Method and kit for chromatographic separation of hemoglobin A.sub.1c
First Claim
1. A method for the separation of hemoglobin A1c from other glycosylated and nonglycosylated hemoglobins and the Schiff base precursor to hemoglobin A1c 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 A1c, 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 A1c 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 A1c over said hemoglobin A and said other nonglycosylated hemoglobins, and(e) recovering the eluate from step (d).
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Abstract
An ion exchange method and kit for the separation of hemoglobin A1c 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 A1c 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.
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Citations
33 Claims
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1. A method for the separation of hemoglobin A1c from other glycosylated and nonglycosylated hemoglobins and the Schiff base precursor to hemoglobin A1c in a sample of human blood which comprises:
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(a) lysing the red blood cells contained in said sample to form a hemolysate containing said hemoglobin A1c, 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 A1c 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 A1c over said hemoglobin A and said other nonglycosylated hemoglobins, and (e) recovering the eluate from step (d). - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28)
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29. A method for determining the level of hemoglobin A1c in a sample of human blood containing other glycosylated hemoglobins and the Schiff base precursor to hemoglobin A1c which comprises:
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(a) lysing the red blood cells contained in said sample to form a hemolysate containing said hemoglobin A1c, 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 A1c 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 A1c over said hemoglobin A and said other nonglycosylated hemoglobins, and (e) analyzing the eluate from step (d) for hemoglobin A1c.
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30. A method for determining the level of hemoglobin A1c in a sample of human blood containing other glycosylated hemoglobins and the Schiff base precursor to hemoglobin A1c which comprises:
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(a) lysing the red blood cells contained in said sample to form a hemolysate containing said hemoglobin A1c, 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 A1c 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 A1c over said hemoglobin A and said other nonglycosylated hemoglobins, and (e) analyzing the eluate from step (d) for hemoglobin A1c.
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31. A kit for use in an assay for determining the hemoglobin A1c content in a sample of human blood without interference from other glycosylated or nonglycosylated hemoglobins or the Schiff base precursor to hemoglobin A1c, said kit comprising:
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(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. - View Dependent Claims (32, 33)
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Specification