Method for immunochromatographic analysis

• US 5,039,607 A
• Filed: 05/17/1988
• Issued: 08/13/1991
• Est. Priority Date: 05/17/1988
• Status: Expired due to Term

• Current Assignee: DADE Behring Beteiligungs GmbH (Siemens AG)
• Original Assignee: Syntec Incorporated (Atos SE)
• Inventors: Ullman, Edwin F., Shanafelt, Armen B., Ghazarossian, Vartan, Skold, Carl N.
• Primary Examiner(s): Saunders, David A.
• Application Number:
  US07/194,708
• Time in Patent Office: 1,183 Days
• Field of Search: 435/7, 435/805, 435/7.5, 435/7.8, 435/7.9, 435/7.92, 435/7.93, 435/970, 435/971, 435/972, 436/501, 436/504, 436/518, 436/530, 436/541, 436/807, 436/810, 436/815, 436/816, 436/819, 436/822, 422/56, 422/58
• US Class Current: 435/7.5
• 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 435/97 Test strip or test slide
• CPC Class Codes: Y10S 435/971 Capture of complex after antigen-antibody reaction
• CPC Class Codes: Y10S 435/972 Modified antibody, e.g. hybrid, bifunctional
• CPC Class Codes: Y10S 435/975 Kit
• CPC Class Codes: Y10S 436/807 Apparatus included in process claim, e.g. physical support structures
• CPC Class Codes: Y10S 436/81 Tube, bottle, or dipstick
• CPC Class Codes: Y10S 436/815 Test for named compound or class of compounds
• CPC Class Codes: Y10S 436/816 Alkaloids, amphetamines, and barbiturates
• CPC Class Codes: Y10S 436/819 Multifunctional antigen or antibody
• CPC Class Codes: Y10S 436/822 Identified hapten

Abstract

A method is disclosed for conducting an assay for an analyte. The method comprises causing a specific binding pair member in a first aqueous medium to become bound to a first bibulous member by contacting a portion of the first bibulous member with the medium. The first bibulous member is in liquid receiving relationship with an absorbent member. The contacting is carried out under conditions wherein the first medium traverses the first bibulous member and at least a portion of the absorbent member by capillary action. The method further comprises causing the first bibulous member to come into liquid receiving relationship with a second bibulous member. A reagent in a second aqueous medium is absorbed by and preferably becomes a non-diffusively bound to the second bibulous member in relation to the presence of analyte in the first medium. This is accomplished by contacting a portion of the first bibulous member with the second aqueous medium under conditions wherein the second aqueous medium traverses the first bibulous member by capillary action and at least a portion of the second bibulous member. Next, the presence of the reagent on at least a portion of the second bibulous member is determined.

Claims

1. A method for conducting an assay for an analyte, which comprises:

causing a specific binding pair (sbp) member in a first aqueous medium suspected of containing an analyte to become bound to a specific binding partner non-diffusively bound to at least a portion of a first bibulous member by contacting a portion of said first bibulous member with said medium, said first bibulous member being in liquid receiving relationship with an absorbent member, under conditions wherein said first medium traverses said first bibulous member and at least a portion of said absorbent member by capillary action;

causing said first bibulous member to come into liquid receiving relationship with a second bibulous member;

causing a reagent, which is capable of binding to said sbp member or a binding partner thereto, in a second aqueous medium to be specifically bound by a specific binding partner non-diffusively bound on said second bibulous member in relation to the presence of said analyte in said first medium by contacting a portion of said first bibulous member with said second aqueous medium, under conditions wherein said second aqueous medium traverses said first bibulous member by capillary action and at least a portion of said second bibulous member; and

determining the presence of said reagent on at least a portion of said second bibulous member, the presence of said reagent indicating the presence of said analyte.

2. The method of claim 1 wherein said liquid receiving relationship between said first bibulous member and said absorbent member is terminated prior to the contact of said first bibulous member with said second medium.

3. The method of claim 1 wherein a specific binding partner capable of binding said sbp member is non-diffusively bound to at least a portion of said first bibulous member.

4. The method of claim 1 wherein said sbp member and said analyte bind together to form a complex and said complex becomes bound to said specific binding partner non-diffusively bound on said first bibulous member.

5. The method of claim 1 where said sbp member is said analyte.

6. A method for conducting an assay for digoxin, which comprises:

contacting an end portion of a bibulous strip having a zone distal from said end portion, said zone having an antibody (Ab₁) for an organic molecule of molecular weight less than 1500 non-diffusively bound thereto with a first aqueous medium containing a sample suspected of containing digoxin, wherein a conjugate comprising an antibody for digoxin bound to said organic molecule is present in said first medium or diffusively bound on said bibulous strip, under conditions wherein said first medium traverses said bibulous strip and at least a portion of said absorbent member by capillary action and said conjugate becomes non-diffusively bound to said Ab₁ and digoxin, if present, becomes bound to said conjugate;

terminating the liquid receiving relationship between said bibulous strip and said absorbent member;

providing in combination in a second aqueous medium said end portion of said bibulous strip and a reagent comprising a digoxin analog bound to an enzyme, said bibulous strip being in liquid receiving relationship with an immunochromatographic strip having non-diffusively bound thereto an antibody for digoxin, under conditions wherein said second medium traverses said bibulous strip and at least a portion of said immunochromatographic strip by capillary action, with the proviso that said bibulous strip is in liquid receiving relationship with said absorbent member and said immunochromatographic strip at substantially different times; and

permitting a signal producing system including said enzyme to produce a signal defining a border on said immunochromatographic strip, the position of said border being related to the amount of analyte in said sample.

7. A method for conducting an assay for digoxin, said method comprising:

contacting an end portion of a bibulous strip to which is non-diffusively bound a digoxin analog with a first aqueous medium containing a sample suspected of containing digoxin, wherein an antibody for digoxin (Ab_D) is present in said first medium or diffusively bound on said bibulous strip, said bibulous strip being in liquid receiving relationship with an absorbent member, under conditions wherein said first medium traverses said bibulous strip and at least a portion of said absorbent member by capillary action and said first antibody becomes non-diffusively bound to said digoxin analog when digoxin is not present in said sample;

terminating the liquid receiving relationship between said bibulous strip and said absorbent member;

providing in combination in a second aqueous medium said end portion of said bibulous strip and a conjugate comprising an antibody for said Ab_D bound to an enzyme, said bibulous strip being in liquid receiving relationship with an immunochromatographic strip having non-diffusively bound thereto a receptor for said antibody for Ab_D, under conditions wherein said second medium traverses said bibulous strip is in liquid receiving relationship with said absorbent member and said immunochromatographic strip at substantially different times; and

permitting a signal producing system including said enzyme to produce a signal defining a border on said immunochromatographic strip, the position of said border being related to the amount of analyte in said first medium.

8. A method for conducting an assay for an analyte, said method comprising:

contacting an end portion of a bibulous strip to which is non-diffusively bound an antibody for digoxin with a first aqueous medium containing a sample suspected of containing digoxin, said bibulous strip being in liquid receiving relationship with an absorbent member, under conditions wherein said first medium traverses said bibulous strip and at least a portion of said absorbent member by capillary action;

terminating the liquid receiving relationship between said bibulous strip and said absorbent member;

providing in combination in a second aqueous medium said end portion of said bibulous strip and a conjugate comprising a digoxin analog bound to an enzyme, said bibulous strip being in liquid receiving relationship with an immunochromatographic strip having non-diffusively bound thereto a receptor for conjugate, under conditions wherein said second medium traverses said bibulous strip and at least a portion of said immunochromatographic strip by capillary action, with the proviso that said bibulous strip is in liquid receiving relationship with said absorbent member and said immunochromatographic strip at substantially different times; and

permitting a signal producing system including said enzyme to produce a signal defining a border on said immunochromatographic strip, the position of said border being related to the amount of analyte in said first medium.

9. A method for conducting an assay for an analyte, said method comprising:

providing in combination in a first aqueous medium (1) a portion of a first bibulous member, at least a portion thereof having a specific binding pair (sbp) member non-diffusively bound thereto and (2) a sample suspected of containing an analyte with the proviso that, when said analyte is not capable of binding to said sbp member, a first reagent capable of binding to said analyte and to said sbp member is present in said medium or diffusively bound on said first bibulous member, said first bibulous member being in liquid receiving relationship with an absorbent member, under conditions wherein said first medium traverses said first bibulous member and at least a portion of said absorbent member by capillary action;

bringing said first bibulous member into liquid receiving relationship with a second bibulous member;

providing in combination in a second aqueous medium a portion of said first bibulous member and a second reagent, said second reagent being capable of binding to said sbp member or said first reagent in relation to the presence of said analyte in said first medium, said second bibulous member having non-diffusively bound to at least a portion thereof a specific binding partner for said second reagent, under conditions wherein said second medium traverses at least a portion of said first bibulous member and at least a portion of said second bibulous member by capillary action; and

determining the presence said second reagent bound to second bibulous member, the presence thereof being related to the amount of said analyte in said sample.

10. The method of claim 9 wherein said liquid receiving relationship between said first bibulous member and said absorbent member is terminated prior to contacting said second bibulous member with said medium.

11. The method of claim 9 wherein said second bibulous member is an immunochromatograph and said second reagent includes an enzyme and said amount of said second reagent bound to said immunochromatographic member is determined by the location on said immunochromatographic member of a border defined by a signal produced by a signal producing system including said enzyme.

12. The method of claim 9 wherein said analyte is a drug.

13. The method of claim 9 wherein said analyte is digoxin.

14. The method of claim 9 wherein said first bibulous member is a paper strip.

15. The method of claim 9 wherein said first reagent comprises (1) a second sbp member complementary to said analyte and (2) a third sbp member complementary to said first sbp member.

16. The method of claim 9 wherein said first reagent comprises an antibody for said analyte conjugated to an organic molecule of molecular weight less than 1500.

17. The method of claim 9 wherein said first reagent comprises an antibody for said analyte and a molecule selected from the group consisting of fluorescein, LSD, biotin, morphine, benzoylecgonine, avidin, intrinsic factor, and folate binding protein.

18. The method of claim 16 wherein said sbp member is an antibody for said organic molecule.

19. The method of claim 9 wherein said absorbent member is a strip of bibulous material.

20. The method of claim 9 wherein said absorbent material has a liquid holding capacity to absorb all of the liquid in said first aqueous medium.

21. The method of claim 9 wherein said second reagent is comprised of said analyte bound to a label.

22. The method of claim 21 wherein said label is an enzyme.

23. The method of claim 11 wherein said immunochromatographic member is a strip of bibulous material.

24. The method of claim 9 wherein said binding partner for said second reagent is an antibody.

25. The method of claim 9 wherein said sbp member is an analyte analog.

26. The method of claim 9 wherein said first reagent is an antibody for said analyte.

27. The method of claim 9 wherein said second reagent is comprised of an antibody for said first reagent bound to a label.

28. The method of claim 27 wherein said label is an enzyme.

29. The method of claim 9 wherein said specific binding partner is a receptor for an immunoglobulin.

30. The method of claim 11 wherein said immunochromatographic member is contacted with an enzyme substrate solution to determine said border.

31. The method of claim 9 wherein said sbp member is antibody for said analyte.

32. A method for conducting an assay for an analyte, said method comprising:

providing in combination in a first aqueous medium (1) a portion of a bibulous member to at least a portion of which is non-diffusively bound a first specific binding pair (sbp) member and (2) a sample suspected of containing an analyte, wherein a conjugate comprising a second sbp member capable of binding said analyte and a third sbp member complementary to said first sbp member is present in said medium or diffusively bound on said bibulous member, said bibulous member being in liquid receiving relationship with an absorbent member, under conditions wherein said first medium traverses said bibulous member and at least a portion of said absorbent member by capillary action and said conjugate becomes non-diffusively bound to said bibulous member;

terminating the liquid receiving relationship between said bibulous member and said absorbent member;

providing in combination in a second aqueous medium a portion of said bibulous member and a reagent comprising an analyte analog bound to, or capable of becoming bound to, a member of a signal producing system, said bibulous member being in liquid receiving relationship with an immunochromatographic member having non-diffusively bound thereto a binding partner for said reagent, under conditions wherein said second medium traverses said bibulous member and at least a portion of said immunochromatographic member by capillary action, with the proviso that said bibulous member is in liquid receiving relationship with said absorbent member and said immunochromatographic member at substantially different times; and

permitting said signal producing system to produce a signal defining a border on said immunochromatographic member, the position of said border being related to the amount of analyte in said first medium.

33. The method of claim 32 wherein said analyte is a drug.

34. The method of claim 32 wherein said analyte is digoxin.

35. The method of claim 32 wherein said bibulous member is a paper strip.

36. The method of claim 32 wherein said conjugate comprises (a) as the second sbp member an antibody for said analyte and (b) as the third sbp member a protein.

37. The method of claim 32 wherein said conjugate comprises (a) as the second sbp member an antibody for said analyte and b) as the third sbp member an organic molecule of molecular weight less than 1500.

38. The method of claim 32 wherein said conjugate comprises a) as the second sbp member an antibody for said analyte and b) as the third sbp member a molecule selected from the group consisting of fluorescein, LSD, biotin, morphine, benzoylecgonine, avidin, intrinsic factor, folate binding protein, and enzymes.

39. The method of claim 37 wherein said first sbp member is an antibody for said organic molecule.

40. The method of claim 32 wherein said absorbent member is a strip of bibulous material.

41. The method of claim 32 wherein said absorbent material has a liquid holding capacity to absorb all of the liquid in said first aqueous medium.

42. The method of claim 32 wherein said reagent is a conjugate of said analyte and a label.

43. The method of claim 42 wherein said label is an enzyme.

44. The method of claim 32 wherein said immunochromatographic member is a strip of bibulous material.

45. The method of claim 32 wherein said binding partner for said second reagent is an antibody.

46. The method of claim 32 wherein said immunochromatographic member is contacted with a developer solution to determine said border.

47. A method for conducting an assay for an analyte, said method comprising:

providing in combination in a first aqueous medium (1) a portion of a bibulous member to which is non-diffusively bound an analyte analog and (2) a sample suspected of containing an analyte, wherein a first reagent capable of binding said analyte is present in said medium or diffusively bound on said bibulous member, said bibulous member being in liquid receiving relationship with an absorbent member, under conditions wherein said first medium traverses said bibulous member and at least a portion of said absorbent member by capillary action and said first reagent becomes non-diffusively bound to said bibulous member when said analyte is not present in said first medium;

terminating the liquid receiving relationship between said bibulous member and said absorbent member;

providing in combination in a second aqueous medium a portion of said bibulous member and a second reagent, said second reagent being bound to, or capable of becoming bound to, a member of a signal producing system and being capable of binding to said first reagent when said first reagent is diffusively bound to said bibulous member, said bibulous member being in liquid receiving relationship with an immunochromatographic member having non-diffusively bound thereto a specific binding partner for said second reagent, under conditions under which said second medium traverses said bibulous member and at least a portion of said immunochromatographic member by capillary action, with the proviso that said bibulous member is in liquid receiving relationship with said absorbent member and said immunochromatographic member at substantially different times; and

permitting said signal producing system to produce a signal defining a border on said immunochromatographic member, the position of said border being related to the amount of analyte in said first medium.

48. The method of claim 47 wherein said analyte is a drug.

49. The method of claim 47 wherein said analyte is digoxin.

50. The method of claim 47 wherein said bibulous member is a paper strip.

51. The method of claim 47 wherein said first reagent is an antibody for said analyte.

52. The method of claim 47 wherein said absorbent member is a strip of bibulous material.

53. The method of claim 47 wherein said absorbent material has a liquid holding capacity to absorb all of the liquid in said first aqueous medium.

54. The method of claim 47 wherein said second reagent is an antibody for an antibody for said analyte conjugated to a label.

55. The method of claim 54 wherein said label is an enzyme.

56. The method of claim 47 wherein said immunochromatographic member is a strip of bibulous material.

57. The method of claim 47 wherein said specific binding partner for said second reagent is a receptor for an immunoglobulin.

58. The method of claim 47 wherein said immunochromatographic member is contacted with a developer solution to determine said border.

59. A method for conductinq an assay for an analyte, said method comprising:

providing in combination in a first aqueous medium (1) a portion of a bibulous member to which is non-diffusively bound an antibody for said analyte and (2) a sample suspected of containing an analyte, said bibulous member being in liquid receiving relationship with an absorbent member, under conditions wherein said first medium traverses said bibulous member and at least a portion of said absorbent member by capillary action;

terminating the liquid receiving relationship between said bibulous member and said absorbent member;

providing in combination in a second aqueous medium a portion of said bibulous member and a reagent, said reagent being bound to, or capable of becoming bound to, a member of a signal producing system and being capable of binding to said antibody for said analyte, said bibulous member being in liquid receiving relationship with an immunochromatographic member having non-diffusively bound thereto a specific binding partner for said reagent, under conditions under which said second medium traverses said bibulous member and at least a portion of said immunochromatographic member by capillary action, with the proviso that said bibulous member is in liquid receiving relationship with said absorbent member and said immunochromatographic member at substantially different times; and

permitting said signal producing system to produce a signal defining a border on said immunochromatographic member, the position of said border being related to the amount of analyte in said first medium.

60. The method of claim 59 wherein said reagent is a conjugate of an analyte analog and an enzyme.