REDUCING LEUKOCYTE INTERFERENCE IN COMPETITIVE IMMUNOASSAYS

• US 20110117581A1
• Filed: 11/17/2009
• Published: 05/19/2011
• Est. Priority Date: 11/17/2009
• 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 8,389,293 B2
• Time in Patent Office: Days
• US Class Current: 435/7.32
• CPC Class Codes: G01N 33/54313 the carrier being characterised by its particulate form
• CPC Class Codes: G01N 33/54393 Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
• CPC Class Codes: Y10T 436/25125 Digestion or removing interfering materials

Abstract

The invention is directed to methods and devices for reducing interference from leukocytes in an competitive analyte immunoassays. In one embodiment, the invention is to a method comprising the steps of (a) amending a biological sample such as a whole blood sample with sacrificial beads opsonized for leukocytes; and (b) performing a competitive immunoassay on the amended sample to determine the concentration of said analyte in said sample. Preferably, the sample is amended with IgG-coated sacrificial beads.

Claims

1. A kit for performing a competitive immunoassay for a sample analyte suspected of being present in a blood sample, comprising:

sacrificial beads opsonized to leukocytes,

a labeled analyte, and

an immobilized antibody to the sample analyte and to the labeled analyte.

2. The kit of claim 1, wherein the sample analyte is selected from the group consisting of digoxin, phenobarbital, phenyloin, theophylline, valproic acid, and vancomycin.

3. The kit of claim 1, wherein the sacrificial beads comprise substrate beads coated with non-human IgG or fragments thereof.

4. The kit of claim 3, wherein the substrate beads are formed of a material selected from the group consisting of polystyrene, polyacrylic acid, and dextran.

5. The kit of claim 1, wherein the sacrificial beads comprise substrate beads coated with a material or fragment thereof selected from the group consisting of a protein, a bacterium, a virus and a xenobiotic.

6. The kit of claim 1, wherein the sacrificial beads are stabilized bacterium or bacterial spores.

7. The kit of claim 1, wherein the sacrificial beads have an average particle size of from 0.01 μ

m to 20 μ

m.

8. The kit of claim 1, wherein the sacrificial beads have an average particle size of from 0.1 μ

m to 5 μ

m.

9. The kit of claim 1, wherein the sacrificial beads are present in an amount sufficient to provide a dissolved sacrificial bead concentration of at least 10⁴ beads per microliter of sample.

10. The kit of claim 1, wherein the immobilized 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.

11. The kit of claim 1, wherein the immobilized antibody is attached to an assay bead, and the assay bead is attached to an amperometric electrode.

12. The kit of claim 1, wherein the sacrificial beads and the labeled analyte are in one or more dissolvable dry reagent coatings.

13. The kit of claim 1, 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.

14. The kit of claim 1, wherein said labeled analyte is labeled with a label selected from the group consisting of a fluorescein, a ferrocene, and a p-aminophenol.

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

(a) a sample inlet for receiving the blood sample;

(b) a metering chamber for metering the blood sample to form a metered sample;

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

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

(e) one or more pumping elements for moving the metered sample, dissolved sacrificial beads, and the labeled analyte to the electrode.

16. The cartridge of claim 15, wherein the sample analyte is selected from the group consisting of digoxin, phenobarbital, phenyloin, theophylline, valproic acid, and vancomycin.

17. The cartridge of claim 15, wherein the sacrificial beads comprise substrate beads coated with non-human IgG or fragments thereof.

18. The cartridge of claim 17, wherein the substrate beads are formed of a material selected from the group consisting of polystyrene, polyacrylic acid, and dextran.

19. The cartridge of claim 15, wherein the sacrificial beads have an average particle size of from 0.01 μ

m to 20 μ

m.

20. The cartridge of claim 15, wherein the sacrificial beads are present in an amount sufficient to provide a dissolved sacrificial bead concentration of at least 10⁴ beads per microliter of sample.

21. The cartridge of claim 15, wherein the one or more dry reagent coating layers are disposed within the metering chamber.

22. A method of performing a competitive immunoassay for a sample analyte suspected of being present in a blood sample, comprising:

(a) exposing a blood sample suspected of containing the sample analyte to sacrificial beads opsonized for leukocytes, a labeled analyte and an immobilized antibody to the sample analyte and to the labeled analyte; and

(b) determining the amount of labeled analyte complexed to said immobilized antibody.

23. The method of claim 22, wherein the sample analyte is selected from the group consisting of digoxin, phenobarbital, phenyloin, theophylline, valproic acid, and vancomycin.

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

25. The method of claim 24, wherein the substrate beads are formed of a material selected from the group consisting of polystyrene, polyacrylic acid, and dextran.

26. The method of claim 22, wherein the sacrificial beads comprise substrate beads coated with a material or fragment thereof selected from the group consisting of a protein, a bacterium, a virus and a xenobiotic.

27. The method of claim 22, wherein the sacrificial beads are stabilized bacterium or bacterial spores.

28. The method of claim 22, wherein the sacrificial beads have an average particle size of from 0.01 μ

m to 20 μ

m.

29. The method of claim 22, wherein the sacrificial beads have an average particle size of from 0.1 μ

m to 5 μ

m.

30. The method of claim 22, wherein the sacrificial beads are present in an amount sufficient to provide a dissolved sacrificial bead concentration of at least 10⁴ beads per microliter of sample.

31. The method of claim 22, wherein the immobilized 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.

32. The method of claim 22, wherein the immobilized antibody is attached to an assay bead, and the assay bead is attached to an amperometric electrode.

33. The method of claim 22, wherein the exposing comprises dissolving the sacrificial beads and the labeled analyte into the blood sample from one or more dissolvable dry reagent coatings.

34. The method of claim 22, 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.

35. The method of claim 22, wherein said labeled analyte is labeled with a label selected from the group consisting of a fluorescein, a ferrocene, and a p-aminophenol.

36. The method of claim 22, wherein said blood sample is whole blood amended with an anticoagulant.

37. A method of performing a competitive immunoassay for a sample analyte suspected of being present in a blood sample, comprising:

(a) exposing a blood sample comprising leukocytes and suspected of containing the sample analyte to sacrificial beads opsonized for the leukocytes under conditions effective for the leukocytes to preferentially phagocytose the sacrificial beads;

(b) contacting the blood sample with an immobilized antibody in the presence of labeled analyte under conditions effective to form a first complex comprising said antibody and said sample analyte and a second complex comprising said antibody and said labeled analyte; and

(c) determining sample analyte concentration based on the amount of the second complex formed.

38. The method of claim 37, wherein the sample analyte is selected from the group consisting of digoxin, phenobarbital, phenyloin, theophylline, valproic acid, and vancomycin.

39. The method of claim 37, wherein the sacrificial beads comprise substrate beads coated with non-human IgG or fragments thereof.

40. The method of claim 39, wherein the substrate beads are formed of a material selected from the group consisting of polystyrene, polyacrylic acid, and dextran.

41. The method of claim 37, wherein the sacrificial beads have an average particle size of from 0.01 μ

m to 20 μ

m.

42. The method of claim 37, wherein the sacrificial beads are present in an amount sufficient to provide a dissolved sacrificial bead concentration of at least 10⁴ beads per microliter of sample.

43. The method of claim 37, wherein the immobilized 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.

44. The method of claim 37, wherein said blood sample is whole blood amended with an anticoagulant.

45. The method of claim 37, wherein the blood sample is mixed with the sacrificial beads, then mixed with the immobilized antibody, and then mixed with the labeled analyte.

46. The method of claim 37, wherein the blood sample is mixed with the sacrificial beads, the immobilized antibody, and the labeled analyte at substantially the same time.

47. The method of claim 37, wherein the blood sample is mixed with the immobilized antibody, and the labeled analyte at substantially the same time.

48. The method of claim 37, wherein the blood sample is mixed with the sacrificial beads and the labeled analyte at substantially the same time, and before the blood sample is mixed with the immobilized antibody.

49. A method of substantially ameliorating white blood cell accumulation on a competitive immunosensor comprising antibody-coated beads to a sample analyte immobilized on an electrode comprising, the method comprising the steps of:

(a) mixing a sample suspected of containing the sample analyte with sacrificial beads to form an amended sample wherein white blood cells in the sample preferentially seek to phagocytose the sacrificial beads, and

(b) contacting the amended sample with an immunosensor in the presence of labeled analyte.

50. A method of performing a competitive immunoassay for a sample analyte suspected of being present in a blood sample, comprising:

(a) mixing a blood sample suspected of containing a sample analyte with an excess of sacrificial beads and a labeled analyte to form an amended sample wherein white blood cells in the sample preferentially seek to phagocytose the sacrificial beads;

(b) contacting the amended sample with an immunosensor comprising antibody-coated beads to the sample analyte immobilized on an electrode to form a first complex between the antibody-coated beads and the sample analyte and a second complex between the antibody-coated beads and the labeled analyte;

(c) washing said blood sample from said immunosensor; and

(d) determining the amount of labeled analyte on said immunosensor and relating the amount of said labeled analyte to the concentration of the sample analyte in the sample.

51. A test cartridge for performing a competitive immunoassay for a sample analyte suspected of being present in a blood sample, the test cartridge comprising an immunosensor in a conduit, said immunosensor having an immobilized antibody to the sample analyte, and said conduit having a dry reagent coating disposed therein, the dry reagent coating comprising sacrificial beads and a labeled analyte and being configured to dissolve into said blood sample.