US4389491A
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Method and kit for chromatographic separation of hemoglobin A1c
Assignee: Bio Rad Laboratories Inc
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Filing: 1982-04-26
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Granted: 1983-06-21
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Expires: 2002-04-26
Abstract
Abstract An ion exchange method and kit for the separation of hemoglobin A 1c from other hemoglobin components in a sample of human blood. The sample is lysed, then used to impregnate a weak cation exchanger. Two buffer solutions are then passed through the column in succession, the first having an alkali metal ion concentration of from about 0.02M to about 0.05M and the second having an alkali metal ion concentration of from about 0.06M to about 0.11M. The second eluate contains substantially all of the hemoglobin A 1c and substantially none of the other hemoglobin components in the original sample. Analysis of the second eluate thus provides a reliable indication of the long-term glucose level in the blood of a patient, and hence the patient's ability to regulate the quantity of glucose ingested. The hemolysate with the Schiff base precursor is introduced directly to the chromatographic column.
Inventors
- 👤 Mark S. Hanamoto
- 👤 Steve K. Tanaka
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-09 04:26
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/72; G01N33/721; G01N33/723
Claims Analysis
Total Claims: 37
Claim 1
Independent Claim
188 words
A method for the separation of hemoglobin A 1c from other glycosylated and nonglycosylated hemoglobins and the Schiff base precursor to hemoglobin A 1c in a sample of human blood which comprises: (a) lysing the red blood cells contained in said sample to form a hemolysate containing said hemoglobin A 1c , said glycosylated and nonglycosylated hemoglobins, and said Schiff base precursor, (b) impregnating a weak cation exchanger with said hemolysate, (c) passing through said exchanger a first buffer solution with ions of an alkali metal dissolved therein at a concentration of from about 0.02 M to about 0.05 M to dissociate said Schiff base precursor into glucose and hemoglobin A and to preferentially elute said glucose and said other glycosylated hemoglobins over said hemoglobin A, said hemoglobin A 1c and said other nonglycosylated hemoglobins, (d) passing through said exchanger a second buffer solution containing ions of an alkali metal dissolved therein at a concentration of from about 0.06 M to about 0.11 M to preferentially elute said hemoglobin A 1c over said hemoglobin A and said other nonglycosylated hemoglobins, and (e) recovering the eluate from step (d).
Claim 2
Independent Claim
188 words
A method for the separation of hemoglobin A 1c from other glycosylated and nonglycosylated hemoglobins and the Schiff base precursor to hemoglobin A 1c in a sample of human blood which comprises: (a) lysing the red blood cells contained in said sample to form a hemolysate containing said hemoglobin A 1c , said glycosylated and nonglycosylated hemoglobins, and said Schiff base precursor, (b) impregnating a weak cation exchanger with said hemolysate, (c) passing through said exchanger a first buffer solution with ions of an alkali metal dissolved therein at a concentration of from about 0.02 M to about 0.05 M to dissociate said Schiff base precursor into glucose and hemoglobin A and to preferentially elute said glucose and said other glycosylated hemoglobins over said hemoglobin A, said hemoglobin A 1c and said other nonglycosylated hemoglobins, (d) passing through said exchanger a second buffer solution containing ions of an alkali metal dissolved therein at a concentration of from about 0.06 M to about 0.11 M to preferentially elute said hemoglobin A 1c over said hemoglobin A and said other nonglycosylated hemoglobins, and (e) recovering the eluate from step (d).
Claim 3
Dependent Claim
42 words
A method according to claim 1, in which said cations on said exchanger are alkali metal ions, and said alkali metal ions in said first and second buffer solution are identical in type to each other and to those on said exchanger.
Claim 4
Dependent Claim
38 words
A method according to claim 2, in which said alkali metal ions on said exchanger and in said first and second buffer solutions are identical and have an atomic weight equal to or less than that of potassium.
Claim 5
Dependent Claim
36 words
A method according to claim 2, in which said alkali metal ions on said exchanger and in said first and second buffer solutions are identical and are selected from the group consisting of sodium and potassium.
Claim 6
Dependent Claim
26 words
A method according to claim 2, in which said alkali metal ions on said exchanger and in said first and second buffer solutions are sodium ions.
Claim 7
Dependent Claim
40 words
A method according to claim 1, in which said exchanger is a copolymer of methacrylic acid and divinylbenzene and from about 30% to about 50% of the active sites on said exchanger are occupied by ions of an alkali metal.
Claim 8
Dependent Claim
28 words
A method according to claim 1, in which the concentration of alkali metal ions in said first buffer solution is from about 0.03 M to about 0.04 M.
Claim 9
Dependent Claim
28 words
A method according to claim 1, in which the concentration of alkali metal ions in said second buffer solution is from about 0.07 M to about 0.09 M.
Claim 10
Dependent Claim
88 words
A method according to claim 1, in which said exchanger is a copolymer of methacrylic acid and divinylbenzene and from about 30% to about 50% of the active sites on said exchanger are occupied by alkali metal ions, the concentration of alkali metal ions in said first buffer solution is from about 0.03 M to about 0.04 M, the concentration of alkali metal ions in said second buffer solution is from about 0.07 M to about 0.09 M and all of said alkali metal ions are sodium ions.
Claim 11
Independent Claim
19 words
A method according to any of claims 5-9 in which said first and second buffer solutions are phosphate buffers.
Claim 12
Independent Claim
36 words
A method according to any of claims 5-9 in which the pH of said first buffer solution and the pH of said second buffer solution are each within the range of about 6.3 to about 7.3.
Claim 13
Dependent Claim
30 words
A method according to claim 1, in which said resin is a polymer of methacrylic acid with a particle size within the range of about 100 to about 400 mesh.
Claim 14
Dependent Claim
32 words
A method according to claim 1, in which said resin is a copolymer of methacrylic acid and divinylbenzene with a particle size within the range of about 200 to about 400 mesh.
Claim 15
Dependent Claim
26 words
A method according to claim 1, in which steps (c) and (d) are performed at a temperature of from about 14° C. to about 35° C.
Claim 16
Dependent Claim
26 words
A method according to claim 1, in which steps (c) and (d) are performed at a temperature of from about 17° C. to about 30° C.
Claim 17
Dependent Claim
125 words
A method according to claim 1, in which all of said alkali metal ions are sodium ions, said first and second buffer solutions are phosphate buffers, each with a pH within the range of about 6.3 to about 7.3, said exchanger is a copolymer of methacrylic acid and divinylbenzene on which about 35% to about 45% to whose active sites are occupied by said sodium ions, the concentration of said sodium ions in said first buffer solution is from about 0.03 M to about 0.04 M, the concentration of said sodium ions in said second buffer solution is from about 0.07 M to about 0.09 M and steps (c) and (d) are performed at a temperature of from about 20° C. to about 28° C.
Claim 18
Dependent Claim
36 words
A method according to claim 1, in which step (a) is accomplished by adding said sample to an aqueous detergent solution and incubating the resulting mixture at approximately room temperature for at least about ten minutes.
Claim 19
Dependent Claim
51 words
A method according to claim 1, in which an effective amount of a dihydroxyboryl compound is present in at least one member selected from the group consisting of said hemolysate prior to step (b), said first buffer solution prior to step (c), and said second buffer solution prior to step (d).
Claim 20
Dependent Claim
66 words
A method according to claim 1, in which step (a) is achieved by combining said sample with an aqueous detergent solution, and a dihydroxyboryl compound is added to said detergent solution prior to step (a), to said first buffer solution prior to step (c), or to said second buffer solution prior to step (d), at a concentration of from about 0.01 M to about 1.00 M.
Claim 21
Dependent Claim
47 words
A method according to claim 1, in which step (a) is achieved by combining said sample with an aqueous detergent solution, and a dihydroxyboryl compound is added to said detergent solution prior to step (a) at a concentration of from about 0.1 M to about 1.0 M.
Claim 22
Dependent Claim
65 words
A method according to claim 1, in which step (a) is achieved by combining said sample with an aqueous detergent solution, and a dihydroxyboryl compound is added to said detergent solution prior to step (a) at a concentration of from about 0.1 M to about 1.0 M, and the pH of said hemolysate is maintained at a value ranging from about 4.5 to about 6.5.
Claim 23
Dependent Claim
58 words
A method according to claim 1, in which step (a) is achieved by combining said sample with an aqueous detergent solution, and a dihydroxyboryl compound is added to said detergent solution prior to step (a) at a concentration of from about 0.1 M to about 1.0 M, and the pH of said hemolysate is maintained at about 5.5.
Claim 24
Dependent Claim
34 words
A method according to claim 1, in which a dihydroxyboryl compound is added to said first buffer solution prior to step (c) at a concentration of from about 0.01 M to about 0.10 M.
Claim 25
Dependent Claim
69 words
A method according to claim 1, in which step (a) is achieved by combining said sample with an aqueous detergent solution, and a dihydroxyboryl compound is added to said detergent solution prior to step (a) at a concentration of from about 0.1 M to about 1.0 M, and to said first buffer solution prior to step (c) at a concentration of from about 0.01 M to about 0.10 M.
Claim 26
Dependent Claim
88 words
A method according to claim 1, in which step (a) is achieved by combining said sample with an aqueous detergent solution, and a dihydroxyboryl compound is added to said detergent solution prior to step (a) at a concentration of from about 0.1 M to about 1.0 M, and the pH of said hemolysate is maintained at a value ranging from about 4.5 to about 6.5, and further dihydroxyboryl compound is added to said first buffer solution at a concentration of from about 0.01 M to about 0.10 M.
Claim 27
Dependent Claim
81 words
A method according to claim 1, in which step (a) is achieved by combining said sample with an aqueous detergent solution, and a dihydroxyboryl compound is added to said detergent solution prior to step (b) at a concentration of from about 0.1 M to about 1.0 M, and the pH of said hemolysate is maintained at about 5.5, and further dihydroxyboryl compound is added to said first buffer solution at a concentration of from about 0.01 M to about 0.10 M.
Claim 28
Independent Claim
27 words
A method according to any of claims 18-26 in which said dihydroxyboryl compound is selected from the group consisting of boric acid and lower alkyl boronic acids.
Claim 29
Independent Claim
16 words
A method according to any of claims 18-26 in which said dihydroxyboryl compound is boric acid.
Claim 30
Independent Claim
224 words
A method for determining the level of hemoglobin A 1c in a sample of human blood containing other glycosylated hemoglobins and the Schiff base precursor to hemoglobin A 1c which comprises: (a) lysing the red blood cells contained in said sample to form a hemolysate containing said hemoglobin A 1c , said glycosylated and nonglycosylated hemoglobins, and said Schiff base precursor, (b) impregnating a cation exchanger consisting essentially of a copolymer of methacrylic acid and divinylbenzene about 30% to about 50% of whose active sites are occupied by ions of an alkali metal, the remainder occupied by hydrogen ions, with said hemolysate, (c) passing through said exchanger a first buffer solution with ions of an alkali metal dissolved therein at a concentration of from about 0.02 M to about 0.05 M to dissociate said Schiff base precursor into glucose and hemoglobin A and to preferentially elute said glucose and said other glycosylated hemoglobins over said hemoglobin A, said hemoglobin A 1c and said other nonglycosylated hemoglobins, (d) passing through said exchanger a second buffer solution containing ions of an alkali metal dissolved therein at a concentration of from about 0.06 M to about 0.11 M to preferentially elute said hemoglobin A 1c over said hemoglobin A and said other nonglycosylated hemoglobins, and (e) analyzing the eluate from step (d) for hemoglobin A 1c .
Claim 31
Independent Claim
224 words
A method for determining the level of hemoglobin A 1c in a sample of human blood containing other glycosylated hemoglobins and the Schiff base precursor to hemoglobin A 1c which comprises: (a) lysing the red blood cells contained in said sample to form a hemolysate containing said hemoglobin A 1c , said glycosylated and nonglycosylated hemoglobins, and said Schiff base precursor, (b) impregnating a cation exchanger consisting essentially of a copolymer of methacrylic acid and divinylbenzene about 30% to about 50% of whose active sites are occupied by ions of an alkali metal, the remainder occupied by hydrogen ions, with said hemolysate, (c) passing through said exchanger a first buffer solution with ions of an alkali metal dissolved therein at a concentration of from about 0.02 M to about 0.05 M to dissociate said Schiff base precursor into glucose and hemoglobin A and to preferentially elute said glucose and said other glycosylated hemoglobins over said hemoglobin A, said hemoglobin A 1c and said other nonglycosylated hemoglobins, (d) passing through said exchanger a second buffer solution containing ions of an alkali metal dissolved therein at a concentration of from about 0.06 M to about 0.11 M to preferentially elute said hemoglobin A 1c over said hemoglobin A and said other nonglycosylated hemoglobins, and (e) analyzing the eluate from step (d) for hemoglobin A 1c .
Claim 32
Independent Claim
311 words
A method for determining the level of hemoglobin A 1c in a sample of human blood containing other glycosylated hemoglobins and the Schiff base precursor to hemoglobin A 1c which comprises: (a) lysing the red blood cells contained in said sample to form a hemolysate containing said hemoglobin A 1c , said glycosylated and nonglycosylated hemoglobins, and said Schiff base precursor, by adding said sample to an aqueous solution of a detergent consisting essentially of a polyoxyethylene ether surfactant and further containing from about 0.1 M to about 1.0 M boric acid adjusted to pH of about 5.5, (b) impregnating a cation exchanger with said hemolysate, said cation exchanger consisting essentially of a copolymer of methacrylic acid and divinylbenzene with a particle size within the range of about 100 to about 400 mesh and about 30% to about 50% of whose active sites are occupied by sodium ions, the remainder occupied by hydrogen ions, (c) passing through said exchanger a first phosphate buffer solution with sodium ions dissolved therein at a concentration of from about 0.03 M to about 0.04 M and further containing from about 0.01 M to about 0.03 M boric acid, the pH of said buffer solution ranging from about 6.5 to about 7.0, to dissociate said Schiff base precursor into glucose and hemoglobin A and to preferentially elute said glucose and said other glycosylated hemoglobins over said hemoglobin A, said hemoglobin A 1c and said other nonglycosylated hemoglobins, (d) passing through said exchanger a second phosphate buffer solution with sodium ions dissolved therein at a concentration of from about 0.07 M to about 0.09 M, the pH of said buffer solution ranging from about 6.5 to about 7.0, to preferentially elute said hemoglobin A 1c over said hemoglobin A and said other nonglycosylated hemoglobins, and (e) analyzing the eluate from step (d) for hemoglobin A 1c .
Claim 33
Independent Claim
311 words
A method for determining the level of hemoglobin A 1c in a sample of human blood containing other glycosylated hemoglobins and the Schiff base precursor to hemoglobin A 1c which comprises: (a) lysing the red blood cells contained in said sample to form a hemolysate containing said hemoglobin A 1c , said glycosylated and nonglycosylated hemoglobins, and said Schiff base precursor, by adding said sample to an aqueous solution of a detergent consisting essentially of a polyoxyethylene ether surfactant and further containing from about 0.1 M to about 1.0 M boric acid adjusted to pH of about 5.5, (b) impregnating a cation exchanger with said hemolysate, said cation exchanger consisting essentially of a copolymer of methacrylic acid and divinylbenzene with a particle size within the range of about 100 to about 400 mesh and about 30% to about 50% of whose active sites are occupied by sodium ions, the remainder occupied by hydrogen ions, (c) passing through said exchanger a first phosphate buffer solution with sodium ions dissolved therein at a concentration of from about 0.03 M to about 0.04 M and further containing from about 0.01 M to about 0.03 M boric acid, the pH of said buffer solution ranging from about 6.5 to about 7.0, to dissociate said Schiff base precursor into glucose and hemoglobin A and to preferentially elute said glucose and said other glycosylated hemoglobins over said hemoglobin A, said hemoglobin A 1c and said other nonglycosylated hemoglobins, (d) passing through said exchanger a second phosphate buffer solution with sodium ions dissolved therein at a concentration of from about 0.07 M to about 0.09 M, the pH of said buffer solution ranging from about 6.5 to about 7.0, to preferentially elute said hemoglobin A 1c over said hemoglobin A and said other nonglycosylated hemoglobins, and (e) analyzing the eluate from step (d) for hemoglobin A 1c .
Claim 34
Dependent Claim
97 words
A kit for use in an assay for determining the hemoglobin A 1c content in a sample of human blood without interference from other glycosylated or nonglycosylated hemoglobins or the Schiff base precursor to hemoglobin A 1c , said kit comprising: (a) a weak cation exchanger, (b) a first buffer solution with ions of an alkali metal dissolved therein at a concentration of from about 0.02 M to about 0.05 M, and (c) a second buffer solution with ions of an alkali metal dissolved therein at a concentration of from about 0.06 M to about 0.11 M.
Claim 35
Dependent Claim
97 words
A kit for use in an assay for determining the hemoglobin A 1c content in a sample of human blood without interference from other glycosylated or nonglycosylated hemoglobins or the Schiff base precursor to hemoglobin A 1c , said kit comprising: (a) a weak cation exchanger, (b) a first buffer solution with ions of an alkali metal dissolved therein at a concentration of from about 0.02 M to about 0.05 M, and (c) a second buffer solution with ions of an alkali metal dissolved therein at a concentration of from about 0.06 M to about 0.11 M.
Claim 36
Dependent Claim
16 words
A kit according to claim 31, further comprising a hemolysis reagent comprising an aqueous detergent solution.
Claim 37
Dependent Claim
56 words
A kit according to claim 31, further comprising a hemolysis reagent comprising an aqueous detergent solution containing a dihydroxyboryl compound at a concentration of from about 0.1 M to about 1.0 M, and in which said first buffer solution further contains a dihydroxyboryl compound at a concentration of from about 0.01 M to about 0.10 M.
References Cited (Backward Citations)
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Cited
US4243534A
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