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Prochloraz hapten, prochloraz artificial antigen and prochloraz antibody, and preparation methods and application thereof

A technology of artificial antigen and prochloraz, which is applied in the preparation methods of peptides, chemical instruments and methods, animal/human proteins, etc., to achieve the effects of high sensitivity, easy control of reaction conditions and enhanced immunogenicity

Active Publication Date: 2019-12-31
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, there is no relevant report on the prochloraz hapten in China

Method used

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  • Prochloraz hapten, prochloraz artificial antigen and prochloraz antibody, and preparation methods and application thereof
  • Prochloraz hapten, prochloraz artificial antigen and prochloraz antibody, and preparation methods and application thereof
  • Prochloraz hapten, prochloraz artificial antigen and prochloraz antibody, and preparation methods and application thereof

Examples

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preparation example Construction

[0034] In a second aspect, the present invention provides a method for preparing the above-mentioned prochloraz hapten, which includes the following steps:

[0035] 1) 3-(3-hydroxyl-2,4,6-trichloro)-propionic acid reacts with 1,2-dichloroethane to obtain intermediate 1, which has the structural formula

[0036] 2) The intermediate 1 is reacted with n-propylamine to obtain the intermediate 2, and the intermediate 2 has the structural formula

[0037]

[0038] 3) reacting the intermediate 2 with bis(trichloromethyl)carbonate and imidazole to obtain the prochloraz hapten.

[0039] Preferably, the step 1) includes the following steps: take 2.69 g of 3-(3-hydroxy-2,4,6-trichloro)-propionic acid and add 60 mL of 1,2-dichloroethane to dissolve, add KOH 0.57 g , reacted at 60°C for 6h, stopped the reaction, washed with water, and evaporated to dryness to obtain intermediate 1.

[0040] Preferably, the step 2) includes the following steps: adding 40 mL of n-propylamine, 0.31 g o...

Embodiment 1

[0060] A kind of preparation method of prochloraz hapten, it comprises the steps:

[0061] 1) Take 2.69g of 3-(3-hydroxy-2,4,6-trichloro)-propionic acid and add 60mL of 1,2-dichloroethane to dissolve, add KOH 0.57g, react at 60°C for 6h, stop the reaction, add water Washing, evaporation to dryness to obtain intermediate 1;

[0062] 2) Add 40mL of n-propylamine, 0.31g of anhydrous sodium iodide, and 1.38g of potassium carbonate to intermediate 1, heat and reflux for 4h, stop the reaction, rotary steam, remove n-propylamine, add water to shake, extract with ethyl acetate, collect The organic phase was recrystallized with n-hexane-dichloromethane to obtain intermediate 2;

[0063] 3) Take 1.2g of intermediate 2 and add 80mL of dichloromethane to dissolve, add 1.1g of bis(trichloromethyl)carbonate and 0.3mL of triethylamine, stir at room temperature for 3h, add 0.27g of imidazole, continue stirring for 7h, stop the reaction, Water was added for extraction and washing, and evapor...

Embodiment 2

[0065] A preparation method of prochloraz artificial antigen, the steps are as follows:

[0066] Dissolve 17 mg of the prochloraz hapten prepared in Example 1 in 1 mL of DMF, dissolve and clarify, add 15.3 mg of carbodiimide and 11.2 mg of N-hydroxysuccinimide, mix well, and react for 4 hours to obtain liquid A ; Dissolve 50 mg of bovine serum albumin (BSA) in 0.05mol / L PB buffer to obtain liquid B; add liquid A dropwise to liquid B, react at 4°C for 24 hours, and dialyze and purify with 0.02mol / L PBS for 3 days , the medium was changed 3 times a day to obtain the prochloraz artificial antigen coupled with bovine serum albumin, which was aliquoted and stored at -20°C.

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Abstract

The invention discloses a prochloraz hapten, a prochloraz artificial antigen and a prochloraz antibody, and preparation methods and application thereof. The prochloraz hapten provided by the inventionretains the characteristic structure of prochloraz to the greatest extent, so the immunogenicity of the prochloraz hapten is obviously enhanced; and the prochloraz hapten has carboxyl groups capableof being coupled with carrier protein. When a prochloraz immunizing antigen obtained by coupling the prochloraz hapten with carrier protein is used for immunizing an animal, the immune response of theanimal is better stimulated, so the antibody with stronger specificity and higher sensitivity is generated; and detection results show that the prochloraz antibody has a sensitivity of 0.1 [mu]g / L, is low in cross reaction rate with other pesticides and provides a basis for subsequent establishment of various immunoassay methods for prochloraz.

Description

technical field [0001] The invention belongs to the field of food safety detection. More specifically, the present invention relates to prochloraz haptens, artificial antigens and antibodies and their preparation methods and applications. Background technique [0002] Prochloraz is a broad-spectrum fungicide that can prevent and control various diseases on field crops, fruits and vegetables, turf and ornamental plants; in addition, prochloraz can also be used for post-harvest citrus, banana and other fruits. Product preservation has a wide range of applications. All countries have strict regulations on the dosage of prochloraz. For example, CAC, the European Union and Japan have all stipulated the MRL values ​​of prochloraz in raw milk, which are 0.05, 0.4, and 0.05 mg / kg respectively. my country’s national standard GB 2763-2016 The "National Food Safety Standard Maximum Residue Limits of Pesticides in Food" also stipulates the residue limits of prochloraz in different food...

Claims

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Application Information

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IPC IPC(8): C07D233/56C07K14/765C07K14/77C07K14/795C07K1/113C07K16/44G01N33/53G01N33/531G01N33/577
CPCC07D233/56C07K14/765C07K14/77C07K14/795C07K16/44G01N33/53G01N33/531G01N33/577
Inventor 万宇平崔廷婷崔海峰张瑜何方洋马玉华崔娜赵正苗
Owner BEIJING KWINBON BIOTECH
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