Assay

• US 20060030050A1
• Filed: 08/03/2005
• Published: 02/09/2006
• Est. Priority Date: 08/03/2004
• Status: Active Grant

• Current Assignee: Axis-Shield Diagnostics Limited (Abbott Laboratories Incorporated)
• Original Assignee: Axis-Shield Diagnostics Limited (Abbott Laboratories Incorporated)
• Inventors: Milne, Ken, Lawlor, Margaret
• Granted Patent: US 7,592,182 B2
• Time in Patent Office: Days
• US Class Current: 436/67
• CPC Class Codes: G01N 2800/042 Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
• CPC Class Codes: G01N 2800/22 Haematology
• CPC Class Codes: G01N 33/54313 the carrier being characterised by its particulate form
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/6842 Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
• CPC Class Codes: G01N 33/6893 related to diseases not provided for elsewhere
• CPC Class Codes: G01N 33/723 Glycosylated haemoglobin

Abstract

Compositions and assay methods for and relating to the detection of glycated hemoglobin are provided. In particular, the presence and level of glycated hemoglobin can be detected in a blood sample. The compositions comprise microparticles which have zinc coating for use in the improved binding of proteins exhibiting a zinc binding domain, in particular glycated hemoglobin.

Claims

1. An assay method for determining of the level of glycated hemoglobin within a sample, comprising:

providing a sample comprising hemoglobin;

denaturing the hemoglobin to effect a conformational change which causes exposure of a zinc binding site;

capturing the hemoglobin molecules using zinc coupled to a support medium; and

determining the amount of glycated hemoglobin bound by the coupled zinc.

2. The assay method of claim 1, wherein denaturation is caused by detergents.

3. The assay method of claim 1, wherein the detergents are one or more of detergents selected from the group consisting of cholate, N-octyl-glucoside, N-octyl-thioglucoside, triton X-100, zwittergent3-14 and bile salts.

4. The assay method of claim 1, wherein capture of the hemoglobin by the coupled zinc molecules is performed in the presence of detergent.

5. The assay method of claim 1, wherein the zinc is coupled to a microparticle.

6. The assay method of claim 5, wherein the microparticle is a bead selected from the group consisting of a latex bead, a polystyrene bead, a silica bead, a chelating sepharose bead and a magnetic bead.

7. The assay method of claim 1, wherein the zinc coating provided on the microparticle is provided by zinc chloride.

8. The assay method of claim 5, wherein the microparticle comprises dextran or polyethylene glycol.

9. The assay method of claim 1, wherein a chelator facilitates the coupling of the zinc to the support medium.

10. The assay method of claim 9 wherein the chelator is zinc binding chelating agent.

11. The assay method of claim 9, wherein the chelator is selected from the group consisting of ethylenediamine tetraacetic acid, trisodium calcium diethylenetriamin-pentaacetate, disodium calcium cyclohexanediaminetetraacetate, sodium calcium edetate, DMSA, DMPS, ethylenediamine, diethylenetriamine, aminoethylethanolamine ethyleneamines and combinations thereof.

12. The assay method of claim 1, wherein the sample comprises whole blood, red blood cells, blood hemolysates or blood extracts.

13. The assay method of claim 12, wherein the sample comprises whole blood cells which are lysed prior to denaturation.

14. The assay method of claim 12, wherein the red bloods cells are lysed using detergents.

15. The assay method of claim 12, wherein the red bloods cells are lysed using heat.

31. A kit comprising zinc-coated microparticles and instructions for the performance of the assay method of claim 1.

16. An assay method for the determination of the level of glycated hemoglobin within a sample, comprising:

providing a blood sample;

extracting the hemoglobin from the sample and partially denaturing the hemoglobin;

exposing the hemoglobin to microparticles coated with zinc to allow binding of hemoglobin and glycated hemoglobin to the microparticles; and

determining the amount of glycated hemoglobin bound to the microparticles.

17. The assay method of claim 16, wherein hemoglobin is extracted from the sample by hemolysis of the sample to release the cell bounded hemoglobin.

18. The assay method of claim 16, wherein the partial denaturation of the hemoglobin during the treatment step is performed with detergents.

19. The assay method of claim 18, wherein the detergents are one or more of detergents selected from the group consisting of cholate, N-octyl-glucoside, N-octyl-thioglucoside, triton X, zwittergent3-14 and bile salts.

20. The assay method of claim 16, wherein the step of exposing the hemoglobin to the zinc coated microparticles is performed at a pH of about 7.

21. The assay method of claim 16, wherein the determination of the amount of glycated hemoglobin present is performed by contacting the glycated hemoglobin with signal-generating molecules which selectively bind to glycated hemoglobin.

22. An assay method for the in-vitro diagnosis and monitoring of diabetes mellitus through the assessment of glycated hemoglobin levels, the method comprising:

providing a blood sample;

hemolysing the blood sample to release cell bounded hemoglobin and partially denature the hemoglobin;

capturing glycated hemoglobin on microparticles coated with zinc;

exposing the captured glycated hemoglobin on the microparticles to signal generating molecules which bind to the glycated hemoglobin;

removing signal generating molecules not bound to glycated hemoglobin; and

quantifying the amount of bound signal generating molecules to determine the percentage level of glycated hemoglobin present in the sample.

23. The assay method of claim 22, wherein the step of determining the percentage level of glycated hemoglobin present in the sample comprises:

capturing the microparticles on a suitable base medium;

washing the microparticles to remove unbound material; and

labelling the glycated hemoglobin with a signal generating molecule comprising a monoclonal antibody specific for glycated hemoglobin, the monoclonal antibody having a label conjugated thereto.

24. A method of isolating hemoglobin from a biological sample, the method comprising:

providing a biological sample;

capturing the hemoglobin on microparticles which are coated with zinc; and

eluting the microparticle-bound hemoglobin from the zinc coated microparticles.

25. The method of claim 24, further comprising the step of hemolysing the sample and partially denaturing the hemoglobin present in the sample prior to the capturing step.

26. The method of claim 24, wherein the biological sample comprises blood.

27. The method of claim 24, wherein the amount of glycated hemoglobin present in the sample is quantified.

28. An assay for the selective binding of at least one target protein bearing a zinc binding site, comprising:

providing a sample;

exposing the sample to zinc coated microparticles;

capturing the target proteins on the zinc coated microparticles;

isolating the microparticles;

detecting the presence of target proteins bound to the microparticles; and

optionally purifying the target protein from the microparticles.

29. The assay method of claim 28, wherein the sample is a biological sample.

30. A zinc coated microparticle.