Immunoassay method, device, and test kit
First Claim
1. A method of detecting an analyte in a test fluid, comprising the steps of:
- providing a reaction chamber having nonoverlapping first and second reaction surfaces, the reaction chamber being adapted to receive and retain a predetermined volume of the test fluid in fluid communication with the first and second reaction surfaces, the first reaction surface having an analyte binding partner immobilized thereon, the analyte binding partner having an analyte conjugate reversibly bound thereto, the analyte conjugate comprising a ligand/marker conjugated to an analyte component, the analyte conjugate having a higher disassociation constant than the analyte with reference to the immobilized analyte binding partner, and the second reaction surface having a ligand/marker binding partner immobilized thereon;
introducing a volume of the test fluid into the reaction chamber in fluid communication with the first and second reaction surfaces;
retaining the volume of test fluid in the reaction chamber to permit two reactions to occur;
a first reaction between analyte and analyte binding partner at the first reaction surface, the analyte thereby proportionally displacing analyte conjugate into the volume of test fluid, and a second reaction between the displaced analyte conjugate and ligand/marker binding partner at the second reaction surface, the displaced analyte conjugate thereby becoming sequestered on the second reaction surface; and
,thereafter measuring the activity of sequestered ligand/marker on the second reaction surface, the measured activity being a function of the analyte concentration that is referable to standards and controls.
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Accused Products
Abstract
An immunoassay device includes one or more reaction chambers. Each reaction chamber is adapted to receive and retain a volume of test fluid in fluid communication with nonoverlapping first and second reaction surfaces. To the first reaction surface is immobilized analyte binding partner that is in turn saturated with analyte conjugate: analyte component conjugated to one or more components, termed ligand/marker, that serve ligand and marker functions as described herein. The analyte conjugate has a higher disassociation constant with reference to the immobilized analyte binding partner than does the analyte to be assayed. To the second reaction surface is immobilized ligand/marker binding partner.
A test fluid sample is introduced into the reaction chamber and retained therein to permit two reactions to occur. In a first reaction between analyte and analyte binding partner at the first reaction reaction surface, analyte proportionately displaces analyte conjugate into the test fluid sample. In a second reaction the displaced analyte conjugate becomes sequestered at the second reaction surface by bonding with immobilized ligand/marker binding partner. Thereafter the marker activity of sequestered analyte analog is measured, the measured activity being a function of the analyte concentration that is referable to standards and controls.
A test kit includes the immunoassay device in combination with comparative test results.
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Citations
48 Claims
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1. A method of detecting an analyte in a test fluid, comprising the steps of:
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providing a reaction chamber having nonoverlapping first and second reaction surfaces, the reaction chamber being adapted to receive and retain a predetermined volume of the test fluid in fluid communication with the first and second reaction surfaces, the first reaction surface having an analyte binding partner immobilized thereon, the analyte binding partner having an analyte conjugate reversibly bound thereto, the analyte conjugate comprising a ligand/marker conjugated to an analyte component, the analyte conjugate having a higher disassociation constant than the analyte with reference to the immobilized analyte binding partner, and the second reaction surface having a ligand/marker binding partner immobilized thereon; introducing a volume of the test fluid into the reaction chamber in fluid communication with the first and second reaction surfaces; retaining the volume of test fluid in the reaction chamber to permit two reactions to occur;
a first reaction between analyte and analyte binding partner at the first reaction surface, the analyte thereby proportionally displacing analyte conjugate into the volume of test fluid, and a second reaction between the displaced analyte conjugate and ligand/marker binding partner at the second reaction surface, the displaced analyte conjugate thereby becoming sequestered on the second reaction surface; and
,thereafter measuring the activity of sequestered ligand/marker on the second reaction surface, the measured activity being a function of the analyte concentration that is referable to standards and controls. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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- 15. A device for detecting an analyte in a test fluid, comprising a reaction chamber having nonoverlapping first and second reaction surfaces, the reaction chamber being adapted to receive and retain a predetermined volume of the test fluid in fluid communication with the first and second reaction surfaces, the first reaction surface having an analyte binding partner immobilized thereon, the analyte binding partner having an analyte conjugate reversibly bound thereto, the analyte conjugate comprising a ligand/marker conjugated to an analyte component, the analyte conjugate having a higher disassociation constant than the analyte with reference to the immobilized analyte binding partner, and the second reaction surface having a ligand/marker binding partner immobilized thereon.
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28. A method of detecting an analyte in a test fluid, comprising the steps of:
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providing a reaction chamber having nonoverlapping first and second reaction surfaces, the reaction chamber being adapted to receive and retain a volume on the order of 100 to 200 μ
l of the test fluid in fluid communication with the first and second reaction surfaces, the first reaction surface having an analyte binding partner immobilized thereon, the analyte binding partner having an analyte conjugate reversibly bound thereto, the analyte conjugate comprising a ligand/marker conjugated to an analyte component, the analyte conjugate having a higher disassociation constant than the analyte with reference to the immobilized analyte binding partner, and the second reaction surface having a ligand/marker binding partner immobilized thereon;introducing a volume of the test fluid into the reaction chamber in fluid communication with the first and second reaction surfaces; retaining the volume of test fluid in the reaction chamber to permit two reactions to occur;
a first reaction between analyte and analyte binding partner at the first reaction surface, the analyte thereby proportionally displacing analyte conjugate into the volume of test fluid, and a second reaction between the displaced analyte conjugate and ligand/marker binding partner at the second reaction surface, the displaced analyte conjugate thereby becoming sequestered on the second reaction surface; and
,thereafter measuring the activity of sequestered ligand/marker on the second reaction surface, the measured activity being a function of the analyte concentration that is referable to standards and controls. - View Dependent Claims (29, 30)
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31. The method of detecting an analyte in a test fluid, comprising the steps of:
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providing a reaction chamber detachably mounted on an insoluble support, the reaction chamber having nonoverlapping first and second reaction surfaces, the reaction chamber being adapted to receive and retain a volume on the order of 10 to 50 μ
l of the test fluid in fluid communication with the first and second reaction surfaces, the first reaction surface having an analyte binding partner immobilized thereon, the analyte binding partner having an analyte conjugate reversibly bound thereto, the analyte conjugate comprising a ligand/marker conjugated to an analyte component, the analyte conjugate having a higher disassociation constant than the analyte with reference to the immobilized analyte binding partner, and the second reaction surface having a ligand/marker binding partner immobilized thereon;introducing a volume of the test fluid into the reaction chamber in fluid communication with the first and second reaction surfaces; retaining the volume of test fluid in the reaction chamber to permit two reactions to occur;
a first reaction between analyte and analyte binding partner at the first reaction surface, the analyte thereby proportionally displacing analyte conjugate into the volume of test fluid, and a second reaction between the displaced analyte conjugate and ligand/marker binding partner at the second reaction surface, the displaced analyte conjugate thereby becoming sequestered on the second reaction surface; and
,thereafter measuring the activity of sequestered ligand/marker on the second reaction surface, the measured activity being a function of the analyte concentration that is referable to standards and controls. - View Dependent Claims (32, 33, 34, 35, 36)
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37. A device for detecting an analyte in a test fluid, comprising a reaction chamber having nonoverlapping first and second reaction surfaces, the reaction chamber being adapted to receive and retain a volume on the order of 100 to 200 μ
- l of the test fluid in fluid communication with the first and second reaction surfaces, the first reaction surface having an analyte binding partner immobilized thereon, the analyte binding partner having an analyte conjugate reversibly bound thereto, the analyte conjugate comprising a ligand/marker conjugated to an analyte component, the analyte conjugate having a higher disassociation constant than the analyte with reference to the immobilized analyte binding partner, and the second reaction surface having a ligand/marker binding partner immobilized thereon.
- View Dependent Claims (38, 39)
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40. The device for detecting an analyte in a test fluid, comprising a reaction chamber detachably mounted on an insoluble support, the reaction chamber having nonoverlapping first and second reaction surfaces, the reaction chamber being adapted to receive and retain a volume on the order of 10 to 50 μ
- l of the test fluid in fluid communication with the first and second reaction surfaces, the first reaction surface having an analyte binding partner immobilized thereon, the analyte binding partner having an analyte conjugate reversibly bound thereto, the analyte conjugate comprising a ligand/marker conjugated to an analyte component, the analyte conjugate having a higher disassociation constant than the analyte with reference to the immobilized analyte binding partner, and the second reaction surface having a ligand/marker binding partner immobilized thereon.
- View Dependent Claims (41, 42, 43, 44, 45)
- 46. A test kit comprising a device for detecting an analyte in a test fluid, the device comprising a reaction chamber having nonoverlapping first and second reaction surfaces, the reaction chamber being adapted to receive and retain a volume of the test fluid in fluid communication with the first and second reaction surfaces, the first reaction surface having an analyte binding partner immobilized thereon, the analyte binding partner having an analyte conjugate reversibly bound thereto, the analyte conjugate comprising a ligand/marker conjugated to an analyte component, the analyte conjugate having a higher disassociation constant than the analyte with reference to the immobilized analyte binding partner, and the second reaction surface having a ligand/marker binding partner immobilized thereon, in combination with comparative test results that associate ligand/marker activity with specific concentrations of analyte in test fluid.
Specification