Immunochromatographic assay device

• US 5,877,028 A
• Filed: 03/31/1993
• Issued: 03/02/1999
• Est. Priority Date: 05/29/1991
• Status: Expired due to Term

• Current Assignee: Beckman Coulter Incorporated (Danaher Corporation)
• Original Assignee: Smithkline Diagnostics, Inc. (GSK plc)
• Inventors: Piasio, Roger N., Prouty, Karen, Chandler, Howard M.
• Primary Examiner(s): Chin, Christopher L.
• Application Number:
  US08/040,430
• Time in Patent Office: 2,162 Days
• Field of Search: 435/7.1, 435/7.92, 435/7.93, 435/7.94, 435/7.95, 435/287.1, 435/287.2, 435/287.7, 435/287.9, 435/970, 435/975, 435/810, 435/805, 436/514, 436/501, 436/578, 436/169, 436/810, 436/805, 422/56, 422/58, 422/60, 422/59
• US Class Current: 436/514
• CPC Class Codes: B01D 15/3804 Affinity chromatography
• CPC Class Codes: B01L 2300/0825 Test strips
• CPC Class Codes: B01L 2400/0406 capillary forces
• CPC Class Codes: B01L 3/5023 with a sample being transported to, and subsequently stored in an absorbent for analysis
• CPC Class Codes: B01L 3/5055 Hinged, e.g. opposable surfaces
• CPC Class Codes: G01N 30/02 Column chromatography
• CPC Class Codes: G01N 30/30 of temperature
• CPC Class Codes: G01N 30/463 for multidimensional chromatography
• CPC Class Codes: G01N 30/90 Plate chromatography, e.g. thin layer or paper chromatography
• CPC Class Codes: G01N 33/5302 Apparatus specially adapted for immunological test procedures
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 33/54388 based on lateral flow
• CPC Class Codes: G01N 33/558 using diffusion or migration of antigen or antibody immunochromatographic test strips G01N33/54387
• CPC Class Codes: Y10S 435/805 Test papers
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 435/97 Test strip or test slide
• CPC Class Codes: Y10S 435/975 Kit
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10S 436/81 Tube, bottle, or dipstick

Abstract

A chromatographic assay device for use with immunoassays allows rapid and convenient assays of analytes of biological interest, and permits extractions to be carried out in situ, avoiding the use of separate extraction vessels. The device has a wide dynamic range and avoids interference from particulates or colored components. In one form, the device comprises: (1) a first opposable component comprising a sample preparation zone adapted to receive a sample to be assayed; and (2) a second opposable component comprising a chromatographic medium. The first and second opposable components can be brought into opposition so as to cause the sample preparation zone to apply the sample to be tested to the chromatographic medium. Preferably, the analyte is detected with a visually detectable label. Other variations of the device vary the arrangement of components to provide optimal chromatography for a variety of analytes, as well as to permit bidirectional chromatography; still other variations are suitable for competitive immunoassays. The devices can be incorporated in test kits, and assay methods, both sandwich and competitive, using the devices are also disclosed.

Claims

1. A chromatographic assay device for detection or determination of an analyte in a sample comprising:

(a) a first opposable component including;

(i) a chromatographic medium having first and second ends and a detection zone, the chromatographic medium having a reagent bound at the detection zone, the reagent binding specifically to an analyte to be detected or determined;

(ii) a detector application pad either containing a mobile reagent for detection or determination of the analyte, the mobile reagent to be subsequently applied to the chromatographic medium during the performance of an assay or for application of the mobile reagent to the pad during the performance of the assay, in operable contact with the first end of the chromatographic medium;

(iii) a conductor for allowing the passage of fluid in operable contact with the detector application pad and in direct contact with the first end of the chromatographic medium; and

(iv) an absorber for absorbing fluid in operable contact with the second end of the chromatographic medium; and

(b) a second opposable component including a sample preparation zone for receiving a sample, the second opposable component being attachable to the first opposable component so that the first and second opposable components are brought into opposition from a position in which they are not in opposition and fluid is transferred from the second opposable component to the first opposable component by pressure;

wherein the first and second opposable components are configured so that a sample can be applied to the sample preparation zone when the first and second opposable components are not in opposition and bringing the first and second opposable components into opposition results in the sample preparation zone being in contact with the conductor to apply the sample to be tested to the conductor for flow through the conductor and then to the first end of the chromatographic medium through the detector application pad to add the reagent for detection of the analyte to the sample, the fluid flow being aided by absorption of fluid from the absorber, so that the analyte is detected or determined within the chromatographic medium at the detection zone after migration by binding of the reagent for detection of the analyte to the analyte bound to the detection zone, the assay being either a sandwich assay or a competitive assay and the reagent for detection of the analyte being an analyte analog for a competitive assay and a labeled primary or second antibody for a sandwich assay.

2. The chromatographic assay device of claim 1 wherein the sample preparation zone on the second opposable component contains at least one reagent for treatment of the sample.

3. The chromatographic assay device of claim 1 wherein the reagent for detection is a first specific binding partner to the analyte in a form that can be resolubilized by addition of an aqueous liquid to the detector application pad, the first specific binding partner being labeled with a detectable label, and the detection zone is substantially smaller in area than the area of the chromatographic medium, the detection zone containing a second specific binding partner to the analyte immobilized thereto, such that a ternary complex comprising the first specific binding partner, the analyte, and the second specific binding partner forms at the detection zone if analyte is present in the sample.

4. The chromatographic assay device of claim 3 wherein the detectable label is a visually detectable label.

5. The chromatographic assay device of claim 1 wherein the first and second opposable components are joined by a hinge that is impermeable to an aqueous liquid.

6. A test kit for the detection and/or the determination of an analyte in a sample comprising, in separate containers:

(a) the chromatographic assay device of claim 1; and

(b) the reagent for detection of the analyte that is a specific binding partner for the analyte labeled with a detectable label to be applied to the detector application pad.

7. A test kit for the detection and/or the determination of an analyte in a sample comprising, in separate containers:

(a) the chromatographic assay device of claim 3; and

(b) an aqueous liquid for resolubilizing the first specific binding partner to the analyte labeled with a detectable label, to be applied to the detector application pad.

8. A method for the detection or the determination of an analyte in an aqueous sample comprising the steps of:

(a) applying the aqueous sample to the sample application pad of the chromatographic assay device of claim 3;

(b) bringing the first and second components of the chromatographic assay device into opposition, such that the sample comprises an aqueous liquid resolubilizing the labeled specific binding partner in the detector application pad, and such that the sample and the resolubilized labeled specific binding partner are applied to the conductor;

(c) allowing the sample and the labeled specific binding partner to move through the conductor and then through at least a portion of the chromatographic medium to reach the detection zone so that the labeled specific binding partner gives a detectable indication of the presence or quantity of the analyte in the test sample; and

(d) observing or measuring the labeled specific binding partner at the detection zone in order to detect or determine the analyte as a ternary complex at the detection zone.

9. A chromatographic assay device for detection or determination of an analyte in a sample comprising:

(a) a first opposable component including;

(i) a chromatographic medium having first and second ends and a detection zone and having a reagent binding specifically to an analyte to be detected or determined, the reagent being bound at the detection zone;

(ii) a first detector application pad in operable contact with the first end of the chromatographic medium, the first detector application pad containing a first specific binding partner to the analyte in a form that can be resolubilized by the addition of an aqueous liquid to the first detector application pad, the first specific binding partner being labeled with a detectable label;

(iii) a conductor for allowing the passage of fluid in operable contact with the first detector application pad so that the first detector application pad bridges the conductor and the first end of the chromatographic medium to allow fluid flow from the conductor through the first detector application pad and to the first end of the chromatographic medium; and

(iv) an absorber for absorbing fluid in operable contact with the second end of the chromatographic medium; and

(b) a second opposable component attachable to the first opposable component so that the first and second opposable components are brought into opposition and fluid is transferred from the second opposable component to the first opposable component by pressure, the second opposable component including;

(i) a sample preparation zone for receiving a sample; and

(ii) a second detector application pad in operable contact with the sample preparation zone, the second detector application pad containing a second specific binding partner for the analyte in a form that can be resolubilized by the addition of a sample to the sample preparation zone, the second specific binding partner being labeled with a detectable label, the second detector application pad being located adjacent to the sample preparation zone on the second opposable component such that application of the sample to the sample preparation zone resolubilizes the second specific binding partner so that the sample preparation zone contains a mixture of the sample and the second specific binding partner;

wherein the first and second opposable components are configured so that a sample can be applied to the sample preparation zone on the second opposable component when the first and second opposable components are not in opposition and so that bringing the first and second opposable components into opposition results in the sample preparation zone on the second opposable component being in contact with the conductor on the first opposable component to apply the sample to be tested and the second specific binding partner to the conductor for flow through the conductor and then to the first end of the chromatographic medium through the first detector application pad to add the first specific binding partner to the sample and the second specific binding partner, the flow being aided by absorption of fluid by the absorber, the analyte being detected or determined at the detection zone by binding of the labeled first or second specific binding partners to the analyte bound to the detection zone.

10. The chromatographic assay device of claim 9 wherein the sample preparation zone on the second opposable component contains at least one reagent for treatment of a sample.

11. The chromatographic assay device of claim 9 wherein the first and second specific binding partners for the analyte in the first and second detector application pads are identical and the detectable labels labeling the first and second specific binding partners are identical.

12. The chromatographic assay device of claim 11 wherein the identical detectable labels are visually detectable labels.

13. The chromatographic assay device of claim 9 wherein the detection zone is substantially smaller in area than the chromatographic medium, the detection zone containing a third specific binding partner to the analyte immobilized thereto, such that a ternary complex comprising:

(1) one of the first and second specific binding partners;

(2) the analyte; and

(3) the immobilized third specific binding partner forms at the detection zone if analyte is present in the sample.

14. A test kit for the detection and/or the determination of an analyte in a sample comprising, in separate containers:

(a) the chromatographic assay device of claim 9; and

(b) an aqueous liquid for resolubilizing at least one of the first specific binding partner to the analyte and the second specific binding partner to the analyte, the aqueous liquid to be applied to at least one of the first detector application pad on the first opposable component and the second detector application pad on the second opposable component.

15. A method for the detection or the determination of an analyte in an aqueous sample comprising the steps of:

(a) applying the sample to the sample preparation zone on the second opposable component of the chromatographic assay device of claim 9 when the first and second opposable components are not in opposition;

(b) subsequent to applying the sample, brining the first and second opposable components of the chromatographic assay device into opposition, such that;

(i) the sample preparation zone on the second opposable component applies the mixture of the sample and the second specific binding partner to the conductor on the first opposable component;

(ii) the mixture of the sample and the second specific binding partner flows through the conductor to reach the first detector application pad on the first opposable component; and

(iii) the mixture of the sample and the second specific binding partner is applied to the first detector application pad to resolubilize the first specific binding partner to form a mixture of the sample, the first specific binding partner, and the second specific binding partner;

(c) then allowing the mixture of the sample, the first labeled specific binding partner, and the second labeled specific binding partner to move from the first detector application pad and then through at least a portion of the chromatographic medium to reach the detection zone so that at least one of the first and second labels specific binding partners gives a detectable indication of the presence or quantity of analyte in the test sample by binding to the detection zone, the flow through the chromatographic medium being aided by absorption of fluid by the absorber; and

(d) then observing or measuring at least one of the first or second specific binding partners bound to the detection zone in order to detect or determine the analyte as a ternary complex.

16. A chromatographic assay device for detection determination of an analyte in a sample comprising:

(a) a first opposable component including;

(i) a chromatographic medium having first and second ends and having a reagent binding specifically to an analyte to be detected or determined, the reagent being bound to the detection zone; and

(ii) a conductor for allowing the passage of fluid in operable contact with the first end of the chromatographic medium; and

(b) a second opposable component attachable to the first opposable component so that the first and second opposable component are brought into opposition from a position in which they are not in opposition and fluid is transferred from the second opposable component to the first opposable component, the second opposable component including;

(i) a sample preparation zone for receiving a sample; and

(ii) an absorber for absorbing fluid separated from the sample preparation zone on the second opposable component;

wherein the first and second opposable components are configured so that a sample can be applied to the sample preparation zone on the second opposable component when the first and second opposable components are not in opposition and so that bringing the first and second opposable components into opposition results in the sample preparation zone on the second opposable component coming into operable contact with the conductor on the first opposable component to apply the sample to be tested to the conductor for flow therethrough and into the first end of the chromatographic medium, and results into the absorber coming into operable contact with the second end of the chromatographic medium to withdraw fluid from the second end of the chromatographic medium to aid fluid flow so that the analyte is detected or determined by binding of the labeled reagent to the analyte bound to the detection zone.

17. A chromatographic assay device for detection or the determination of an analyte in a sample comprising:

(a) a first opposable component including;

(i) a sample preparation zone for receiving a sample;

(ii) a chromatographic medium having first and second ends and a detection zone and having a reagent binding specifically to an analyte to be detected or determined, the reagent being bound to the detection zone; and

(iii) a conductor allowing the passage of fluid in operable contact with the sample preparation zone and with the first end of the chromatographic medium so that the conductor bridges the sample preparation zone and the chromatographic medium to allow fluid flow from the sample preparation zone through the conductor and to the first end of the chromatographic medium; and

(b) a second opposable component attachable to the first opposable component so that the first and second opposable components are brought into opposition from a position in which they are not in opposition and fluid is transferred from the second opposable component to the first opposable component by pressure, the second opposable component including;

(i) an applicator for applying fluid to the sample preparation zone on the first opposable component when the first and second opposable components are brought into opposition and containing a specific binding partner for an analyte labeled with a detectable label in a form that can be resolubilized by the addition of an aqueous liquid to the applicator; and

(ii) an absorber for absorbing fluid therein separated from the applicator when the first and second opposable components are not in opposition;

wherein the first and second opposable components are configured so that a sample can be applied to the sample preparation zone on the first opposable component when the first and second opposable components are not in opposition and so that bringing the first and second opposable components into opposition results in the applicator on the second opposable component coming into operable contact with the sample preparation zone on the first opposable component such that, when a sample has been added to the sample preparation zone, the labeled specific binding partner for the analyte is resolubilized, and results in the absorber on the second opposable component coming into operable contact with the second end of the chromatographic medium on the first opposable component to withdraw fluid from the chromatographic medium to aid fluid flow so that the analyte is detected or determined by binding of the labeled specific binding partner for the analyte to the analyte bound to the detection zone.

18. A chromatographic assay device for the detection or determination of an analyte in a sample comprising:

(a) a first opposable component including;

(i) a chromatographic medium having first and second ends and a detection zone and having a reagent binding specifically to an analyte to be detected bound at the detection zone;

(ii) a conductor for allowing the passage of fluid in operable contact with the first end of the chromatographic medium; and

(b) a second opposable component attachable to the first opposable component so that the first and second opposable components are brought into opposition from a position in which they are not in opposition and fluid is transferred from the second opposable component to the first opposable component by pressure, the second opposable component including;

(i) a first applicator for applying fluid to the conductor on the first opposable component when the first and second opposable components are brought into opposition;

(ii) a second applicator separated on the second opposable component from the first applicator for applying fluid to the conductor on the first opposable component when the first and second opposable components are brought into opposition; and

(iii) an absorber for absorbing fluid therein separated on the second opposable component from the first applicator and the second applicator, the first applicator and the second applicator being located on the second opposable component so that they are not in operable contact with the each other when the first and second opposable components are not in opposition, the absorber being located on the second opposable component such that it is in operable contact with the second end of the chromatographic medium when the first and second opposable components are brought into opposition;

wherein the first and second opposable components are configured so that bringing the first and second opposable components into opposition results in the conductor on the first opposable component coming into operable contact with the first applicator on the second opposable component and results in the conductor coming into operable contact with the second applicator on the second opposable component, thereby resulting in the first and second applicators coming into operable indirect contact with each other, so that the contents of the first and second applicators flow through the conductor and the chromatographic medium, the fluid flow being aided by absorption of fluid by the absorber, so that the analyte is detected or determined by binding of a labeled reagent to the analyte bound to the detection zone.

19. The chromatographic assay device of claim 18 wherein the first applicator on the second opposable component includes a sample application pad for receiving a sample when the first and second opposable components are not in opposition and the second applicator on the second opposable component includes a detector application pad either containing a labeled reagent that binds specifically to the analyte for application of the labeled reagent that binds specifically to the analyte to the conductor on the first opposable component or for application of the labeled reagent that binds specifically to the analyte to the detector application pad during the performance of the assay, whereby, when the first and second opposable components are brought into opposition, the contents of the sample application pad and the detector application pad are applied to the conductor on the first opposable component.

20. The chromatographic assay device of claim 19 wherein the reagent for detection of the analyte is a first specific binding partner to the analyte in a form that can be resolubilized by addition of an aqueous liquid to the detector application pad, the first specific binding partner being labeled with a detectable label, and the detection zone is substantially smaller in area than the chromatographic medium, the detection zone containing a second specific binding partner to the analyte immobilized thereto, such that a ternary complex comprising the first specific binding partner, the analyte, and the second specific binding partner forms at the detection zone if analyte is present in the sample.

22. A test kit for the detection and/or determination of an analyte in a sample comprising, in separate containers:

(a) the chromatographic assay device of claim 19; and

(b) the reagent for detection of the analyte, to be applied to the second applicator on the second opposable component.

23. A test kit for the detection and/or the determination of an analyte in a sample comprising, in separate containers:

(a) the chromatographic assay device of claim 20; and

(b) an aqueous liquid for resolubilizing the specific binding partner for the analyte labeled with a detectable label, to be applied to the detector application pad.

24. A method for the detection or determination of an analyte in an aqueous sample comprising the steps of:

(a) applying the sample to the first applicator of the chromatographic assay device of claim 20;

(b) bringing the first and second opposable components of the chromatographic assay device into opposition such that;

(i) the sample comprises the aqueous liquid resolubilizing the specific binding partner in the detector application pad;

(ii) the sample and the resolubilized labeled specific binding partner are applied to the first end of the chromatographic medium; and

(iii) the absorber is placed in operable contact with the second end of the chromatographic medium;

(c) allowing the sample and the labeled specific binding partner to move through at least a portion of the chromatographic medium to reach the detection zone so that the labeled specific binding partner gives a detectable indication of the presence or quantity of the analyte; and

(d) observing or measuring the labeled specific binding partner at the detection zone in order to detect or determine the analyte as a ternary complex.

21. The chromatographic assay device of claim wherein the detectable label is a visually detectable label.

25. A chromatographic assay device for the detection or the determination of an analyte in a sample comprising:

(a) a first opposable component including;

(i) a chromatographic medium having first and second ends and a detection zone and having a reagent binding specifically to an analyte to be detected or determined, the reagent being bound at the detection zone;

(ii) a conductor for allowing the passage of fluid in operable contact with the first end of the chromatographic medium; and

(iii) a detector application pad containing a labeled reagent that binds specifically to the analyte for detection of the analyte in direct contact with the conductor and located on the first opposable component such that it is in indirect contact with the first end of the chromatographic medium for applying the reagent for detection of the analyte to the chromatographic medium; and

(b) a second opposable component attachable to the first opposable component so that the first and second opposable components are brought into opposition from a position in which they are not in opposition and fluid is transferred from the second opposable component to the first opposable component by pressure, the second opposable component including;

(i) a sample application pad for application of a sample thereto; and

(ii) an absorber for absorbing fluid separated from the sample application pad on the second opposable component;

wherein the first and second opposable components are configured so that a sample can be applied to the sample application pad on the second opposable component when the first and second opposable components are not in opposition and so that bringing the first and second opposable components into opposition results in;

(1) the sample application pad on the second opposable component applying the sample to the detector application pad on the first opposable component and thus to the first end of the chromatographic medium through the conductor on the first opposable component in direct contact with the detector application pad; and

(2) the absorber on the second opposable component being in operable contact with the second end of the chromatographic medium on the first opposable component so that the fluid flow is aided by absorption of fluid by the absorber, the analyte being detected or determined on the chromatographic medium after migration by binding of the labeled reagent to the analyte bound to the detection zone.

26. The chromatographic assay device of claim 25 wherein the reagent for detection of the analyte is a first specific binding partner to the analyte in a form that can be resolubilized by addition of an aqueous liquid to the detector application pad, the first specific binding partner being labeled with a detectable label, and the chromatographic medium further comprises a detection zone substantially smaller in area than the chromatographic medium, the detection zone containing a second specific binding partner to the analyte immobilized thereto, such that a ternary complex comprising the first specific binding partner, the analyte, and the second specific binding partner forms in the detection zone if analyte is present in the sample.

27. A method for the detection or determination of an analyte in an aqueous sample comprising the steps of:

(a) applying the sample to the sample application pad of the chromatographic assay device of claim 26;

(b) bringing the first and second components of the chromatographic assay device into opposition, such that;

(i) the sample comprises the aqueous liquid resolubilizing the specific binding partner in the detector application pad;

(ii) the sample and the resolubilized label specific binding partner are applied to the first end of the chromatographic medium; and

(iii) the absorber of the second opposable component is brought into operable contact with the second end of the chromatographic medium;

(c) allowing the sample and the labeled specific binding partner to move through at least a portion of the chromatographic medium to reach the detection zone so that the label specific binding partner gives a detectable indication of the presence or quantity of the analyte; and

(d) observing or measuring the labeled specific binding partner at the detection zone in order to detect or determine the analyte as a ternary complex.

28. A chromatographic assay device for the detection or the determination of an analyte in a sample comprising:

(a) a first opposable component including;

(i) a chromatographic medium having first and second ends and a detection zone and having a reagent binding specifically to an analyte to be detected or determined, the reagent being bound to the detection zone; and

(ii) a detector application pad containing a labeled reagent for detection of the analyte that binds specifically to the analyte in direct contact with the first end of the chromatographic medium for applying the reagent for detection of the analyte to the chromatographic medium; and

(b) a second opposable component attachable to the first opposable component so that the first and second opposable components are brought into opposition from a position in which they are not in opposition and fluid is transferred from the second opposable component to the first opposable component by pressure, the second opposable component including;

(i) a sample application pad for receiving a sample; and

(ii) an absorber for absorbing fluid separated on the second opposable component from the sample application pad;

wherein the first and second opposable components are configured so that a sample can be applied to the sample application pad on the second opposable component when the first and second opposable components are not in opposition so that bringing the first and second opposable components into opposition causes the detector application pad on the first opposable component and the sample application pad on the second opposable component to come into contact except for the region of the detector application pad directly adjacent to the first end of the chromatographic medium to transfer fluid from the sample application pad to the detector application pad while minimizing the transfer of fluid from the sample application pad directly to the chromatographic medium; and

wherein bringing the first and second opposable components into opposition causes;

(1) the sample to be tested to be applied to the detector application pad on the first opposable component and then to the first end of the chromatographic medium; and

(2) the absorber on the second opposable component to be brought into operable contact with the second end of the chromatographic medium so that fluid flow is aided by absorption of fluid by the absorber, the analyte being detected or determined after migration by binding of the labeled reagent to the analyte bound to the detection zone.

29. The chromatographic assay device of claim 28 wherein the labeled reagent for detection of the analyte is a first specific binding partner to the analyte in a form that can be resolubilized by addition of an aqueous liquid to the detector application pad, the first specific binding partner being labeled with a detectable label, and the chromatographic medium further comprises a detection zone substantially smaller in area than the chromatographic medium, the detection zone containing a second specific binding partner to the analyte immobilized thereto, such that a ternary complex comprising the first specific binding partner, the analyte, and the second specific binding partner forms at the detection zone if analyte is present in the sample.

30. A method for the detection or determination of an analyte in an aqueous test sample comprising the steps of:

(a) applying the sample to the sample application pad on the second opposable component of the chromatographic assay device of claim 29 when the first and second opposable components are not in opposition;

(b) subsequent to applying the sample, bringing the first and second opposable components of the chromatographic assay device into opposition, such that;

(i) the sample comprises the aqueous liquid resolubilizing the labeled specific binding partner in the detector application pad on the first opposable component;

(ii) the sample and the resolubilized labeled specific binding partner are applied to the first end of the chromatographic medium as a result of the contact between the sample application pad and the detector application pad on the first opposable component; and

(iii) the absorber on the second opposable component is brought into operable contact with the second end of the chromatographic medium on the first opposable component to aid fluid flow from the sample application pad and the detector application pad through the chromatographic medium;

(c) then allowing the sample and the labeled specific binding partner to move through at least a portion of the chromatographic medium to reach the detection zone so that the labeled specific binding partner gives a detectable indication of the presence or quantity of the analyte in the test sample by binding to the detection zone; and

(d) then observing or measuring the labeled specific binding partner bound at the detection zone in order to detect or determine the analyte as a ternary complex.

31. A chromatographic assay device for detection or determination of an analyte in a sample comprising:

(a) a first opposable component including;

(i) a chromatographic medium having first and second ends and a detection zone and having a reagent binding specifically to an analyte to be detected or determined, the reagent being bound to the detection zone; and

(ii) a conductor located on the first opposable component for allowing the passage of fluid such that it is not in operable contact with the first end of the chromatographic medium when the first opposable component and the second opposable component are not in opposition; and

(b) a second opposable component attachable to the first opposable component so that the first and second opposable components are brought into opposition from a position in which they are not in opposition and fluid is transferred by pressure from the second opposable component to the first opposable component, the second opposable component including;

(i) a first applicator for applying fluid to the conductor on the first opposable component when the first and second opposable components are brought into opposition;

(ii) a second applicator separated on the second opposable component from the second applicator for applying fluid to the conductor on the first opposable component when the first and second opposable components are brought into opposition; and

(iii) an absorber for absorbing fluid separated on the second opposable component from the first and second applicators, the first and second applicators being positioned on the second opposable component such that they are not in operable contact with each other when the first and second opposable components are not in opposition;

wherein the first and second opposable components are configured so that bringing the first and second opposable components into opposition;

(1) results in the conductor on the first opposable component coming into operable contact with the first applicator on the second opposable component, results in the conductor coming into operable contact with the second applicator on the second opposable component, and results in the second applicator coming into operable contact with the first end of the chromatographic medium, thereby placing the first and second applicators into operable indirect contact with each other to apply the contents of the first and second applicators to the chromatographic medium so that the contents of the first and second applicators flow through the chromatographic medium; and

(2) results in the absorber on the second opposable component coming into operable contact with the second end of the chromatographic medium so that absorption of fluid by the absorber aids fluid flow, the analyte being detected or determined on the chromatographic medium after migration by binding of the labeled reagent to the analyte bound to the detection zone.

32. The chromatographic assay device of claim 31 wherein the first applicator on the second opposable component includes a sample application pad for application of a sample thereto when the first and second opposable components are not in opposition and the second applicator on the second opposable component includes a detector application pad either containing a detection reagent including a labeled reagent binding specifically to the analyte or for application of the detection reagent including the labeled reagent binding specifically to the analyte during the performance of the assay for application of the detection reagent to the chromatographic medium, whereby, when the first and second opposable components are brought into opposition, the contents of the sample application pad and the detector application pad are applied to the chromatographic medium through the conductor.

33. The chromatographic assay device of claim 32 wherein the detection reagent is a first specific binding partner to the analyte in a form that can be resolubilized by addition of an aqueous liquid to the detector application pad, the first specific binding partner being labeled with a detectable label, and the chromatographic medium further comprises a detection zone substantially smaller in area than the chromatographic medium, the detection zone containing a second specific binding partner to the analyte immobilized thereto, such that a ternary complex comprising the first specific binding partner, the analyte, and the second specific binding partner forms at the detection zone if analyte is present in the sample.

34. The chromatographic assay device of claim 33 wherein the detectable label is a visually detectable label.

35. A test kit for the detection and/or determination of an analyte in a sample comprising, in separate containers:

(a) the chromatographic assay device of claim 32; and

(b) the labeled reagent binding specifically to the analyte, to be applied to the second applicator on the second opposable component.

36. A method for the detection or determination of an analyte in an aqueous test sample comprising the steps of:

(a) applying the sample to the first applicator on the second opposable component of the chromatographic assay device of claim 32 when the first and second opposable components are not in opposition;

(b) subsequent to applying the sample, bringing the first and second opposable components of the chromatographic assay device into opposition such that;

(i) the sample comprises the aqueous liquid resolubilizing the labeled specific binding partner in the detector application pad on the second opposable component;

(ii) the sample and the resolubilized labeled specific binding partner are applied to the first end of the chromatographic medium through the conductor on the first opposable component; and

(iii) the absorber on the second opposable component is brought into operable contact with the second end of the chromatographic medium to aid fluid flow from the sample application pad and the detector application pad through the conductor and the chromatographic medium;

(c) then allowing the sample and the labeled specific binding partner to move through at least a portion of the chromatographic medium to reach the detection zone so that the labeled specific binding partner gives a detectable indication of the presence or quantity of the analyte by binding to the detection zone; and

(d) then observing or measuring the labeled specific binding partner bound to the detection zone in order to detect or determine the analyte as a ternary complex.

37. A chromatographic assay device for the detection or determination of an analyte in a sample comprising:

(a) a first opposable component including;

(i) a chromatographic medium having first and second ends and a detection zone and having a reagent binding specifically to an analyte to be detected or determined bound to the detection zone;

(ii) a conductor for allowing the passage of fluid in operable contact with the first end of the chromatographic medium; and

(iii) an absorber for absorbing fluid in operable contact with the second end of the chromatographic medium; and

(b) a second opposable component attachable to the first opposable component so that the first and second opposable components are brought into opposition and fluid is transferred by pressure from the second opposable component to the first opposable component, the second opposable component including an applicator for applying fluid to the conductor on the first opposable component when the first and second opposable components are brought into opposition, the applicator divided into two sectors;

(i) a first sector containing a first specific binding partner for the analyte in a form that can be resolubilized by the addition of an aqueous liquid to the applicator where the first and second opposable components are not in opposition, the first specific binding partner being labeled with a detectable label; and

(ii) a second sector lacking the first specific binding partner for the analyte;

wherein the first and second opposable components are configured so that bringing the first and second opposable components into opposition places the first sector, but not the second sector of the applicator on the second opposable component into direct contact with the conductor on the first opposable component, the second sector of the applicator being in indirect contact with the conductor through the first sector, to apply the contents of the first sector of the applicator to the chromatographic medium, and subsequent to the application of the contents of the first sector of the applicator to the chromatographic medium, to apply the content of the second sector of the applicator to the chromatographic medium, the absorber withdrawing fluid from the chromatographic medium to aid fluid flow, the analyte being detected or determined on the chromatographic medium after migration by binding of the labeled specific binding partner to the analyte bound to the detection zone.

38. The chromatographic assay device of claim 37 wherein the detection zone is substantially smaller in area than the chromatographic medium, the detection zone containing a second specific binding partner to the analyte immobilized thereto, such that a ternary complex comprising the first specific binding partner, the analyte, and the second specific binding partner forms at the detection zone if analyte is present in the sample.

39. The chromatographic assay device of claim 38 wherein the detectable label is a visually detectable label.

40. A method for the detection or determination of an analyte in an aqueous test sample comprising the steps of:

(a) applying the sample to the applicator on the second opposable component of the chromatographic assay device of claim 38 when the first and second opposable components are not in opposition so that the sample is applied to both the first and second sectors of the applicator;

(b) subsequent to applying the sample to the applicator, bringing the first and second opposable components of the chromatographic assay device into opposition, such that;

(i) the sample applied to the first sector of the applicator on the second opposable component comprises the aqueous liquid resolubilizing the labeled specific binding partner in the first sector of the applicator;

(ii) the sample and the resolubilized labeled specific binding partner present in the first sector of the applicator are applied to the first end of the chromatographic medium through the conductor on the first opposable component; and

(iii) subsequent to the application of the sample and the resolubilized labeled specific binding partner from the first sector of the applicator to the first end of the chromatographic medium, the sample present in the second sector of the applicator is applied to the first end of the chromatographic medium through the conductor;

(c) then allowing the sample and the resolubilized labeled specific binding partner from the first sector of the applicator, followed by the sample from the second sector of the applicator, to move through at least a portion of the chromatographic medium to reach the detection zone so that the label of the labeled specific binding partner gives a detectable indication of the presence or quantity of the analyte by binding to the detection zone and so that the sample from the second sector of the applicator washes at least some labeled specific binding partner unbound to analyte from at least a portion of the chromatographic medium, the flow of fluid through the chromatographic medium being aided by withdrawal of fluid from the chromatographic medium by the absorber; and

(d) then observing or measuring the label bound to the detection zone in order to detect or determine the analyte as a ternary complex.

41. A chromatographic assay device for the detection and/or determination of an immunologically monovalent analyte in a sample by a competitive immunoassay comprising:

(a) a first opposable component including;

(i) a chromatographic medium having a first end and a second end and a detection zone, the detection zone being a discrete area substantially smaller than the area of the chromatographic medium, and having immobilized in the detection zone, analyte or an immunological analog thereof;

(ii) a first conductor for allowing the passage of fluid in operable contact with the first end of the chromatographic medium; and

(iii) a second conductor for allowing the passage of fluid in operable contact with the second end of the chromatographic medium;

(b) a second opposable component attachable to the first opposable component so that the first and second opposable components are brought into opposition from a position in which they are not in opposition and fluid is transferred from the second opposable component to the first opposable component, the second opposable component including a first applicator for applying fluid to the first conductor on the first opposable component when the first and second opposable components are brought into opposition, the first applicator containing a first specific binding partner to the analyte in a form that can be resolubilized by addition of a first aqueous liquid to the first applicator; and

(c) a third opposable component attachable to the first opposable component so that the first and third opposable components are brought into opposition from a position in which they are not in opposition and fluid is transferred from the third opposable component to the first opposable component, the third opposable component including;

(i) a second applicator for applying fluid to the second conductor on the first opposable component when the first and third opposable components are brought into opposition, the second applicator containing a second specific binding partner to the analyte in a form that can be resolubilized by the addition of a second aqueous liquid to the second applicator, the second specific binding partner being labeled with a detectable label; and

(ii) an absorber for absorbing fluid separated on the third opposable component from the second applicator;

wherein the first and second opposable components are configured so that bringing the first and second opposable components into opposition places the first conductor on the first opposable component in operable contact with the first applicator on the second opposable component so that the contents of the first applicator are applied to the chromatographic medium through the first conductor and are drawn through at least a portion of the chromatographic medium; and

wherein the first and third opposable components are configured so that bringing the first and third opposable components into opposition places the absorber on the third opposable component in operable contact with the first conductor on the first opposable component to withdraw fluid from the chromatographic medium through the first conductor and causes the second applicator on the third opposable component to come into operable contact with the second conductor on the first opposable component so that the contents of the second applicator are applied to the chromatographic medium and are drawn through at least a portion of the chromatographic medium overlapping the portion through which the contents of the first applicator applied to the chromatographic medium are drawn, the chromatographic assay being performed so that the analyte is detected at the detection zone by binding of the labeled reagent to the detection zone.

42. The chromatographic assay device of claim 41 wherein the first and second specific binding partners are each antibody specific for the analyte.

43. The chromatographic assay device of claim 41 wherein the immobilized analyte or analogue thereof comprises analyte covalently linked to a protein lacking specific binding activity for the analyte.

44. The chromatographic assay device of claim 41 wherein the detectable label is a visually detectable label.

45. A test kit for the detection and/or determination of an immunologically monovalent analyte in a sample comprising, in separate containers:

(a) the chromatographic assay device of claim 41; and

(b) an aqueous liquid for resolubilizing the second specific binding partner to the analyte.

46. A method for detecting and/or determining an immunologically monovalent analyte in an aqueous sample, comprising the steps of:

(a) applying the sample to the first applicator of the chromatographic assay device of claim 41, the sample comprising the first aqueous liquid;

(b) applying a reconstitution fluid to the second applicator, the reconstitution fluid comprising the second aqueous liquid;

(c) bringing the first and second opposable components of the chromatographic assay device into opposition, such that the sample and the resolubilized first specific binding partner to the analyte are applied to the first conductor and then to the first end of the chromatographic medium;

(d) allowing the sample and the resolubilized first specific binding partner to move through at least a portion of the chromatographic medium to reach the detection zone;

(e) separating the first and second opposable components so that they are no longer in opposition;

(f) bringing the first and third opposable components into opposition such that the resolubilized labeled second specific binding partner is applied to the second conductor and then to the second end of the chromatographic medium;

(g) allowing the resolubilized labeled second specific binding partner to move through at least a portion of the chromatographic medium overlapping the portion of the chromatographic medium through which the sample and the resolubilized first specific binding partner are drawn and including the detection zone so that, in the presence of analyte in the test sample, the labeled second specific binding partner binds to the analyte or immunological analog thereof immobilized in the detection zone; and

(h) observing and/or measuring the labeled second specific binding partner bound to the detection zone to detect and/or determine the analyte.

47. The method of claim 46 further comprising the step of incubating the chromatographic assay device following applying the sample of the first applicator in order to promote the reaction between the analyte and the first specific binding partner.

48. A method for detecting and/or determining an immunologically monovalent analyte in an aqueous sample, comprising the steps of:

(a) applying a first reconstitution fluid to the first applicator of the chromatographic assay device of claim 41;

(b) applying the sample to the first applicator of the chromatographic assay device of claim 45, the combination of the first reconstitution fluid and the sample comprising the first aqueous liquid;

(c) applying a second reconstitution fluid to the second applicator, the second reconstitution fluid comprising the second aqueous liquid;

(d) bringing the first and second opposable components of the chromatographic assay device into opposition, such that the sample and the resolubilized first specific binding partner to the analyte are applied to the first conductor and then to the first end of the chromatographic medium;

(e) allowing the sample and the resolubilized first specific binding partner to move through at least a portion of the chromatographic medium to reach the detection zone;

(f) separating the first and second opposable components so that they are no longer in opposition;

(g) bringing the first and third opposable components into opposition, such that the resolubilized labeled second specific binding partner is applied to the second conductor and then to the second end of the chromatographic medium;

(h) allowing the resolubilized labeled second specific binding partner to move through at least a portion of the chromatographic medium overlapping the portion of the chromatographic medium through which the sample and the resolubilized first specific binding partner are drawn and including the detection zone, so that, in the presence of analyte in the test sample, the labeled second specific binding partner binds the analyte or immunological analog thereof immobilized in the detection zone; and

(i) observing and/or measuring the labeled second specific binding partner bound to the detection zone in order to detect and/or determine the analyte.

49. The method of claim 48 further comprising the step of incubating the chromatographic assay device following applying the sample to the first applicator in order to promote the reaction between the analyte and the first specific binding partner.

50. A chromatographic assay device for the detection and/or determination of an immunologically monovalent analyte in a sample by a competitive immunoassay comprising:

(a) a first opposable component including;

(i) a chromatographic medium having a first end and a second end and having immobilized thereon, in separate discrete and non-overlapping areas, each area being substantially smaller than the area of the chromatographic medium;

(A) a specific binding partner for the analyte; and

(B) a secondary specific binding partner, the secondary specific binding partner capable of binding a first member of an additional specific binding pair that specifically binds a second member of the additional specific binding pair that is covalently linked to an analyte analog. neither member of the additional specific binding pair having affinity for the analyte, the secondary specific binding partner being located closer to the first end of the chromatographic medium than the first specific binding partner;

(ii) a first conductor in operable contact with the first end of the chromatographic medium; and

(iii) a second conductor in operable contact with the second end of the chromatographic medium;

(b) a second opposable component including;

(i) an applicator containing an analyte analog, the analyte analog comprising analyte covalently linked to a second member of the additional specific binding pair lacking affinity for the analyte and bindable by the secondary specific binding partner, the member of the additional specific binding pair being labeled with a detectable label, the analyte analog being in a form that can be resolubilized by the addition of an aqueous liquid to the applicator; and

(ii) an absorber separated from the applicator;

wherein the first and second opposable components are configured so that bringing the first and second opposable components into opposition places the second conductor in operable contact with the applicator so that the contents of the applicator are applied to the chromatographic medium and are drawn through at least a portion of the chromatographic medium, and places the absorber in operable contact with the first conductor to withdraw fluid from the chromatographic medium.

51. The chromatographic assay device of claim 50 further comprising a cover hingedly attached to the first opposable component so that it can be folded over the first and second opposable components when they are opposed, the cover having an aperture cut therein to permit viewing of at least a portion of the chromatographic medium when the first and second opposable components are opposed and the cover is folded over the first and second opposable components.

52. The chromatographic assay device of claim 50 wherein the first specific binding partner is an antibody specific for the analyte and additional specific binding pair members are both antibodies.

53. The chromatographic assay device of claim 50 wherein the detectable label is a visually detectable label.

54. The chromatographic assay device of claim 50 wherein the analyte analogue comprises analyte covalently linked to an immunoglobulin.

55. The chromatographic assay device of claim 50 wherein the area of the secondary specific binding partner immobilized on the chromatographic medium is divided into at least two discrete and non-overlapping bands, with the quantity of secondary specific binding partner in each band being determined so that the quantity of analyte analogue binding to the detection zone, and thus the concentration of analyte in the test sample, is indicated by the number of bands to which the analyte analogue binds.

56. A test kit for the detection and/or determination of an immunologically monovalent analyte in a sample comprising, in separate containers:

(a) the chromatographic assay device of claim 50; and

(b) an aqueous liquid for resolubilizing the second specific binding partner for the analyte.

57. A method for detecting and/or determining an immunologically monovalent analyte in an aqueous sample, comprising the steps of:

(a) applying a reconstitution fluid to the applicator of the chromatographic assay device of claim 50, the reconstitution fluid comprising the aqueous liquid for reconstituting the second specific binding partner;

(b) applying the sample to the first conductor of the chromatographic assay device;

(c) allowing the sample to move through at least a portion of the chromatographic medium, including the first specific binding partner for the analyte immobilized in a discrete area of the chromatographic medium;

(d) bringing the first and second opposable components into opposition such that the resolubilized labeled analyte analogue is applied to the second conductor and then to the second end of the chromatographic medium;

(e) allowing the resolubilized labeled analyte analogue to move through at least a portion of the chromatographic medium overlapping the portion of the chromatographic medium through which the sample is drawn, including both discrete areas of immobilized reagents, so that when analyte is present in the test sample, the labeled analyte analogue binds the secondary specific binding partner; and

(f) observing and/or measuring the labeled analyte analogue in the discrete area containing the immobilized secondary specific binding partner to detect and/or determine the analyte.

58. A method for detecting and/or determining an immunologically monovalent analyte in an aqueous sample, comprising the steps of:

(a) applying a reconstitution fluid to the applicator of the chromatographic assay device of claim 55, the reconstitution fluid comprising the aqueous liquid for resolubilizing the second specific binding partner;

(b) applying the sample to the first conductor of the chromatographic assay device;

(c) allowing the sample to move through at least a portion of the chromatographic medium, including the first specific binding partner for the analyte immobilized in a discrete area of the chromatographic medium;

(d) bringing the first and second opposable components into opposition such that the resolubilized labeled analyte analogue is applied to the second conductor and then to the second end of the chromatographic medium;

(e) allowing the resolubilized labeled analyte analogue to move through at least a portion of the chromatographic medium overlapping the portion of the chromatographic medium through which the sample is drawn, including both discrete areas of immobilized reagents, so that when analyte is present in the test sample, the labeled analyte analogue binds the secondary specific binding partner; and

(f) observing and/or measuring the labeled analyte analogue in the two or more bands within the discrete area containing the immobilized secondary specific binding partner to detect and/or determine the analyte, the number of bands to which labeled analyte analogue is bound increasing with analyte concentration.

59. A chromatographic assay device for the detection and/or determination of an immunologically monovalent analyte in a sample by a competitive immunoassay comprising:

(a) a first opposable component including;

(i) a chromatographic medium having a first end and a second end and having immobilized thereon, in separate discrete and non-overlapping areas, each area being smaller than the area of the chromatographic medium;

(A) a specific binding partner for the analyte; and

(B) a secondary specific binding partner, the secondary specific binding partner capable of binding a first member of an additional specific binding pair that specifically binds a second member of the additional specific binding pair that is covalently linked to an analyte analog neither member of the additional specific binding pair having affinity for the analyte, the specific binding partner for the analyte being located closer to the first end of the chromatographic medium;

(ii) a first conductor in operable contact with the first end of the chromatographic medium, the first conductor capable of functioning as a first applicator; and

(iii) a second conductor in operable contact with the second end of the chromatographic medium;

(b) a second opposable component including;

(i) a second applicator containing an analyte analog, the analyte analog comprising analyte covalently linked to a second member of the additional specific binding pair lacking affinity for the analyte and bindable by the secondary specific binding partner, the member of the specific binding pair being labeled with a detectable label, the analyte analog being in a form that can be resolubilized by the addition of an aqueous liquid to the applicator; and

(ii) a first absorber separated from the second applicator; and

(c) a third opposable component including a second absorber;

wherein the first and second opposable components are configured so that bringing the first and second opposable components into opposition places the second applicator into operable contact with the first conductor and places the first absorber into operable contact with the second conductor; and

wherein the first and third opposable components are configured so that bringing the first and third opposable components into opposition places the second absorber into direct contact with the first conductor and with the chromatographic medium to withdraw fluid therefrom.

60. The assay device of claim 59 wherein the detectable label is a visually detectable label.

61. A method for detecting and/or determining an immunologically monovalent analyte in an aqueous sample, comprising the steps of:

(a) applying the sample to the first conductor of the chromatographic assay device of claim 59;

(b) applying a reconstitution fluid to the second applicator of the chromatographic assay device to resolubilize the labeled analyte analogue in the second applicator;

(c) allowing the sample to move through at least a portion of the chromatographic medium, including the specific binding partner for the analyte and the secondary specific binding partner immobilized thereon;

(d) bringing the third and first opposable components into opposition to place the second absorber into direct contact with the first conductor and with the chromatographic medium to withdraw fluid therefrom;

(e) bringing the second and first opposable components into opposition to place the second applicator into operable contact with the first conductor to place the first absorber into operable contact with the second conductor to apply the resolubilized labeled analyte analogue to the first conductor and then to the first end of the chromatographic medium;

(f) allowing the resolubilized labeled analyte analogue to move through at least the portion of the chromatographic medium including the specific binding partner for the analyte and the secondary specific binding partner; and

(g) observing and/or measuring the labeled analyte analogue bound to the secondary specific binding partner in order to detect and/or determine the analyte.

62. A chromatographic assay device for the detection and/or determination of an immunologically monovalent analyte in a sample by a competitive immunoassay comprising:

(a) a first opposable component including

(i) a chromatographic medium having a first end and a second end and having immobilized thereon, in separate discrete non-overlapping areas, each area being substantially smaller than the area of the chromatographic medium;

(A) a substance capable of specifically binding biotin selected from the group consisting of avidin, streptavidin, anti-biotin antibody, and derivatives thereof; and

(B) a secondary specific binding partner capable of specifically binding a three-component complex, the three-component complex comprising;

(1) analyte;

(2) a member of a specific binding pair lacking specific binding affinity for the analyte and capable of binding the secondary specific binding partner, the member covalently conjugated to the analyte; and

(3) a detectable label bound to the member of the specific binding pair, the secondary specific binding partner binding the member of the specific binding pair lacking specific binding affinity for the analyte within the three-component complex;

(ii) a first conductor in operable contact with the first end of the chromatographic medium; and

(iii) a second conduction in operable contact with the second end of the chromatographic medium;

(b) a second opposable component including a first applicator containing a first specific binding partner to the analyte in a form that can be resolubilized by the addition of an aqueous sample to the first applicator, the first specific binding partner being covalently linked to biotin, the first specific binding partner not capable of being bound by the secondary specific binding partner; and

(c) a third opposable component including;

(i) a second applicator containing the three-component complex, the complex being in a form that can be resolubilized by the addition of a second aqueous liquid to the second applicator; and

(ii) an absorber separated from the second applicator;

wherein the first and second opposable components are configured so that bringing the first and second opposable components into opposition places the first conductor in operable contact with the first applicator so that the contents of the first applicator are applied to the chromatographic medium and are drawn through at least a portion of the chromatographic medium; and

wherein the first and third opposable components are configured so that bringing the first and third opposable components into opposition places the absorber in contact with the first conductor to withdraw fluid from the chromatographic medium, and causes the second applicator come into operable contact with the second conductor so that the contents of the second applicator are applied to the chromatographic medium and are drawn through at least a portion of the chromatographic medium overlapping the portion through which the contents of the first applicator are drawn.

63. The chromatographic assay device of claim 62 wherein the first specific binding partner is an anti-analyte antibody.

64. The chromatographic assay device of claim 63 wherein the member of the specific binding pair in the three-component complex is rabbit immunoglobulin G and the second specific binding partner is goat anti-rabbit IgG.

65. The chromatographic assay device of claim 62 wherein the detectable label is a visually detectable label.

66. The chromatographic assay device of claim 62 wherein the substance capable of specifically binding biotin is streptavidin.

67. The chromatographic assay device of claim 62 wherein the area of the secondary specific binding partner immobilized on the chromatographic medium is divided into at least two discrete and non-overlapping bands, with the quantity of secondary specific binding partner in each band being determined so that the quantity of three-component complex binding to the detection zone, and thus the original analyte concentration in the test sample, is indicated by the number of bands to which the three-component complex binds.

68. A test kit for the detection and/or determination of an immunologically monovalent analyte in a sample comprising, in separate containers:

(a) the chromatographic assay device of claim 62; and

(b) an aqueous liquid for resolubilizing the three-component complex.

69. A method for detecting and/or determining an immunologically monovalent analyte in an aqueous sample, comprising the steps of:

(a) applying the sample to the first applicator of the chromatographic assay device of claim 62, the sample comprising the first aqueous liquid;

(b) applying a reconstitution fluid to the second applicator, the reconstitution fluid comprising the second aqueous liquid;

(c) bringing the first and second opposable components of the chromatographic assay device into opposition such that the sample and the resolubilized first specific binding partner to the analyte are applied to the first conductor and then to the first end of the chromatographic medium;

(d) allowing the sample and the resolubilized first specific binding partner to move through at least a portion of the chromatographic medium to at least the area of immobilized substance that specifically binds biotin;

(e) separating the first and second opposable components so that they are no longer in opposition;

(f) bringing the first and third opposable components into opposition, such that the resolubilized three-component complex is applied to the second conductor and then to the second end of the chromatographic medium;

(g) allowing the resolubilized three-component complex to move through at least a portion of the chromatographic medium overlapping the portion of the chromatographic medium through which the sample and the resolubilized first specific binding partner were drawn so that, in the presence of analyte in the test sample, the three-component complex is bound to the secondary specific binding partner immobilized in one of the separate discrete non-overlapping areas; and

(h) observing and/or measuring the labeled specific binding partner in the one of the separate discrete non-overlapping areas in order to detect and/or determine the analyte.

70. The method of claim 69 further comprising the step of incubating the chromatographic assay device following applying the sample to the first applicator in order to promote the reaction between the analyte and the first specific binding partner.

71. A method for detecting and/or determining an immunologically monovalent analyte in an aqueous sample, comprising the steps of:

(a) applying a first reconstitution fluid to the first applicator of the chromatographic assay device of claim 62;

(b) applying the sample to the first applicator of the chromatographic assay device of claim 62, the combination of the first reconstitution fluid and the sample comprising the first aqueous liquid;

(c) applying a second reconstitution fluid to the second applicator, the second reconstitution fluid comprising the second aqueous liquid;

(d) bringing the first and second opposable components of the chromatographic assay device into opposition such that the sample and the resolubilized first specific binding partner to the analyte are applied to the first conductor and then to the first end of the chromatographic medium;

(e) allowing the sample and the resolubilized first specific binding partner to move through at least a portion of the chromatographic medium to at least the area of immobilized substance that specifically binds biotin;

(f) separating the first and second opposable components so that they are no longer in opposition;

(g) bringing the first and third opposable components into opposition, such that the resolubilized three-component complex is applied to the second conductor and then to the second end of the chromatographic medium;

(h) allowing the resolubilized three-component complex to move through at least a portion of the chromatographic medium overlapping the portion of the chromatographic medium through which the sample and the resolubilized first specific binding partner are drawn so that, in the presence of analyte in the test sample, the three-component complex is bound to the secondary specific binding partner immobilized in one of the separate discrete non-overlapping areas; and

(i) observing and/or measuring the labeled specific binding partner in one of the separate discrete non-overlapping areas in order to detect and/or determine the analyte.

72. The method of claim 71 further comprising the step of incubating the chromatographic assay device following applying the sample to the first applicator in order to promote the reaction between the analyte and the first specific binding partner.

73. A method for the detection and/or determination of an immunologically monovalent analyte in a test sample comprising the steps of:

(a) applying the sample to the first applicator of the chromatographic assay device of claim 67, the sample comprising the first aqueous liquid;

(b) applying a reconstitution fluid to the second applicator, the reconstitution fluid comprising the second aqueous liquid;

(c) bringing the first and second opposable components of the chromatographic assay device into opposition such that the sample and the resolubilized first specific binding partner to the analyte are applied to the first conductor and then to the first end of the chromatographic medium;

(d) allowing the sample and the resolubilized first specific binding partner to move through at least a portion of the chromatographic medium to at least the area of immobilized substance that specifically binds biotin;

(e) separating the first and second opposable components so that they are no longer in opposition;

(f) bringing the first and third opposable components into opposition, such that the resolubilized three-component complex is applied to the second conductor and then to the second end of the chromatographic medium;

(g) allowing the resolubilized three-component complex to move through at least a portion of the chromatographic medium overlapping the portion of the chromatographic medium through which the sample and the resolubilized first specific binding partner were drawn so that, in the presence of analyte in the test sample, the three-component complex is bound to the secondary specific binding partner immobilized in one of the separate discrete non-overlapping areas; and

(h) observing and/or measuring the labeled analyte analog in the two or more bands within one of the separate discrete non-overlapping areas containing the immobilized secondary specific binding partner to detect and/or determine the analyte, the number of bands to which labeled analyte analog is bound increasing with analyte concentration.

74. A chromatographic assay device for the detection and/or determination of an immunologically monovalent analyte in a sample by a competitive immunoassay comprising:

(a) a first opposable component including;

(i) a chromatographic medium having a first end and a second end, and having immobilized thereon in separate and non-overlapping discrete areas each substantially smaller than the area of the chromatographic medium;

(A) an analyte analog capable of binding a specific binding partner for the analyte; and

(B) a secondary specific binding partner that is capable of binding a specific binding pair member that has affinity for the analyte, the secondary specific binding partner itself lacking binding affinity for the analyte; and

(ii) a conductor in operable contact with the first end of the chromatographic medium; and

(b) a second opposable component including an applicator containing a specific binding partner to the analyte in a form that can be resolubilized by addition of an aqueous liquid to the applicator, the specific binding partner contained in the applicator being labeled with a detectable label;

wherein the first and second opposable components are configured so that bringing the first and second opposable components into opposition places the conductor in operable contact with the applicator so that the contents of the applicator are applied to the chromatographic medium and are drawn through at least a portion of the chromatographic medium.

75. The chromatographic assay device of claim 74 wherein the specific binding partner contained in the applicator is antibody specific for the analyte.

76. The chromatographic assay device of claim 74 wherein the analyte analogue comprises analyte covalently linked to a protein lacking specific binding activity for the analyte or for the specific binding partner for the analyte.

77. The chromatographic assay device of claim 75 wherein the secondary specific binding partner binds the antibody specific for the analyte on the basis of species-specific interactions not involving the antigen-combining site of the antibody for the analyte.

78. The chromatographic assay device of claim 74 wherein the detectable label is a visually detectable label.

79. The chromatographic assay device of claim 74 wherein the first opposable component further includes an absorber in operable contact with the second end of the chromatographic medium.

80. The chromatographic assay device of claim 74 wherein the second opposable component further includes an absorber separated from the applicator, the absorber being positioned on the second opposable component so that bringing the first and second opposable components into opposition places the absorber into operable contact with the second end of the chromatographic medium.

81. The chromatographic assay device of claim 74 wherein the area of the secondary specific binding partner immobilized on the chromatographic medium is divided into at least two discrete and non-overlapping bands, with the quantity of secondary specific binding partner in each band being determined so that the quantity of labeled specific binding partner for the analyte binding to the detection zone, and thus the quantity of analyte in the test sample, is indicated by the number of bands to which the labeled specific binding partner for the analyte binds.

82. A method for detecting and/or determining an immunologically monovalent analyte in an aqueous sample, comprising the steps of:

(a) applying the sample to the applicator of the chromatographic assay device of claim 74, the sample comprising the aqueous liquid;

(b) bringing the first and second opposable components of the chromatographic assay device into opposition, such that the sample and the resolubilized labeled specific binding partner to the analyte are applied to the conductor and then to the first end of the chromatographic medium;

(c) allowing the sample and the resolubilized labeled specific binding partner to move through at least a portion of the chromatographic medium, the portion including at least the separate discrete and non-overlapping areas of the analyte analogue and the secondary specific binding partner; and

(d) observing and/or measuring the labeled specific binding partner in the discrete area of the secondary specific binding partner to detect and/or determine the analyte.

83. A method for detecting and/or determining an immunologically monovalent analyte in an aqueous sample, comprising the steps of:

(a) applying the sample to the applicator of i the chromatographic assay device of claim 81, the sample comprising the aqueous liquid;

(b) bringing the first and second opposable components of the chromatographic assay device into opposition, such that the sample and the resolubilized labeled specific binding partner to the analyte are applied to the conductor and then to the first end of the chromatographic medium;

(c) allowing the sample and the resolubilized labeled specific binding partner to move through at least a portion of the chromatographic medium, the portion including at least the separate discrete and non-overlapping areas of the analyte analogue and the secondary specific binding partner; and

(d) observing and/or measuring the labeled specific binding partner in the two or more bands within the discrete area containing the immobilized secondary specific binding partner to detect and/or determine the analyte, the number of bands to which labeled analyte analogue is bound increasing with analyte concentration.