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Method for measuring glycated hemoglobin

A technology for glycosylated hemoglobin and hemoglobin, which is applied in biochemical equipment and methods, microbial determination/inspection, measurement devices, etc., and can solve problems such as affecting the measured value.

Active Publication Date: 2016-03-23
HITACHI CHEM DIAGNOSTICS SYST CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] With regard to the method involving the action of protease on glycosylated hemoglobin, the action of glycosylated peptide oxidase on the produced glycosylated peptide, and the measurement of produced hydrogen peroxide, in the case of simultaneous measurement using an automatic analyzer or the like in addition to the measurement of glycosylated hemoglobin In this case, the protease in the glycosylated hemoglobin assay reagent may act on other reagents and may affect the measured value

Method used

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  • Method for measuring glycated hemoglobin
  • Method for measuring glycated hemoglobin
  • Method for measuring glycated hemoglobin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0105] [Example 1] Construction of a protein having glycated hemoglobin oxidase activity by introducing site-specific amino acid substitutions

[0106] Inoculate the Escherichia coli XL1-BlueMRF' strain expressing glycosylated peptide oxidase FPOX-9 deposited under the accession number FERMBP-11026 into 3 mL of LB medium containing 50 mg / L ampicillin, and culture it overnight at 37°C with shaking . The culture solution was centrifuged at 8000rpm for 2 minutes to collect the bacterial cells. Use "WizardPlusSVMiniprepsDNAPurification" manufactured by Promega Company to extract the expression plasmid pTrc-FPOX-9 comprising the DNA consisting of the nucleotide sequence represented by SEQIDNO:36 from the obtained thalline, said nucleotide sequence expresses the expression plasmid pTrc-FPOX-9 having the expression sequence represented by SEQIDNO: 2 represents the amino acid sequence of the glycated peptide oxidase FPOX-9.

[0107] Using the method described in International Public...

Embodiment 2

[0137] [Example 2] Correlation between the method for measuring glycated hemoglobin of the present invention and the method for measuring glycated hemoglobin using the HPLC method (KO500 method) and the hemoglobin-SLS method

[0138] Each of the mutants FPOX-19, FPOX-20, FPOX-32, and FPOX-42 obtained in Example 1 was used as an enzyme, and its HbA1c concentration had been determined by the HPLC method (KO500 method) and the hemoglobin-SLS method. Hemolyzed samples derived from human blood cells determined by both were used as test samples, and changes in absorbance for each sample were determined using the following reagents and measurement procedures. In the measurement of the hemoglobin concentration by the hemoglobin-SLS method, the hemoglobin B-test Wako (manufactured by Wako Pure Chemical Industries, Ltd.) was used.

[0139] (Reagents for activity measurement)

[0140] Solution A: 0.1 mol / L MOPS buffer (pH6.8)

[0141] Solution B: 24 mmol / L solution of DA-67 in DMF

[...

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PUM

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Abstract

Provided are a method for measuring glycated hemoglobin in a sample, characterized in that glycated hemoglobin in a sample is directly oxidized, and a substance produced thereby, or a substance consumed thereby, is measured; and a method for measuring glycated hemoglobin in a sample, characterized in that, using an enzyme, glycated hemoglobin in a sample is directly oxidized, and a substance produced thereby, or a substance consumed thereby, is measured. This method for measuring glycated hemoglobin is useful in diagnosing lifestyle-related diseases such as diabetes.

Description

technical field [0001] The present invention relates to a method for determining glycated hemoglobin in a sample. Background technique [0002] Glycated proteins are contained in biological samples such as body fluids including blood in living bodies, hair, and the like. The concentration of glycated protein present in blood depends on the concentration of sugar dissolved in serum such as glucose, and recently, in the field of clinical diagnosis, hemoglobin A1c (hereinafter, HbA1c; non-patented The determination of the concentration of Document 1) is being used in the diagnosis and monitoring of diabetes. As a method for measuring this HbA1c, an instrumental analysis method using high performance liquid chromatography (HPLC) (Non-Patent Document 2), an immunoassay method using an antigen-antibody reaction (for example, Non-Patent Document 3), etc. are known. Yes, but in recent years, enzyme assays have been developed, and for example, methods using protease and glycosylate...

Claims

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Application Information

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IPC IPC(8): C12Q1/26
CPCC12Q1/26G01N33/723G01N33/725
Inventor 小川德之楳原芙美木村豪秀村田幸作桥本涉
Owner HITACHI CHEM DIAGNOSTICS SYST CO LTD
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