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High-concentration glucose removal method and kit for determining serum 1,5 anhydro-D-glucitol based on pyranose oxidase method

An anhydroglucitol and glucose technology, which is applied in biochemical equipment and methods, and the determination/inspection of microorganisms, etc., can solve problems such as the inability of glucose elimination methods to be applied.

Active Publication Date: 2012-03-21
浙江夸克生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since pyranose oxidase does not produce any oxidation on 1,5-anhydroglucitol-6 phosphate, this glucose elimination method cannot be applied to the determination of 1,5-anhydroglucitol by pyranose oxidase method

Method used

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  • High-concentration glucose removal method and kit for determining serum 1,5 anhydro-D-glucitol based on pyranose oxidase method
  • High-concentration glucose removal method and kit for determining serum 1,5 anhydro-D-glucitol based on pyranose oxidase method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Precisely formulated glucose scavenging reagent (hereinafter referred to as "Clearing Reagent 1") with the following composition: pH6.50 MES buffer is 20mmol / L, GK is 1KU / L, G6PD is 5KU / L, NADP is 5mmol / L, and PK is 0.5KU / L, PEP is 1mmol / L, ATP is 0.2mmol / L. Perform a glucose clearance test to determine the effectiveness of this method. Add (1) deionized water, (2) test solution containing 304μmol / L 1,5-AG, (3) test solution containing 60mmol / L glucose, and (4) test solution containing 304μmol / L glucose into 4 test tubes. L 1,5-AG and 60mmol / L glucose test solution each 0.10ml, and then respectively inject 1.50ml of the above glucose removal reagent, after heating at 37℃ for 5 minutes, each tube containing 4-aminoantipyrine ( 4-AAP) 2mmol / L, PROD50KU / L, peroxidase (HRP) 6KU / L, 3-hydroxy-2,4,6-tribromobenzoic acid (TBHBA) 2.0mmol / L color developer each 0.75 Milliliter, continue heating at 37°C for 5 minutes, and measure the absorbance at 510 nm to obtain the results in ...

Embodiment 2

[0023] Precisely formulated glucose scavenging reagent (hereinafter referred to as "Clearing Reagent 2") with the following composition: pH6.50 MES buffer is 1000mmol / L, GK is 25KU / L, G6PD is 100KU / L, NADP is 30mmol / L, and PK is 10KU / L, PEP is 2mmol / L, ATP is 1mmol / L. The glucose clearance test was performed as in Example 1 to determine the effectiveness of this method. Add (1) deionized water, (2) test solution containing 304μmol / L 1,5-AG, (3) test solution containing 60mmol / L glucose, and (4) test solution containing 304μmol / L glucose into 4 test tubes. 0.10ml each of 1,5-AG and 60mmol / L glucose to be tested, then add 1.50ml of glucose scavenging reagent, after heating at 37℃ for 4 minutes, add 0.75ml each of the color reagent consistent with Example 1 , Continue heating at 37°C for 5 minutes, and measure the absorbance at 510 nm to obtain the results in Table 3.

[0024] table 3

[0025] Test solution Absorbance values (1) Deionized water 0.033 (2) 304μmol / L1,5-AG ...

Embodiment 3

[0028] Precisely formulated glucose scavenging reagent (hereinafter referred to as "clearing reagent 3") with the following composition: pH6.50 MES buffer is 2000mmol / L, GK is 50KU / L, G6PD is 200KU / L, NADP is 60mmol / L, and PK is 20KU / L, PEP is 3mmol / L, ATP is 2mmol / L. The glucose clearance test was performed as in Example 1 to determine the effectiveness of this method. Add (1) deionized water, (2) test solution containing 304μmol / L 1,5-AG, (3) test solution containing 60mmol / L glucose, and (4) test solution containing 304μmol / L glucose into 4 test tubes. 0.10ml each of 1,5-AG and 60mmol / L glucose to be tested, then add 1.50ml of glucose scavenging reagent, after heating at 37℃ for 3 minutes, add 0.75ml each of the color reagent consistent with Example 1 , Continue heating at 37°C for 5 minutes, and measure the absorbance at 510 nm to obtain the results in Table 4.

[0029] Table 4

[0030] Test solution Absorbance values (1) Deionized water 0.037 (2) 304μmol / L1,5-AG...

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Abstract

The invention discloses a high-concentration glucose removal method for determining serum 1,5 anhydro-D-glucitol based on a pyranose oxidase method, which comprises the following steps: preparing a 2-morpholinoethanesulfonic acid (MES) buffer solution having a pH value of 6.0-9.0, wherein the MES buffer solution contains a certain amount of glucokinase (GK), glucose-6-phosphate dehydrogenase (G6PD), nicotinamide adenine dinucleotide phosphate (NADP), pyruvate kinase (PK), phosphoenolpyruvate (PEP) and adenosine triphosphate (ATP); and adding into a serum sample containing glucose (Glu), and reacting at 20-50 DEG C for 3-5 minutes, wherein the GK converts the Glu into glucose-6-phosphoric acid (G6P) in the presence of the ATP, and adenosine diphosphate (ADP) is generated at the same time; the G6P is further converted into glucose-6-phosphate lactone (G6PL) by the G6PD in the presence of the NADP+; and the ADP generated in the reaction is converted into ATP again under the action of thePK in the presence of the PEP. The glucose removal agent prepared by the method can be stably stored at 2-8 DEG C for 14 months, and can be used for a clinical biochemical autoanalyzer extensively used at present.

Description

Technical field [0001] The invention relates to a high-concentration glucose removal method and a kit suitable for the pyranose oxidase method to determine serum 1,5-anhydroglucitol. Background technique [0002] 1,5-Anhydroglucitol (1,5-AG), also known as 2-deoxyglucose and 1,5-sorbitan. Clinical data shows that the monitoring of serum 1,5-AG can accurately reflect the degree of diabetes control in a relatively short period of time, and it is a good indicator in diabetes monitoring. [0003] Since the diagnostic significance of 1,5-anhydroglucitol for diabetes has been recognized in the clinic, there have been anion exchange chromatography, high performance liquid chromatography (HPLC), liquid chromatography mass spectrometry (LC-MS), pyran Many methods such as sugar oxidase analysis method are applied to the determination of serum 1,5-AG. The first three methods require derivatization of 1,5-AG and complex sample pre-processing, as well as special and expensive instruments, whi...

Claims

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Application Information

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IPC IPC(8): C12Q1/54C12Q1/48C12Q1/32
Inventor 商纯尔陈青松
Owner 浙江夸克生物科技有限公司
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