MAGNETIC BEADS FOR REDUCING LEUKOCYTE INTERFERENCE IN IMMUNOASSAYS

• US 20110306070A1
• Filed: 06/14/2010
• Published: 12/15/2011
• Est. Priority Date: 06/14/2010
• Status: Active Grant

• Current Assignee: Abbott Point Of Care Incorporated (Abbott Laboratories Incorporated)
• Original Assignee: Abbott Point Of Care Incorporated (Abbott Laboratories Incorporated)
• Inventors: Campbell, John Lewis Emerson, Davis, Graham
• Granted Patent: US 8,394,325 B2
• Time in Patent Office: Days
• US Class Current: 435/7.93
• CPC Class Codes: G01N 33/54326 Magnetic particles
• CPC Class Codes: Y10T 436/25125 Digestion or removing interfering materials

Abstract

Methods and devices for reducing interference from leukocytes in an analyte immunoassay are provided. In one embodiment, a method is provided comprising the steps of amending a biological sample with magnetic sacrificial beads opsonized to leukocytes, binding leukocytes in the sample to the magnetic sacrificial beads, and magnetically retaining the beads out of contact from an immuno sensor.

Claims

1. A method of reducing interference from leukocytes in an analyte immunoassay, comprising:

contacting a biological sample containing leukocytes with magnetic sacrificial beads opsonized to said leukocytes under conditions sufficient to bind at least a portion of said leukocytes in the biological sample to the magnetic sacrificial beads; and

magnetically retaining the magnetic sacrificial beads substantially out of contact from an immuno sensor.

2. The method of claim 1, wherein the immunoassay is a sandwich assay.

3. The method of claim 1, wherein the immunoassay is a competitive assay.

4. The method of claim 1, wherein the biological sample is a blood sample.

5. The method of claim 1, wherein the biological sample is a whole blood sample.

6. The method of claim 1, wherein the biological sample is amended with an anticoagulant.

7. The method of claim 1, wherein the magnetic sacrificial beads comprise a magnetite (Fe₃O₄) core coated with a styrene-acrylic acid copolymer.

8. The method of claim 1, wherein the magnetic sacrificial beads comprise magnetic beads coated with non-human IgG or fragments thereof.

9. The method of claim 1, wherein the magnetic sacrificial beads have an average particle size of from 0.01 μ

m to 20 μ

m.

10. The method of claim 1, wherein the magnetic sacrificial beads have an average particle size of from 0.1 μ

m to 5 μ

m.

11. The method of claim 1, wherein the magnetic sacrificial beads have an average particle size of from 3 μ

m to 5 μ

m.

12. The method of claim 1, wherein the magnetic sacrificial beads are in one or more dissolvable dry reagent coatings.

13. The method of claim 1, wherein the retaining is achieved with a permanent magnet.

14. The method of claim 1, wherein the retaining is achieved with an electromagnet.

15. The method of claim 1, wherein at least 50 wt. % of the magnetic sacrificial beads are retained out of contact from the immuno sensor.

16. The method of claim 1, wherein at least 75 wt. % of the magnetic sacrificial beads are retained out of contact from the immuno sensor.

17. A method of performing an analyte sandwich immunoassay in a blood sample, comprising:

introducing a blood sample containing leukocytes into a holding chamber in a test cartridge, wherein a portion of the holding chamber is coated with magnetic sacrificial beads opsonized to leukocytes;

contacting the blood sample with the magnetic sacrificial beads under conditions sufficient to bind at least a portion of the leukocytes in the biological sample to the magnetic sacrificial beads;

moving the blood sample into a conduit, wherein a magnetic field localized to at least a portion of the conduit attracts and retains the magnetic sacrificial beads on at least a portion of the wall of the conduit thereby substantially preventing the magnetic sacrificial beads from coming into contact with an immunosensor;

contacting the blood sample with the immunosensor to form a sandwich comprising an immobilized capture antibody, a sample analyte and a signal antibody;

optionally washing the blood sample from the immunosensor; and

detecting the presence of the analyte based on a signal provided by the signal antibody.

18. The method of claim 17, further comprising:

washing the blood sample from the immunosensor; and

contacting the immunosensor with a reagent capable of reacting with said signal antibody to produce the signal at the immunosensor related to the presence of the sample analyte in the blood sample.

19. The method of claim 17, wherein the blood sample is a whole blood sample.

20. The method of claim 17, wherein the blood sample is amended with an anticoagulant.

21. The method of claim 17, wherein the magnetic sacrificial beads comprise a magnetite (Fe₃O₄) core coated with a styrene-acrylic acid copolymer.

22. The method of claim 17, wherein the magnetic sacrificial beads comprise magnetic beads coated with non-human IgG or fragments thereof.

23. The method of claim 17, wherein the magnetic sacrificial beads have an average particle size of from 0.01 μ

m to 20 μ

m.

24. The method of claim 17, wherein the magnetic sacrificial beads have an average particle size of from 0.1 μ

m to 5 μ

m.

25. The method of claim 17, wherein the magnetic sacrificial beads have an average particle size of from 3 μ

m to 5 μ

m.

26. The method of claim 17, wherein the magnetic sacrificial beads are in one or more dissolvable dry reagent coatings.

27. The method of claim 17, wherein the magnetic field is generated by a permanent magnet.

28. The method of claim 17, wherein the magnetic field is generated by an electromagnet.

29. The method of claim 17, wherein the magnetic field is generated by a magnet embedded in the test cartridge.

30. The method of claim 17, wherein at least 50 wt. % of the magnetic sacrificial beads are retained out of contact from the immuno sensor.

31. The method of claim 17, wherein at least 75 wt. % of the magnetic sacrificial beads are retained out of contact from the immuno sensor.

32. The method of claim 17, wherein the sample analyte is selected from the group consisting of troponin I, troponin T, BNP, creatine kinase MB, procalcitonin, proBNP, NTproBNP, myoglobin, PSA, D-dimer, CRP, HCG, NGAL, myeloperoxidase and TSH.

33. The method of claim 17, wherein the immobilized capture antibody is attached to a sensor selected from the group consisting of an amperometric electrode, a potentiometric electrode, a conductimetric electrode, an optical wave guide, a surface plasmon resonance sensor, an acoustic wave sensor and a piezoelectric sensor.

34. The method of claim 17, wherein the immobilized capture antibody is attached to an assay bead, and the assay bead is attached to an amperometric electrode.

35. A method of performing an analyte competitive immunoassay in a blood sample, comprising:

introducing a blood sample containing leukocytes into a holding chamber in a test cartridge, wherein a portion of the holding chamber is coated with magnetic sacrificial beads opsonized to leukocytes;

amending the blood sample with a labeled analyte;

contacting the blood sample with the magnetic sacrificial beads under conditions sufficient to bind at least a portion of the leukocytes in the biological sample to the magnetic sacrificial beads;

applying a magnetic field localized to at least a portion of a conduit to attract and retain the magnetic sacrificial beads on at least a portion of a wall of the conduit thereby substantially preventing the magnetic sacrificial beads from coming into contact with an immuno sensor;

contacting the blood sample with an immunosensor to form a competitive ratio of bound sample analyte to bound labeled analyte;

optionally washing the blood sample from the immunosensor; and

detecting the presence of the analyte based on the competitive ratio as determined by a signal provided by the bound labeled analyte.

36. The method of claim 35, further comprising:

washing the blood sample from the immunosensor; and

contacting the immunosensor with a reagent capable of reacting with the labeled analyte to produce a signal at the immunosensor inversely related to the presence of sample analyte in the blood sample.

37. The method of claim 35, wherein the blood sample is a whole blood sample.

38. The method of claim 35, wherein the blood sample is amended with an anticoagulant.

39. The method of claim 35, wherein the magnetic sacrificial beads comprise a magnetite (Fe₃O₄) core coated with a styrene-acrylic acid copolymer.

40. The method of claim 35, wherein the magnetic sacrificial beads comprise magnetic beads coated with non-human IgG or fragments thereof.

41. The method of claim 35, wherein the magnetic sacrificial beads have an average particle size of from 0.01 μ

m to 20 μ

m.

42. The method of claim 35, wherein the magnetic sacrificial beads have an average particle size of from 0.1 μ

m to 5 μ

m.

43. The method of claim 35, wherein the magnetic sacrificial beads have an average particle size of from 3 μ

m to 5 μ

m.

44. The method of claim 35, wherein the magnetic sacrificial beads are in one or more dissolvable dry reagent coatings.

45. The method of claim 35, wherein the magnetic field is generated by a permanent magnet.

46. The method of claim 35, wherein the magnetic field is generated by an electromagnet.

47. The method of claim 35, wherein the magnetic field is generated by a magnet embedded in the test cartridge.

48. The method of claim 35, wherein at least 50 wt. % of the magnetic magnetic sacrificial beads are retained out of contact with the immuno sensor.

49. The method of claim 35, wherein at least 75 wt. % of the magnetic magnetic sacrificial beads are retained out of contact from the immuno sensor.

50. The method of claim 35, wherein the sample analyte is selected from the group consisting of digoxin, phenobarbital, phenyloin, theophylline, valproic acid, and vancomycin.

51. The method of claim 35, wherein the labeled analyte is in one or more dissolvable dry reagent coatings.

52. The method of claim 35, wherein said labeled analyte is labeled with a label selected from the group consisting of a radiolabel, an enzyme, a chromophore, a fluorophore, a chemiluminescent species, an ionophore and an electroactive species.

53. The method of claim 35, wherein said labeled analyte is labeled with a label selected from the group consisting of a fluorescein, a ferrocene, and a p-aminophenol.

54. A cartridge for performing a competitive immunoassay for a sample analyte suspected of being present in a blood sample, the cartridge comprising:

a sample inlet for receiving the blood sample;

a metering chamber for metering the blood sample to form a metered sample;

one or more dry reagent coating layers comprising magnetic sacrificial beads opsonized to leukocytes and a labeled analyte;

an electrode comprising an immobilized antibody to the sample analyte and to the labeled analyte; and

one or more pumping elements for moving the metered sample and the labeled analyte to the electrode.

55. The cartridge of claim 54, further comprising a magnet for positioning the magnetic sacrificial beads.

56. A cartridge for performing a non-competitive immunoassay for a sample analyte suspected of being present in a blood sample, the cartridge comprising:

a sample inlet for receiving the blood sample;

a metering chamber for metering the blood sample to form a metered sample;

one or more dry reagent coating layers comprising magnetic sacrificial beads opsonized to leukocytes and a signal antibody to the analyte;

an electrode comprising an immobilized antibody to the sample analyte; and

one or more pumping elements for moving the metered sample to the electrode.

57. The cartridge of claim 56, further comprising a magnet for positioning the magnetic sacrificial beads.

58. A kit for performing an analyte immunoassay in a whole blood sample, comprising:

magnetic sacrificial beads opsonized to leukocytes, wherein the magnetic sacrificial beads are movable by a magnetic field;

a magnet for retaining the magnetic sacrificial beads; and

an immunosensor, wherein leukocytes in the whole blood sample are bindable to said magnetic sacrificial beads, and wherein the magnetic field is capable of retaining at least a portion of said magnetic sacrificial beads out of contact from said immuno sensor.

59. The kit of claim 58, wherein the immunoassay is a sandwich assay.

60. The kit of claim 58, wherein the immunoassay is a competitive assay.

61. The kit of claim 58, wherein the whole blood sample is amended with an anticoagulant.

62. The kit of claim 58, wherein the magnetic sacrificial beads comprise a magnetite (Fe₃O₄) core coated with a styrene-acrylic acid copolymer.

63. The kit of claim 58, wherein the magnetic sacrificial beads comprise magnetic beads coated with non-human IgG or fragments thereof.

64. The kit of claim 58, wherein the magnetic sacrificial beads have an average particle size of from 0.01 μ

m to 20 μ

m.

65. The kit of claim 58, wherein the magnetic sacrificial beads have an average particle size of from 0.1 μ

m to 5 μ

m.

66. The kit of claim 58, wherein the magnetic sacrificial beads have an average particle size of from 3 μ

m to 5 μ

m.

67. The kit of claim 58, wherein the magnetic sacrificial beads are in one or more dissolvable dry reagent coatings.

68. The kit of claim 58, wherein the magnet is a permanent magnet.

69. The kit of claim 58, wherein the magnet is an electromagnet.

70. The kit of claim 58, wherein at least 50 wt. % of the magnetic sacrificial beads are retained out of contact from the immuno sensor.

71. The kit of claim 58, wherein at least 75 wt. % of the magnetic sacrificial beads are retained out of contact from the immuno sensor.