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Detection kit using glycosylated hemoglobin enzyme method

A technology for glycosylated hemoglobin and detection kits, which is applied in biochemical equipment and methods, material analysis by observing the effect on chemical indicators, and microbial determination/inspection, etc., and can solve problems such as high demand for enzymes and long time. , to achieve the effect of less enzyme amount, lower production cost and shorter reaction time

Active Publication Date: 2009-02-04
NINGBO MEDICAL SYSTEM BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, using the existing fructosyl amino acid oxidase to catalyze this reaction, the time is still a bit long, and the demand for the amount of enzyme is relatively high

Method used

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  • Detection kit using glycosylated hemoglobin enzyme method
  • Detection kit using glycosylated hemoglobin enzyme method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Obtaining of highly active fructose-valine oxidase FVO-m-21.

[0060] 1. Using the FVO gene coding sequence of Corynebacterium sp.2-4-1 as a template, error-prone PCR amplification was performed.

[0061] 1. Primer sequence:

[0062] Forward primer: 5'-TTGTTCGGATCCATGTCCTCCACCGCTAC-3'

[0063] Reverse primer: 5'-TTGTTCAAGCTTCTAGGAGAACCGGCCCG-3'

[0064] 2. Error-prone PCR reaction system and reaction conditions:

[0065] PCR reaction system:

[0066] 100μL system contains:

[0067] 10mM Tris-HCl pH 8.30, 50mM KCl, 6.5mM MgCl 2 ,

[0068] 0.15mM MnCl 2 , 0.2mM dGTP / dATP, 0.8mM dTTP / dCTP,

[0069] 2.2μg SSB Protein, 0.5μM forward / reverse primer, 10ng template DNA

[0070] 2.5 units of Taq DNA polymerase.

[0071] PCR reaction conditions:

[0072] 94°C for 5min; 94°C for 30sec, 55°C for 30sec, 72°C for 2min, 35 cycles; 72°C for 10min; 4°C forever.

[0073] 3. Take 3 μL of the error-prone PCR product and electrophoresis on 1% agarose gel. It can be seen that the...

Embodiment 2

[0085] Using distilled water as a solvent, the formulations of the glycosylated hemoglobin enzymatic detection kit were prepared according to the conventional reagent preparation method, as shown in Table 1.

[0086] Table 1 The formulations of the enzymatic detection kit for glycosylated hemoglobin

[0087]

Embodiment 3

[0089] Whole blood was collected from patients (the number of patients was 35), and the erythrocytes were recovered by natural sedimentation. 20 μL of sedimented erythrocytes were taken, and 500 μL of the hemolysis buffer of the kit of the present invention was added thereto. Mix 30 μL of the obtained hemolysis sample with 40 μL of reagent R1a and 40 μL of reagent R1b, incubate at 37° C. for 3 minutes, 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. Among them, the Hb concentration can be obtained from the absorbance at a wavelength of 751 nm, and the HbA1c concentration can be obtained from the absorbance at a wavelength of 571 nm.

[0090] Calculation formula

[0091]

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Abstract

The invention relates to an enzymatic test kit for glycosylated hemoglobin, which is composed of hemolysis buffer solution, reagent R1a, reagent R1b and reagent 2; wherein, the hemolysis buffer solution is 10 to 1000mmol / L N- cycloethyl-2-aminoethanesulfonic acid, 10 to 1000mmol / L MOPS and 1 to 100g / L polyethylene glycol oxide dodecyl ether; the reagent R1a is 100 to 10000 kU / L metalloproteinase, 0.1 to 10mmol / L 2-(iodophenyl)-3-(2, 4- dinitrobenzene)-5-(2, 4-disulfophenyl)-2H tetrazole sodium salt; the reagent R1b is 0.1 to 10mmol / L MES and 0.5 to 50mmol / L CaCl2; the reagent 2 is 1 to 100kU / L fructose valine oxidase with high activity, 30 to 600kU / L POD, 0.01 to 0.5mmol / L DA-64 and 10 to 1000mmol / L Tris-HCl. The test kit coincides with the characteristics of rapidness, large batch size and economy which are required by the glycosylated hemoglobin clinical tests.

Description

technical field [0001] The invention relates to the technical field of biological preparations, in particular to an enzymatic detection kit for glycosylated hemoglobin. Background technique [0002] The World Health Organization estimates that 180 million people worldwide have diabetes. Glycated hemoglobin (glycosylated Hb) is a very important inspection index for diabetic patients. HbA1c is glycated Hb in which the α amino group at the N-terminal of the β chain of Hb is glycosylated, and among glycated Hb, it is used as a particularly important index for the diagnosis of diabetes and the like. At present, HbA1c determination methods mainly include ion exchange chromatography, high performance liquid chromatography, affinity chromatography, radioimmunoassay, enzyme immunoassay and latex immunoagglutination method, etc. The above methods may require professional expensive equipment, Or the detection accuracy, anti-interference, speed, etc. are not ideal, which limits its la...

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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