Rapid diagnostic device, assay and multifunctional buffer

• US 20030049857A1
• Filed: 06/06/2002
• Published: 03/13/2003
• Est. Priority Date: 06/07/2001
• Status: Active Grant

• Current Assignee: MedMira Incorporated (MedMira Holding AG)
• Original Assignee: MedMira Incorporated (MedMira Holding AG)
• Inventors: Chan, Hermes K.W.
• Granted Patent: US 7,531,362 B2
• Time in Patent Office: Days
• US Class Current: 436/170
• 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: Y10T 436/10 Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
• CPC Class Codes: Y10T 436/108331 Preservative, buffer, anticoagulant or diluent

Abstract

An improved rapid diagnostic device, assay and multifunctional buffer reagent are provided for the detection of a target analyte in a fluid test sample. The 2-step assay utilizes a dual component flow-through device comprising a test unit and a post-filter unit capable of receiving the fluid sample and multifunctional buffer, respectively. The test unit comprises a reaction zone containing immobilized capture reagent that can specifically bind to the target analyte, an absorbent zone supporting the reaction zone, and optionally, a blood separation zone in lateral fluid communication with the reaction zone. The post-filter unit comprises a label zone permeated with a dried indicator reagent which is capable of being placed in transient fluid communication with the reaction zone of the test unit during the assay procedure. The rapid diagnostic assay system reduces the number of assay reagents, method steps and time required for performance compared to other conventional assays.

Claims

1. A device for determining the presence or absence of a target analyte in a fluid test sample, comprising:

a test unit comprising a reaction zone in vertical communication with an absorbent zone, wherein the reaction zone contains immobilized capture reagent capable of binding with a target analyte of interest to form a two-membered complex of a specific binding interaction; and

a post-filter unit comprising a label zone containing a dried indicator reagent, wherein the indicator reagent is capable of binding to a member of the specific binding interaction to produce a visually detectable signal following resolubilization thereof by buffer reagent; and

wherein the reaction zone of the test unit and the label zone of the post-filter unit are capable of being disposed in transient fluid communication with each other so as to allow direct passage of resolubilized indicator reagent from the label zone to the reaction zone following application of buffer reagent to the label zone.

2. The device according to claim 1, wherein the specific binding interaction is an antibody-antigen interaction.

3. The device according to claim 1, wherein the indicator reagent is capable of binding to a target analyte at a site which does not interfere with the specific binding interaction between the target analyte and the capture reagent.

4. The device according to claim 1, wherein the indicator reagent is capable of binding to the capture reagent at a site which interferes with the specific binding interaction between the target analyte and the capture reagent.

5. The device according to claim 2, wherein the target analyte is an antigen and the capture reagent is a monoclonal antibody or an affinity purified polyclonal antibody for the antigen.

6. The device according to claim 1, wherein the reaction zone is comprised of a material which has a pore size permitting separation and filtration of unbound components from the fluid test sample and a thickness which permits an adequate amount of capture reagent to be immobilized thereto.

7. The device according to claim 6, wherein the material has a pore size ranging from about 0.1 to 12.0 microns.

8. The device according to claim 7, wherein the material has a pore size ranging from about 0.2 to 0.8 microns.

9. The device according to claim 6, wherein the thickness of the material ranges from about 0.05 mm to about to 3.0 mm.

10. The device according to claim 9, wherein the thickness of the material ranges from about 0.1 to about 1.0 mm.

11. The device according to claim 6, wherein the material is a nitrocellulose membrane.

12. The device according to claim 1, wherein the reaction zone contains two or more different capture reagents immobilized thereto in discernable and separate areas so that multiple target analytes in a single fluid test sample can be analyzed simultaneously.

13. The device according to claim 1, wherein the reaction zone further comprises an immobilized control reagent in a discernable and separate area from the capture reagent.

14. The device according to claim 1, wherein the absorbent zone is separated from the reaction zone by an intervening spacer layer having one or more openings defined therein to permit fluid communication between the reaction zone and the absorbent zone.

15. The device according to claim 14, wherein the spacer layer is a rigid or semi-rigid fluid-resistant material.

16. The device according to claim 1, wherein the absorbent zone comprises one or more layers of a material which is capable of wicking fluid by capillary action and absorbing a substantial volume of fluid.

17. The device according to claim 16, wherein two or more layers comprise identical or different materials.

18. The device according to claim 16, wherein the material is cellulose acetate.

19. The device according to claim 1, wherein the label zone comprises a filter material having a pore size capable of allowing dried indicator reagent to be effectively resolubilized by buffer reagent and transferred to the reaction zone by laminar fluid flow.

20. The device according to claim 19, wherein the filter material is glass fiber material.

21. The device according to claim 1, wherein the indicator reagent comprises a direct label.

22. The device according to claim 21, wherein the direct label is colloidal gold.

23. The device according to claim 1, wherein said test unit and said post-filter unit are contained in a suitable housing.

51. A method for determining the presence or absence of a target analyte in a fluid test sample, comprising the steps of:

depositing the fluid test sample onto the reaction zone of the test unit of the device defined in claim 1;

bringing the label zone of the post-filter unit and the reaction zone of the test unit into fluid communication therewith;

applying buffer reagent to the post-filter unit to reconstitute the dried indicator reagent and wash any unbound reactants from the reaction zone; and

removing the post-filter unit to observe a test result depicted by a presence or absence of a visually detectable signal on the reaction zone.

52. The method according to claim 51, wherein the buffer reagent is a multifunctional buffer comprising:

a biological buffer to maintain the pH between 7.0 to 10.0;

at least one surfactant to reduce non-specific binding of assay reagents while simultaneously avoiding inhibition of a specific binding interaction;

a high molecular weight polymer as a dispersing and suspending reagent having a molecular weight in a range of from about 2×

10² to about 2×

10⁶ D;

a pH stabilizer to maintain the pH of the multifunctional buffer within a range of about pH 7.0 to 10.0;

an ionic salt to reduce non-specific binding of antibodies;

at least one preservative to reduce bacterial and microbial growth; and

a calcium chelator to prevent a whole blood test sample from clotting;

wherein the biological buffer, the surfactant, the high molecular weight polymer, the pH stabilizer, the ionic salt, the preservative and the calcium chelator are all in effective concentrations.

24. A device for determining the presence or absence of a target analyte in a whole blood test sample, comprising:

a test unit comprising a reaction zone in vertical communication with an absorbent zone, wherein the reaction zone contains immobilized capture reagent capable of binding with a target analyte of interest to form a two-membered complex of a specific binding interaction;

a blood separation zone in lateral communication with the reaction zone wherein the blood separation zone has a first end defining a region for receiving a whole blood test sample, and a second end in direct communication with the reaction zone; and

a post-filter unit comprising a label zone containing a dried indicator reagent, wherein the indicator reagent is capable of binding to a member of the specific binding interaction to produce a visually detectable signal following resolubilization thereof by buffer reagent; and

wherein the reaction zone of the test unit and the label zone of the post-filter unit are capable of being disposed in transient fluid communication with each other so as to allow direct passage of resolubilized indicator reagent from the label zone to the reaction zone following application of buffer reagent to the label zone.

25. The device according to claim 24, wherein the blood separation zone comprises a material capable of selectively retaining an effective amount of red blood cells (RBC) from the whole blood test sample to generate a substantially RBC-free fluid portion which can flow with unimpaired movement from the first end of the blood separation zone to the reaction zone.

26. The device according to claim 25, wherein the material is a glass fiber matrix.

27. The device according to claim 25, wherein the material comprises a hydrophobic carrier capable of reducing seepage of the whole blood test sample and the RBC-free fluid portion as it migrates along the blood separation zone.

28. The device according to claim 24, wherein the test unit and the post-filter unit are housed in a suitable container.

53. A method for determining the presence or absence of a target analyte in a whole blood test sample, comprising the steps of:

depositing the whole blood test sample onto the first end of the blood separation zone of the device defined in claim 24;

bringing the label zone of the post-filter unit and the reaction zone of the test unit into fluid communication therewith;

applying buffer reagent to the post-filter unit to reconstitute the dried indicator reagent and wash any unbound reactants from the reaction zone; and

removing the post-filter unit to observe a test result depicted by a presence or absence of a visually detectable signal on the reaction zone.

54. The method according to claim 53, wherein the buffer reagent is a multifunctional buffer comprising:

a biological buffer to maintain the pH between 7.0 to 10.0;

at least one surfactant to reduce non-specific binding of assay reagents while simultaneously avoiding inhibition of a specific binding interaction;

a high molecular weight polymer as a dispersing and suspending reagent having a molecular weight in a range of from about 2×

10² to about 2×

10⁸ D;

a pH stabilizer to maintain the pH of the multifunctional buffer within a range of about pH 7.0 to 10.0;

an ionic salt to reduce non-specific binding of antibodies;

at least one preservative to reduce bacterial and microbial growth; and

a calcium chelator to prevent a whole blood test sample from clotting;

wherein the biological buffer, the surfactant, the high molecular weight polymer, the pH stabilizer, the ionic salt, the preservative and the calcium chelator are all in effective concentrations.

29. A multifunctional buffer comprising:

a biological buffer to maintain the pH between 7.0 to 10.0;

at least one surfactant to reduce non-specific binding of assay reagents while simultaneously avoiding inhibition of a specific binding interaction;

a high molecular weight polymer as a dispersing and suspending reagent having a molecular weight in a range of from about 2×

10² to about 2×

10⁸ D;

a pH stabilizer to maintain the pH of the multifunctional buffer within a range of about pH 7.0 to 10.0;

an ionic salt to reduce non-specific binding of antibodies;

at least one preservative to reduce bacterial and microbial growth; and

a calcium chelator to prevent a whole blood test sample from clotting;

wherein the biological buffer, the surfactant, the high molecular weight polymer, the pH stabilizer, the ionic salt, the preservative and the calcium chelator are all in effective concentrations.

30. The multifunctional buffer according to claim 29, wherein the concentration of the biological buffer ranges from about 5 to 100 mM.

31. The multifunctional buffer according to claim 30, wherein the concentration of the biological buffer ranges from about 5 to 30 mM.

32. The multifunctional buffer according to claim 31, wherein the concentration of the biological buffer is about 5 mM.

33. The multifunctional buffer according to claim 38, wherein the biological buffer is a phosphate buffer.

34. The multifunctional buffer according to claim 29, wherein the concentration of the surfactant ranges from about 0.01 to about 0.50% (w/v).

35. The multifunctional buffer according to claim 34, wherein the concentration of the surfactant ranges from about 0.05 to about 0.10% (w/v).

36. The multifunctional buffer according to claim 35, wherein the concentration of the surfactant is about 0.07% (w/v).

37. The multifunctional buffer according to claim 29, wherein the surfactant is Triton®

X-100.

38. The multifunctional buffer according to claim 29, wherein the concentration of ionic salt is from about 0 to about 300 mM.

39. The multifunctional buffer according to claim 38, wherein the concentration of ionic salt is from about 50 to about 200 mM.

40. The multifunctional buffer according to claim 29, wherein the ionic salt is sodium chloride.

41. The multifunctional buffer according to claim 29, wherein the polymer is polyvinylpyrrolidone having a molecular weight range from about 10 kD to about 1500 kD.

42. The multifunctional buffer according to claim 41, wherein the concentration of polyvinylpyrrolidone polymer is about 0.1 to about 3.0% (w/v).

43. The multifunctional buffer according to claim 42, wherein the concentration of polyvinylpyrrolidone polymer is about 0.5 to about 2.5% (w/v).

44. The multifunctional buffer according to claim 43, wherein the concentration of polyvinylpyrrolidone polymer is about 1.4% (w/v).

45. The multifunctional buffer according to claim 29, wherein the calcium chelator is EDTA.

46. The multifunctional buffer according to claim 45, wherein the concentration of EDTA is from about 5.0 to 100.0 mM.

47. The multifunctional buffer according to claim 46, wherein the concentration of EDTA is from about 10.0 to 50.0 mM.

48. The multifunctional buffer according to claim 47, wherein the concentration of EDTA is about 20.0 mM.

49. The multifunctional buffer according to claim 29, wherein the pH stabilizer is trizma hydrochloride.

50. The multifunctional buffer according to claim 49, wherein the concentration of the pH stabilizer is from about 20 to 30 mM.

55. A test kit for use in the detection of a target analyte in a fluid test sample or a whole blood test sample suspected of containing the analyte, comprising:

a device for determining the presence or absence of a target analyte in a whole blood test sample, comprising;

a test unit comprising a reaction zone in vertical communication with an absorbent zone, wherein the reaction zone contains immobilized capture reagent capable of binding with a target analyte of interest to form a two-membered complex of a specific binding interaction; and

optionally comprising a blood separation zone in lateral communication with the reaction zone wherein the blood separation zone has a first end defining a region for receiving a whole blood test sample, and a second end in direct communication with the reaction zone; and

a post-filter unit comprising a label zone containing a dried indicator reagent, wherein the indicator reagent is capable of binding to a member of the specific binding interaction to produce a visually detectable signal following resolubilization thereof by buffer reagent; and

wherein the reaction zone of the test unit and the label zone of the post-filter unit are capable of being disposed in transient fluid communication with each other so as to allow direct passage of resolubilized indicator reagent from the label zone to the reaction zone following application of buffer reagent to the label zone; and

buffer reagent to reconstitute the dried indicator reagent and wash any unbound reactants from the reaction zone.

56. The test kit according to claim 55, wherein the buffer reagent is a multifunctional buffer comprising:

a biological buffer to maintain the pH between 7.0 to 10.0;

at least one surfactant to reduce non-specific binding of assay reagents while simultaneously avoiding inhibition of a specific binding interaction;

a high molecular weight polymer as a dispersing and suspending reagent having a molecular weight in a range of from about 2×

10² to about 2×

10⁶ D;

a pH stabilizer to maintain the pH of the multifunctional buffer within a range of about pH 7.0 to 10.0;

an ionic salt to reduce non-specific binding of antibodies;

at least one preservative to reduce bacterial and microbial growth; and

a calcium chelator to prevent a whole blood test sample from clotting;

wherein the biological buffer, the surfactant, the high molecular weight polymer, the pH stabilizer, the ionic salt, the preservative and the calcium chelator are all in effective concentrations.

57. The test kit according to claim 55, further comprising:

instructions for performing the diagnostic assay; and

a pair of pipettes for separately applying the test sample and the buffer reagent to the test unit and the post-filter unit, respectively.

58. The test kit according to claim 55, wherein the test unit and the post-filter unit are housed in a suitable container.