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Dicofol hapten, dicofol artificial antigen, dicofol antibody, and preparation method and application of dicofol antibody

A technology of dicofol and artificial antigen, which is applied to the preparation method of peptides, chemical instruments and methods, and carboxylate preparation, etc., which can solve the problems of reduced antibody affinity and specificity, increased difficulty in identifying hapten molecular characteristic structures, and unfavorable Exposure of the characteristic structure of the hapten molecule to achieve enhanced immunogenicity, easy control of reaction conditions, and good affinity

Inactive Publication Date: 2020-06-26
广州勤邦生物技术有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Invention patents CN107907697A and CN110607283A both adopt the method of derivatizing the -OH of dicofol into a spacer arm with -COOH, and then coupling with the carrier protein, so that the distance between the modification site and the characteristic structure of the dicofol molecule is relatively close, which is not conducive to the molecular characteristics of the hapten The exposure of the structure makes it more difficult for the animal's immune system to recognize the characteristic structure of the hapten molecule, and the affinity and specificity of the antibody decrease

Method used

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  • Dicofol hapten, dicofol artificial antigen, dicofol antibody, and preparation method and application of dicofol antibody
  • Dicofol hapten, dicofol artificial antigen, dicofol antibody, and preparation method and application of dicofol antibody
  • Dicofol hapten, dicofol artificial antigen, dicofol antibody, and preparation method and application of dicofol antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] A kind of preparation method of dicofol hapten, the steps are as follows:

[0034] Take 1.7g of o-chlorophenylacetic acid and add 100mL of carbon tetrachloride to dissolve, add 1.12g of chlorobenzene, stir well, add 1.47g of chloral, cool to 10-15°C, add dropwise 10mL of 10% sulfuric acid solution, continue Stir the reaction for 4 hours. After the reaction is completed, add 100mL of 0-5°C cold water, transfer to a separatory funnel, oscillate, stand to separate layers, extract the water phase with chloroform three times, 80mL each time, combine the organic phase; carbon tetrachloride Add water and wash again, combine with chloroform extract and evaporate to dryness to obtain red oil, which is purified by silica gel column, eluted and separated with a mixed solvent of petroleum ether and ethyl acetate with a volume ratio of 6:1, and dicofol hapten is obtained.

Embodiment 2

[0036] A kind of preparation method of dicofol artificial antigen, the steps are as follows:

[0037]Take 14 mg of the dicofol hapten prepared in Example 1, add 1 mL of dimethyl sulfoxide (DMSO) to dissolve, add 8.2 mg of N-hydroxysuccinimide (NHS) and 15.7 mg of carbodiimide (EDC), and react at room temperature for 3 h , to obtain hapten solution A; take bovine serum albumin (BSA) 50mg, add 0.05mol / L PB buffer to dissolve, and obtain B; add A dropwise to B, react at 4°C for 12h, and use 0.02mol / L PBS dialysis and purification for 3 days, changing the medium 3 times a day to obtain dicofol artificial antigen coupled with bovine serum albumin, aliquoted and stored at -20°C.

Embodiment 3

[0039] A kind of preparation method of dicofol artificial antigen, the steps are as follows:

[0040] Take 8.1 mg of dicofol hapten prepared in Example 1, add 1 mL of 1,4-dioxane to dissolve, add NHS 6.1 mg, EDC 9.7 mg, and react at room temperature for 3 hours to obtain hapten solution A; take ovalbumin (OVA ) 50mg, add 0.05mol / LPB buffer solution to dissolve to obtain liquid B; add liquid A dropwise to liquid B, react at 4°C for 12h, dialysis and purify with 0.02mol / L PBS for 3 days, change the liquid 3 times a day, and obtain the same Ovalbumin-coupled dicofol artificial antigen, aliquoted and stored at -20°C.

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Abstract

The invention discloses a dicofol hapten, a dicofol artificial antigen, a dicofol antibody, and a preparation method and application of the dicofol antibody. According to the invention, dicofol haptenprovided by the invention retains the characteristic structure of dicofol to the greatest extent, so that the immunogenicity of the dicofol hapten is obviously enhanced; the dicofol hapten has carboxyl capable of being coupled with a carrier protein, and is coupled with the carrier protein to obtain a dicofol artificial antigen, and the dicofol artificial antigen immunizes animals, so that generation of antibody with strong specificity and high sensitivity by stimulating animal immune response is facilitated; and the detection results show that the sensitivity of the dicofol antibody can reach 0.1 [mu]g / L, the cross reaction rate of the dicofol antibody and other organochlorine pesticides is low, and a foundation is provided for establishing various immunoassay methods of dicofol subsequently.

Description

technical field [0001] The invention belongs to the field of food safety detection. More specifically, the present invention relates to dicofol haptens, artificial antigens and antibodies and their preparation methods and applications. Background technique [0002] Dicofol (dicofol) is an organochlorine acaricide, which can prevent and control mites on crops such as cotton, fruit trees, and vegetables. It has strong contact and stomach poisoning effects on adults, nymphs, and eggs. long term. Studies have shown that the drug has certain toxicity to humans and animals. The main symptoms of poisoning are headache, dizziness, sweating, chest tightness, mydriasis, blurred vision, nausea, vomiting, and diarrhea. Local exposure can cause contact dermatitis. Dicofol has been banned in my country, and the national standard GB2763-2019 "National Food Safety Standard Maximum Residue Limits of Pesticides in Food" also stipulates that the residue limit of dicofol in fruits is 1mg / kg. ...

Claims

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Application Information

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IPC IPC(8): C07C59/56C07C51/367C07K14/765C07K14/77C07K14/795C07K1/10C07K1/107C07K16/44G01N33/53
CPCC07C59/56C07C51/367C07K14/765C07K14/77C07K14/795C07K16/44G01N33/53G01N33/184
Inventor 王兆芹马玉华万宇平甘桂湘赵正苗陶燕飞
Owner 广州勤邦生物技术有限公司
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