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Preparation and application of pendimethalin hapten and antigen

A pendimethalin and hapten technology, which is applied in the preparation of hydrazone, animal/human protein, serum albumin, etc., to achieve high specificity, rapid detection, and high sensitivity

Active Publication Date: 2019-01-25
ZHENGZHOU TOBACCO RES INST OF CNTC +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Example 1 Preparation of pendimethalin hapten

[0027] 1. Synthesis of pendimethalin hapten (see attached figure 1 )

[0028] Take 0.60 g of 3-(1-ethylpropylamine)-6-methyl-2,4-dinitrobenzaldehyde, add 50 mL of acetonitrile to dissolve, after clarification, add dropwise the solution of 0.32 g of 3-hydrazinopropionic acid 5 mL of methanol solution, stirred at room temperature for 3 h; stop the reaction, remove the organic solvent by rotary evaporation, add water, add 0.45 g of sodium hydroxide, dissolve and clarify, add 80 mL of ethyl acetate for extraction, separate the organic phase, add dilute hydrochloric acid to the water phase Adjust the pH to 4, add 50 mL of chloroform for extraction, wash with water, concentrate, and recrystallize with ethanol / petroleum ether at a volume ratio of 1:1 to obtain 0.70 g of the hapten product with a yield of 90.1%.

[0029] 2. Identification of pendimethalin hapten

[0030] The above haptens were identified by H NMR spectroscopy, ...

Embodiment 2

[0031] Example 2 Preparation of pendimethalin antigen

[0032] 1. Synthesis of pendimethalin immunogen

[0033] The pendimethalin hapten was conjugated with bovine serum albumin (BSA) to obtain the immunogen.

[0034]Take 15 mg hapten and dissolve it in 1 mL N,N-dimethylformamide (DMF); add 8 mg carbodiimide (EDC) and stir at room temperature for 24 h to obtain reaction solution A; weigh BSA 30 mg, fully dissolved in 4 mL 0.1 mol / L phosphate buffer (PB, pH 7.0), slowly added the reaction solution A to the protein solution drop by drop, and stirred at room temperature for 24 h, with 0.01 mol / L Phosphate buffered saline (PBS) dialyzed at 4 °C for 3 days, and the dialysate was changed 3 times a day to remove unreacted small molecular substances to obtain immunogens, 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 20 mg of hapte...

Embodiment 3

[0040] Example 3 Preparation of Pendimethalin Monoclonal Antibody

[0041] 1. Obtaining hybridoma cells

[0042] 1) First immunization: Fully emulsify the pendimethalin hapten-BSA conjugate (immunogen) with an equal amount of Freund's complete 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 discloses preparation and application of a pendimethalin hapten and antigen. Preparation of the pendimethalin hapten and antigen is characterized in that the pendimethalin hapten is prepared from 3-(1-ethyl aminopropylamino)-6-methyl-2,4-dinitrobenzaldehyde and 3-hydrazino propionic acid which are reacted; the pendimethalin antigen is obtained by coupling the pendimethalin hapten andcarrier protein. The prepared antigen presents a specific pendimethalin antigenic determinantantigen epitope, so that screening out a high-specificity pendimethalin monoclonal antibody becomes possible; the generated antibody is high in specificity and high in sensitivity, and can be used for building an enzyme-linked immunosorbent assay method and a colloidal gold test strip rapid determinationmethod, so that pendimethalin in tobacco and food is fast detected.

Description

technical field [0001] The invention relates to the preparation and application of pendimethalin hapten and antigen, and belongs to the technical field of pesticide immunochemistry. Background technique [0002] Pendimethalin is a selective herbicide for upland crops, which can be widely used in corn, soybean, peanut, cotton, direct-directed upland rice, potato, tobacco, vegetables and other crop fields for weeding. At present, pendimethalin is the third largest herbicide in the world, second only to glyphosate and paraquat in sales, and it is also the largest selective herbicide in the world. Therefore, it is widely used in agricultural production in our country. At the same time, the residue of pendimethalin in crops has also received high attention. my country stipulates that the maximum residue limit (MRL) of pendimethalin in rice is 0.2 mg / kg, and the MRL in brown rice, corn and cottonseed is 0.1 mg / kg, the MRL in leeks, cabbage, spinach, celery, Chinese cabbage and ot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07C251/86C07C249/16C07K14/435C07K14/765C07K14/77C07K14/795G01N33/532G01N33/535G01N33/558G01N33/577
CPCC07C251/86C07K14/435C07K14/765C07K14/77C07K14/795C07K19/00G01N33/532G01N33/535G01N33/558G01N33/577
Inventor 陈黎范子彦刘惠民唐纲岭崔华鹏樊美娟潘立宁鲁亚辉朱亮吴小胜
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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