Methods and devices for quantitation of glycated protein

• US 20030068830A1
• Filed: 01/31/2002
• Published: 04/10/2003
• Est. Priority Date: 01/31/2001
• Status: Active Grant

• Current Assignee: Biohermes
• Original Assignee: Scripps Laboratories, Inc. (Scripps Health)
• Inventors: McCroskey, Ralph P., Melton, Cameron E.
• Granted Patent: US 7,195,923 B2
• Time in Patent Office: Days
• US Class Current: 436/518
• CPC Class Codes: G01N 2400/00 Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
• CPC Class Codes: G01N 33/54387 Immunochromatographic test strips
• CPC Class Codes: G01N 33/54388 based on lateral flow
• CPC Class Codes: G01N 33/54391 based on vertical flow
• 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 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 and 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 of quantitation of glycated protein in a sample which comprises:

(a) contacting a solid support matrix which comprises a negatively charged group and a hydroxyboryl compound and which has a measurement area, with an aliquot of biological sample sufficient to cover said measurement area;

(b) contacting said solid support matrix with an aliquot of a first buffer sufficient to rinse off unbound protein wherein said first buffer has a pH selected to allow both glycated and non-glycated protein to be bound to said solid support matrix;

(c) quantitating protein bound to said measurement area using measurement of a selected property of said protein to give a first bound protein reading;

(d) contacting said solid support matrix with an aliquot of a second buffer sufficient to rinse off unbound protein wherein said second buffer has a pH selected to allow glycated protein to be bound to said solid support matrix but where non-glycated protein is not substantially bound to said solid support matrix;

(e) quantitating protein bound to said measurement area using measurement of the property measured in step (c) to give a second bound protein reading; and

(f) calculating percentage of glycated protein using said first and second bound protein readings.

2. A method according to claim 1 wherein the property measured is an optical reading.

3. A method according to claim 2 wherein the optical reading is absorbance or reflectance at a specified wavelength.

4. A method according to claim 3 wherein the glycated protein is glycated hemoglobin.

5. A method according to claim 3 wherein the glycated protein is glycated albumin.

35. A diagnostic device for quantitation of glycated hemoglobin utilizing the method according to claim 1.

6. A method for quantitation of amount of glycated protein in a biological sample which compromises:

(a) contacting a solid support matrix which comprises a negatively charged group and a hydroxyboryl compound and which has a measurement area with an aliquot of a biological sample sufficient to cover said measurement area;

(b) contacting said solid support matrix with an aliquot of a first buffer sufficient to rinse off unbound protein, wherein said first buffer has a pH of about 5.0 to about 7.0;

(c) quantitating protein bound to said measurement area to give a first bound protein reading;

(d) contacting said solid support matrix with an aliquot of second buffer sufficient to rinse off unbound protein, wherein said buffer has a pH of about 8.0 to about 10.0;

(e) quantitating protein bound to said measurement area to give a second bound protein reading; and

(f) calculating percentage of glycated protein in said sample using said first bound protein reading and said second bound protein reading.

7. A method according to claim 6 wherein said first and second bound protein readings measure the same property.

8. A method according to claim 7 wherein the property measured is an optical reading.

9. A method according to claim 8 wherein the optical reading is absorbance or reflectance at a specified wavelength.

10. A method according to claim 9 wherein the glycated protein is glycated hemoglobin.

11. A method according to claim 9 wherein the glycated protein is glycated albumium.

36. A diagnostic device for quantitation of glycated protein utilizing the method according to claim 6.

12. A method for quantitation of glycated hemoglobin in a biological sample which comprises;

(a) adding said sample to a sample application site which is in communication with a solid support matrix which comprises a negatively charged group and a dihydroxyboryl compound and which has a measurement area;

(b) adding an aliquot of a first buffer at said sample application site, wherein said first buffer has a pH between about 5.0 and about 7.0;

(c) making a first optical reading of said measurement area at a wavelength at which hemoglobin absorbs light;

(d) adding an aliquot of a second buffer at said sample application site, wherein said second buffer has a pH between about 8.0 and about 10.0;

(e) making a second optical reading of said measurement area at a wavelength at which hemoglobin absorbs light; and

(f) calculating the percentage of glycated hemoglobin in said blood sample using said first and second optical readings.

13. A method according to claim 12 wherein said dihydroxyboryl compound has the formula

14. A method according to claim 13 wherein R is selected from the group consisting of phenyl, m-aminophenyl, hydrogen, ethyl, 1-propyl and 2-methyl-1-butyl.

15. A method according to claim 14 wherein R is m-aminophenyl.

16. A method according to claim 13 wherein the negatively charged group is selected from the group consisting of carboxylate, sulfate, sulfonate, sulfinate and phosphate.

17. A method according to claim 16 wherein the negatively charged group is carboxylate.

18. A method according to claim 12 wherein said solid support matrix is selected from the group consisting of cellulose, nitrocellulose, cellulose acetate, polyacrylamide, agarose polyacrylaminde copolymer, agarose, starch, nylon, nylon polyesters, dextran, cross-linked dextran, dextran acrylamide copolymer, cross-linked hydroxyethylmethacrylate substituted cross-linked polystyrenes, and polyvinylalcohol.

19. A method according to claim 18 wherein said solid support matrix is carboxy cellulose.

20. A method according to claim 12 wherein said first buffer is selected from the group consisting of MES, MOPS and HEPES.

21. A method according to claim 12 wherein said second buffer is ammonium acetate or taurine.

26. A method according to claim 21 wherein the negatively charged group is selected from the group consisting of carboxylate, sulfate, sulfonate, sulfinate and phosphate.

27. A method according to claim 26 wherein the negatively charged group is carboxylate.

37. A diagnostic device for quantitation of glycated protein utilizing the method according to claim 12.

22. A method for quantitation of non-hemoglobin glycated protein in a biological sample which comprises:

(a) adding said sample to a sample application site which is in communication with a solid support matrix which comprises a negatively charged group and a dihydroxyboryl compound and which has a measurement area;

(b) adding an aliquot of a first buffer to said sample application site, wherein said first buffer has a pH between about 5.0 and about 7.0;

(c) making a first optical reading of said measurement area at a wavelength at which said labeling agent absorbs light;

(d) adding an aliquot of a second buffer to said sample application site wherein said second buffer has a pH between about 8.0 and about 10.0;

(e) making a second optical reading of said measurement area at a wavelength at which said labeling agent absorbs light; and

(f) calculating the percentage of glycated protein in said sample using said first and second optical readings.

23. A method according to claim 22 wherein said dihydroxyboryl compound has the formula:

24. A method according to claim 23 wherein R is selected from the group consisting of phenyl, m-amino phenyl, hydrogen, ethyl, 1-propyl and 2-methyl-1-butyl.

25. A method according to claim 23 wherein R is m-aminophenyl.

28. A method according to claim 22 wherein said solid support matrix is selected from the group consisting of cellulose, nitrocellulose, cellulose acetate, polyacrylamide, agarose polyacrylaminde copolymer, agarose, starch, nylon, nylon polyesters, dextran, cross-linked dextran, dextran acrylamide copolymer, cross-linked hydroxyethylmethacrylate substituted cross-linked polystyrenes, and polyvinylalcohol.

29. A method according to claim 28 wherein said solid support matrix is carboxy cellulose.

30. A method according to claim 22 wherein said first buffer is selected from the group consisting of MES, MOPS and HEPES.

31. A method according to claim 22 wherein said second buffer is ammonium acetate or taurine.

32. A method according to claim 22 wherein said sample is labeled with a protein specific labeling agent.

33. A method according to claim 22 wherein said sample is serum or plasma.

34. A method according to claim 33 wherein said non-hemloglobin glycated protein is glycated albumin.

38. A diagnostic device for quantitation of glycated protein utilizing the method according to claim 22.

39. A diagnostic device according to claim 22 wherein said glycated non-hemoglobin protein is albumin.

40. A kit comprising said diagnostic device according to any of claims 34 to claim 39, a first buffer having a pH of about 5.0 to about 7.0 and a second buffer having a pH of about 8.0 to about 10.0.