REDUCING LEUKOCYTE INTERFERENCE IN NON-COMPETITIVE IMMUNOASSAYS

• US 20110117580A1
• 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: Moss, Adam Roger, Campbell, John Lewis Emerson, Davis, Graham
• Granted Patent: US 8,377,669 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 analyte immunoassay, and in particular in non-competitive 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; and (b) performing a non-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 sandwich immunoassay for an analyte suspected of being present in a blood sample, comprising sacrificial beads opsonized to leukocytes, an immobilized first antibody to the analyte and a labeled second antibody to the analyte.

2. The kit of claim 1, wherein the analyte is selected from the group consisting of BNP, proBNP, NTproBNP, cTnI, TnT, HCG, TSH, PSA, D-dimer, CRP, myoglobin, NGAL, CKMB and myeloperoxidase.

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 first 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 first 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 second labeled antibody are in one or more dissolvable dry reagent coatings.

13. The kit of claim 1, wherein said labeled second antibody is labeled with a label selected from the group consisting of a radiolabel, an enzyme, a chromophore, a fluorophore and a chemiluminescent species.

14. The kit of claim 1, wherein said labeled second antibody is labeled with an enzyme selected from the group consisting of alkaline phosphatase, glucosidase, diaphorase, horse raddish peroxidase and glucose oxidase.

15. A cartridge for performing a sandwich immunoassay for an 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 antibody to the analyte;

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

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

16. The cartridge of claim 15, wherein the analyte is selected from the group consisting of BNP, proBNP, NTproBNP, cTnI, TnT, HCG, TSH, PSA, D-dimer, CRP, myoglobin, NGAL, CKMB and myeloperoxidase.

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 sandwich immunoassay for an analyte suspected of being present in a blood sample comprising:

(a) exposing a blood sample suspected of containing the analyte to sacrificial beads opsonized to leukocytes;

an immobilized first antibody to the analyte; and

a labeled second antibody to form a complex comprising said first antibody, said analyte and said second antibody; and

(b) determining the amount of complexed label.

23. The method of claim 22, wherein the sacrificial beads contact the blood sample under conditions effective for leukocytes in the sample to preferentially phagocytose the sacrificial beads.

24. The method of claim 22, wherein the analyte is selected from the group consisting of BNP, proBNP, NTproBNP, cTnI, TnT, HCG, TSH, PSA, D-dimer, CRP, myoglobin, NGAL, CKMB and myeloperoxidase.

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

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

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

m to 20 μ

m.

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

m to 20 μ

m.

29. 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.

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

31. The method of claim 22, wherein the blood sample is mixed with the sacrificial beads, then mixed with the immobilized first antibody, and then mixed with the labeled second antibody.

32. The method of claim 22, wherein the blood sample is mixed with the sacrificial beads, the immobilized first antibody, and the labeled second antibody at substantially the same time.

33. The method of claim 22, wherein the blood sample is mixed with the immobilized first antibody and the labeled second antibody at substantially the same time.

34. The method of claim 22, wherein the blood sample is mixed with the sacrificial beads and the labeled second antibody at substantially the same time, and before the blood sample is mixed with the immobilized first antibody.

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

(a) mixing a sample suspected of containing an 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.

36. The method of claim 35, 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.

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

(a) mixing a blood sample suspected of containing an analyte with an excess of sacrificial beads 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 analyte immobilized on an electrode forming a sandwich between said antibody-coated bead, said analyte and a second labeled antibody;

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

(d) determining the amount of said sandwiched label with said immunosensor and relating the amount of said label to the concentration of the analyte in the sample.

38. A method of performing a sandwich immunoassay for an analyte suspected of being present in a blood sample, comprising:

(a) mixing a blood sample suspected of containing an analyte with an excess of opsonized sacrificial beads 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 antibody-coated beads to the analyte under conditions effective to form a sandwich between said antibody-coated bead, said analyte and a second labeled antibody;

(c) washing said blood sample from said antibody-coated beads; and

(d) determining the amount of said sandwiched label and relating the amount of said label to the concentration of the analyte in the sample.

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