Barrier-controlled assay device

• US 5,607,863 A
• Filed: 12/07/1993
• Issued: 03/04/1997
• Est. Priority Date: 05/29/1991
• Status: Expired due to Fees

• Current Assignee: Smithkline Diagnostics, Inc. (GlaxoSmithKline PLC)
• Original Assignee: Smithkline Diagnostics, Inc. (GlaxoSmithKline PLC)
• Inventors: Chandler, Howard M.
• Primary Examiner(s): Housel, James C.
• Assistant Examiner(s): CHIN, CHRISTOPHER L
• Application Number:
  US08/163,860
• Time in Patent Office: 1,183 Days
• Field of Search: 436/518, 436/165, 436/170, 436/810, 436/514, 435/310, 435/805, 435/969, 435/970, 435/7.92, 435/7.93, 435/7.94, 422/56, 422/57, 422/58, 422/61, 422/104
• US Class Current: 436/518
• 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 33/54386 Analytical elements
• CPC Class Codes: G01N 33/54387 Immunochromatographic test strips
• 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 435/969 Multiple layering of reactants
• CPC Class Codes: Y10S 435/97 Test strip or test slide
• CPC Class Codes: Y10S 436/81 Tube, bottle, or dipstick

Abstract

An assay device for detection and/or determination of an analyte in a test sample uses a barrier containing an aperture to control the application of reagents to the device for greater reproducibility of results. In its simplest form, the device comprises: (1) a chromatographic medium having a first end, a second end, and first and second surfaces, and having a specific binding partner for the analyte immobilized thereto in a detection zone; (2) at least one absorber in operable contact with at least one of the first and second ends; and (3) a substantially fluid-impermeable barrier adjacent to the first surface of the chromatographic medium, the barrier having at least one aperture therethrough for application of liquid to the chromatographic medium, the barrier at least partially blocking application of liquid to the chromatographic medium. The device can be adapted for sandwich or competitive assays and can be used to perform amplified assays, such as those using silver amplification or enzyme amplification. Various arrangements of components within the device are possible, and elements such as filters can be accommodated.

Claims

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

(a) a first opposable component including;

(i) a chromatographic medium having a first end, a second end, and first and second surfaces, and having a specific binding partner for the analyte immobilized thereon in a detection zone between the first and second ends of the chromatographic medium;

(ii) at least one absorber in operable contact with at least one of the first and second ends of the chromatographic medium; and

(iii) a substantially fluid-impermeable barrier layered on top of the first surface of the chromatographic medium and having an aperture for application of liquid to the chromatographic medium, the barrier at least partially blocking application of liquid to the chromatographic medium; and

(b) a second opposable component containing at least one reactant for applying the at least one reactant directly or indirectly to the chromatographic medium through the aperture;

wherein the first and second opposable components are configured so that bringing the first and second opposable components into opposition results in the second opposable component applying the at least one reactant directly or indirectly to the chromatographic medium through the aperture.

2. The assay device of claim 1 wherein the first and second opposable components can be placed in opposition so that pressure facilitates application of the at least one reactant to the chromatographic medium through the aperture.

3. A chromatographic assay device for detection and/or determination of an analyte in a test sample comprising:

(a) a first opposable component including;

(i) a first opposable component including a chromatographic medium having a first end, a second end, and first and second surfaces, and having a specific binding partner for the analyte immobilized thereon in a detection zone between the first and second ends of the chromatographic medium;

(ii) at least one absorber in operable contact with at least one of the first and second ends of the chromatographic medium;

(iii) a substantially fluid-impermeable barrier layered on top of the first surface of the chromatographic medium and having an aperture for application of liquid to the chromatographic medium, the barrier at least partially blocking application of liquid to the chromatographic medium; and

(iv) an applicator layered on top of the barrier, the applicator containing a labeled specific binding partner to the analyte in a form that can be resolubilized by the addition of an aqueous liquid to the applicator, the applicator being positioned such that the barrier is located between the applicator and the chromatographic medium and such that an aqueous liquid applied to the applicator is drawn through the aperture after resolubilizing the labeled specific binding partner and then is drawn through the chromatographic medium by the at least one absorber so that the analyte and the labeled specific binding partner can form a ternary complex at the detection zone on the chromatographic medium; and

(b) a second opposable component including a sample preparation zone;

wherein the first and second opposable components are configured so that bringing the first and second opposable components into opposition results in the sample preparation zone being in contact with the applicator so that sample in the sample preparation zone is applied to the applicator for resolubilization of the resolubilizable labeled specific binding partner and chromatography through the chromatographic medium.

4. The assay device of claim 3 comprising at least two absorbers, one in operable contact with the first end of the chromatographic medium and one in operable contact with the second end of the chromatographic medium, wherein the detection zone is substantially smaller than the area of the chromatographic medium, and wherein the chromatographic medium further includes a control zone of analyte or analogue thereof immobilized thereon in an area substantially smaller than the area of the chromatographic medium and not overlapping with the detection zone, the control and detection zones positioned with respect to the aperture and the absorber such that the detection zone is between the aperture and one of the absorbers and the control zone is between the aperture and the other absorber.

5. The assay device of claim 3 wherein the label of the labeled specific binding partner is a visually detectable label.

6. The assay device of claim 3 wherein the sample preparation zone contains at least one reagent for treatment of the sample distinct from the labeled specific binding partner for the analyte.

7. A method for the detection and/or determination of an analyte in an aqueous test sample comprising:

(a) applying the sample to the sample preparation zone of the assay device of claim 6;

(b) incubating the sample in the sample preparation zone so that the at least one reagent for treatment of the sample can react with the sample to produce a treated sample;

(c) bringing the first and second opposable components into opposition so that the treated sample is transferred from the sample preparation zone to the applicator;

(d) allowing the treated sample transferred to the applicator to resolubilize the labeled specific binding partner in the applicator to form a solution in the applicator containing the transferred treated sample and the resolubilized labeled specific binding partner;

(e) allowing the solution containing the treated sample and the resolubilized labeled specific binding partner to enter the chromatographic medium through the aperture in the barrier and then to flow through at least the portion of the chromatographic medium including the detection zone; and

(f) detecting and/or determining the analyte in the test sample by observing and/or measuring the labeled specific binding partner in the detection zone.

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

(a) applying the sample to the sample preparation zone of the assay device of claim 3;

(b) bringing the first and second opposable components into opposition so that the sample is transferred from the sample preparation zone to the applicator;

(c) allowing the sample transferred to the applicator to resolubilize the labeled specific binding partner in the applicator to form a solution in the applicator containing the transferred sample and the resolubilized labeled specific binding partner;

(d) allowing the solution containing the sample and the labeled specific binding partner to enter the chromatographic medium through the aperture and the barrier and then to flow through at least the portion of the chromatographic medium including the detection zone; and

(e) detecting and/or determining the analyte in the test sample by observing and/or measuring the labeled specific binding partner bound to the detection zone.

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

(a) a first opposable component including;

(i) a chromatographic medium having a first end, a second end, and first and second surfaces, and having a specific binding partner to the analyte immobilized thereon in a detection zone between the first and second ends of the chromatographic medium;

(ii) at least one absorber in operable contact with at least one of the first and second ends of the chromatographic medium; and

(iii) a substantially fluid-impermeable barrier layered on top of the first surface of the chromatographic medium and having an aperture for application of liquid to the chromatographic medium, the barrier at least partially blocking application of liquid to the chromatographic medium; and

(b) a second opposable component including an applicator for application of a sample thereto, the applicator containing a labeled specific binding partner to the analyte in a form that can be resolubilized by the addition of an aqueous liquid to the applicator;

wherein the first and second opposable components are configured so that bringing the first and second opposable components into opposition results in the applicator being in operable contact with the barrier so that the sample and the resolubilized labeled specific binding partner in the applicator are applied through the aperture in the barrier to the chromatographic medium for chromatography through the chromatographic medium.

10. The assay device of claim 9 comprising at least two absorbers, one in operable contact with the first end of the chromatographic medium and one in operable contact with the second end of the chromatographic medium, wherein the detection zone is substantially smaller than the area of the chromatographic medium, and wherein the chromatographic medium further includes a control zone of analyte or analogue thereof immobilized thereon in an area substantially smaller than the area of the chromatographic medium and not overlapping with the detection zone, the control and detection zones positioned with respect to the aperture and the absorber such that the detection zone is between the aperture and one of the absorbers and the control zone is between the aperture and the other absorber.

11. The assay device of claim 9 wherein the label of the labeled specific binding partner is a visually detectable label.

12. The assay device of claim 9 wherein the applicator contains at least one reagent for treatment of the sample distinct from the labeled specific binding partner for the analyte.

13. The assay device of claim 9 wherein the first and second opposable components can be placed in opposition so that pressure facilitates application of the sample and the resolubilized detection reagent to the chromatographic medium through the aperture.

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

(a) applying the sample to the applicator of the assay device of claim 9;

(b) allowing the sample to resolubilize the labeled specific binding partner in the applicator to form a solution in the applicator containing the sample and the resolubilized labeled specific binding partner;

(c) bringing the first and second opposable components into opposition so that the solution containing the sample and the resolubilized labeled specific binding partner is applied to the chromatographic medium through the aperture in the barrier;

(d) allowing the solution containing the sample and the labeled specific binding partner to flow through at least a portion of the chromatographic medium including the detection zone; and

(e) detecting and/or determining the analyte in the test sample by observing and/or measuring the labeled specific binding partner bound at the detection zone.

15. An assay device for detection and/or determination of an analyte in a test sample comprising:

(a) a first opposable component including;

(i) a chromatographic medium having a first end, a second end, and first and second surfaces, and having a specific binding partner to the analyte immobilized thereon in a detection zone between the first and second ends of the chromatographic medium;

(ii) at least one absorber in operable contact with at least one of the first and second ends of the chromatographic medium;

(iii) a substantially fluid-impermeable barrier layered on top of the first surface of the chromatographic medium and having an aperture for application of liquid to the chromatographic medium, the barrier at least partially blocking application of liquid to the chromatographic medium; and

(iv) a filter for removing particulates layered on top of the barrier;

the chromatographic medium, the barrier, and the filter being positioned so that the barrier is located between the filter and the chromatographic medium in a fluid flow path transverse to the direction of flow along the chromatographic medium; and

(b) a second opposable component including an applicator for application of a sample thereto, the applicator containing a labeled specific binding partner to the analyte in a form that can be resolubilized by the addition of an aqueous liquid to the applicator;

wherein the first and second opposable components are configured so that bringing the first and second opposable components into opposition results in the applicator being in operable contact with the filter so that the sample and the resolubilized labeled specific binding partner in the applicator are applied to the filter and then through the aperture in the barrier to the chromatographic medium for chromatography through the chromatographic medium.

16. The assay device of claim 15 comprising at least two absorbers, one in operable contact with the first end of the chromatographic medium and one in operable contact with the second end of the chromatographic medium, wherein the detection zone is substantially smaller than the area of the chromatographic medium, and wherein the chromatographic medium further includes a control zone of analyte or analogue thereof immobilized thereon in an area substantially smaller than the area of the chromatographic medium and not overlapping with the detection zone, the control and detection zones positioned with respect to the aperture and the absorber such that the detection zone is between the aperture and one of the absorbers and the control zone is between the aperture and the other absorber.

17. The assay device of claim 15 wherein the label of the labeled specific binding partner is a visually detectable label.

18. The assay device of claim 15 wherein the applicator contains at least one reagent for treatment of the sample distinct from the labeled specific binding partner for the analyte.

19. The assay device of claim 15 wherein the filter removes fecal matter.

20. The assay device of claim 15 wherein the first and second opposable components can be placed in opposition so that pressure facilitates application of the sample and the resolubilized labeled detection reagent to the chromatographic medium through the aperture.

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

(a) applying the sample to the applicator of the assay device of claim 15;

(b) allowing the sample to resolubilize the labeled specific binding partner in the applicator to form a solution in the applicator containing the sample and the resolubilized labeled specific binding partner;

(c) bringing the first and second opposable components into opposition so that the solution containing the sample and the resolubilized labeled specific binding partner is applied to the filter;

(d) allowing the solution containing the sample and the labeled specific binding partner to pass through the filter to form a filtered solution;

(e) allowing the filtered solution to enter the chromatographic medium through the aperture in the barrier and to flow through at least the portion of the chromatographic medium including the detection zone; and

(f) detecting and/or determining the analyte in the test sample by observing and/or measuring the labeled specific binding partner bound at the detection zone.

22. An assay device for detection and/or determination of an analyte in a test sample comprising:

(a) a first opposable component including;

(i) a chromatographic medium having a first end, a second end, and first and second surfaces, and having a specific binding partner to the analyte immobilized thereon in a detection zone between the first and the second ends of the chromatographic medium;

(ii) at least one absorber in operable contact with at least one of the first and second ends of the chromatographic medium;

(iii) a substantially fluid-impermeable barrier layered on top of the first surface of the chromatographic medium and having an aperture for application of liquid to the chromatographic medium, the barrier at least partially blocking application of liquid to the chromatographic medium;

(iv) an applicator layered on top of the barrier, the applicator containing a labeled specific binding partner for the analyte in a form that can be resolubilized by the addition of an aqueous liquid to the applicator; and

(v) a filter for removing particulates layered on top of the applicator, the chromatographic medium, the barrier, the applicator, and the filter being positioned so that the applicator is located between the filter and the chromatographic medium in a fluid flow path transverse to the direction of flow along the chromatographic medium; and

(b) a second opposable component including a sample preparation zone;

where the first and second opposable components are configured so that bringing the first and second opposable components into opposition results in the sample preparation zone being in operable contact with the filter so that sample is applied to the filter, and a solution of filtered sample and labeled specific binding partner is then applied to the chromatographic medium through the aperture in the barrier.

23. The assay device of claim 22 comprising at least two absorbers, one in operable contact with the first end of the chromatographic medium and one in operable contact with the second end of the chromatographic medium, wherein the detection zone is substantially smaller than the area of the chromatographic medium, and wherein the chromatographic medium further includes a control zone of analyte or analogue thereof immobilized thereon in an area substantially smaller than the area of the chromatographic medium and not overlapping with the detection zone, the control and detection zones positioned with respect to the aperture and the absorber such that the detection zone is between the aperture and one of the absorbers and the control zone is between the aperture and the other absorber.

24. The assay device of claim 22 wherein the label of the labeled specific binding partner is a visually detectable label.

25. The assay device of claim 22 wherein the sample preparation zone contains at least one reagent for treatment of the sample distinct from the specific binding partner for the analyte.

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

(a) applying the sample to the sample preparation zone of the assay device of claim 25;

(b) incubating the sample in the sample preparation zone so that the at least one reagent for treatment of the sample can react with the sample forming a treated sample;

(c) bringing the first and second opposable components into opposition to transfer the treated sample from the sample preparation zone to the filter;

(d) allowing the treated sample transferred to the filter to pass through the filter producing a filtered treated sample;

(e) allowing the filtered treated sample to enter the applicator to resolubilize the labeled specific binding partner in the applicator, forming a solution in the applicator containing the filtered treated sample and the resolubilized labeled specific binding partner;

(f) allowing the solution containing the filtered treated sample and the resolubilized labeled specific binding partner to enter the chromatographic medium through the aperture in the barrier and to flow through at least the portion of the chromatographic medium including the detection zone; and

(g) detecting and/or determining the analyte in the test sample by observing and/or measuring the labeled specific binding partner bound at the detection zone.

27. The assay device of claim 22 wherein the filter removes fecal matter.

28. The assay device of claim 22 wherein the first and second opposable components can be placed in opposition so that pressure facilitates application of the solution of filtered sample and labeled specific binding partner to the chromatographic medium through the aperture.

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

(a) applying the sample to the sample preparation zone of the assay device of claim 22;

(b) bringing the first and second opposable components into opposition so that the sample is transferred from the sample preparation zone to the filter;

(c) allowing the sample transferred to the filter to pass through the filter to produce a filtered sample;

(d) allowing the filtered sample to enter the applicator to resolubilize the labeled specific binding partner in the applicator forming a solution in the applicator containing the filtered sample and the resolubilized specific binding partner;

(e) allowing the solution containing the filtered sample and the resolubilized labeled specific binding partner to enter the chromatographic medium through the aperture in the barrier and to flow through at least the portion of the chromatographic medium including the detection zone; and

(f) detecting and/or determining the analyte in the test sample by observing and/or measuring the labeled specific binding partner bound to the detection zone.

30. An assay device for detection and/or determination of an analyte in a test sample comprising:

(a) a first opposable component including;

(i) a chromatographic medium having a first end, a second end, and first and second surfaces, and having a specific binding partner to the analyte immobilized thereon in a detection zone between the first end and the second end of the chromatographic medium;

(ii) at least one absorber in operable contact with at least one of the first and second ends of the chromatographic medium;

(iii) a first substantially fluid-impermeable barrier layered on top of the first surface of the chromatographic medium and having an aperture for application of liquid to the chromatographic medium, the first barrier at least partially blocking application of liquid to the chromatographic medium;

(iv) an applicator layered on top of the first barrier, the applicator containing a labeled specific binding partner to the analyte in a form that can be resolubilized by the addition of an aqueous liquid to the applicator;

(v) a second substantially fluid-impermeable barrier layered on top of a first portion of the applicator centrally located on the applicator; and

(vi) a distribution membrane layered on top of the second barrier and in contact with portions of the applicator not supporting the second barrier so that fluid can flow from the distribution membrane to the applicator around the second barrier; and

(b) a second opposable component including a receptacle for a swab that can contain a test sample;

wherein the first and second opposable components are configured so that bringing the first and second opposable components into opposition results in applying a sample to the distribution membrane and then to the chromatographic medium.

31. The assay device of claim 30 comprising at least two absorbers, one in operable contact with the first end of the chromatographic medium and one in operable contact with the second end of the chromatographic medium, wherein the detection zone is substantially smaller than the area of the chromatographic medium, and wherein the chromatographic medium further includes a control zone of analyte or analogue thereof immobilized thereon in an area substantially smaller than the area of the chromatographic medium and not overlapping with the detection zone, the control and detection zones positioned with respect to the aperture and the absorber such that the detection zone is between the aperture and one of the absorbers and the control zone is between the aperture and the other absorber.

32. The assay device of claim 30 wherein the label of the labeled specific binding partner is a visually detectable label.

33. The assay device of claim 30 wherein the first and second opposable components can be placed in opposition so that pressure facilitates application of the sample to the chromatographic medium through the aperture.

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

(a) placing a swab containing the test sample in the receptacle of the assay device of claim 30;

(b) bringing the first and second opposable components of the assay device into opposition so that the test sample is transferred from the swab to the distribution membrane;

(c) allowing the sample to enter the applicator to resolubilize the labeled specific binding partner in the applicator to form a solution in the applicator containing the sample and the resolubilized labeled specific binding partner;

(d) allowing the solution containing the sample and the labeled specific binding partner to enter the chromatographic medium and to flow through at least a portion of the chromatographic medium including the detection zone; and

(e) detecting and/or determining the analyte in the test sample by observing and/or measuring the labeled specific binding partner bound at the detection zone.

35. An assay device for performing a competitive immunoassay for detection and/or determination of an analyte in a test sample, comprising:

(a) a chromatographic medium having a first end, a second end, and first and second surfaces, and having a secondary specific binding partner immobilized thereon in a detection zone between the first and second ends of the chromatographic medium, the secondary specific binding partner capable of binding a member of a specific binding pair that lacks affinity for the analyte;

(b) at least one absorber in operable contact with at least one of the first and second ends of the chromatographic medium;

(c) a substantially fluid-impermeable barrier layered on top of the first surface of the chromatographic medium, the barrier having at least one aperture for application of liquid to the chromatographic medium, the barrier at least partially blocking application of liquid to the chromatographic medium; and

(d) an affinity membrane layered on top of the barrier, the affinity membrane containing a specific binding partner to the analyte immobilized thereto;

the chromatographic medium, absorber, barrier, aperture, and affinity membrane being configured such a liquid applied to the affinity membrane is drawn through the chromatographic medium by the at least one absorber so that a labeled analyte analogue comprising analyte bound to a member of a specific binding pair lacking affinity for the analyte and bindable by the secondary specific binding partner, the analyte analog being labeled with a detectable label, binds at the detection zone on the chromatographic medium if analyte is present in the sample.

36. An assay device for performing a competitive immunoassay for detection and/or determination of an analyte in a test sample comprising:

(a) a first opposable component including;

(i) a chromatographic medium having a first end, a second end, and first and second surfaces, and having a secondary specific binding partner immobilized thereon in a detection zone between the first end and second end of the chromatographic medium, the secondary specific binding partner capable of binding a member of a specific binding pair that lacks affinity for the analyte, the secondary specific binding partner being an antibody specific for a species, class, or subclass of antibody that does not bind the analyte;

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

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

(iv) a substantially fluid-impermeable barrier layered on top of the first surface of the chromatographic medium and having an aperture therethrough for application of liquid to the chromatographic medium, the aperture being substantially smaller in area than the barrier so that liquid can enter the chormatographic medium only through the aperture;

the chromatographic medium, absorbers, barrier and aperture being configured so that the detection zone is located between the aperture and the first end of the chromatographic medium; and

(v) an affinity membrane layered on top of the barrier, the affinity membrane containing a specific binding partner to the analyte immobilized thereto; and

(b) a second opposable component including an applicator, the applicator containing an analyte analogue in a form that can be resolubilized by the addition of an aqueous liquid to the applicator, the analyte analogue comprising analyte covalently bound to a member of a specific binding pair lacking affinity for the analyte and bindable by the secondary specific binding partner, the analyte analogue 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 results in the applicator being in operable contact with the affinity membrane.

37. The assay device of claim 36 wherein the detectable label is a visually detectable label.

38. The assay device of claim 36 wherein the first and second opposable components can be placed in opposition so that pressure facilitates application of the liquid to the chromatographic medium through the aperture.

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

(a) applying the sample to the applicator of the assay device of claim 36;

(b) allowing the sample to resolubilize the labeled analyte analogue in the applicator to form a solution in the applicator containing the test sample and the resolubilized labeled analyte analog;

(c) bringing the first and second opposable components of the assay device into opposition so that the solution containing the test sample and the resolubilized labeled analyte analogue is transferred from the applicator to the affinity membrane;

(d) allowing the solution containing the test sample and the resolubilized analyte analogue to pass through the affinity membrane, and then into the chromatographic medium through the aperture in the barrier and to flow through at least a portion of the chromatographic medium including the detection zone; and

(e) detecting and/or determining the analyte in the test sample by observing and/or measuring the labeled analyte analogue bound to the detection zone by performance of a competitive immunoassay, wherein the quantity of labeled analyte analogue bound is directly correlated with the concentration of analyte in the test sample.

40. An assay device for detection and/or determination of an analyte in a test sample comprising:

(a) a first opposable component including;

(i) a chromatographic medium having a first end and a second end and first and second surfaces, and having immobilized thereon in separate discrete and non-overlapping zones between the first and second ends of the chromatographic medium;

(A) a detection zone with immobilized specific binding partner to the analyte; and

(B) a control zone of analyte or analogue thereof;

the detection zone being located between a point removed from the ends of the chromatographic medium and the second end of the chromatographic medium and the control zone being located between a point removed from the ends of the chromatographic medium and the second end of the chromatographic medium;

(ii) an absorber in operable contact with the first end of the chromatographic medium;

(iii) a first substantially fluid-impermeable barrier layered on top of the first surface of the chromatographic medium and having a first application aperture substantially smaller than the area of the chromatographic medium and located at a point removed from the ends of the chromatographic medium, the first barrier at least partially blocking application of liquid to the chromatographic medium;

(iv) an applicator layered on top of the first barrier, the applicator containing a labeled specific binding partner to the analyte in a form that can be resolubilized by the addition of an aqueous liquid to the applicator, the applicator positioned to apply liquid to the chromatographic medium via the first application aperture; and

(v) a second substantially fluid-impermeable barrier layered on top of the applicator and positioned to at least partially block application of liquid to the applicator and to allow application of liquid to the applicator through a second application aperture substantially smaller than the area of the chromatographic medium and located closer to the second end of the chromatographic medium then to the first end of the chromatographic medium; and

(b) a second opposable component including a sample preparation zone;

wherein the first and second opposable components are configured so that bringing the first and second opposable components into opposition results in the sample preparation zone being in contact with the second barrier so that the sample in the sample preparation zone is applied to the applicator through the second application aperture and traverses substantially the entire length of the applicator in order to apply the sample and the resolubilized labeled specific binding partner to the chromatographic medium through the first application aperture.

41. The assay device of claim 40 wherein the sample preparation zone contains at least one reagent for treatment of the sample.

42. A method for detection and/or determination of an analyte in a test sample comprising:

(a) applying the test sample to the sample preparation zone of the assay device of claim 41;

(b) incubating the sample in the sample preparation zone so the at least one reagent for treatment of the sample can react with the sample forming a treated sample;

(c) bringing the first and second opposable components into opposition so that the treated sample is transferred to the applicator through the second application aperture;

(d) allowing the treated sample transferred to the applicator to resolubilize the labeled specific binding partner in the applicator to form a solution containing the treated sample and the resolubilized labeled specific binding partner;

(e) allowing the solution containing the treated sample and the resolubilized labeled specific binding partner to enter the chromatographic medium and to flow through at least the portion of the chromatographic medium including the detection zone and the control zone; and

(f) detecting and/or determining the analyte in the test sample by observing and/or measuring the labeled specific binding partner bound to the detection zone.

43. The assay device of claim 40 wherein the detectable label is a visually detectable label.

44. The assay device of claim 40 wherein the first and second opposable components can be placed in opposition so that pressure facilitates application of the sample to the chromatographic medium through the first and second apertures.

45. A method for detection and/or determination of an analyte in a test sample comprising:

(a) applying the test sample to the sample preparation zone of the assay device of claim 40;

(b) bringing the first and second opposable components into opposition so that the test sample is transferred to the applicator through the second application aperture;

(c) allowing the sample transferred to the applicator to resolubilize the labeled specific binding partner in the applicator to form a solution containing the sample and the resolubilized labeled specific binding partner;

(d) allowing the solution containing the sample and the resolubilized labeled specific binding partner to enter the chromatographic medium and to flow through at least the portion of the chromatographic medium including the detection zone and the control zone; and

(e) detecting and/or determining the analyte in a test sample by observing and/or measuring the labeled specific binding partner bound at the detection zone.

46. An assay device for detection and/or determination of a hydrophobic analyte in a test sample comprising:

(a) a first opposable component including;

(i) a nylon chromatographic medium having a first end, a second end, and first and second surfaces, the chromatographic medium having an affinity for a hydrophobic analyte sufficient to immobilize the analyte from an aqueous solution containing the analyte on the chromatographic medium;

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

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

(iv) a substantially fluid-impermeable barrier layered on top of the first surface of the chromatographic medium and having an aperture for application of liquid to the chromatographic medium, the barrier at least partially blocking application of liquid to the chromatographic medium; and

(b) a second opposable component including a sample preparation zone;

wherein the first and second opposable components are configured so that bringing the first and second opposable components into opposition results in the sample preparation zone being in contact with the barrier so that the sample in the sample preparation zone is applied to the chromatographic medium and so that analyte in a test sample is immobilized on the chromatographic medium in at least the vicinity of the aperture.

47. The assay device of claim 46 wherein the detectable label is a visually detectable label.

48. The assay device of claim 46 wherein the antigen is a lipopolysaccharide.

49. The assay device of claim 46 wherein the sample preparation zone contains at least one reagent for treatment of the analyte.

50. The assay device of claim 46 wherein the first and second opposable components can be placed in opposition so that pressure facilitates application of the sample to the chromatographic medium through the aperture.

51. An assay device for detection and/or determination of an analyte in a test sample comprising:

(a) a first opposable component including;

(i) a chromatographic medium having a first end, a second end, and first and second surfaces, and having a specific binding partner for the analyte immobilized thereon in a detection zone between the first and second ends of the chromatographic medium;

(ii) a sample preparation zone in operable contact with the first end of the chromatographic medium;

(iii) a first absorber in operable contact with the second end of the chromatographic medium; and

(iv) a substantially fluid-impermeable barrier layered on top of the first surface of the chromatographic medium and having an aperture for application of liquid to the chromatographic medium, the barrier at least partially blocking application of liquid to the chromatographic medium; and

(b) a second opposable component including;

(i) an applicator containing a labeled specific binding partner to the analyte in a form that can be resolubilized by the addition of an aqueous liquid to the applicator, the applicator positioned such that when the first and second opposable components are brought into opposition, the applicator is in contact with the barrier so that the resolubilized labeled specific binding partner is applied through the aperture in the barrier to the chromatographic medium; and

(ii) second and third absorbers positioned such that when the first and second opposable components are brought into opposition, the second absorber and third absorber are in operable contact with portions of the chromatographic medium so that the second and third absorbers remove fluid from the chromatographic medium.

52. The assay device of claim 51 wherein the label is a visually detectable label.

53. The assay device of claim 51 wherein the sample preparation zone contains at least one reagent for treatment of the sample.

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

(a) applying the sample to the sample preparation zone of the assay device of claim 53;

(b) incubating the sample in the sample preparation zone so that the at least one reagent for treatment of the sample can react with the sample to produce a treated sample;

(c) allowing the treated sample to flow through at least the portion of the chromatographic medium including the detection zone;

(d) adding an aqueous liquid to the applicator to resolubilize the labeled specific binding partner to the analyte;

(e) bringing the first and second opposable components into opposition to apply the resolubilized labeled specific binding partner to the chromatographic medium through the aperture in the barrier and to bring the second and third absorbers into operable contact with the chromatographic medium to withdraw fluid therefrom;

(f) allowing the resolubilized labeled specific binding partner to flow through at least the portion of the chromatographic medium including the detection zone to form a ternary complex including labeled specific binding partner at the detection zone if analyte is present in the test sample; and

(g) detecting and/or determining the analyte in the test sample by observing and/or measuring the labeled specific binding partner bound at the detection zone.

55. The assay device of claim 51 wherein the chromatographic medium further includes a control zone of analyte or analogue thereof immobilized thereto in an area not overlapping with the detection zone, the detection zone being positioned between the aperture and the first end of the chromatographic medium and the control zone being positioned between the aperture and the second end of the chromatographic medium.

56. The assay device of claim 51 wherein the first and second opposable components can be placed in opposition so that pressure facilitates application of the resolubilized labeled specific binding partner to the chromatographic medium through the aperture.

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

(a) the assay device of claim 51; and

(b) an aqueous liquid for resolubilizing the labeled specific binding partner in the applicator.

58. A method for the detection and/or determination of an analyte in an aqueous test sample comprising:

(a) applying the sample to the sample preparation zone of the assay device of claim 51;

(b) allowing the sample to flow through at least the portion of the chromatographic medium including the detection zone;

(c) adding an aqueous liquid to the applicator to resolubilize the labeled specific binding partner to the analyte;

(d) bringing the first and second opposable components into opposition to apply the labeled specific binding partner to the chromatographic medium through the aperture in the barrier and to bring the second and third absorbers into operable contact with the chromatographic medium to withdraw fluid therefrom;

(e) allowing the resolubilized labeled specific binding partner to flow through at least the portion of the chromatographic medium including the detection zone to form a ternary complex including labeled specific binding partner at the detection zone if analyte is present in the test sample; and

(f) detecting and/or determining the analyte in the test sample by observing and/or measuring the labeled specific binding partner bound at the detection zone.

59. An assay device for detection and/or determination of an analyte in a test sample comprising:

(a) a first opposable component including;

(i) a chromatographic medium having a first end, a second end, and first and second surfaces, and having a specific binding partner to the analyte immobilized thereon in a detection zone between the first end and the second end of the chromatographic medium;

(ii) an absorber in operable contact with the second end of the chromatographic medium;

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

(iv) a conjugate zone containing a specific binding partner to the analyte labeled with a gold sol in a form that can be resolubilized by the addition of an aqueous liquid to the conjugate zone, the conjugate zone being in direct contact with the conductor;

(v) a sample preparation zone, the sample preparation zone being in direct contact with the conjugate zone, the conductor, the conjugate zone, and the sample preparation zone being positioned so that the conjugate zone bridges the sample preparation zone and the conductor; and

(vi) a substantially fluid-impermeable barrier layered on top of the first surface of the chromatographic medium and having an aperture for application of liquid to the chromatographic medium, the barrier at least partially blocking application of liquid to the chromatographic medium; and

(b) a second opposable component including;

(i) an applicator containing, in a form that can be resolubilized by the addition of an aqueous liquid to the applicator;

(1) a soluble silver salt and (2) a reducing agent, the applicator positioned such that when the first and second opposable components are brought into opposition, the applicator is in contact with the barrier so that the resolubilized silver salt and the reducing agent are applied through the aperture in the barrier to the chromatographic medium; and

(ii) second and third absorbers positioned such that when the first and second opposable components are brought into opposition, the second absorber and third absorber are in operable contact with portions of the chromatographic medium so that the second and third absorbers remove fluid from the chromatographic medium.

60. The assay device of claim 59 wherein the sample preparation zone contains at least one reagent for treatment of the sample.

61. A method for the detection and/or determination of an analyte in an aqueous test sample comprising:

(a) applying the sample to the sample preparation zone of the assay device of claim 60;

(b) incubating the sample and the sample preparation zone so that at least the one reagent for treatment of the sample can react with the sample to produce a treated sample;

(c) allowing the treated sample to flow through the conjugate zone to resolubilize the labeled specific binding partner in the conjugate zone;

(d) allowing the treated sample and the resolubilized labeled specific binding partner to enter the chromatographic medium through the conductor and to flow through at least the portion of the chromatographic medium including the detection zone;

(e) adding an aqueous liquid to the applicator to resolubilize the soluble silver salt and the reducing agent;

(f) bringing the first and second opposable components into opposition to apply the resolubilized silver salt and the reducing agent to the chromatographic medium through the aperture in the barrier and to bring the second and third absorbers into operable contact with the chromatographic medium to withdraw fluid from the chromatographic medium;

(g) allowing the resolubilized silver salt and reducing agent to flow through at least the portion of the chromatographic medium including the detection zone so that silver is deposited around gold in the gold label of the labeled specific binding partner bound at the detection zone to enhance the signal generated by the label; and

(h) detecting and/or determining the analyte in the test sample by observing and/or measuring the labeled specific binding partner bound at the detection zone.

62. The assay device of claim 59 wherein the soluble silver salt is silver lactate and the reducing agent is a quinone.

63. The assay device of claim 59 wherein the first and second opposable components can be placed in opposition so that pressure facilitates application of the resolubilized silver salt and the reducing agent to the chromatographic medium through the aperture.

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

(a) applying the sample to the sample preparation zone of the assay device of claim 59;

(b) allowing the sample to flow through the conjugate zone to resolubilize the labeled specific binding partner in the conjugate zone;

(c) allowing the sample and the resolubilized labeled specific binding partner to enter the chromatographic medium through the conductor and to flow through at least a portion of the chromatographic medium including the detection zone;

(d) adding an aqueous liquid to the applicator to resolubilize the soluble silver salt and the reducing agent;

(e) bringing the first and second opposable components into opposition to apply the resolubilized silver salt and the reducing agent to the chromatographic medium through the aperture in the barrier and to bring the second and third absorbers into operable contact with the chromatographic medium to withdraw fluid from the chromatographic medium;

(f) allowing the resolubilized silver salt and reducing agent to flow through at least the portion of the chromatographic medium including the detection zone so that silver is deposited around gold in the gold label of the labeled specific binding partner bound at the detection zone to enhance the signal generated by the label; and

(g) detecting and/or determining the analyte in the test sample by observing and/or measuring the labeled specific binding partner bound at the detection zone.

65. An assay device for the detection and/or determination of an analyte in a test sample comprising:

(a) a first opposable component including;

(i) a chromatographic medium having a first end, a second end, and first and second surfaces, and having a specific binding partner to the analyte immobilized thereon in a detection zone between the first and second ends of the chromatographic medium;

(ii) an absorber in operable contact with the second end of the chromatographic medium;

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

(iv) a conjugate zone containing a specific binding partner to the analyte labeled with a gold sol in a form that can be resolubilized by the addition of an aqueous liquid to the conjugate zone, the conjugate zone being in direct contact with the conductor;

(v) a sample preparation zone, the sample preparation zone being in direct contact with the conjugate zone, the conductor, conjugate zone and sample preparation zone being positioned so that the conjugate zone bridges the sample preparation zone and the conductor; and

(vi) a substantially fluid-impermeable barrier layered on top of the first surface of the chromatographic medium and having an aperture for application of liquid to the chromatographic medium, the barrier at least partially blocking application of liquid to the chromatographic medium; and

(b) the second opposable component including;

(i) an applicator including two sectors;

(A) a first sector for receiving an aqueous liquid; and

(B) a second sector containing, in a form that can be resolubilized by the addition of an aqueous liquid to the second sector of the applicator;

(1) a soluble silver salt and (2) a reducing agent, the applicator positioned so that when the first and second opposable components are brought into opposition, the first sector of the applicator is in direct contact with the aperture in the barrier and the second sector of the applicator is in indirect contact with the aperture so that the aqueous liquid is applied first to the chromatographic medium to provide a wash, followed by the aqueous silver salt and the reducing agent; and

(ii) second and third absorbers positioned such that when the first and second opposable components are brought into opposition, the second absorber is in operable contact with a portion of the chromatographic medium between the barrier and the first end of the chromatographic medium and the third absorber is in operable contact with a portion of the chromatographic medium between the barrier and the second end of the chromatographic medium so that the second and third absorbers remove fluid from the chromatographic medium.

66. The assay device of claim 65 wherein the sample preparation zone contains at least one reagent for treatment of the sample.

67. A method for the detection and/or determination of an analyte in an aqueous test sample comprising:

(a) applying the sample to the sample preparation zone of the assay device of claim 66;

(b) incubating the sample in the sample preparation zone so that the at least one reagent for treatment of the sample can react with the sample to produce a treated sample;

(c) allowing the treated sample to flow through the conjugate zone to resolubilize the labeled specific binding partner in the conjugate zone;

(d) allowing the treated sample and the resolubilized labeled specific binding partner to enter the chromatographic medium through the conductor and to flow through at least the portion of the chromatographic medium including the detection zone;

(e) adding an aqueous liquid to the first and second sectors of the applicator to resolubilize the soluble silver salt and the reducing agent in the second sector of the applicator;

(f) bringing the first and second components into opposition to apply the aqueous liquid in the first sector of the applicator to the chromatographic medium through the aperture as a wash, followed by the application of the resolubilized silver salt and the reducing agent to the chromatographic medium, and to bring the second and third absorbers into operable contact with the chromatographic medium to withdraw fluid from the chromatographic medium;

(g) allowing the resolubilized silver salt and reducing agent to flow through at least the portion of the chromatographic medium including the detection zone so that silver is deposited around gold in the gold label of the labeled specific binding partner bound at the detection zone to enhance the signal generated by the label; and

(h) detecting and/or determining the analyte in the test sample by observing and/or measuring the labeled specific binding partner bound at the detection zone.

68. The assay device of claim 65 wherein the soluble silver salt is silver lactate and the reducing agent is a quinone.

69. The assay device of claim 65 wherein the first and second opposable components can be placed in opposition so that pressure facilitates application of liquid to the chromatographic medium through the aperture.

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

(a) applying the sample to the sample preparation zone of the assay device of claim 65;

(b) allowing the sample to flow through the conjugate zone to resolubilize the labeled specific binding partner in the conjugate zone;

(c) allowing the sample and the resolubilized label to specific binding partner to enter the chromatographic medium through the conductor and to flow through at least a portion of the chromatographic medium including the detection zone;

(d) adding an aqueous liquid to the first and second sectors of the applicator to resolubilize the soluble silver salt and the reducing agent in the second sector of the applicator;

(e) bringing the first and second components into opposition to apply the aqueous liquid in the first sector of the applicator to the chromatographic medium through the aperture as a wash, followed by the application of the resolubilized silver salt and the reducing agent to the chromatographic medium, and to bring the second and third absorbers into operable contact with the chromatographic medium to withdraw fluid from the chromatographic medium;

(f) allowing the resolubilized silver salt and reducing agent to flow through at least the portion of the chromatographic medium including the detection zone so that silver is deposited around gold in the gold label of the labeled specific binding partner bound at the detection zone to enhance the signal generated by the label; and

(g) detecting and/or determining the analyte in the test sample by observing and/or measuring the labeled specific binding partner bound at the detection zone.

71. An assay device for the detection and/or determination of an analyte in a test sample comprising:

(a) a panel including;

(i) a first opposable component including;

(A) a chromatographic medium having a first end, a second end, and first and second surfaces, and having a specific binding partner to the analyte immobilized thereon in a detection zone between the first end and the second end of the chromatographic medium;

(B) a conjugate zone containing a specific binding partner to the analyte labeled with a gold sol in a form that can be resolubilized by the addition of an aqueous liquid to the conjugate zone, the conjugate zone being in operable contact with the first end of the chromatographic medium;

(C) a conductor in operable contact with the conjugate zone, the conjugate zone bridging the first end of the chromatographic medium and the conductor; and

(D) a substantially fluid-impermeable barrier layered on top of the first surface of the chromatographic medium and having an aperture for application of liquid to the chromatographic medium, the barrier at least partially blocking application of liquid to the chromatographic medium;

(ii) a second opposable component including;

(A) a first receptacle for a swab which may contain a test sample;

(B) a well for addition of at least one extraction reagent to the swab; and

(C) a first absorber separated from the first receptacle and the well; and

(iii) a second receptacle for holding a protrusion of an insert to align the insert with the first and second opposable components;

the first and second opposable components of the panel being configured so that, when they are brought into opposition, the first absorber is brought into contact with a portion of the chromatographic medium, and the first receptacle is brought into contact with the conductor; and

(b) an insert including;

(i) an applicator containing, in a form that can be resolubilized by the addition of an aqueous liquid to the applicator;

(1) a soluble silver salt and (2) a reducing agent;

(ii) a second absorber;

(iii) a third absorber; and

(iv) a protrusion for insertion into the second receptacle of the panel;

the insert configured so that when the protrusion is inserted into the second receptacle of the panel, the applicator is in operable contact with the aperture to apply the contents of the applicator to the chromatographic medium through the aperture, and the second and third absorbers are each in operable contact with a portion of the chromatographic medium to withdraw fluid from the chromatographic medium.

72. The assay device of claim 71 wherein the soluble silver salt is silver lactate and the reducing agent is a quinone.

73. The assay device of claim 71 wherein the first and second opposable components can be placed in opposition so that pressure facilitates application of the liquid to the chromatographic medium through the aperture.

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

(a) placing a swab containing a test sample in the first receptacle of the assay device of claim 71;

(b) applying at least one extraction reagent to the well to extract analyte from the swab;

(c) bringing the first and second opposable components into opposition so that the swab in the receptacle is in contact with the conductor to apply the extracted analyte with the conductor and so that the first absorber is in contact with a portion of the chromatographic medium;

(d) allowing the extracted sample to flow through the conjugate zone to resolubilize the labeled specific binding partner in the conjugate zone;

(e) allowing the extracted sample and the resolubilized labeled specific binding partner to enter the chromatographic medium and to flow through at least the portion of the chromatographic medium including the detection zone;

(f) adding an aqueous liquid to the applicator of the insert to resolubilize the soluble silver salt and the reducing agent;

(g) inserting the protrusion of the insert into the second receptacle of the panel and positioning the insert so that the applicator is in contact with the aperture in the barrier to apply the contents of the applicator to the chromatographic medium through the aperture and so that the second and third absorbers are in contact with the chromatographic medium to withdraw fluid from the chromatographic medium;

(h) allowing the resolubilized silver salt and reducing agent to flow through at least the portion of the chromatographic medium including the detection zone so that the silver is deposited around gold in the gold label of the labeled specific binding partner bound at the detection zone to enhance the signal generated by the label; and

(i) detecting and/or determining the analyte in the test sample by observing and/or measuring the labeled specific binding partner bound at the detection zone.

75. An assay device for the detection and/or determination of an analyte in a test sample comprising:

(a) a panel including;

(i) a first opposable component including;

(A) a chromatographic medium having a first end, a second end, and first and second surfaces and having a specific binding partner to the analyte immobilized thereon in a detection zone between the first and second ends of the chromatographic medium and substantially smaller than the area of the chromatographic medium;

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

(C) a substantially fluid-impermeable barrier layered on top of the first surface of the chromatographic medium and having an aperture for application of liquid to the chromatographic medium, the barrier at least partially blocking application of liquid to the chromatographic medium;

(ii) a second opposable component including a first absorber, the first and second opposable components being configured so that when they are brought into opposition the first absorber is brought into operable contact with a portion of the chromatographic medium so that the first absorber contacts the chromatographic medium on the opposite side of the detection zone from the conductor; and

(iii) a receptacle for holding a protrusion of an insert to align the insert with the first and second opposable components; and

(b) an insert including;

(i) an applicator containing, in a form that can be resolubilized by the addition of an aqueous liquid to the applicator, a substrate for an enzyme label that is bound to a specific binding partner to the analyte, the enzyme label producing an insoluble detectable product by catalysis of a reaction involving the substrate;

(ii) a second absorber;

(iii) a third absorber; and

(iv) a protrusion for insertion into the receptacle of the panel;

the insert configured so that when the protrusion is inserted into the receptacle of the panel, the applicator is in operable contact with the aperture to apply the contents of the applicator to the chromatographic medium through the aperture, and the second and third absorbers are each in operable contact with a portion of the chromatographic medium to withdraw fluid from the chromatographic medium.

76. The assay device of claim 75 wherein the first and second opposable components can be placed in opposition so that pressure facilitates application of the contents of the applicator to the chromatographic medium through the aperture.

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

(a) adding to an aqueous test sample a labeled specific binding partner to the analyte, the label of the labeled specific binding partner being an enzyme catalyzing a reaction producing an insoluble detectable product, to form a sample-label solution;

(b) applying the sample-label solution to the conductor of the assay device of claim 75;

(c) bringing the first and second opposable components into opposition to bring the first absorber into operable contact with a portion of the chromatographic medium;

(d) allowing the sample-label solution to enter the chromatographic medium and to flow through at least the portion of the chromatographic medium including the detection zone;

(e) adding an aqueous liquid to the applicator of the insert to resolubilize the substrate;

(f) inserting the protrusion of the insert into the receptacle of the panel and positioning the insert so that the applicator is in contact with the aperture and the barrier to apply the contents of the applicator to the chromatographic medium through the aperture and so that the second and third absorbers are placed in contact with the chromatographic medium to withdraw fluid from the chromatographic medium;

(g) allowing the resolubilized substrate to flow through at least the portion of the chromatographic medium including the detection zone, so that the enzyme of the enzyme label catalyzes a reaction involving the substrate and depositing an insoluble product as a signal of the label; and

(h) detecting and/or determining the analyte in the test sample by observing and/or measuring the labeled specific binding partner bound at the detection zone based on the insoluble product deposited as a signal.

78. An assay device for the detection and/or determination of an analyte in a test sample comprising:

(a) a panel including;

(i) a first opposable component including;

(A) a chromatographic medium having a first end, a second end, and first and second surfaces, and having a specific binding partner to the analyte immobilized thereon in a detection zone between the first and second ends of the chromatographic medium;

(B) a conjugate zone containing a specific binding partner to the analyte labeled with an enzyme in a form that can be resolubilized by the addition of an aqueous liquid to the conjugate zone, the conjugate zone being in contact with the first end of the chromatographic medium;

(C) a conductor in direct contact with the conjugate zone, the conjugate zone bridging the first end of the chromatographic medium and the conductor; and

(D) a substantially fluid-impermeable barrier layered on top of the first surface of the chromatographic medium and having an aperture for application of liquid to the chromatographic medium, the barrier at least partially blocking application of liquid to the chromatographic medium;

(ii) a second opposable component including;

(A) a first receptacle for a swab containing a test sample;

(B) a well for addition of at least one extraction reagent to the swab; and

(C) a first absorber separated from the first receptacle and the well; and

(iii) a second receptacle for holding a protrusion of an insert to align the insert with the first and second opposable components;

the first and second opposable components being configured so that when they are brought into opposition, the first absorber is brought into contact with a portion of the chromatographic medium so that the first absorber contacts the chromatographic medium on the opposite side of the detection zone from the conductor and the first receptacle is brought into contact with the conductor; and

(b) an insert including;

(i) an applicator containing, in a form that can be resolubilized by the addition of an aqueous liquid to the applicator, a substrate for the enzyme label bound to the specific binding partner to the analyte in the conjugate zone, the enzyme label producing a insoluble detectable product by catalysis of a reaction involving the substrate;

(ii) a second absorber;

(iii) a third absorber; and

(iv) a protrusion for insertion into the second receptacle of the panel;

the protrusion configured such that when the protrusion is inserted into the second receptacle of the panel, the applicator is in contact with the aperture to apply the contents of the applicator to the chromatographic medium through the aperture, and the second and third absorbers are each in contact with a portion of the chromatographic medium to withdraw fluid from the chromatographic medium.

79. The assay device of claim 78 wherein the first and second opposable components can be placed in opposition so that pressure facilitates application of the contents of the applicator to the chromatographic medium through the aperture.

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

(a) placing a swab containing a test sample in the first receptacle of the assay device of claim 78;

(b) applying at least one extraction reagent to the well to extract analyte from the swab;

(c) bringing the first and second opposable components into opposition so that the swab in the first receptacle is in contact with the conductor to apply the extracted analyte to the conductor and so that the first absorber is in contact with a portion of the chromatographic medium;

(d) allowing the extracted sample to flow through the conjugate zone to resolubilize the labeled specific binding partner in the conjugate zone;

(e) allowing the extracted sample and the resolubilized labeled specific binding partner to enter the chromatographic medium and to flow through at least the portion of the chromatographic medium including the detection zone;

(f) adding an aqueous liquid to the applicator of the insert to resolubilize the substrate;

(g) inserting the protrusion of the insert into the second receptacle of the panel and positioning the insert so that the applicator is in contact with the aperture and the barrier to apply the contents of the applicator to the chromatographic medium through the aperture and so that the second and third absorbers are in contact with the chromatographic medium to withdraw fluid from the chromatographic medium;

(h) allowing the resolubilized substrate to flow through at least a portion of the chromatographic medium including the detection zone so that the enzyme of the enzyme label catalyzes a reaction involving the substrate and depositing an insoluble product that is a signal of the label; and

(i) detecting and/or determining the analyte in the test sample by observing and/or measuring the labeled specific binding partner bound at the detection zone by means of the insoluble product deposited as a signal.

81. An assay device for the detection and/or determination of an analyte in a test sample comprising:

(a) a first opposable component including;

(i) the chromatographic medium having a first end, a second end, and first and second surfaces, and having a specific binding partner to the analyte immobilized thereon in a detection zone between the first and second ends of the chromatographic medium;

(ii) at least one absorber in operable contact with the one of the ends of the chromatographic medium;

(iii) a first substantially fluid-impermeable barrier layered on top of the first surface of the chromatographic medium and having a first aperture therethrough for application of liquid to the chromatographic medium;

(iv) a distribution membrane adjacent to the first substantially fluid-impermeable barrier to direct fluid to the first aperture in the first substantially fluid-impermeable barrier, the distribution membrane having a central portion and a portion surrounding the central portion;

(v) a second substantially fluid-impermeable barrier layered on top of the central portion of the distribution membrane;

(vi) an applicator containing a labeled specific binding partner to the analyte in resolubilizable form and layered on top of the second substantially fluid impermeable barrier and extending to make operable contact with the portion surrounding the central portion of the distribution membrane so that fluid can flow from the applicator around the second barrier to the portion of the distribution membrane surrounding the central portion; and

(vii) a surface barrier containing a second aperture therethrough for application of liquid to the applicator; and

(b) a second opposable component including a receptacle for a swab containing a test sample.

82. The assay device of claim 81 wherein the label of the labeled specific binding partner is a visually detectable label.

83. The assay device of claim 81 wherein the first and second opposable components can be placed in opposition so that pressure facilitates application of liquid to the chromatographic medium through the aperture.

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

(a) the assay device of claim 83; and

(b) at least one reagent for the extraction of analyte from the swab.

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

(a) placing a swab containing a test sample in the receptacle of the assay device of claim 83;

(b) applying at least one extraction reagent to the swab in the receptacle to extract analyte from the swab;

(c) bringing the first and second opposable components into opposition to apply the extracted analyte to the second aperture in the surface barrier and then to the applicator and chromatographic medium after passing around the second barrier and through the distribution membrane, thereby resolubilizing the labeled specific binding partner in the applicator;

(d) allowing the sample and the resolubilized labeled specific binding partner to the analyte to flow through at least the portion of the chromatographic medium including the detection zone; and

(e) detecting and/or determining the analyte in the test sample by observing and/or measuring the labeled detection reagent bound at the detection zone.