Methods for assaying percentage of glycated hemoglobin
First Claim
1. A method for directly assaying percentage of glycated hemoglobin A1c in a blood sample without measuring the total hemoglobin in the blood sample in a separate process, said method comprising:
- a) contacting protein fragments containing glycated peptides or glycated amino acids with a fructosyl amino acid oxidase to generate hydrogen peroxide (H2O2), wherein the protein fragments are generated by contacting the blood sample with
1) a lysing buffer which releases hemoglobin from red blood cells in the blood sample;
2) a first oxidizing agent which selectively oxidizes low molecular weight reducing substances;
3) a second oxidizing agent which selectively oxidizes high molecular weight reducing substances, and
4) a protease which digests glycated hemoglobin into glycated peptides or glycated amino acids;
b) measuring the amount of H2O2 generated in step a); and
c) determining percentage of glycated hemoglobin A1c in the sample by correlating the measured value of H2O2 in step b) to a percentage of glycated hemoglobin A1c using a calibration curve without measuring the total hemoglobin in the blood sample separately.
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Abstract
The invention provides enzymatic methods for direct determination of percentage of glycated hemoglobin in blood samples without the need of a separated measurement of total hemoglobin content in blood samples. The methods utilizes one or two different types of oxidizing agents which selectively oxidize low-molecular weight reducing substances and high-molecular weight (mainly hemoglobin) reducing substances in blood samples, coupled with enzymatic reactions catalyzed by proteases, fructosyl amino acid oxidase. The amount of hydrogen peroxide generated in the reaction is measured for determination of percentage of glycated hemoglobin in blood samples. The invention provides kits for performing the methods of the invention.
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Citations
32 Claims
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1. A method for directly assaying percentage of glycated hemoglobin A1c in a blood sample without measuring the total hemoglobin in the blood sample in a separate process, said method comprising:
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a) contacting protein fragments containing glycated peptides or glycated amino acids with a fructosyl amino acid oxidase to generate hydrogen peroxide (H2O2), wherein the protein fragments are generated by contacting the blood sample with
1) a lysing buffer which releases hemoglobin from red blood cells in the blood sample;
2) a first oxidizing agent which selectively oxidizes low molecular weight reducing substances;
3) a second oxidizing agent which selectively oxidizes high molecular weight reducing substances, and
4) a protease which digests glycated hemoglobin into glycated peptides or glycated amino acids;b) measuring the amount of H2O2 generated in step a); and c) determining percentage of glycated hemoglobin A1c in the sample by correlating the measured value of H2O2 in step b) to a percentage of glycated hemoglobin A1c using a calibration curve without measuring the total hemoglobin in the blood sample separately. - 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, 29, 30, 31, 32)
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Specification