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Kit for detecting glycosylated hemoglobin by enzyme method

A technology of glycosylated hemoglobin and kits, which is applied in the field of biological preparations, can solve the problems affecting the accuracy of detection results and the effect of enzymatic hydrolysis to be improved, and achieve the effects of rapid and large-scale sample detection, low cost, and reduced production costs

Active Publication Date: 2009-02-11
NINGBO MEDICAL SYSTEM BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The proteolytic enzymes used to digest glycosylated hemoglobin were alkaline proteases, including metalloprotease, bromelain, papain, trypsin, proteinase K, subtilisin and aminopeptidase, etc. The enzymatic hydrolysis effect needs to be improved, and the enzymatic hydrolysis reaction In addition to glycosylated hemoglobin, other glycosylated proteins and glycosylated peptides can also be hydrolyzed, which affects the accuracy of the test results

Method used

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  • Kit for detecting glycosylated hemoglobin by enzyme method
  • Kit for detecting glycosylated hemoglobin by enzyme method
  • Kit for detecting glycosylated hemoglobin by enzyme method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Obtaining of highly active fructose-valine oxidase FVO-m-21.

[0066] 1. Using the FVO gene coding sequence of Corynebacterium sp.2-4-1 as a template, error-prone PCR amplification was performed.

[0067] 1. Primer sequence:

[0068] Forward primer: 5'-TTGTTCGGATCCATGTCCTCCACCGCTAC-3'

[0069] Reverse primer: 5'-TTGTTCAAGCTTCTAGGAGAACCGGCCCG-3'

[0070] 2. Error-prone PCR reaction system and reaction conditions:

[0071] PCR reaction system:

[0072] 100μL system contains:

[0073] 10mM Tris-HCl pH8.30, 50mM KCl, 6.5mM MgCl 2 ,

[0074] 0.15mM MnCl 2 , 0.2mM dGTP / dATP, 0.8mM dTTP / dCTP,

[0075] 2.2μg SSB Protein, 0.5μM forward / reverse primer, 10ng template DNA

[0076] 2.5 units of Taq DNA polymerase.

[0077] PCR reaction conditions:

[0078] 94°C 5min; 94°C 30sec, 55°C 30sec, 72°C 2min, 35 cycles; 72°C 10min; 4°C forever.

[0079] 3. Take 3 μL of the error-prone PCR product and electrophoresis on 1% agarose gel. It can be seen that there is a product band ...

Embodiment 2

[0091] Using distilled water as the solvent, it was prepared according to the conventional method for preparing reagents, as shown in Table 1.

Embodiment 3

[0092] Example 3 Determination of glycosylated hemoglobin standard solution using the kit of the present invention.

[0093] Whole blood was collected from patients (16 patients), and the erythrocytes were collected by natural sedimentation. 20 μL of the sedimented erythrocyte fraction was collected, and 500 μL of the above-mentioned hemolysis buffer was added thereto. 30 μL of the obtained hemolyzed sample was mixed with 40 μL of reagent R1a and 40 μL of reagent R1b, and incubated at 37° C. for 3 minutes. Add 40 μL neutralization buffer, mix well, and read absorbance A1. Add 20 μL of reagent 2 to the reaction system, incubate at 37° C. for 2 minutes, and read the absorbance A2. ΔA=A2-A1. . The absorbance values ​​at 751nm and 571nm can be selected during measurement. Here, the Hb concentration can be obtained from the absorbance at a wavelength of 751 nm, and the HbAlc concentration can be obtained from the absorbance at a wavelength of 571 nm.

[0094] Calculation formu...

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Abstract

The invention relates to a promega for testing glycolated hemoglobin by using an enzymatic method, which contains the ingredients of hemolysis buffer: 10-1000 mmol / L of N-chclohexyl-2 ethyl amine sulfoacid, 10-1000mmol / L of morpholino propane sulfoacid, and 1-100g / L of polyethylene glycol oxide dodecyl ether; reagent R1a: 10-1000kU / L of pepsin of pig, 0.1-10mmol / L of 2-(iodophenyl)-3-(2, 4-dinitro benzene)-5-(2, 4-sulfophenyl)-2H tetrazole sodium salt, and 0.0001-0.1mol / L of HCl; reagent R1b: 0.1-10mmol / L of morpholino ethane sulfonic acid, and 0.5-50mmol / L of CaCl2; neutralization buffer: 0.0001-0.1mol / L of NaOH reagent, 2:1-1000kU / L of fructose valine oxidase, 30-600kU / L of peroxydase, 0.01-0.5mmol / L of N-(carboxymethyl aminocarbonyl)-4,4 quadri (dimethyl amine)-diphenylamine sodium salt, and 10-1000mmol / L of Tris-HCl. The promega has the zymohydrolysis effect which is better than alkalescence prolease, little dosage, low cost and accurate measuring result.

Description

technical field [0001] The invention relates to the technical field of biological preparations, in particular to a kit for enzymatically detecting glycosylated hemoglobin. Background technique [0002] Glycated hemoglobin (glycated Hb) concentration in blood has been used as an important indicator for the diagnosis and treatment of diabetes in recent years. At present, the determination of glycosylated hemoglobin can be carried out by ion exchange chromatography, high performance liquid chromatography, affinity chromatography, radioimmunoassay, enzyme immunoassay and latex immunoagglutination method. However, these methods either require special instruments, are expensive, or take a long time to detect and have poor measurement accuracy. The recently popular enzymatic detection uses oxidation-reduction reaction and does not require special measuring instruments. It is easy to operate, high in precision and short in time, so it is widely used in biochemical analysis and clin...

Claims

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Application Information

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IPC IPC(8): C12Q1/28C12Q1/26G01N21/78
Inventor 邹炳德姜云飞
Owner NINGBO MEDICAL SYSTEM BIOTECHNOLOGY CO LTD
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