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Preparation method and application of hapten and antigen of butralin

A technology of Zhongbuling and hapten, which is applied in the field of preparation of Zhongbuling hapten and antigen, to achieve high sensitivity, high antibody specificity and rapid detection effect

Active Publication Date: 2018-12-21
ZHENGZHOU TOBACCO RES INST OF CNTC +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no report about the preparation method of Zhongbuling hapten and antigen

Method used

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  • Preparation method and application of hapten and antigen of butralin
  • Preparation method and application of hapten and antigen of butralin

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Experimental program
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Effect test

Embodiment 1

[0020] The preparation of embodiment 1 secbutaline hapten

[0021] 1. Synthesis of Zhongbutaline hapten (see the appendix for the synthetic route figure 1 )

[0022] Take 1.00 g of 4-chloro-3,5-dinitrophenylacetic acid, add 50 mL of ethanol to dissolve, add 0.45 g of sodium carbonate, stir, add 0.31 g of butylamine, heat in an oil bath at 80°C, stir for 3 h, and detect the raw material The basic reaction is complete; stop the reaction, cool to room temperature, rotary evaporate, remove ethanol, add 80 mL of water, adjust the pH value to 4 with 1 mol / L hydrochloric acid, there is a lot of turbidity, add 80 mL of ethyl acetate to extract, wash with water, and apply silica gel The column was eluted and separated with dichloromethane / methanol with a volume ratio of 5:1 to obtain 1.05 g of the hapten product of sec-butyrin, with a yield of 92.11%.

[0023] 2. Identification of Zhongbuling hapten

[0024] NMR identification 1 H NMR (CDCl 3 , 300MHz) δ: 11.01 (1H, d, J=0.000), 8...

Embodiment 2

[0026] Example 2 Preparation of Zhongbuling Antigen

[0027] 1. Synthesis of Zhongbuling immune antigen

[0028] The immunogen was obtained by conjugating the secbutaline hapten with bovine serum albumin (BSA).

[0029] Take 11 mg of sec-butyrin hapten, add 0.2 mL of dimethyl sulfoxide (DMSO) to dissolve, add 20 μL of triethylamine, stir, mix well, add 6 mg of isobutyl chloroformate, stir for 2 h, and obtain the hapten Activation solution A solution; take 50 mg BSA and add 0.1 mol / L PB buffer solution with a pH value of 7.2 to dissolve to obtain B solution, slowly add A solution to B solution, continue to stir for 5 h, stop the reaction, 0.02mol / L L PBS was dialyzed for 3 days, and the medium was changed 3 times a day to obtain the secbutamyl-BSA conjugate, which was the immunogen, which was aliquoted and stored at -20°C.

[0030] 2. Synthesis of Zhongbuling-coated antigen

[0031] The sec-butyrin hapten was coupled with ovalbumin (OVA) to obtain the coating source.

[003...

Embodiment 3

[0035] Example 3 Preparation of secbutaline monoclonal antibody

[0036] 1. Obtaining hybridoma cells

[0037] 1) First immunization: fully emulsify the secbutyrine hapten-BSA conjugate (immunogen) with the same amount of complete Freund's adjuvant, and inject 6-week-old Balb / c mice subcutaneously, 0.2 mL each;

[0038] 2) Booster immunization twice: from the first immunization, booster immunization once every two weeks, with Freund's incomplete adjuvant instead of Freund's complete adjuvant, the method and dosage are the same as the first immunization;

[0039] 3) One week after the last booster immunization, the fundus vein blood was collected to measure the titer and inhibition. When there was inhibition and the titer reached more than 1:10000, the following last immunization was carried out: intraperitoneal injection of 0.1 mL of the immunogen solution without any adjuvant, and then executed three days later Mice, whose spleen was fused with myeloma cells;

[0040] 4) Us...

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Abstract

The invention relates to a preparation method and the application of a hapten and an antigen of butralin. The preparation method is characterized in that the hapten of butralin is obtained through thereaction of 4-chloro-3,5-dinitrophenylacetic acid and butylamine, and the antigen of butralin is obtained by coupling the hapten of butralin with a carrier protein. The antigen prepared by the methodprovided by the invention presents a specific butralin antigenic determinant, which ensures the probability of screening out a high-specificity butralin monoclonal antibody. A generated antibody hashigh specificity and sensitivity, and can be used for establishing an enzyme-linked immunosorbent assay method and a colloidal gold test paper rapid assay method, therefore, rapid detection of butralin in tobacco and food is achieved.

Description

technical field [0001] The present invention relates to a preparation method and application of Zhongbuling hapten and antigen. The invention belongs to the technical field of pesticide immunochemistry. Background technique [0002] Butralin (Butralin) is also known as dimethamine, bibutralin, nifediamine, bidarin, and budling. The chemical name is N-sec-butyl-4-tert-butyl-2,6-dinitro Aniline, the molecular formula is C 14 h 21 N 3 o 4 . Zhongbuling is a dinitroaniline herbicide. The pure product is an orange-yellow crystal. It is easily soluble in organic solvents but hardly soluble in water. It is volatile and easy to decompose when exposed to light. It has low toxicity to humans and animals. The drug is a selective preemergent herbicide, and its effect is similar to that of trifluralin. After the medicament enters the plant, it mainly inhibits the cell division of the meristem, thereby inhibiting the growth of weed shoots and young roots, resulting in the death of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07C227/08C07C229/42C07K14/47C07K14/765C07K14/77C07K14/795G01N33/577G01N33/558
CPCC07C227/08C07K14/47C07K14/765C07K14/77C07K14/795C07K19/00G01N33/558G01N33/577C07C229/42
Inventor 陈黎范子彦刘惠民唐纲岭潘立宁崔华鹏樊美娟鲁亚辉刘玉梅申梁
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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