Pyraclostrobin hapten, artificial antigen and antibody as well as preparation method and application thereof

A technology of pyraclostrobin and artificial antigen, which is applied in chemical instruments and methods, animal/human protein, serum albumin, etc., can solve the problem of incompatibilities in on-site detection, accurate detection and screening, inability to be widely used, and sample pretreatment cumbersome and other problems, to achieve the effect of easy control of reaction conditions, high purity and yield, and good affinity

Pending Publication Date: 2020-11-20
BEIJING WANGER BIOTECH +1
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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 pyraclostrobin 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

Method used

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  • Pyraclostrobin hapten, artificial antigen and antibody as well as preparation method and application thereof
  • Pyraclostrobin hapten, artificial antigen and antibody as well as preparation method and application thereof
  • Pyraclostrobin hapten, artificial antigen and antibody as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] A preparation method of pyraclostrobin hapten, which comprises the steps of:

[0041] 1) Dissolve 4.0 g of methyl 2-(bromomethyl)-4-iodobenzene (methoxy) carbamate in 80 mL of N,N-dimethylformamide (DMF), add 1.12 g of KOH and 1.94g 1-(4-chlorophenyl)-2H-pyrazolin-3-one, after fully stirring, add 0.33g of anhydrous potassium iodide, heat and stir in an oil bath, react at 80°C for 7h, stop the reaction, cool to room temperature, Add 100mL of water and 200mL of ethyl acetate, oscillate, stand still, separate the water phase, add 80mL pure water to the organic phase, oscillate, stand still, separate the water phase, and evaporate the organic phase to dryness to obtain intermediate 1;

[0042] 2) Add 100mL dimethyl sulfoxide to dissolve all the intermediate 1, add 0.48g sodium hydride, 0.77g anhydrous cuprous iodide and 1.21g aminobutyric acid, stir well, react at 100°C for 9h, stop the reaction, and cool to room temperature, add 200mL water, adjust the pH to 5 with 6mol / L...

Embodiment 2

[0044] A preparation method of pyraclostrobin artificial antigen, the steps are as follows:

[0045]Take 17 mg of pyraclostrobin hapten prepared in Example 1, add 1 mL of DMF to dissolve, add 20 μL of triethylamine and 127 μL of isobutyl chloroformate, cool to 0-5 °C, and react for 3 hours to obtain hapten solution A; Take 50mg of bovine serum albumin (BSA), add 0.05mol / L PB buffer solution to dissolve to obtain liquid B; add liquid A dropwise to liquid B, react at 4°C for 12h, and dialyze and purify with 0.02mol / L PBS for 3 days. The medium was changed 3 times a day to obtain the pyraclostrobin artificial antigen coupled with bovine serum albumin, which was aliquoted and stored at -20°C.

Embodiment 3

[0047] A preparation method of pyraclostrobin artificial antigen, the steps are as follows:

[0048] Take 10 mg of the pyraclostrobin hapten prepared in Example 1, add 1 mL of 1,4-dioxane to dissolve, add 8.2 mg of N-hydroxysuccinimide (NHS), 1-(3-dimethylaminopropyl base)-3-Ethylcarbodiimide hydrochloride (EDC) 13mg, react at room temperature for 3h to obtain hapten solution A liquid; take ovalbumin (OVA) 50mg, add 0.05mol / L PB buffer solution to dissolve, and obtain Liquid B: Add liquid A dropwise to liquid B, react at 4°C for 12 hours, dialysis and purify with 0.02mol / L PBS for 3 days, change the liquid 3 times a day, and obtain pyraclostrobin artificial antigen coupled with ovalbumin , aliquoted, and stored at -20°C.

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Abstract

The invention discloses a pyraclostrobin hapten, an artificial antigen, an antibody as well as a preparation method and application thereof. The provided pyraclostrobin hapten retains the characteristic structure of pyraclostrobin to the greatest extent, so that the immunogenicity of the pyraclostrobin hapten is obviously enhanced and the pyraclostrobin hapten has a carboxyl coupled with a carrierprotein; a pyraclostrobin artificial antigen obtained by coupling the pyraclostrobin hapten with a carrier protein is used for immunizing animals; the prepared pyraclostrobin antigen is more beneficial to stimulating animal immune response to generate an antibody with stronger specificity and higher sensitivity, the sensitivity of the pyraclostrobin antibody can reach 0.1 [mu]g/L through detection, the cross reaction rate of the pyraclostrobin antibody and other methoxy acrylate bactericides is low, and a basis is provided for establishing various immunoassay methods of pyraclostrobin subsequently.

Description

technical field [0001] The invention belongs to the field of food safety detection. More specifically, the present invention relates to pyraclostrobin haptens, artificial antigens and antibodies and their preparation methods and applications. Background technique [0002] Pyraclostrobin, also known as pyraclostrobin, is a new type of broad-spectrum methoxyacrylate fungicide, which has a broad bactericidal spectrum, high activity, long duration, low toxicity, and non-target Biosafety and other characteristics, mainly used to prevent and control leaf blight, rust, powdery mildew, downy mildew, blight, anthracnose caused by Ascomycetes, Basidiomycetes, Deuteromycetes and Oomycetes on crops such as fruits and vegetables , scab, brown spot, blight, rice blast and other diseases. my country's national standard GB2763-2019 "National Food Safety Standard Maximum Residue Limits of Pesticides in Food" stipulates the residue limits of pyraclostrobin in different foods. [0003] At p...

Claims

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Application Information

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IPC IPC(8): C07D231/22C07K14/765C07K14/77C07K14/795C07K16/44G01N33/53G01N33/577
CPCC07D231/22C07K14/765C07K14/77C07K14/795C07K16/44G01N33/577G01N33/5308C07K2317/35C07K2317/33
Inventor 王兆芹万宇平何方洋马玉华张瑜崔娜凡静静
Owner BEIJING WANGER BIOTECH
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