Method for detecting carbendazim content in edible fungi by using carbendazim-specific antibody

A technology for antibody detection and carbendazim, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems that have not been reported in the literature, achieve high yield, simple sample pretreatment steps, and good application prospects

Inactive Publication Date: 2016-11-16
TIANJIN NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no literature report on th...

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  • Method for detecting carbendazim content in edible fungi by using carbendazim-specific antibody
  • Method for detecting carbendazim content in edible fungi by using carbendazim-specific antibody
  • Method for detecting carbendazim content in edible fungi by using carbendazim-specific antibody

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Embodiment 1

[0031] (1) Synthesis of carbendazim hapten

[0032] Stir and dissolve 0.5g of 2-aminobenzimidazole in 100mL of acetonitrile in a 250mL beaker, then add 0.564g of succinic anhydride, stir and react at 40°C for 4 hours, a large amount of white precipitate precipitates, filter the reaction solution to obtain a filter residue, and filter the residue Rinse twice with acetonitrile at 40°C to remove unreacted raw materials remaining in the filter residue to obtain a crude product. The crude product was further purified by column chromatography (mobile phase was ethyl acetate:n-hexane=1:3, v / v), and identified by thin layer chromatography and mass spectrometry, the obtained product was 2-succinylbenzimidazole (white powder), its structural analysis see figure 2 .

[0033] (2) Preparation of carbendazim immunogen

[0034]In a 4 mL brown vial, stir and dissolve 18.6 mg hapten in 0.9 mL N,N-dimethylformamide (DMF), then add dropwise a mixed solution of 13.8 mg NHS and 24.7 mg DCC d...

Embodiment 2

[0040] 1. Establish the standard curve of carbendazim by direct competition ELISA method:

[0041] The optimal coating volume of the antibody was 5ug / mL, the optimal dilution factor of the enzyme-labeled antigen was 150 times, and the optimal ion concentration and pH value were 0.01MPBS with pH=7.4. Using the optimized experimental conditions, the standard curve is established with the standard concentration (ug / kg) as the abscissa and the inhibition rate (%) as the ordinate. The results are shown in figure 1 ,Specific steps are as follows:

[0042] (1) Coating: Prepare the antibody with 0.01M carbonic acid buffer (pH=9.6) to make a 5ug / mL coating solution, add 100uL coating solution to each well of the microplate plate, incubate at 4°C for 12h, shake dry, and use Wash 3 times with PBST;

[0043] (2) Adding samples: add 100uLPBS to the blank well; add 50uLPBS and 50uL enzyme-labeled antigen diluted 150 times to the control well; add 50uL standard products of different ...

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Abstract

The invention discloses a method for detecting carbendazim content in edible fungi by using carbendazim-specific antibody. The method includes: stirring and dissolving 2-aminobenzimidazole in acetonitrile, adding succinic anhydride, stirring and reacting at 40 for 4 h, filtering a reaction solution to obtain residue, washing the residue twice with acetonitrile at 40 DEG C to obtain a crude product, subjecting the crude product further to column chromatography purification, and identifying via thin-layer chromatography and mass spectrometry to obtain the product 2-succinyl benzimidazole. A 4-carbon spacer arm is then coupled to 2-amino group of the 2-succinyl benzimidazole to serve as a hapten, and coupling carrier proteins by activated ether method to obtain an artificial antigen; then, a specific polyclonal antibody is acquired by animal immunization and antiserum purification; finally, simple, quick and sensitive direct competitive ELISA (enzyme-linked immuno sorbent assay) method established via the antibody is utilized to quantify carbendazim in edible fungi, and important technical means is provided for monitoring carbendazim content in edible fungi.

Description

[0001] This invention was funded by the "Twelfth Five-Year" National Science and Technology Support Project (2014BAD04B03); the National Natural Science Youth Fund Project (31301487); Tianjin Science and Technology Plan Project (142CDGNC00098). technical field [0002] The invention belongs to the field of immunochemistry and residue analysis of pesticide small molecule compounds, and relates to the synthesis of hapten, the preparation of artificial antigen and polyclonal antibody and the establishment of direct competition ELISA method. Specifically, it is a carbendazim antibody and its application in the detection of edible fungus residues. Background technique [0003] Carbendazim belongs to the class of benzimidazole fungicides, and is widely used in agricultural production due to its high efficiency, low toxicity, and systemic characteristics. Although it is a low-toxic pesticide, considering its long residue period (more than 20 days) and the harm of bioaccumulation to...

Claims

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Application Information

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IPC IPC(8): G01N33/577
CPCG01N33/577
Inventor 宋洋王静马宁宁陈晓明
Owner TIANJIN NORMAL UNIVERSITY
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