US5529899A
Expired / Lapsed
US
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Immunoassay for AH receptor transformed by dioxin-like compounds
Assignee: Cornell Research Foundation Inc
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Filing: 1993-07-27
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Granted: 1996-06-25
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Expires: 2013-07-27
Abstract
Abstract The present invention is directed to a method of detecting dioxin-like compounds in a test sample. The test sample is contacted with a heteromer formed from a plurality of proteins, one of which is an inactive Ah receptor. If dioxin-like compounds are present in the test sample, they will bind to the Ah receptor causing it to dissociate from the heteromer as a complex containing active Ah receptor bound to a dioxin-like compound ligand. The presence of the complex is then detected. The process of the present invention can be practiced utilizing solid phase capture, competitive, and sandwich immunoassay test kit formats.
Inventors
- 👤 Geoffrey D. Wheelock
- 👤 John G. Babish
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 05:04
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/5308; G01N33/566; G01N33/567; G01N2430/00; G01N2430/40; Y; Y10; Y10S; Y10S435/00; Y10S435/97; Y10S435/975; Y10S436/00; Y10S436/807; Y10S436/809
Claims Analysis
Total Claims: 32
Claim 1
Independent Claim
192 words
A method of detecting, in a test sample, a ligand selected from the group consisting of polychlorinated dibenzodioxins, polychlorinated dibenzofurans, polychlorinated biphenyls, and structural analogues thereof comprising: providing a heteromer from a cytosol fraction of mammalian hepatocytes, wherein the heteromer is formed from a plurality of proteins, in which one of the proteins is an Ah receptor in inactive form; contacting the test sample with the heteromer under conditions effective to bind said ligand in the test sample to the Ah receptor, causing the heteromer to undergo transformation, resulting in dissociation from the heteromer of a complex containing active Ah receptor bound to said ligand; and detecting the presence of the complex containing active Ah receptor bound to said ligand and thereby detecting the presence of said ligand in the test sample, wherein said detecting comprises: capturing the complex containing active Ah receptor bound to said ligand on a solid support on which a dioxin responsive element is immobilized; contacting the complex with a labelled antibody so that the labelled antibody binds to the complex; and detecting the complex containing active Ah receptor bound to said ligand by detecting the labelled antibody.
Claim 2
Independent Claim
192 words
A method of detecting, in a test sample, a ligand selected from the group consisting of polychlorinated dibenzodioxins, polychlorinated dibenzofurans, polychlorinated biphenyls, and structural analogues thereof comprising: providing a heteromer from a cytosol fraction of mammalian hepatocytes, wherein the heteromer is formed from a plurality of proteins, in which one of the proteins is an Ah receptor in inactive form; contacting the test sample with the heteromer under conditions effective to bind said ligand in the test sample to the Ah receptor, causing the heteromer to undergo transformation, resulting in dissociation from the heteromer of a complex containing active Ah receptor bound to said ligand; and detecting the presence of the complex containing active Ah receptor bound to said ligand and thereby detecting the presence of said ligand in the test sample, wherein said detecting comprises: capturing the complex containing active Ah receptor bound to said ligand on a solid support on which a dioxin responsive element is immobilized; contacting the complex with a labelled antibody so that the labelled antibody binds to the complex; and detecting the complex containing active Ah receptor bound to said ligand by detecting the labelled antibody.
Claim 3
Dependent Claim
16 words
A method according to claim 1, wherein said detecting provides a quantitative measurement of the complex.
Claim 4
Dependent Claim
24 words
A method according to claim 1, wherein the test sample is an environmental matrix selected from the group consisting of air, water, and soil.
Claim 5
Dependent Claim
18 words
A method according to claim 1, wherein the heteromer is from a cytosol fraction of guinea pig hepatocytes.
Claim 6
Dependent Claim
168 words
A sandwich immunoassay test kit for detecting, in a test sample, a ligand selected from the group consisting of polychlorinated dibenzodioxins, polychlorinated dibenzofurans, polychlorinated biphenyls, and structural analogues thereof comprising: a solid support; a dioxin responsive element having a first region capable of binding to said solid support and a second region capable of binding to a complex containing active Ah receptor bound to said ligand; a heteromer from a cytosol fraction of mammalian hepatocytes, wherein said heteromer is formed from a plurality of proteins, one of which proteins is the Ah receptor in inactive form, wherein, upon contact with a test sample containing said ligand, said ligand binds to the Ah receptor, and said heteromer undergoes transformation, causing dissociation from the heteromer of the complex containing active Ah receptor bound to said ligand; and an antibody with a region capable of binding to the complex containing active Ah receptor bound to said ligand, wherein said antibody is provided with a label to permit detection of said antibody.
Claim 7
Dependent Claim
168 words
A sandwich immunoassay test kit for detecting, in a test sample, a ligand selected from the group consisting of polychlorinated dibenzodioxins, polychlorinated dibenzofurans, polychlorinated biphenyls, and structural analogues thereof comprising: a solid support; a dioxin responsive element having a first region capable of binding to said solid support and a second region capable of binding to a complex containing active Ah receptor bound to said ligand; a heteromer from a cytosol fraction of mammalian hepatocytes, wherein said heteromer is formed from a plurality of proteins, one of which proteins is the Ah receptor in inactive form, wherein, upon contact with a test sample containing said ligand, said ligand binds to the Ah receptor, and said heteromer undergoes transformation, causing dissociation from the heteromer of the complex containing active Ah receptor bound to said ligand; and an antibody with a region capable of binding to the complex containing active Ah receptor bound to said ligand, wherein said antibody is provided with a label to permit detection of said antibody.
Claim 8
Dependent Claim
32 words
A sandwich immunoassay test kit according to claim 5, wherein said dioxin responsive element is prepared by annealing the following nucleotide sequences: 5'GATCCGGAGTTGCGTGAGAAGAGCCA-3' SEQ. ID. No. 6 5'TGGCTCTTCTCACGCAACTCCGGATC-3' SEQ. ID. No. 7
Claim 9
Dependent Claim
25 words
A sandwich immunoassay test kit according to claim 5, wherein the label is a colored, fluorescent, chemiluminescent, radioactive, or enzymatic material conjugated to said antibody.
Claim 10
Dependent Claim
20 words
A sandwich immunoassay test kit according to claim 5, wherein said dioxin responsive element is immobilized on said solid support.
Claim 11
Dependent Claim
21 words
A sandwich immunoassay test kit according to claim 5, wherein the heteromer is from a cytosol fraction of guinea pig hepatocytes.
Claim 12
Dependent Claim
167 words
A competitive immunoassay test kit for detecting, in a test sample, a ligand selected from the group consisting of polychlorinated dibenzodioxins, polychlorinated dibenzofurans, polychlorinated biphenyls, and structural analogues thereof comprising: a solid support; a dioxin responsive element having a first region capable of binding to said solid support and a second region capable of binding to a complex comprising an active Ah receptor bound to said ligand; a heteromer from a cytosol fraction of mammalian hepatocytes, wherein said heteromer is formed from a plurality of proteins, one of which proteins is the Ah receptor in inactive form, wherein, upon contact with a test sample containing said ligand, said ligand binds to the Ah receptor, and said heteromer undergoes transformation, causing dissociation from the heteromer of the complex containing active Ah receptor bound to said ligand; and an analogue with a region capable of binding to said second region of the dioxin responsive element, wherein said analogue is provided with a label to permit detection of said analogue.
Claim 13
Dependent Claim
167 words
A competitive immunoassay test kit for detecting, in a test sample, a ligand selected from the group consisting of polychlorinated dibenzodioxins, polychlorinated dibenzofurans, polychlorinated biphenyls, and structural analogues thereof comprising: a solid support; a dioxin responsive element having a first region capable of binding to said solid support and a second region capable of binding to a complex comprising an active Ah receptor bound to said ligand; a heteromer from a cytosol fraction of mammalian hepatocytes, wherein said heteromer is formed from a plurality of proteins, one of which proteins is the Ah receptor in inactive form, wherein, upon contact with a test sample containing said ligand, said ligand binds to the Ah receptor, and said heteromer undergoes transformation, causing dissociation from the heteromer of the complex containing active Ah receptor bound to said ligand; and an analogue with a region capable of binding to said second region of the dioxin responsive element, wherein said analogue is provided with a label to permit detection of said analogue.
Claim 14
Dependent Claim
25 words
A competitive immunoassay test kit according to claim 10, wherein the label is a colored, fluorescent, chemiluminescent, radioactive, or enzymatic material conjugated to said antibody.
Claim 15
Dependent Claim
20 words
A competitive immunoassay test kit according to claim 10, wherein said dioxin responsive element is immobilized on said solid support.
Claim 16
Dependent Claim
32 words
A competitive immunoassay test kit according to claim 10, wherein said dioxin responsive element is prepared by annealing the following nucleotide sequences: 5'GATCCGGAGTTGCGTGAGAAGAGCCA-3' SEQ. ID. No. 6 5'TGGCTCTTCTCACGCAACTCCGGATC-3' SEQ. ID. No. 7.
Claim 17
Dependent Claim
21 words
A competitive immunoassay test kit according to claim 10, wherein the heteromer is from a cytosol fraction of guinea pig hepatocytes.
Claim 18
Dependent Claim
151 words
A solid phase capture immunoassay test kit for detecting, in a test sample, a ligand selected from the group consisting of polychlorinated dibenzodioxins, polychlorinated dibenzofurans, polychlorinated biphenyls, and structural analogues thereof comprising: a dioxin responsive element affinity layer; a solid support; a heteromer from a cytosol fraction of mammalian hepatocytes, wherein said heteromer is formed from a plurality of proteins, one of which proteins is the Ah receptor in inactive form, wherein, upon contact with a test sample containing said ligand, said ligand binds to the Ah receptor, and said heteromer undergoes transformation, causing dissociation from the heteromer of a complex containing active Ah receptor bound to said ligand, wherein the complex is capable of binding to said affinity layer and to said solid support; and an antibody with a region capable of binding to the complex, wherein said antibody is provided with a label to permit detection of said antibody.
Claim 19
Dependent Claim
151 words
A solid phase capture immunoassay test kit for detecting, in a test sample, a ligand selected from the group consisting of polychlorinated dibenzodioxins, polychlorinated dibenzofurans, polychlorinated biphenyls, and structural analogues thereof comprising: a dioxin responsive element affinity layer; a solid support; a heteromer from a cytosol fraction of mammalian hepatocytes, wherein said heteromer is formed from a plurality of proteins, one of which proteins is the Ah receptor in inactive form, wherein, upon contact with a test sample containing said ligand, said ligand binds to the Ah receptor, and said heteromer undergoes transformation, causing dissociation from the heteromer of a complex containing active Ah receptor bound to said ligand, wherein the complex is capable of binding to said affinity layer and to said solid support; and an antibody with a region capable of binding to the complex, wherein said antibody is provided with a label to permit detection of said antibody.
Claim 20
Dependent Claim
27 words
A solid phase capture immunoassay test kit according to claim 15, wherein the label is a colored, fluorescent, chemiluminescent, radioactive, or enzymatic material conjugated to said antibody.
Claim 21
Dependent Claim
37 words
A solid phase capture immunoassay test kit according to claim 15 further comprising: a plate with a plurality of wells each having said solid support at the well bottom, wherein said solid support is a permeable membrane.
Claim 22
Dependent Claim
64 words
A solid phase capture immunoassay test kit according to claim 17 further comprising: a plurality of affinity columns with open ends and a dioxin responsive element affinity layer within each column between the ends, wherein the columns are spaced so that one end can be received within one well of said plate, whereby material eluted from the affinity layer will pass into the well.
Claim 23
Dependent Claim
23 words
A solid phase capture immunoassay test kit according to claim 15, wherein the heteromer is from a cytosol fraction of guinea pig hepatocytes.
Claim 24
Independent Claim
210 words
A method of detecting, in a test sample, a ligand selected from the group consisting of polychlorinated dibenzodioxins, polychlorinated dibenzofurans, polychlorinated biphenyls, and structural analogues thereof comprising: providing a dioxin responsive element affinity layer; providing a solid support; providing a heteromer from a cytosol fraction of mammalian hepatocytes, wherein the heteromer is formed from a plurality of proteins with one of the proteins being an Ah receptor in inactive form; contacting the test sample with the heteromer under conditions effective to bind said ligand in the test sample to the Ah receptor to cause the heteromer to undergo transformation, resulting in dissociation from the heteromer of a complex containing active Ah receptor bound to said ligand; contacting the complex with said dioxin responsive element affinity layer, whereby the complex binds to the dioxin responsive element; eluting the complex from said dioxin responsive element affinity layer; contacting the complex with said solid support, whereby the complex binds to said solid support; contacting an antibody with the complex bound to said solid support, wherein the antibody has a region capable of binding to the complex and is provided with a label to permit detection of said antibody; and detecting the label to detect the presence of said ligand in the test sample.
Claim 25
Independent Claim
210 words
A method of detecting, in a test sample, a ligand selected from the group consisting of polychlorinated dibenzodioxins, polychlorinated dibenzofurans, polychlorinated biphenyls, and structural analogues thereof comprising: providing a dioxin responsive element affinity layer; providing a solid support; providing a heteromer from a cytosol fraction of mammalian hepatocytes, wherein the heteromer is formed from a plurality of proteins with one of the proteins being an Ah receptor in inactive form; contacting the test sample with the heteromer under conditions effective to bind said ligand in the test sample to the Ah receptor to cause the heteromer to undergo transformation, resulting in dissociation from the heteromer of a complex containing active Ah receptor bound to said ligand; contacting the complex with said dioxin responsive element affinity layer, whereby the complex binds to the dioxin responsive element; eluting the complex from said dioxin responsive element affinity layer; contacting the complex with said solid support, whereby the complex binds to said solid support; contacting an antibody with the complex bound to said solid support, wherein the antibody has a region capable of binding to the complex and is provided with a label to permit detection of said antibody; and detecting the label to detect the presence of said ligand in the test sample.
Claim 26
Dependent Claim
18 words
A method according to claim 20, wherein the heteromer is from a cytosol fraction of guinea pig hepatocytes.
Claim 27
Independent Claim
223 words
A method of detecting, in a test sample, a ligand selected from the group consisting of polychlorinated dibenzodioxins, polychlorinated dibenzofurans, polychlorinated biphenyls, and structural analogues thereof comprising: providing a solid support; providing a heteromer from a cytosol fraction of mammalian hepatocytes, wherein the heteromer is formed from a plurality of proteins with one of the proteins being an Ah receptor in inactive form; contacting the test sample with the heteromer under conditions effective to bind said ligand in the test sample to the Ah receptor, causing the heteromer to undergo transformation, resulting in dissociation from the heteromer of a complex containing active Ah receptor bound to said ligand; contacting a dioxin responsive element with the solid support, whereby a first region of the dioxin responsive element binds to the solid support; contacting the complex with the dioxin responsive element bound to said solid support under conditions causing the complex to bind to the second region of the dioxin responsive element; contacting an analogue with the dioxin responsive element bound to the solid support under conditions allowing the analogue to compete with the complex for binding to said second region of the dioxin responsive element, wherein the analogue is provided with a label to permit detection of the analogue; detecting the label to indirectly detect the presence of said ligand in the test sample.
Claim 28
Independent Claim
223 words
A method of detecting, in a test sample, a ligand selected from the group consisting of polychlorinated dibenzodioxins, polychlorinated dibenzofurans, polychlorinated biphenyls, and structural analogues thereof comprising: providing a solid support; providing a heteromer from a cytosol fraction of mammalian hepatocytes, wherein the heteromer is formed from a plurality of proteins with one of the proteins being an Ah receptor in inactive form; contacting the test sample with the heteromer under conditions effective to bind said ligand in the test sample to the Ah receptor, causing the heteromer to undergo transformation, resulting in dissociation from the heteromer of a complex containing active Ah receptor bound to said ligand; contacting a dioxin responsive element with the solid support, whereby a first region of the dioxin responsive element binds to the solid support; contacting the complex with the dioxin responsive element bound to said solid support under conditions causing the complex to bind to the second region of the dioxin responsive element; contacting an analogue with the dioxin responsive element bound to the solid support under conditions allowing the analogue to compete with the complex for binding to said second region of the dioxin responsive element, wherein the analogue is provided with a label to permit detection of the analogue; detecting the label to indirectly detect the presence of said ligand in the test sample.
Claim 29
Dependent Claim
18 words
A method according to claim 22, wherein the heteromer is from a cytosol fraction of guinea pig hepatocytes.
Claim 30
Independent Claim
229 words
A method of detecting, in a test sample, a ligand selected from the group consisting of polychlorinated dibenzodioxins, polychlorinated dibenzofurans, polychlorinated biphenyls, and structural analogues thereof comprising: providing a solid support; providing a heteromer from a cytosol fraction of mammalian hepatocytes, wherein the heteromer is formed from a plurality of proteins with one of the proteins being an Ah receptor in inactive form; contacting the test sample with the heteromer under conditions effective to bind said ligand in the test sample to the Ah receptor, causing the heteromer to undergo transformation, resulting in dissociation from the heteromer of a complex containing active Ah receptor bound to said ligand; contacting a dioxin responsive element with the solid support, whereby a first region of the dioxin responsive element binds to the solid support; contacting the complex with the dioxin responsive element bound to said solid support under conditions causing the complex containing active Ah receptor bound to the ligand to bind to a second region of the dioxin responsive element; contacting an antibody with the complex bound to the second region of the dioxin responsive element, wherein the antibody is capable of binding to the complex containing active Ah receptor bound to said ligand and the antibody has a label to permit detection of said antibody; and detecting the label to detect the presence of said ligand in the test sample.
Claim 31
Independent Claim
229 words
A method of detecting, in a test sample, a ligand selected from the group consisting of polychlorinated dibenzodioxins, polychlorinated dibenzofurans, polychlorinated biphenyls, and structural analogues thereof comprising: providing a solid support; providing a heteromer from a cytosol fraction of mammalian hepatocytes, wherein the heteromer is formed from a plurality of proteins with one of the proteins being an Ah receptor in inactive form; contacting the test sample with the heteromer under conditions effective to bind said ligand in the test sample to the Ah receptor, causing the heteromer to undergo transformation, resulting in dissociation from the heteromer of a complex containing active Ah receptor bound to said ligand; contacting a dioxin responsive element with the solid support, whereby a first region of the dioxin responsive element binds to the solid support; contacting the complex with the dioxin responsive element bound to said solid support under conditions causing the complex containing active Ah receptor bound to the ligand to bind to a second region of the dioxin responsive element; contacting an antibody with the complex bound to the second region of the dioxin responsive element, wherein the antibody is capable of binding to the complex containing active Ah receptor bound to said ligand and the antibody has a label to permit detection of said antibody; and detecting the label to detect the presence of said ligand in the test sample.
Claim 32
Dependent Claim
18 words
A method according to claim 24, wherein the heteromer is from a cytosol function of guinea pig hepatocytes.
References Cited (Backward Citations)
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