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Preparation methods and application of pendimethalin hapten and antigen

A pendimethalin and hapten technology, which is applied in the preparation methods of peptides, the preparation of organic compounds, the preparation of cyanide reactions, etc., to achieve the effects of high sensitivity, rapid detection and high specificity

Active Publication Date: 2019-01-18
ZHENGZHOU TOBACCO RES INST OF CNTC +2
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There is no report about the preparation method of pendimethalin hapten and antigen at present

Method used

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  • Preparation methods and application of pendimethalin hapten and antigen
  • Preparation methods and application of pendimethalin hapten and antigen
  • Preparation methods and application of pendimethalin hapten and antigen

Examples

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Embodiment 1

[0023] Example 1 Preparation of pendimethalin hapten

[0024] 1. Synthesis of pendimethalin hapten (see attached figure 1 )

[0025] 1) Take 1.00 g of N-(1-ethylpropyl)-3,4-dimethylaniline, add 80 mL of acetone to dissolve, add 0.35 g of potassium hydroxide, add 1.21 g of ethyl 4-bromobutyrate, and heat Reflux reaction for 3 h, TLC detection, all the raw materials were reacted, stop the reaction, cool to room temperature, rotary evaporate, remove acetone, add 60 mL water, add 50 mL dichloromethane for extraction, dry the organic phase with anhydrous sodium sulfate, evaporate to dryness and concentrate , put on a silica gel column, use petroleum ether / ethyl acetate with a volume ratio of 10:1 for elution and separation, and obtain 1.51 g of intermediate alkylated N-(1-ethylpropyl)-3,4-dimethylaniline, Yield 94.97%;

[0026] 2) Take 1.50 g of the intermediate alkylated N-(1-ethylpropyl)-3,4-dimethylaniline, add 5 mL of concentrated sulfuric acid to dissolve and clarify, slowl...

Embodiment 2

[0031] Example 2 Preparation of pendimethalin antigen

[0032] 1. Synthesis of pendimethalin immunogen

[0033] The pendimethalin hapten is conjugated with human serum albumin (HSA) to obtain the immunogen.

[0034] Take pendimethalin hapten 10 mg, add dimethyl sulfoxide (DMSO) to dissolve, add carbodiimide (EDC) 5.7 mg, stir, clarify, add N-hydroxysuccinimide (NHS) 3.8 mg, Stir and activate at room temperature for 3 h to obtain liquid A; take 50 mg of HAS and add 8 mL of 0.1 mol / L PB buffer solution to dissolve to obtain liquid B, slowly add liquid A to liquid B dropwise, and stir at room temperature for 5 h. Stop the reaction, dialyze with 0.02 M PBS buffer for 3 days, and change the medium three times a day to obtain the pendimethalin-HSA immunogen, which is aliquoted and stored at -20°C.

[0035] 2. Synthesis of Pendimethalin Coating Source

[0036] The pendimethalin hapten was coupled with ovalbumin (OVA) to obtain the coating source.

[0037] Take 6 mg of pendimethal...

Embodiment 3

[0040] Example 3 Preparation of Pendimethalin Monoclonal Antibody

[0041] 1. Obtaining hybridoma cells

[0042] 1) First immunization: fully emulsify the pendimethalin hapten-HSA conjugate (immunogen) with the same amount of complete Freund's adjuvant, and inject 6-week-old Balb / c mice subcutaneously, 0.2 mL each;

[0043] 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;

[0044] 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;

[0045] 4...

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Abstract

The invention provides preparation methods and application of pendimethalin hapten and antigen. The pendimethalin hapten is characterized by being prepared by taking N-(1-ethylpropyl)-3,4-dimethylaniline and 4-ethyl bromobutyrate to react to generate alkylated N-(1-ethylpropyl)-3,4-dimethylaniline, taking the alkylated N-(1-ethylpropyl)-3,4-dimethylaniline and concentrated nitric acid to react togenerate nitro-alkylated N-(1-ethylpropyl)-3,4-dimethylaniline, and then reacting with potassium hydroxide; the pendimethalin antigen is prepared by coupling the pendimethalin hapten and carrier protein. The antigen prepared by the invention has a specific pendimethalin antigen determinant, so that a high-specificity pendimethalin monoclonal antibody is possibly screened. The produced antibody hashigh specificity and high sensitivity and can be used for establishing an enzyme-linked immunosorbent assay method and a colloidal gold test strip determination method, so that rapid detection of pendimethalin in tobaccos and foods is realized.

Description

technical field [0001] The invention relates to a preparation method and application of pendimethalin hapten and antigen. The invention belongs to the technical field of pesticide immunochemistry. Background technique [0002] Pendimethalin belongs to dinitroaniline herbicides, and its mechanism of action is to inhibit meristem cell division. The part of pendimethalin absorbed by dicotyledons is hypocotyl, and the part absorbed by monocots is sprout. Pendimethalin is suitable for corn, soybean, wheat, peanut, cotton and other dryland crops to control annual grass and broad-leaved weeds such as crabgrass, foxtail and purslane. Tobacco is mainly used for chemical bud suppression and tobacco weed control. According to the classification standard of acute toxicity of pesticides in China, pendimethalin is classified as a low toxicity compound. According to the toxic classification standard of the US Environmental Protection Agency EPA, pendimethalin belongs to Class III, a lo...

Claims

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Application Information

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IPC IPC(8): C07C227/20C07C229/18C07K14/765C07K14/77C07K1/107C07K16/44G01N33/531G01N33/544G01N33/558
CPCC07C227/08C07C227/16C07C227/20C07K14/765C07K14/77C07K16/44C07K19/00G01N33/531G01N33/544G01N33/558C07C229/18
Inventor 陈黎范子彦刘惠民唐纲岭崔华鹏樊美娟赵乐武侠书鲁亚辉曹东山
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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