 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 selected 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 to form a reaction mixture, such that specific binding occurs between said first and second reactants and said analyte, thereby forming a ternary complex; contacting the reaction mixture with a solid phase capable of selectively binding said ternary complex, thereby removing said complex from the reaction mixture; and measuring the amount of reporter activity in the bound complex or in solution and therefrom determining the presence and/or concentration of said analyte.
Claim 3
Dependent Claim
20 words
The method of claim 1 wherein the step of contacting comprises contacting said reaction mixture with a cellulose acetate membrane.
Claim 4
Dependent Claim
19 words
The method of claim 1 wherein the step of contacting comprises filtering the reaction mixture through the solid phase.
Claim 5
Dependent Claim
26 words
The method of claim 1 wherein said analyte is selected from the group consisting of drugs, vitamins, hormones, DNA, proteins, metabolites, cells, haptens, viruses and microorganisms.
Claim 6
Dependent Claim
29 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, peptides and non-immunoglobulin antibody-binding proteins.
Claim 7
Dependent Claim
22 words
The method of claim 1 wherein said reporter is selected from the group consisting of enzymes, fluorophores, radioisotopes, luminescers and dye particles.
Claim 8
Dependent Claim
19 words
The method of claim 1 wherein said first and second reactants are antibodies and said analyte is an antigen.
Claim 9
Dependent Claim
17 words
The method of claim 1 wherein the said polymer is characterized by a lower critical solution temperature.
Claim 10
Dependent Claim
24 words
The method of claim 1 wherein said polymer is selected from the group consisting of N-alkylacrylamides, N-arylacrylamides, alkyl acrylates, aryl acrylates and combinations thereof.
Claim 11
Dependent Claim
13 words
The method of claim 1 wherein said polymer is poly-N-isopropylacrylamide or derivatives thereof.
Claim 12
Dependent Claim
15 words
The method of claim 1 wherein said polymer is a copolymer formed from selected monomers.
Claim 13
Dependent Claim
16 words
The method of claim 11 wherein said copolymer is formed from N-isopropylacrylamide, N-n-butylacrylamide and N-acryloxysuccinimide monomers.
Claim 14
Dependent Claim
47 words
The method of claim 11 wherein said copolymer is formed from N-isopropylacrylamide monomers and N-acryloxysuccinimide monomers copolymerized with monomers selected from the group consisting of n-amyl acrylate, iso-amyl acrylate, n-octyl acrylate, methyl acrylate, ethyl acrylate, hexadecyl acrylate, 3,5,5-trimethylhexyl acrylate, N-tert-butylacrylamide, N-decylacrylamide, N-tert-octylacrylamide, N-benzylacrylamide, N-isobutoxymethylacrylamide, and diacetone acrylamide.
Claim 15
Independent Claim
158 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 selected monomer to form a monomer/reactant conjugate; copolymerizing said monomer/reactant conjugate with additional monomers to yield a 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 to form a reaction mixture, such that specific binding occurs between said first and second reactants and said analyte, thereby forming a ternary complex; contacting the reaction mixture with a solid phase capable of selectively binding ternary complex, thereby removing said complex from the reaction mixture; and measuring the amount of reporter activity in the bound complex or in solution and therefrom determining the presence and/or concentration of said analyte.
Claim 16
Independent Claim
158 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 selected monomer to form a monomer/reactant conjugate; copolymerizing said monomer/reactant conjugate with additional monomers to yield a 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 to form a reaction mixture, such that specific binding occurs between said first and second reactants and said analyte, thereby forming a ternary complex; contacting the reaction mixture with a solid phase capable of selectively binding ternary complex, thereby removing said complex from the reaction mixture; and measuring the amount of reporter activity in the bound complex or in solution and therefrom determining the presence and/or concentration of said analyte.
Claim 17
Independent Claim
118 words
A method for determining the presence and/or concentration of an analyte in a biological fluid sample, comprising: contacting a polymer/reactant conjugate capable of specifically binding with said analyte with a solid phase capable of selectively binding with said polymer; contacting the polymer/reactant-solid phase with a biological fluid sample suspected of containing said analyte such that specific binding occurs between said polymer/reactant and said analyte; contacting said solid phase having the polymer/reactant/ analyte complex bound thereto with a reporter/reactant conjugate capable of specifically binding with said analyte, such that specific binding occurs between said reporter/reactant and said analyte; and measuring the amount of reporter activity in the bound complex and therefrom determining the presence and/or concentration of said analyte.
Claim 18
Independent Claim
118 words
A method for determining the presence and/or concentration of an analyte in a biological fluid sample, comprising: contacting a polymer/reactant conjugate capable of specifically binding with said analyte with a solid phase capable of selectively binding with said polymer; contacting the polymer/reactant-solid phase with a biological fluid sample suspected of containing said analyte such that specific binding occurs between said polymer/reactant and said analyte; contacting said solid phase having the polymer/reactant/ analyte complex bound thereto with a reporter/reactant conjugate capable of specifically binding with said analyte, such that specific binding occurs between said reporter/reactant and said analyte; and measuring the amount of reporter activity in the bound complex and therefrom determining the presence and/or concentration of said analyte.
Claim 19
Independent Claim
148 words
A method for determining the presence and/or concentration of an analyte in a biological fluid sample, comprising: conjugating a first reactant to a selected polymer to form a polymer/reactant conjugate; conjugating a second reactant to a reporter to form a reporter/reactant conjugate, said second reactant being capable of specifically binding with said analyte and said first reactant; admixing in solution said polymer/reactant, reporter/reactant and said biological fluid sample suspected of containing said analyte to form a reaction mixture, such that competitive binding occurs between said polymer/reactant and said analyte for said second reactant; contacting said reaction mixture with a solid phase capable of selectively binding the polymer/reactant and the polymer/reactant-reporter reactant complex, thereby removing said complex and said polymer/reactant from the reaction mixture; and measuring the amount of reporter activity in the bound complex or in the solution and therefrom determining the presence and/or concentration of the analyte.
Claim 20
Independent Claim
148 words
A method for determining the presence and/or concentration of an analyte in a biological fluid sample, comprising: conjugating a first reactant to a selected polymer to form a polymer/reactant conjugate; conjugating a second reactant to a reporter to form a reporter/reactant conjugate, said second reactant being capable of specifically binding with said analyte and said first reactant; admixing in solution said polymer/reactant, reporter/reactant and said biological fluid sample suspected of containing said analyte to form a reaction mixture, such that competitive binding occurs between said polymer/reactant and said analyte for said second reactant; contacting said reaction mixture with a solid phase capable of selectively binding the polymer/reactant and the polymer/reactant-reporter reactant complex, thereby removing said complex and said polymer/reactant from the reaction mixture; and measuring the amount of reporter activity in the bound complex or in the solution and therefrom determining the presence and/or concentration of the analyte.
Claim 21
Independent Claim
161 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 capable of specifically binding with one of the analytes to selected polymers; conjugating a plurality of selected second reactants capable of specifically binding with one of the analytes to one or more selected reporters to form multiple reporter/reactant conjugates; admixing in solution the multiple polymer/reactant, multiple reporter/reactant, and the biological fluid sample suspected of containing one or more analytes, such that specific binding occurs between the reactants and the analytes, thereby forming a plurality of ternary complexes; contacting the admixed polymer/reactant, reporter/reactant, and analyte containing solution with a solid phase capable of selectively binding the ternary complexes, thereby removing the complexes from the reaction mixture; and measuring the activity of each selected reporter associated with the bound complexes or in the solution and therefrom determining the presence and/or concentration of each of the analytes.
Claim 22
Independent Claim
161 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 capable of specifically binding with one of the analytes to selected polymers; conjugating a plurality of selected second reactants capable of specifically binding with one of the analytes to one or more selected reporters to form multiple reporter/reactant conjugates; admixing in solution the multiple polymer/reactant, multiple reporter/reactant, and the biological fluid sample suspected of containing one or more analytes, such that specific binding occurs between the reactants and the analytes, thereby forming a plurality of ternary complexes; contacting the admixed polymer/reactant, reporter/reactant, and analyte containing solution with a solid phase capable of selectively binding the ternary complexes, thereby removing the complexes from the reaction mixture; and measuring the activity of each selected reporter associated with the bound complexes or in the solution and therefrom determining the presence and/or concentration of each of the analytes.
Claim 23
Independent Claim
184 words
A method for conducting multiple analyses on a biological fluid sample suspected of containing one or more analytes, comprising: conjugating a plurality of selected first reactants to a plurality of selected polymers having varied affinity for a solid phase to form multiple polymer/reactant conjugates, each of said first reactants being capable of specifically binding with one of the analytes; conjugating a plurality of selected second reactants capable of specifically binding with one of the analytes to one or more reporters to form multiple reporter/reactant conjugates; admixing in solution the multiple polymer/reactant, multiple reporter/reactant, and the biological fluid sample suspected of containing one or more analytes, such that specific binding occurs between the reactants and the analytes, thereby forming a plurality of ternary complexes; contacting the admixed polymer/reactant, reporter/reactant, and analyte sample-containing solution with a solid phase capable of selectively binding the ternary complexes, thereby removing the complexes from the reaction mixture; selectively eluting said ternary complexes from the solid phase; and measuring the reporter activity associated with each of the eluted complexes, and therefrom determining the presence and/or concentration of each of the analytes.
Claim 24
Independent Claim
184 words
A method for conducting multiple analyses on a biological fluid sample suspected of containing one or more analytes, comprising: conjugating a plurality of selected first reactants to a plurality of selected polymers having varied affinity for a solid phase to form multiple polymer/reactant conjugates, each of said first reactants being capable of specifically binding with one of the analytes; conjugating a plurality of selected second reactants capable of specifically binding with one of the analytes to one or more reporters to form multiple reporter/reactant conjugates; admixing in solution the multiple polymer/reactant, multiple reporter/reactant, and the biological fluid sample suspected of containing one or more analytes, such that specific binding occurs between the reactants and the analytes, thereby forming a plurality of ternary complexes; contacting the admixed polymer/reactant, reporter/reactant, and analyte sample-containing solution with a solid phase capable of selectively binding the ternary complexes, thereby removing the complexes from the reaction mixture; selectively eluting said ternary complexes from the solid phase; and measuring the reporter activity associated with each of the eluted complexes, and therefrom determining the presence and/or concentration of each of the analytes.
Claim 25
Dependent Claim
23 words
The method of claim 18 wherein the ternary complexes are selectively eluted using an ionic detergent, a non-ionic detergent, or a chaotropic agent.
Claim 26
Independent Claim
197 words
A method for determining the presence and/or concentration of an analyte in a biological fluid sample, comprising: conjugating a first reactant to a selected polymer to form a polymer/reactant conjugate; conjugating a second reactant to a reporter to form a reporter/reactant conjugate, said second reactant being capable of competing for specifically binding to the first reactant with an analyte; admixing in solution said polymer/reactant, reporter/reactant and said biological fluid sample suspected of containing said analyte to form a reaction mixture, such that competitive binding occurs between said reporter/reactant and said analyte for said first reactant; contacting said reaction mixture with a solid phase capable of selectively binding the polymer/reactant, and polymer/reactant-reporter/reactant complexes and the polymer/reactant, thereby removing the complexes and the polymer/reactant from the reaction mixture to form a resultant solution; immobilizing a third reactant to a second solid phase to form a reactant activated solid phase, said third reactant capable of specifically binding the reporter in the reporter/reactant conjugate; contacting the resultant solution with the reactant-activated solid phase, thereby removing the reporter/ reactant from the resultant solution; and measuring the reporter activity in the bound reporter/reactant and therefrom determining the presence and/or concentration of the analyte.
Claim 27
Independent Claim
197 words
A method for determining the presence and/or concentration of an analyte in a biological fluid sample, comprising: conjugating a first reactant to a selected polymer to form a polymer/reactant conjugate; conjugating a second reactant to a reporter to form a reporter/reactant conjugate, said second reactant being capable of competing for specifically binding to the first reactant with an analyte; admixing in solution said polymer/reactant, reporter/reactant and said biological fluid sample suspected of containing said analyte to form a reaction mixture, such that competitive binding occurs between said reporter/reactant and said analyte for said first reactant; contacting said reaction mixture with a solid phase capable of selectively binding the polymer/reactant, and polymer/reactant-reporter/reactant complexes and the polymer/reactant, thereby removing the complexes and the polymer/reactant from the reaction mixture to form a resultant solution; immobilizing a third reactant to a second solid phase to form a reactant activated solid phase, said third reactant capable of specifically binding the reporter in the reporter/reactant conjugate; contacting the resultant solution with the reactant-activated solid phase, thereby removing the reporter/ reactant from the resultant solution; and measuring the reporter activity in the bound reporter/reactant and therefrom determining the presence and/or concentration of the analyte.
References Cited (Backward Citations)
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