Preparation method and application of flumetralin hapten and antigen

A flumeetamide and hapten technology, which is applied in the field of preparation of flumemetrid hapten and antigen, achieves the effects of rapid detection, high antibody specificity and high sensitivity

Active Publication Date: 2019-01-15
ZHENGZHOU TOBACCO RES INST OF CNTC +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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

Examples

Experimental program
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Effect test

Embodiment 1

[0021] Example 1 The preparation of flumetriamine hapten

[0022] 1. Synthesis of flumetriamine hapten

[0023] Take 1.00 g of 2-chloro-1,3-dinitro-5-trifluoromethylbenzene, add 20 mL of absolute ethanol to dissolve to obtain liquid A; another 3-amino-3-(2-chloro-6- Add 10 mL of absolute ethanol to dissolve 0.88 g of fluorophenyl)-propionic acid, add 1 mL of aqueous solution containing 0.37 g of sodium bicarbonate to obtain liquid B, add liquid A dropwise to liquid B, and react at room temperature for 3 h; TLC After detection, the basic reaction of the raw material is complete; stop the reaction, spin evaporate, remove ethanol, add 80 mL of water to dissolve, adjust the pH value to 6 with 1 mol / L hydrochloric acid, add 80 mL of ethyl acetate to shake and separate, wash the organic phase with water, and spin evaporate , on a silica gel column, eluted and separated with n-hexane and ethyl acetate at a volume ratio of 10:1 to obtain the intermediate 3-(2-chloro-6-fluorophenyl)-3...

Embodiment 2

[0028] Example 2 Preparation of flumetriamine antigen

[0029] 1. Synthesis of flumetriamine immune antigen

[0030] The immunogen was obtained by conjugating the flumetonide hapten with bovine serum albumin (BSA).

[0031] Take 18 mg of flumetriamine hapten, add 0.3 mL of dimethylformamide (DMF) to dissolve, clarify, add carbodiimide (EDC) 8.6 mg, stir, clarify, add N-hydroxysuccinimide (NHS) 5.2 mg, stir and activate at room temperature for 3 h to obtain liquid A; take 50 mg of BSA, add 8 mL of 0.05 mol / L PB buffer solution with a pH value of 7.2 to dissolve to obtain liquid B, slowly add liquid A to liquid B, The reaction was stirred at room temperature for 5 h, the reaction was stopped, and the 0.02 M PBS buffer was dialyzed for 3 days, and the medium was changed three times a day to obtain flumetramide-BSA immunogen, which was aliquoted and stored at -20°C.

[0032] 2. Synthesis of flumetriamine-coated antigen

[0033] Flumetriamine hapten was conjugated with ovalbumin...

Embodiment 3

[0037] Example 3 Preparation of Flumetriamine Monoclonal Antibody

[0038] 1. Animal immunity

[0039] The immunogen obtained in the above steps was injected into Balb / c mice at an immunization dose of 150 μg / mouse to make them produce antiserum.

[0040] 2. Cell fusion and cloning

[0041] After the test result of mouse serum was higher, the splenocytes were taken and fused with SP2 / 0 myeloma cells at a ratio of 8:1 (quantity ratio), and the cell supernatant was measured by indirect competition ELISA, and the positive wells were screened. Positive wells were cloned by limiting dilution until a hybridoma cell line secreting flumetramide monoclonal antibody was obtained.

[0042] 3. Cell cryopreservation and recovery

[0043] The monoclonal hybridoma cell line was made into 1×10 6 cells / mL for long-term storage in liquid nitrogen. When recovering, take out the cryopreservation tube, put it into a 37°C water bath to thaw quickly, remove the cryopreservation solution by cent...

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Abstract

The invention provides a preparation method and application of flumetralin hapten and antigen. The preparation method is characterized in that the flumetralin hapten is prepared by reacting 2-chloro-1,3-dinitro-5-trifluoromethylbenzene with 3-amino-3-(2-chloro-6-fluorophenyl)-propionic acid to produce 3-(2-chloro-6-fluorophenyl)-3-(2,6-dinitro-4-trifluoromethyl-phenylamino)-propionic acid and thenfurther reacting 3-(2-chloro-6-fluorophenyl)-3-(2,6-dinitro-4-trifluoromethyl-phenylamino)-propionic acid with iodoethane; and the flumetralin antigen is obtained by coupling the flumetralin hapten to a carrier protein. The antigen prepared in the invention presents a specific flumetralin antigenic determinant, making it possible to screen out highly-specific flumetralin monoclonal antibodies. The produced antibodies have high specificity and high sensitivity, and can be used for establishing an enzyme-linked immunosorbent assay and a colloidal gold test paper rapid-determination method so asto realize rapid detection of flumetralin in tobacco and food.

Description

technical field [0001] The invention relates to a preparation method and application of a flumetonide hapten and an antigen. The invention belongs to the technical field of pesticide immunochemistry. Background technique [0002] Flumetralin is a contact and local systemic dinitroaniline plant growth regulator that inhibits tobacco lateral buds. It is an excellent tobacco bud suppressant. In 1977, it was successfully developed by Ciba-Geigy in Switzerland. In 1990, it was officially registered in my country under the trade name Prime, with the registration number PDll6-90. It is a new type of high-efficiency sprout inhibitor that is popular in the world. It is suitable for flue-cured tobacco, open flame flue-cured tobacco, Maryland tobacco, sun-cured tobacco, and cigar tobacco. Zhejiang Research Institute of Chemical Industry in China was the first to conduct research and development, and successfully developed it in 1998, and obtained temporary registration of pesticides...

Claims

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Application Information

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IPC IPC(8): C07C227/18C07C229/34C07K14/765C07K14/77C07K14/795C07K14/435G01N33/577G01N33/558
CPCC07C227/18C07K14/435C07K14/765C07K14/77C07K14/795C07K19/00G01N33/558G01N33/577C07C229/34
Inventor 陈黎范子彦刘惠民唐纲岭赵乐崔华鹏樊美娟何方洋李旭鲁亚辉
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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