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Anti-dextran monoclonal antibody D24 and application thereof in determination of enzyme activity of dextranase in sugar product

A technology of dextranase activity and monoclonal antibody, which is applied in the field of sugar production, can solve the problems of no significant change in content and the estimation of dextranase activity, and achieves the effect of low detection limit and simple operation.

Active Publication Date: 2021-08-03
GUANGDONG ACAD OF SCI INST OF BIOENGINEERING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, after enzymolysis of dextran by dextranase, the enzymatic hydrolysis product is still dextran, and its content has not changed significantly (because the enzymatic hydrolysis process only cuts the α-1,6-glucosidic bond, the sugar chain is divided into two, but the total mass is basically unchanged), so it is impossible to directly use the public method to calculate the dextranase activity by measuring the change of dextran content before and after enzymolysis

Method used

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  • Anti-dextran monoclonal antibody D24 and application thereof in determination of enzyme activity of dextranase in sugar product
  • Anti-dextran monoclonal antibody D24 and application thereof in determination of enzyme activity of dextranase in sugar product
  • Anti-dextran monoclonal antibody D24 and application thereof in determination of enzyme activity of dextranase in sugar product

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1: Preparation of anti-dextran monoclonal antibody D24

[0043] BALB / c mice aged 6-8 weeks (purchased from the Experimental Animal Center of Southern Medical University) with strong physique and good growth conditions were inoculated with liquid paraffin intraperitoneally, 0.5 mL per mouse, and two weeks later, cultured with serum-free Dextran hybridoma cell line D24 (preservation number: CGMCC No.21002) diluted with base (purchased from ThermoFisher scientific), 5×10 per mouse 5Each / 0.2mL. After an interval of 8 days, the production of ascites in the mice was observed every day. When the ascites was as large as possible and the mice were on the verge of death, the mice were killed, and the ascites was sucked out under aseptic conditions. Stand at room temperature for 30 minutes, centrifuge at 10,000 rpm for 10 minutes, collect the supernatant, and freeze it at -70°C for later use.

[0044] The purification scheme was octanoic acid-ammonium sulfate precipit...

Embodiment 2

[0045] Example 2: Sensitivity test of anti-dextran monoclonal antibody D24 and α-glucan monoclonal antibody to dextran molecular weight

[0046] 100 mg / L dextran T-2000 (molecular weight 2,000,000), dextran T-500 (molecular weight 500,000), dextran T- 70 (molecular weight 70000), dextran T-40 (molecular weight 40000), dextran T-20 (molecular weight 20000), dextran T-10 (molecular weight 10000) solution.

[0047] α-glucan monoclonal antibody is prepared by the method described in Chinese patent application 201010606983.5 α-glucan antigen and specific monoclonal antibody preparation method, specifically, α-glucan monoclonal antibody is obtained from the injection deposit number It is purified from mouse ascites fluid of the hybridoma cell line D9 of CCTCC C2010107 (the patent application 201310625649.8 published by our laboratory is also described in the kit for the quantitative detection of α-glucan by immunoturbidimetric monoclonal antibody).

[0048] 0.3mg / mL anti-dextran mo...

Embodiment 3

[0053] Embodiment 3: A kind of method for measuring dextranase enzyme activity in white granulated sugar

[0054] (1) Drawing of standard curve

[0055] Prepare standard series solutions with dextranase activity of 0, 0.000002U / mL, 0.000004U / mL, 0.000006U / mL, 0.000008U / mL, 0.00001U / mL respectively.

[0056] Prepare 200 mg / L dextran T-2000 solution with pH 5.5, 0.1 mol / L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution as solvent. Take six 5mL EP tubes, add 1mL dextran T-2000 solution and 1mL of the above-mentioned standard series solution respectively, react at 65°C for 30 minutes, and place in a boiling water bath for 5 minutes; the reaction solution 1 is obtained, cooled to room temperature, and set aside.

[0057] Prepare a 0.3 mg / mL solution of anti-dextran monoclonal antibody D24 that is sensitive to changes in the molecular weight of dextran with pH 7.2 and 0.1mol / L PBS buffer. Take 1.5mL of anti-dextran monoclonal antibody D24 solution and measu...

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Abstract

The invention discloses an anti-dextran monoclonal antibody D24 and application of the anti-dextran monoclonal antibody D24 in determination of dextranase activity in a sugar product, and belongs to the technical field of sugar refining. The anti-dextran monoclonal antibody D24 disclosed by the invention is generated by a dextran hybridoma cell strain D24 with the preservation number of CGMCC No. 21002. The anti-dextran monoclonal antibody D24 can be used for determining the enzyme activity of dextranase in a sugar product. The invention provides a method for determining the enzyme activity of dextranase in a sugar product. The method is simple to operate and low in detection limit.

Description

technical field [0001] The invention belongs to the technical field of sugar production, and in particular relates to an anti-dextran monoclonal antibody D24 and its application in measuring the activity of dextranase in sugar products. Background technique [0002] Dextranase (EC 3.2.1.11) is an enzyme capable of hydrolyzing dextran α-1,6 glycosidic linkages. Dextran is a kind of poly D-glucose, the glucose residues of which are linked by α-1,6-glycosidic bonds into the main chain, and the branch chains are composed of glucose residues or glucose chains (simple or complex) with α-1,2-, α-1,3- or α-1,4-glycosidic bonds are connected to the main chain. Dextran is an impurity that generally exists in the sugar production process, and it has many adverse effects on the sugar production process. Using dextranase can hydrolyze dextran and eliminate its adverse effects on the sugar production process. The material temperature in some sections of the sugar making process is high...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/44G01N33/573G01N33/577
CPCC07K16/44G01N33/573G01N33/577
Inventor 常国炜梁达奉柳颖刘桂云
Owner GUANGDONG ACAD OF SCI INST OF BIOENGINEERING
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