Positive detection lateral-flow apparatus and method for small and large analytes

• US 6,699,722 B2
• Filed: 04/13/2001
• Issued: 03/02/2004
• Est. Priority Date: 04/14/2000
• Status: Expired due to Term

• Current Assignee: Quantrx Biomedical Corporation
• Original Assignee: A-Fem Medical Corporation
• Inventors: Wang, Huiying, Hyatt, Timothy P., Bauer, Jeffrey S.
• Primary Examiner(s): Le, Long V.
• Assistant Examiner(s): Counts, Gary W.
• Application Number:
  US09/835,304
• Publication Number:
  US 20020142291A1
• Time in Patent Office: 1,054 Days
• Field of Search: 435/5, 435/7.1, 435/7.42, 435/810, 435/287.1, 436/514, 436/518, 436/541, 436/808, 436/109, 422/56, 422/58, 422/61, 422/57
• US Class Current: 436/518
• 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: G01N 33/94 involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
• CPC Class Codes: G01N 33/946 CNS-stimulants, e.g. cocaine, amphetamines

Abstract

Methods and devices for the detection and/or quantification of an analyte in a sample are provided. These are positive detection methods and devices, in that the more analyte is present in the sample, the stronger the signal that is provided. Devices of the invention include a mobilization zone including a mobile or mobilizable detectable analyte analog, a sample application area, primary and secondary capture areas each including an immobilized binding partner having a binding affinity for the analyte being tested for a detectable analyte analog. The mobilization zone, sample application area, primary and secondary capture area are in fluid continuous contact with each other. In these devices, the first immobilized binding partner has an equal or lower apparent affinity for the analyte than it has for the detectable analyte analog. Methods of this invention involve introducing a sample (which is suspected of containing the analyte to be tested for) to a device such as those described herein, and permitting the sample to migrate from the application area to and through the first and secondary binding zones. A detectable tracer conjugate is also permitted to migrate through the device, usually slightly behind the sample so that any analyte in the sample contacts the first binding partner before the conjugate. Results of such methods are read based on the presence and/or intensity of the detectable signal given by conjugate that binds in the second capture area.

Claims

1. A method for detecting and/or quantitating an analyte in a liquid sample, comprising:

(a) providing a test strip comprising

a mobilization zone;

a mobile or mobilizable detectable tracer in the mobilization zone;

a sample application area;

a primary capture area comprising a first immobilized binding partner having a binding affinity for the analyte and a binding affinity for the detectable tracer; and

a secondary capture area comprising a second immobilized binding partner having a binding affinity for the detectable tracer;

wherein the sample application area, mobilization zone, primary capture area and secondary capture area are in a path of liquid flow from the sample application area distally through the mobilization zone to the primary capture area and then to the secondary capture area, wherein a distal flow of analyte reaches the primary capture area before a distal flow of tracer reaches the primary capture area;

(b) contacting the liquid sample with the sample application area of the test strip, and allowing the liquid sample to mobilize the detectable tracer such that the detectable tracer migrates with the liquid sample, wherein the detectable tracer has a weight greater than the analyte to permit analyte flow to move at a faster rate than the tracer flow along the path of liquid flow, such that the tracer flow reaches the primary capture zone after analyte flow in the liquid sample reaches the primary capture area;

wherein the analyte flow that reaches the primary capture zone occupies first immobilized binding partner such that subsequent binding of detectable tracer to first immobilized binding partner is inhibited, whereby unbound detectable tracer continues along the path of liquid flow distally to bind to second immobilized binding partner and provide a signal from the secondary capture area that indicates the presence of the analyte in the liquid sample.

3. The test strip of claim 1, further comprising at least one filter pad in the path of liquid flow.

4. The test strip of claim 3, wherein the filter pad is pretreated with at least one reagent to enhance the sensitivity of the assay device.

5. The test strip of claim 4, wherein the at least one reagent is selected from the group consisting of buffers, detergents, and anticoagulants.

6. The test strip of claim 1, wherein the first and second immobilized binding partners are antibodies, antigens, haptens, lectins or receptors.

7. The test strip of claim 1, wherein the first and second immobilized binding partners for the analyte are identical.

8. The test strip of claim 1, wherein the first and second immobilized binding partners are each anti-analyte antibodies.

9. The test strip of claim 1, wherein the detectable tracer comprises an analyte analog.

10. The test strip of claim 1, wherein the detectable tracer comprises a visually detectable label.

11. The test strip of claim 1, wherein the detectable tracer comprises a detectable tracer for an analyte selected from the group consisting of an antigen of an infectious disease, an antigen to an antibody of an infectious disease, a hormone, a growth factor, a therapeutic drug, a drug of abuse, a product of the metabolism of a drug of abuse, and a hapten.

12. The test strip of claim 11, wherein the detectable tracer comprises a detectable tracer for an analyte comprising an antibody selected from the group consisting of an antibody to Human Immunodeficiency Virus (HIV), an antibody to Human T-Cell Lymphotropic Virus (HTLV), an antibody to Helicobacter pylori, an antibody to hepatitis, an antibody to measles, an antibody to mumps, and an antibody to rubella.

13. The test strip of claim 11, wherein the detectable tracer comprises a detectable tracer for an analyte comprising a therapeutic drug or drug of abuse or products of the metabolism of a drug of abuse, wherein the analyte is selected from the group consisting of tetrahydrocannabinol, nicotine, cotinine, ethanol, theophylline, phenytoin, acetaminophen, lithium, diazepam, nortryptyline, secobarbital, and phenobarbitol, methamphetamine and fragments, mimetics, analogs or derivatives thereof.

14. The test strip of claim 13, wherein the detectable tracer comprises a detectable tracer conjugate for an analyte that is a product of metabolism of a drug of abuse, and the product of metabolism comprises cotinine.

15. The test strip of claim 11, wherein the detectable tracer comprises a detectable tracer conjugate for an analyte comprising a hormone, and the hormone is selected from the group consisting of testosterone, estradiol, estriol, 17-hydroxyprogesterone, progesterone, thyroxine, thyroid stimulating hormone, follicle stimulating hormone, and luteinizing hormone, and fragments, mimetics, analogs or derivatives thereof.

16. The test strip of claim 1, wherein a quantity of the second specific binding partner in the secondary capture area is such that the quantity of detectable tracer binding to the secondary capture area, and by correlation the amount of the analyte in the liquid sample, is indicated by an intensity of a detection signal of the detectable tracer in the secondary capture area.

17. The test strip of claim 1, wherein the secondary binding area of the test strip is divided into at least two discrete and non-overlapping bands, with a quantity of the second specific binding partner in each band being such that quantity of tracer binding to the secondary capture area, and by correlation the amount of the analyte in a tested sample, is indicated by a number of bands to which the tracer molecule binds.

18. The method of claim 1, further comprising quantifying an amount of analyte in the liquid sample, wherein the amount of analyte in the liquid sample determines an intensity of the signal from the tracer in the secondary capture area.

19. The method of claim 1, wherein the liquid sample migrates along the test strip in the path of liquid flow by capillary action.

20. The method of claim 1, wherein the analyte has a molecular weight of about 100-1,000 Daltons.

21. The method of claim 1, wherein the analyte has a molecular weight of greater than 1,000 Daltons.

22. The method of claim 1, wherein the detectable tracer comprises an analyte or analyte analog linked to a detectable molecule by a linker.

23. The method of claim 22, wherein the linker is selected to increase the weight of the detectable tracer.

24. The method of claim 23, wherein the linker comprises bovine serum albumin (BSA).

2. A test strip for determining presence and/or amount of an analyte in a liquid sample comprising:

a mobilization zone;

a mobile or mobilizable detectable tracer in the mobilization zone;

a sample application area;

a primary capture area comprising a first immobilized binding partner having a binding affinity for the analyte and a binding affinity for the detectable tracer; and

a secondary capture area comprising a second immobilized binding partner having a binding affinity for the detectable tracer;

wherein the sample application area, mobilization zone, primary capture area and secondary capture area are in a path of liquid flow from the sample application area through the mobilization zone to the primary capture area and then to the secondary capture area, wherein the detectable tracer has a weight greater than a weight of the analyte such that a tracer flow migrates more slowly than an analyte flow along the path of liquid flow, whereby the analyte flow reaches the primary capture area before the tracer flow reaches the primary capture area.

25. The test strip of claim 2, further comprising at least one filter pad in the path of liquid flow.

26. The test strip of claim 25, wherein the filter pad is pretreated with at least one reagent to enhance the sensitivity of the assay device.

27. The test strip of claim 26, wherein the at least one reagent is selected from the group consisting of buffers, detergents, and anticoagulants.

28. The test strip of claim 2, wherein the first and second immobilized binding partners are antibodies, antigens, lectins, receptors or haptens.

29. The test strip of claim 2, wherein the first and second immobilized binding partner for the analyte are identical.

30. The test strip of claim 2, wherein the first and second immobilized binding partner are each anti-analyte antibodies.

31. The test strip of claim 2, wherein the detectable tracer is a tracer conjugate comprising an analyte or an analyte analog.

32. The test strip of claim 2, wherein the detectable tracer comprises a visually detectable label.

33. The test strip of claim 2, wherein the detectable tracer comprises a detectable tracer for an analyte selected from the group consisting of an antigen of an infectious disease, an antigen to an antibody of an infectious disease, a hormone, a growth factor, a therapeutic drug, a drug of abuse, a product of the metabolism of a drug of abuse, and a hapten.

34. The test strip of claim 2, wherein the detectable tracer comprises a detectable tracer for an analyte comprising an antibody selected from the group consisting of an antibody to Human Immunodeficiency Virus (HIV), an antibody to Human T-Cell Lymphotropic Virus (HTLV), an antibody to Helicobacter pylori, an antibody to hepatitis, an antibody to measles, an antibody to mumps, and an antibody to rubella.

35. The test strip of claim 2, wherein the detectable tracer comprises a detectable tracer for an analyte comprising a therapeutic drug or drug of abuse or products of the metabolism of a drug of abuse, wherein the analyte is selected from the group consisting of tetrahydrocannabinol, nicotine, cotinine, ethanol, theophylline, phenytoin, acetaminophen, lithium, diazepam, nortryptyline, secobarbital, and phenobarbitol, methamphetamine and fragments, mimetics, analogs or derivatives thereof.

36. The test strip of claim 2, wherein the detectable tracer comprises a detectable tracer for an analyte that is a product of metabolism of a drug of abuse, and the product of metabolism comprises cotinine.

37. The test strip of claim 33, wherein the detectable tracer comprises a detectable tracer for an analyte comprising a hormone, and the hormone is selected from the group consisting of testosterone, estradiol, estriol, 17-hydroxyprogesterone, progesterone, thyroxine, thyroid stimulating hormone, follicle stimulating hormone, and luteinizing hormone, and fragments, mimetics, analogs or derivatives thereof.

38. The test strip of claim 2, wherein a quantity of the second specific binding partner in the secondary capture area is such that the quantity of detectable tracer binding to the secondary capture area, and by correlation the amount of the analyte in the liquid sample, is indicated by an intensity of a detection signal of the detectable tracer in the secondary capture area.

39. The test strip of claim 2, wherein the secondary binding area of the test strip is divided into at least two discrete and non-overlapping bands, with a quantity of the second specific binding partner in each band being such that quantity of tracer binding to the secondary capture area, and by correlation the amount of the analyte in a tested sample, is indicated by a number of bands to which the tracer molecule binds.