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) an oxidizing agent which is a tetrazolium salt, and
3) a protease which digests glycated hemoglobin into glycated peptides or glycated amino acids, wherein the protease is a neutral protease of Aspergillus or Bacillus origin and generates glycated glycine, glycated valine, glycated lysine residue, or a glycated peptide comprising glycated glycine, glycated valine or glycated lysine residue;
wherein the fructosyl amino acid oxidase comprises the amino acid sequence set forth in SEQ ID NO;
1;
b) contacting H2O2 generated in step a) with a color forming substance in the presence of a peroxidase to generate a measurable signal; and
c) determining percentage of glycated hemoglobin A1c by correlating the measured value in step b) to a percentage of glycated hemoglobin A1c in a calibration curve without measuring the total hemoglobin in the blood sample separately.
2 Assignments
0 Petitions
Accused Products
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, and peroxidase. The invention provides kits for performing the methods of the invention.
-
Citations
21 Claims
-
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) an oxidizing agent which is a tetrazolium salt, and
3) a protease which digests glycated hemoglobin into glycated peptides or glycated amino acids, wherein the protease is a neutral protease of Aspergillus or Bacillus origin and generates glycated glycine, glycated valine, glycated lysine residue, or a glycated peptide comprising glycated glycine, glycated valine or glycated lysine residue;
wherein the fructosyl amino acid oxidase comprises the amino acid sequence set forth in SEQ ID NO;
1;b) contacting H2O2 generated in step a) with a color forming substance in the presence of a peroxidase to generate a measurable signal; and c) determining percentage of glycated hemoglobin A1c by correlating the measured value in step b) to a percentage of glycated hemoglobin A1c in 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)
-
Specification