Membrane assay using focused sample application

• US 4,818,677 A
• Filed: 12/03/1987
• Issued: 04/04/1989
• Est. Priority Date: 12/03/1987
• Status: Expired due to Term

• Current Assignee: Quidel Corporation
• Original Assignee: Monoclonal Antibodies, Inc. (Quidel Corporation)
• Inventors: Hay-Kaufman, Martha L., Danisch, Robert, Becker, Rosette
• Primary Examiner(s): WARDEN, ROBERT J
• Application Number:
  US07/128,257
• Time in Patent Office: 488 Days
• Field of Search: 435/7, 435/805, 435/810, 435/4, 435/5, 435/6, 436/810, 436/813, 422/56, 422/58
• US Class Current: 435/4
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 33/76 Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
• CPC Class Codes: Y10S 435/805 Test papers
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 436/81 Tube, bottle, or dipstick
• CPC Class Codes: Y10S 436/813 Cancer

Abstract

Methods and kits are described for performing immunoassays. The kit includes a reaction cell having a microporous membrane and an absorbent capable of drawing liquid sample through the membrane. An applicator is provided for applying a small volume of the liquid sample to the membrane by contacting a port of the applicator to the membrane and allowing the sample to flow therethrough by capillary action. Analyte in the sample is immobilized, typically by immunoadsorption, and the immobilized analyte may then be visualized using conventional signal producing systems, such as color, fluorescence, and luminescent systems.

Claims

1. An assay method for determining the presence of an analyte in a liquid sample, said method employing:

a reaction cell including a microporous membrane and an absorbent in liquid receiving relationship with the membrane;

an applicator separate from the reaction cell and having a port with an open area in the range from about 0.5 mm² to 20 mm², said area being substantially less than the area of the membrane; and

a reagent system including components capable of interacting with analyte immobilized within the microporous membrane to produce a visual signal;

said method comprising;

applying to the membrane of the reaction cell in a predetermined order (1) a preselected volume of sample, and (2) the components of the reagent system, wherein at least one of the sample and the components is applied using the applicator, the port of the applicator being contacted with the membrane to limit outward diffusion of the analyte within the membrane, whereby a visual signal is produced having the dimensions of the port; and

observing the entire surface of the membrane to determine if the visual signal has been produced.

2. An assay method as in claim 1, wherein the applicator is a capillary tube and the sample or component is drawn from the tube by capillary action without pressurization.

3. An assay method as in claim 1, wherein the membrane and the absorbent in the reaction cell are separated by a porous, non-absorbent layer which remains substantially free from color development, whereby color which develops in the absorbent is visually blocked from the membrane.

4. An assay method as in claim 1, wherein the sample volume is in the range from about 1 μ

l to 1000 μ

l.

5. An assay method as in claim 1, wherein the reagent system produces a color signal.

6. An assay method as in claim 5, wherein the reagent system includes an enzyme and enzyme substrate.

7. An assay method as in claim 1, wherein the reagent system produces a fluorescent signal.

8. An assay method as in claim 1, wherein the applicator port has an area in the range from about 1 mm² to 10 mm².

9. An assay method as in claim 8, wherein the applicator port is generally circular having a diameter in the range from 1 mm to 3 mm.

10. An assay method as in claim 1, wherein the membrane includes an immobilized substance capable of specifically binding the analyte.

11. An assay method as in claim 10, wherein the substance is an antibody specific for the analyte.

12. An assay method as in claim 1, wherein the membrane is substantially free from specifically binding substances and blocked against non-specific binding.

13. An assay method as in claim 1, wherein c control sample is applied to a second area on the membrane prior to addition of the components of the reagent system required for production of the visual signal, whereby the signal resulting from the liquid sample may be compared to that resulting from the control sample.

14. An assay method as in claim 1, wherein the analyte is selected from the group consisting of antigens, antibodies, haptens, drugs, hormones macromolecules, toxins, bacteria, viruses, enzymes, tumor markers, and nucleic acids.

15. An assay method as in claim 1, wherein the applicator is a capillary tube.

16. An assay method as in claim 1, wherein the applicator is a focusing tube.

17. An assay method as in claim 16, wherein the focusing tube includes at least two ports, wherein each of the ports will contact a different area on the membrane.

18. An assay method as in claim 17, wherein different samples are applied through each of the ports.

19. An assay method as in claim 17, wherein different reagent system components are applied through each of the ports.

45. An assay method as in claim 1, wherein the exposed membrane area is in the range from about 0.2 cm² to 2 cm².

20. An assay method for determining the presence of an analyte in a liquid sample, said method comprising:

passing a predetermined volume of the sample through a limited area in the range from about 0.5 mm² to 20 mm² on a microporous membrane capable of immobilizing the analyte, wherein the exposed area of the membrane is sufficiently greater than the limited area to allow visualization of the sample on the limited area in contrast to the remaining area of the membrane;

applying to the entire membrane surface in a predetermined order components of a reagent system capable of interacting with analyte immobilized on the microporous membrane to produce a visual signal within said well-defined area only; and

observing the membrane to determine if a visual signal has been produced within the limited area.

21. An assay method as in claim 20, wherein the sample is passed through the membrane by contacting one face of the membrane with a capillary tube holding a predetermined volume of sample and absorbing the sample with an absorbent placed against the other face of the membrane.

22. An assay method as in claim 20, wherein the membrane includes an immobilized substance capable of specifically binding the analyte.

23. An assay method as in claim 22, wherein the substance is an antibody specific for the analyte.

24. An assay method as in claim 20, wherein the membrane is substantially free from specifically-binding substances.

25. An assay method as in claim 20, wherein the sample volume is in the range from about 1 μ

l to 1000 μ

l.

26. An assay method as in claim 20, wherein the reagent system produces a color signal within said limited area.

27. An assay method as in claim 26, wherein the reagent system includes an enzyme and enzyme substrate.

28. An assay method as in claim 20, wherein the reagent system produces a fluorescent signal.

29. An assay method as in claim 20, wherein the analyte is selected from the group consisting of antigens, antibodies, haptens, drugs, hormones, macromolecules, toxins, bacteria, viruses, enzymes, tumor markers, and nucleic acids.

30. An assay method as in claim 20, wherein the sample is passed through the membrane by contacting one face of the membrane with a focusing tube and applying liquid sample through the focusing tube, said sample being absorbed by an absorbent placed against the other face of the membrane.

46. An assay method as in claim 20, wherein the exposed membrane area is in the range from about 0.2 cm² to 2 cm².

31. An assay kit comprising:

a reaction cell including a microporous membrane and an absorbent in liquid receiving relationship with the membrane;

an applicator separate from the reaction cell and having a port with an open area in the from range from about 0.5 mm² to 20 mm², said area being substantially less than the area of the membrane;

a reagent system including components capable of interacting with analyte immobilized within the microporous membrane to produce a visual signal; and

instructions to apply to the membrane of the reaction cell in a predetermined order (1) a preselected volume of sample, and (2) the components of the reagent system, wherein at least one of the sample and the components is applied using the applicator with the port in contact with the membrane to limit outward diffusion of the sample within the membrane; and

to observe the membrane to determine if a visual signal has been produced.

32. An assay kit as in claim 31, wherein the applicator is a capillary tube.

33. An assay kit as in claim 31, wherein the membrane and the absorbent in the reaction cell are separated by a porous, non-absorbent layer which remains substantially free from color development, whereby color which develops in the absorbent is visually blocked from the membrane.

34. An assay kit as in claim 31, wherein the reagent system produces a color signal.

35. An assay kit as in claim 34, wherein the reagent system includes an enzyme and enzyme substrate.

36. An assay kit as in claim 31, wherein the reagent system produces a fluorescent signal.

37. An assay kit as in claim 31, wherein the membrane includes an immobilized substance capable of specifically binding the analyte.

38. An assay kit as in claim 37, wherein the substance is an antibody specific for the analyte.

39. An assay kit as in claim 31, wherein the membrane is substantially free from specifically-binding substances.

40. An assay kit as in claim 31, wherein the analyte is selected from the group consisting of antigens, antibodies, haptens, drugs, hormones, macromolecules, toxins, bacteria, viruses, enzymes, tumor markers, and nucleic acids.

41. An assay kit as in claim 31, wherein the applicator is a focusing tube.

42. An assay kit as in claim 41, wherein the applicator tube includes at least two ports, wherein each of the ports will contact a different area on the membrane.

43. An assay kit as in claim 42, wherein the instructions state to apply different samples through each of the ports.

44. An assay kit as in claim 42, wherein the instructions state to apply different reagent system components through each of the ports.

47. An assay kit as in claim 31, wherein the membrane has an exposed area in the range from about 0.2 cm² to 2 cm².