Determination of glycated protein

• US 7,695,973 B2
• Filed: 02/07/2007
• Issued: 04/13/2010
• Est. Priority Date: 01/31/2001
• Status: Expired due to Term

• Current Assignee: Biohermes
• Original Assignee: Scripps Laboratories, Inc. (Scripps Health)
• Inventors: McCroskey, Ralph P., Melton, Cameron E.
• Primary Examiner(s): Jung; Unsu
• Application Number:
  US11/672,467
• Publication Number:
  US 20070190658A1
• Time in Patent Office: 1,161 Days
• Field of Search: 427/308, 427/324, 427/339, 427/415, 427/439
• US Class Current: 436/87
• CPC Class Codes: G01N 2400/00 Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
• CPC Class Codes: G01N 33/54386 Analytical elements
• 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 33/723 Glycosylated haemoglobin
• CPC Class Codes: Y10S 435/803 Physical recovery methods, e.g. chromatography, grinding
• CPC Class Codes: Y10S 435/805 Test papers
• CPC Class Codes: Y10S 435/815 by sorption
• CPC Class Codes: Y10S 435/817 Enzyme or microbe electrode
• CPC Class Codes: Y10S 435/962 Prevention or removal of interfering materials or reactants or other treatment to enhance results, e.g. determining or preventing nonspecific binding
• CPC Class Codes: Y10S 435/967 Standards, controls, materials, e.g. validation studies, buffer systems
• CPC Class Codes: Y10S 435/97 Test strip or test slide
• CPC Class Codes: Y10S 435/973 Simultaneous determination of more than one analyte
• CPC Class Codes: Y10S 436/805 Optical property
• 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 436/825 Pretreatment for removal of interfering factors from sample
• CPC Class Codes: Y10T 436/104998 Glucose, ketone, nitrate standard or control
• CPC Class Codes: Y10T 436/105831 Protein or peptide standard or control [e.g., hemoglobin, etc.]
• CPC Class Codes: Y10T 436/106664 Blood serum or blood plasma standard or control
• CPC Class Codes: Y10T 436/108331 : Preservative, buffer, anticoagulant or diluent
• CPC Class Codes: Y10T 436/112499 : with sample on test slide
• CPC Class Codes: Y10T 436/201666 : Carboxylic acid
• CPC Class Codes: Y10T 436/203332 : Hydroxyl containing
• CPC Class Codes: Y10T 436/25 : including sample preparation
• CPC Class Codes: Y10T 436/25125 : Digestion or removing interfering materials
• CPC Class Codes: Y10T 436/25375 : Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
• CPC Class Codes: Y10T 436/255 : including use of a solid sorbent, semipermeable membrane, or liquid extraction

Abstract

The present invention provides methods for quantitation of glycated protein in a biological sample using a solid support matrix by making a first bound protein measurement total bound protein under conditions where both glycated and non-glycated protein bind to the support in making a second bound protein measurement under conditions where glycated protein is bound to the support and non-glycated protein is not substantially bound. Diagnostic devices and kits comprising the methods of the present invention are also provided.

Claims

1. A method for ascertaining the extent to which a protein in a sample has been glycated, comprising:

providing a biological sample containing both glycated and nonglycated forms of a protein;

contacting the biological sample with a solid support at a first pH and binding both glycated and nonglycated forms of the protein to negatively charged groups immobilized on the solid support;

obtaining a first measurement indicative of the total amount of the glycated and nonglycated forms of the protein bound to the solid support at the first pH;

then changing the first pH on the solid support to a second pH, thereby removing both the glycated form of the protein and the nonglycated form of the protein from the negatively charged groups and inmiediately rebinding only the glycated form of the protein to hydroxyboryl groups immobilized on the solid support;

obtaining a second measurement indicative of the amount of the glycated form of the protein bound to the solid support at the second pH; and

relating the first measurement to the second measurement to ascertain the extent to which the protein in the sample has been glycated.

2. The method of claim 1 further comprising contacting the solid support with an aliquot of a buffer solution sufficient to rinse off excess biological sample, including both unbound glycated and nonglycated forms of the protein, prior to obtaining the first measurement.

3. The method of claim 1, wherein the negatively charged groups are selected from the group consisting of sulfinate, sulfonate, sulfate and phosphate.

4. The method of claim 1, wherein the first pH is achieved by applying a buffer of about pH 5.0 to 7.0.

5. The method of claim 1, wherein the second pH is achieved by applying a buffer of about pH 8.0 to 10.0.

6. The method of claim 1, wherein the protein is hemoglobin.

7. The method of claim 1, wherein the protein is albumin.

8. The method of claim 1, wherein the sample comprises blood.

9. The method of claim 1, wherein the sample comprises serum.

10. The method of claim 1, wherein the sample comprises plasma.

11. The method of claim 1, wherein the first and second measurements measure an optical property of the protein.

12. The method of claim 1, wherein the first and second measurements are optical readings at a predetermined wavelength.

13. The method of claim 1, wherein the first and second measurements measure a protein label.

14. The method of claim 1, wherein the hydroxyboryl group is of the type

15. The method of claim 1, wherein the solid support is selected from the group consisting of cellulose, nitrocellulose, cellulose acetate, polyacrylamide, agarose polyacrylamide copolymer, agarose, starch, nylon, nylon polyesters, dextran, cross-linked dextran, dextran acrylamide copolymer, cross-linked hydroxyethylmethacrylate, substituted cross-linked polystyrenes, polyvinylalcohol, wool, metal oxides, porous ceramics coated with hydrophilic organic polymers and glass.

16. A method for ascertaining the extent to which albumin in a sample has been glycated, comprising:

providing a strip-type device comprising;

(1) a solid support matrix having a measurement area; and

(2) negatively charged groups and dihydroxyboryl groups immobilized on the solid support matrix, wherein said negatively charged groups are capable of binding both glycated and nonglycated albumin at a first pH between about 5.0 and about 7.0, and said dihydroxyboryl groups are capable of binding glycated albumin at a second pH between about 8.0 and about 10.0;

adding the sample to the solid support matrix at the first pH, thereby binding both glycated and nonglycated albumin to the negatively charged groups on the solid support matrix, and then performing a first measurement on the measurement area indicative of the total amount of glycated and nonglycated albumin bound to the solid support matrix;

changing the pH on the solid support matrix to the second pH, thereby removing both the nonglycated albumin and the glycated albumin from the negatively charged groups, after which removal the glycated albumin immediately binds to the dihydroxyboryl groups on the solid support matrix independent of incubation time, and then performing a second measurement on the measurement area indicative of the amount of glycated albumin bound to the solid support matrix; and

relating the first measurement to the second measurement to ascertain the extent to which the albumin in the sample has been glycated.

17. The method of claim 16, wherein the negatively charged groups are selected from the group consisting of sulfate, sulfinate, sulfonate and phosphate.

18. The method of claim 16, wherein said dihydroxyboryl groups have the formula:

19. The method of claim 16, wherein the solid support matrix is selected from the group consisting of cellulose, nitrocellulose, cellulose acetate, polyacrylamide, agarose polyacrylamide copolymer, agarose, starch, nylon, nylon polyesters, dextran, cross-linked dextran, dextran acrylamide copolymer, cross-linked hydroxyethylmethacrylate, substituted cross-linked polystyrenes, polyvinylalcohol, wool, metal oxides, porous ceramics coated with hydrophilic organic polymers and glass.

20. The method of claim 16, wherein said first pH is achieved with a buffer selected from the group consisting of 2(N-morpholino) ethanesulfonic acid (“

MES”

), 3(N-morpholino)propanesulfonic acid (“

MOPS”

) and N-2-hydroxyethylpiperazine-N′

-2-ethanesulfonic acid (“

HEPES”

).

21. The method of claim 16, wherein said second pH is achieved with a buffer selected from the group consisting of ammonium acetate or taurine.

22. The method of claim 16, wherein the sample comprises blood, plasma or serum.

23. A method for ascertaining the extent to which hemoglobin in a sample has been glycated, comprising:

providing a strip-type device comprising;

(1) a solid support matrix having a measurement area; and

(2) negatively charged groups and dihydroxyboryl groups immobilized on the solid support matrix, wherein said negatively charged groups are capable of binding both glycated and nonglycated hemoglobin at a first ph between about 5.0 and about 7.0, and said dihydroxyboryl groups are capable of binding glycated hemoglobin at a second pH between about 8.0 and about 10.0;

adding the sample to the solid support matrix at the first pH, thereby binding both glycated and nonglycated hemoglobin to the negatively charged groups on the solid support matrix, and then performing a first measurement on the measurement area indicative of the total amount of glycated and nonglycated hemoglobin bound to the solid support matrix;

changing the pH on the solid support matrix to the second pH, thereby removing both the nonglycated hemoglobin and the glycated hemoglobin from the negatively charged groups, after which removal the glycated hemoglobin immediately binds to the dihydroxyboryl groups on the solid support matrix independent of incubation time, and then performing a second measurement on the measurement area indicative of the amount of glycated hemoglobin bound to the solid support matrix; and

relating the first measurement to the second measurement to ascertain the extent to which the hemoglobin in the sample has been glycated.

24. The method of claim 23, wherein said negatively charged groups are selected from the group consisting of sulfate, sulfinate, sulfonate and phosphate.

25. The method of claim 23, wherein said dihydroxyboryl groups have the formula:

26. The method of claim 23, wherein the solid support matrix is selected from the group consisting of cellulose, nitrocellulose, cellulose acetate, polyacrylamide, agarose polyacrylamide copolymer, agarose, starch, nylon, nylon polyesters, dextran, cross-linked dextran, dextran acrylamide copolymer, cross-linked hydroxyethylmethacrylate, substituted cross-linked polystyrenes, polyvinylalcohol, wool, metal oxides, porous ceramics coated with hydrophilic organic polymers and glass.

27. The method of claim 23, wherein said first pH is achieved with a buffer selected from the group consisting of 2(N-morpholino) ethanesulfonic acid (“

MES”

), 3(N-morpholino)propanesulfonic acid (“

MOPS”

) and N-2-hydroxyethylpiperazine-N′

-2-ethanesulfonic acid (“

HEPES”

).

28. The method of claim 23, wherein said second pH is achieved with a buffer selected from the group consisting of ammonium acetate or taurine.

29. The method of claim 23, wherein the sample comprises blood, plasma or serum.