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Difenoconazole hapten, artificial antigen and antibody and preparation method and application thereof

A technology of difenoconazole and artificial antigens, which is applied in chemical instruments and methods, animal/human proteins, instruments, etc. It can solve the problems of inconsistency in on-site detection, accurate detection and screening, inability to be widely used, cumbersome sample pretreatment, etc. problems, to achieve the effects of easy control of reaction conditions, high purity and yield, and good affinity

Active Publication Date: 2022-07-08
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, domestic and foreign detection of difenoconazole mainly adopts gas chromatography, gas chromatography-mass spectrometry and liquid chromatography tandem mass spectrometry and other analytical methods, which have disadvantages such as cumbersome sample pretreatment, long detection time, and expensive instruments, so in our country It cannot be widely used, and does not meet the requirements of on-site detection "accurate detection and screening of a large number of samples in a short period of time and at low cost"

Method used

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  • Difenoconazole hapten, artificial antigen and antibody and preparation method and application thereof
  • Difenoconazole hapten, artificial antigen and antibody and preparation method and application thereof
  • Difenoconazole hapten, artificial antigen and antibody and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] A kind of preparation method of difenoconazole hapten, it comprises the steps:

[0035] 1) Dissolve 4.2 g of difenoconazole impurity 1 in 100 mL of 1,2-dichloroethane, add 0.12 g of p-toluenesulfonic acid, stir well, add 1.61 g of liquid bromine dropwise at room temperature, and add dropwise in batches , firstly add half of it dropwise, after the color becomes lighter, add the other half dropwise, and continue the reaction for 2h; after the reaction is over, add 100 mL of saturated saline, shake it fully, let it stand, separate the water phase, add 100 mL of water, shake it, let it stand, The aqueous phase was separated, and the organic phase was concentrated and evaporated to dryness to obtain Intermediate 1;

[0036] 2) Add 100 mL of N,N-dimethylformamide to dissolve all intermediate 1, add 2.14 g of KOH, stir well, add 0.47 g of anhydrous sodium iodide, and then dropwise add 2 mL of an aqueous solution containing 1.8 g of aminopropionic acid , heated in an oil bath,...

Embodiment 2

[0038] A preparation method of difenoconazole artificial antigen, the steps are as follows:

[0039] Take 19 mg of the difenoconazole hapten prepared in Example 1, add 1 mL of N,N-dimethylformamide (DMF) to dissolve, add 13.1 mg of N-hydroxysuccinimide (NHS), 1-(3- Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) 27mg, react at room temperature for 3h to obtain hapten solution A; take bovine serum albumin (BSA) 50mg, add 0.05mol / L PB The buffer solution was dissolved to obtain solution B; A was added dropwise to solution B, reacted at 4°C for 12 h, dialyzed and purified with 0.02mol / L PBS for 3 days, and the solution was changed 3 times a day to obtain benzene conjugated with bovine serum albumin. Difenoconazole artificial antigen, aliquoted and stored at -20°C.

Embodiment 3

[0041] A preparation method of difenoconazole artificial antigen, the steps are as follows:

[0042]Take 12 mg of the difenoconazole hapten prepared in Example 1, add 1 mL of dimethyl sulfoxide (DMSO) to dissolve, add 11 mg of 1-hydroxybenzotriazole (HOBT) and 11 mg of EDC, and react at room temperature for 3 hours to obtain the hapten Solution A solution; take ovalbumin (OVA) 50mg, add 0.05mol / L PB buffer to dissolve to obtain B solution; add A dropwise to B solution, react at 4°C for 12h, and dialysis and purify with 0.02mol / L PBS 3 On the next day, the medium was changed 3 times a day to obtain the artificial antigen of difenoconazole coupled with ovalbumin, which was packaged and stored at -20°C.

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Abstract

The invention discloses a difenoconazole hapten, an artificial antigen and an antibody as well as a preparation method and application thereof. The difenoconazole hapten provided by the invention not only retains the characteristic structure of The immunogenicity of the difenoconazole hapten is obviously enhanced, and it has a carboxyl group that can be coupled with the carrier protein; the difenoconazole artificial antigen obtained by coupling the difenoconazole hapten with the carrier protein is used to deimmunize Animals are more conducive to stimulating the immune response of animals to produce antibodies with higher specificity and higher sensitivity. The sensitivity of difenoconazole antibodies can reach 0.05 μg / L after detection, and the cross-reaction rate with other triazole pesticides is low. It provides a basis for the subsequent establishment of various immunoassay methods for difenoconazole.

Description

technical field [0001] The invention belongs to the field of food safety detection. More specifically, the present invention relates to difenoconazole haptens, artificial antigens and antibodies, and preparation methods and applications thereof. Background technique [0002] Difenoconazole, also known as oxadiazole, is a broad-spectrum and highly efficient triazole systemic fungicide, which is a 14α-sterol demethylation inhibitor, which can interfere with mycelial growth and inhibit pathogens The spores germinate, ultimately inhibiting fungal growth. Difenoconazole has protective and therapeutic effects and is widely used in cereals, oilseeds, fruits, vegetables, peanuts, sugar beets, tea, ginseng and other crops to control pests and diseases, mainly for the control of gray rot, downy mildew and powdery mildew , and can also inhibit strawberry and pepper anthracnose. However, because it is directly sprayed on crops, people are vulnerable to its harm. Therefore, my country...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D405/06C07K14/765C07K14/77C07K14/795C07K16/44G01N33/53
CPCC07D405/06C07K14/765C07K14/77C07K14/795C07K16/44G01N33/5308
Inventor 万宇平崔廷婷王兆芹何方洋崔海峰赵正苗吴小胜
Owner BEIJING KWINBON BIOTECH
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