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Carbendazim monoclonal antibody, preparation method and application thereof

A technology of monoclonal antibody and carbendazim, applied in biochemical equipment and methods, chemical equipment and methods, methods based on microorganisms, etc., can solve the problems of failing to meet EU standards

Inactive Publication Date: 2013-09-18
PEOPLES REPUBLIC OF CHINA BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although carbendazim is a low-toxicity pesticide, it is widely used. The dehydrated mushrooms I exported to Germany have been found to have "carbendazim" residues many times, at about 1~10mg / kg, which does not meet the EU standard

Method used

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  • Carbendazim monoclonal antibody, preparation method and application thereof
  • Carbendazim monoclonal antibody, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Obtaining the Hybridoma Cell Line MC-3 CGMCC No. 4575 and Identification of the Anti-Carbendazim Monoclonal Antibody It Produced

[0035] 1. Obtaining the hybridoma cell line MC-3 CGMCC No. 4575

[0036] 1. Amino modification of carbendazim

[0037] The carbendazim was amino-modified by the ethylene glycol method. The specific method is as follows: Dissolve 5 mg carbendazim in 0.65 mL dimethylformamide, add 350 μL 0.05 mmol / L sodium periodate within 2 minutes, react in the dark for 30 minutes at room temperature, add 130 μL 1mol / L ethylene di Alcohol, shake well for 5 minutes to get amino-modified carbendazim ML.

[0038] 2. Preparation of carbendazim complete antigen ML-KLH

[0039] Take 1130 μL of amino-modified carbendazim ML, add 650 μL of 15 mg / mL keyhole limpet hemocyanin, that is, KLH (preparation buffer is 0.1 mol / L sodium bicarbonate at pH 8.2), react at room temperature for 1 hour, add total 0.7 times the volume of 0.4 mol / L sodium borohydride ...

Embodiment 2

[0073] Example 2 Establishing an indirect competitive ELISA method with the monoclonal antibody secreted by the hybridoma cell line MC-3

[0074] 1. Preparation of carbendazim standard substance: carbendazim standard substance, using high-purity water to prepare standard solutions with the following concentrations (in ppm): 10, 5, 1, 0.5, 0.1, 0.

[0075] 2. Antibody solution preparation: the monoclonal antibody prepared in Example 1 was labeled with horseradish peroxidase, and then the horseradish peroxidase-labeled monoclonal antibody was diluted with phosphate buffer to a working concentration of 1:20000, Then add 1% bovine serum albumin (BSA).

[0076] 3. Preparation of washing solution (10×PBST): 0.1mol / L pH7.5 phosphate buffer containing 0.2% (volume percentage) Tween-20 and 80g / L sodium chloride. When in use, dilute the solution 10 times with pure water before use.

[0077] 4. Substrate solution preparation B solution: use 0.1mol / L sodium acetate-citric acid buffer...

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Abstract

The invention discloses a carbendazim monoclonal antibody, a preparation method and an application thereof. The carbendazim monoclonal antibody is prepared by a mouse hybridoma cell strain MC-3 with a preservation number of CGMCC N0.4575 and the preparation method is as follows: (1) introducing ninth amino acid residue of carbendazim into one amino group to obtain the carbendazim modified by the amino and coupling the carbendazim modified by the amino with a carrier protein to obtain a complete antigen A; (2) immunizing a mouse by the complete antigen A obtained in step (1), taking splenocyte of the immune mice to fuse with a mouse myeloma cell to screen out a positive hybridoma cell strain; extensively culturing the positive hybridoma cell strain and injecting the positive cell strain into a homologous mouse abdominal cavity to induce to generate ascitic fluid and to obtain more carbendazim monoclonal antibodies. The Carbendazim monoclonal antibody can be used for detecting the carbendazim.

Description

technical field [0001] The invention relates to a carbendazim monoclonal antibody and its preparation method and application. Background technique [0002] Carbendazim (carbendazim, CAS: 10605-21-7) is a broad-spectrum benzimidazole fungicide, which has control effects on various crop diseases caused by fungi (such as Deuteromycetes, Polyascomycetes) . Carbendazim is a crystalline powder with a melting point of 302~307°C (decomposition), a vapor pressure of 0.09mPa (20°C), a density of 1.45 (20°C), and a solubility in water of 29mg / L (pH4, 24°C). The solubility in dimethylformamide is 5g / L (24°C), slightly soluble in organic solvents, stable for at least two years below 50°C, slowly decomposes in alkaline solution, and decomposes faster as the pH value increases. Stable in acid. Its chemical structural formula is as shown in formula I: [0003] (formula Ⅰ) [0004] Although carbendazim is a low-toxicity pesticide, it is widely used. The dehydrated mushrooms exported t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/44C12N5/20G01N33/577C12R1/91
Inventor 王金花曾静李小林卢晓宇韩深张蓉刘韦华魏海燕
Owner PEOPLES REPUBLIC OF CHINA BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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