Lateral flow, non-bibulous membrane assay protocols

• US 4,943,522 A
• Filed: 08/10/1988
• Issued: 07/24/1990
• Est. Priority Date: 06/01/1987
• Status: Expired due to Term

• Current Assignee: Quidel Corporation
• Original Assignee: Quidel Corporation
• Inventors: Eisinger, Robert W., Sargeant, Robert B., Katz, David H., Khalil, Mohammed H.
• Primary Examiner(s): Kepplinger, Esther M.
• Assistant Examiner(s): Spiegel, Carol A.
• Application Number:
  US07/230,642
• Time in Patent Office: 713 Days
• Field of Search: 422/55-61, 422/70, 422/101, 422/102, 424/11, 435/7, 435/5, 435/805, 435/810, 436/514-520, 436/512, 436/523, 436/531, 436/535, 436/807, 436/808, 436/810, 210/431
• US Class Current: 435/7.25
• CPC Class Codes: G01N 33/54386 Analytical elements
• CPC Class Codes: G01N 33/54388 based on lateral flow
• CPC Class Codes: G01N 33/558 using diffusion or migration of antigen or antibody immunochromatographic test strips G01N33/54387
• CPC Class Codes: Y10S 435/805 Test papers
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 436/807 Apparatus included in process claim, e.g. physical support structures
• CPC Class Codes: Y10S 436/808 Automated or kit
• CPC Class Codes: Y10S 436/81 Tube, bottle, or dipstick

Abstract

A method and apparatus for conducting specific binding pair assays, such as immunoassays, is described. A porous membrane capable of non-bibulous lateral flow is used as assay substrate; a member of the binding pair is affixed in an indicator zone defined in the substrate. The sample is applied at a position distant from the indicator zone and permitted to flow laterally through the zone; any analyte in the sample is complexed by the affixed specific binding member, and detected. A novel method of detection employs entrapment of observable particle in the complex. Blood is a particularly preferred sample as the red blood cells can be used as the observable particles for detection of the complex.

Claims

1. Apparatus for determining the presence or absence or the approximate amount of an analyte in a liquid sample which method comprises:

a non-bibulous lateral flow membrane;

wherein said membrane has on its surface;

a liquid sample application zone defined to receive said liquid sample;

at least one indicator zone spaced laterally apart from the application zone on said surface, wherein immobilized in said indicator zone is a member of a binding pair capable of binding said analyte or a derivative of said analyte; and

wherein the lateral spacing of the application zone and the indicator zone is configured so as to cause the liquid sample introduced at the application zone to flow by non-bibulous lateral flow through the indicator zone;

so as to permit the immobilized member to react with said analyte or derivative thereof so that the presence or absence or approximate amount of analyte is determined in the indicator zone.

2. The apparatus of claim 1 which further includes an absorption zone in lateral contact with said indicator zone so as to cause said liquid sample to flow through the indicator zone and into the absorption zone.

3. The apparatus of claim 2 wherein the absorption zone comprises a contiguous portion of the non-bibulous lateral flow membrane.

4. The apparatus of claim 2 wherein the absorption zone comprises a bibulous absorption body in contact with the non-bibulous lateral flow membrane.

5. The apparatus of claim 1 wherein the membrane is elongated in the application zone and the indicator zone are linearly spaced along the non-bibulous lateral flow membrane.

6. The apparatus of claim 5 which comprises a multiplicity of indicator zones linearly spaced along the non-bibulous lateral flow membrane.

7. The apparatus of claim 1 which comprises a multiplicity of indicator zones spaced radially around the liquid sample application zone.

8. The apparatus of claim 7 wherein the indicator zones are equidistant from the liquid sample application zone.

9. The apparatus of claim 1 wherein the member of the binding pair immobilized in the indicator zone is an antibody or fragment thereof.

10. The apparatus of claim 9 wherein the antibody or fragment thereof immobilized in the indicator zone is immunoreactive with a blood group antigen.

11. The apparatus of claim 1 wherein the member of the binding pair immobilized in the indicator zone is an antigen.

12. The apparatus of claim 1 which further contains a sample receiving pad in contact and on the top surface of the liquid sample application zone on the non-bibulous lateral flow membrane.

13. The apparatus of claim 12 wherein said sample receiving pad contains a reagent reactive with analyte to form a derivative of said analyte which derivative is reactive with the member of the binding pair that is immobilized in the indicator zone.

14. The apparatus of claim 13 wherein the reagent is an antibody or antigen or fragment thereof.

15. The apparatus of claim 1 which further comprises a breakable container of liquid reagent, said container positioned so as to release the reagent laterally through the non-bibulous lateral flow membrane and through the indicator zone without passing through the liquid sample application zone.

16. The apparatus of claim 15 which further includes a means to break said container.

17. An apparatus for measuring the presence or absence or approximate quantity of an analyte in a liquid sample which apparatus comprises, in combination:

a non-bibulous lateral flow membrane;

a housing for holding the membrane, said holding being so constructed as to form;

means defining an application zone on the non-bibulous lateral flow membrane for receiving a liquid sample in said application zone;

at least on indicator zone laterally apart from said application zone on said membrane to which is immobilized a member of a binding pair, said member being reactive with with analyte or a derivative or said analyte; and

reading access means for permitting the indicator zone to be read, thus permitting measuring the presence or absence or approximate quantity of analyte by reading said indicator zone.

18. The apparatus of claim 17 which further comprises a bibulous absorbent body;

and

means for positioning a portion of the non-bibulous lateral flow membrane into contact with the absorbent body for permitting liquid to flow from the membrane to the absorbent body.

19. The apparatus of claim 17 further comprising a sample receiving pad on top of and contacting the liquid sample application zone.

20. The apparatus of claim 19 wherein said sample receiving pad contains a reagent capable of reacting with analyte to form a derivative of said analyte which is reactive with the member of the binding pair that is immobilized in the indicator zone.

21. The apparatus of claim 17 further comprising:

at least one breakable container of liquid reagent,

said container positioned to permit liquid to flow from the container, upon being broken in use, into the non-bibulous lateral flow membrane and through the indicator zone without passing through the liquid sample application zone.

22. A method for determining the presence or approximate amount of analyte in a sample comprising the steps of:

placing the sample on the liquid sample application zone of a non-bibulous lateral flow membrane which comprises a liquid sample application zone and at least one indicator zone spaced apart from said application zone laterally on the surface of said membrane, there being immobilized in said indicator zone a member of a binding pair capable of binding the analyte or a derivative thereof,

said sample being of sufficient quantity to cause sample liquid to flow laterally from the application zone through said indicator zone to permit the binding of analyte or derivative thereof with the member of the binding pair immobilized in the indicator zone; and

assessing the binding of the analyte or derivative thereof in the indicator zone, to determine the presence, absence or approximate amount of analyte.

23. The method of claim 22 wherein the member of the binding pair immobilized in the indicator zone is an antibody or fragment thereof and the analyte is an antigen which contains an epitope reactive with the antibody or fragment thereof.

24. The method of claim 23 wherein the antigen is a soluble antigen.

25. The method of claim 24 wherein the antigen is selected from the group consisting of, tissue-specific cell surface markers, tissue-shared cell surface markers, viral-associated cell surface markers, tumor-specific cell surface markers, bacterial polysaccharides, viral coat proteins, hormones, drugs, and antibodies.

26. The method of claim 24 wherein the antigen is selected from the group consisting of apolipoprotein B and apolipoprotein A1.

27. The method of claim 23 wherein the antigen is bound to the surface of a particle.

28. The method of claim 27 wherein the particle is a cell.

29. The method of claim 27 wherein the particle is selected from the group consisting of red blood cells, platelets, tumor cells, bacteria, viruses, and viral particles.

30. The method of claim 29 wherein the particle is a red blood cell.

31. The method of claim 22 wherein the derivative of the analyte is a conjugate of said analyte with a member complementary to the member of the binding pair immobilized in the indicator zone.

32. The method of claim 31 wherein the member conjugated to analyte is further conjugated to label.

33. The method of claim 31 wherein said member complementary to the binding member immobilized in the indicator zone is applied to the application zone along with the analyte so as to effect conjugation of said member to the analyte in the application zone.

34. A method to detect the formation of a specific binding pair complex in an indicator zone of a non-bibulous lateral flow membrane said membrane having a pore size of 1-250 microns and having on its surface a liquid sample application zone defined to receive said liquid sample and at least one indicator zone spaced laterally apart from the application zone;

wherein immobilized in said indicator zone is a member of a binding pair bound to its a complementary member of said binding pair so as to form a complex,

which method comprises applying to the surface of said membrane detectable particles of sufficient size to be entrapped by said complex and causing said particles to flow laterally through said indicator zone, and

detecting the presence of the particles in the indicator zone as a means of detecting the formation of the specific binding pair complex.

35. The method of claim 34 wherein the particles are red blood cells.

36. The method of claim 34 wherein the particles are colored latex.

37. The method of claim 34 wherein said detectable particles are present in a sample providing an analyte, which analyte is a member of said complex.

38. The method of claim 37 wherein said sample is whole blood.

39. A method for determining the presence or absence or approximate amount of an analyte in a liquid sample comprising the steps of:

applying the sample to the liquid sample application zone of a non-bibulous lateral flow membrane having a pore size of 1-250 microns which membrane comprises said application zone and at least one indicator zone spaced laterally apart from said application zone on the surface of said membrane, there being immobilized in said indicator zone a member of a binding pair capable of binding the analyte or a derivative thereof,

said sample being of sufficient quantity to cause sample liquid to flow laterally from the application zone through said indicator zone; and

detecting the binding of the analyte or derivative thereof in the indicator zone;

wherein said detecting is effected by entrapping detectable particles in a complex formed by the binding of the analyte or derivative thereof with the member of the binding pair immobilized in the indicator zone; and

assessing the entrapment of the particles in the indicator zone to determine the presence or absence of approximate amount of analyte.

40. The method of claim 39 wherein the particles are added to the liquid sample before said sample is applied to the application zone.

41. The method of claim 39 wherein the particles are suspended in liquid and applied to the liquid sample application zone after the sample has been applied to said application zone.

42. The method of claim 39 wherein the particles are present in the sample.

43. The method of claim 39 wherein the particles are red blood cells.

44. The method of claim 39 wherein the particles are colored latex particles.

45. A method of type blood, which method comprises applying a sample of whole blood to the liquid sample application zone of a non-bibulous lateral flow membrane, wherein the membrane comprises:

said application zone defined to receive said whole blood sample, and

at least one indicator zone spaced laterally apart from the application zone, said indicator zone having immobilized therein an antibody or fragment thereof immunoreactive with a blood typing antigen,

said whole blood sample being of sufficient volume to permit the whole blood to flow through the indicator zone, and

applying sufficient clear liquid to the membrane at a position apart from the indicator zone so as to laterally flow through the indicator zone to remove unbound reed blood cells from said indicator zone and

detecting the presence or absence of red color in the indicator zone so as to determine the presence or absence of a blood typing antigen immunoreactive with the antibody or fragment thereof immobilized in the indicator zone.

46. The method of claim 45 wherein the membrane comprises an indicator zone in which is immobilized an antibody or fragment thereof having reactivity with group A antigen and an indicator zone in which is immobilized an antibody or fragment thereof having reactivity with group B antigen.