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Preparation method and application of carbendazim hapten and antigen

A carbendazim and hapten technology, applied in the field of preparation of carbendazim haptens and antigens, can solve the problems of inability to perform large-scale on-site detection, poor timeliness, and cumbersome processing, and achieve rapid detection, good affinity, and crossover. The effect of low response rate

Inactive Publication Date: 2019-01-25
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

The instrument method has the advantages of high detection sensitivity and strong specificity, but the pretreatment of the detection sample is cumbersome and time-consuming, and the sample needs to be extracted and purified. At the same time, the instrument detection method requires expensive large-scale instruments and equipment. Operation and management, unable to conduct large-scale on-site testing, poor timeliness, difficult to promote

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Preparation of carbendazim hapten

[0025] 1. Synthesis of carbendazim hapten (see the synthetic route figure 1 )

[0026] Dissolve 1.05 g (10.5 mmol) of succinic anhydride in 100 mL of acetonitrile, add 1.33 g (10 mmol) of 2-aminobenzimidazole in portions under heating and reflux, continue to reflux for 1 hour, cool to room temperature, filter to obtain a solid, and , washed with acetonitrile and dried to obtain 2.00 g of carbendazim hapten with a yield of 86%.

[0027] 2. Identification of carbendazim hapten

[0028] 1H NMR (DMSO-d6): 11.95 (br, 3H), 7.43 (d,d, J1 = 5.53 Hz, J2 =5.61 Hz), 7.07 (d,d, J1 = 5.53 Hz, J2 = 5.61 Hz) , 2.70 (d, J = 6.61 Hz, 2H), 2.58 (d, J = 6.61 Hz, 2H).

[0029] In the spectrum, chemical shifts δ=2.70 and 2.58 are resonance absorption peaks of methylene hydrogen on the spacer arm. The existence of these peaks proves that the spacer arm coupling is successful and the structure of the carbendazim hapten is correct.

Embodiment 2

[0030] Example 2 Preparation of carbendazim antigen

[0031] 1. Synthesis of carbendazim immunogen

[0032] The carbendazim hapten was conjugated with bovine serum albumin (BSA) to obtain the immunogen.

[0033] Take 15 mg of carbendazim hapten, dissolve it in 1 mL of N,N-dimethylformamide (DMF), add 0.22 mL of oxalyl chloride, and stir overnight to obtain hapten activation solution A; take bovine serum albumin ( BSA) 50 mg, add 3.6 mL of 0.05 M borate buffer solution to dissolve to obtain liquid B, add liquid A dropwise to liquid B, stir at 4°C for 5 h, stop the reaction, dialyze with 0.01 mol / L PBS at 4°C for 3 d. Change the dialysate 3 times a day. Aliquoted to obtain the carbendazim-BSA antigen and stored at -20°C for future use.

[0034] 2. Synthesis of carbendazim coating agent

[0035] The carbendazim hapten was coupled with ovalbumin (OVA) to obtain the coating source.

[0036] Take 13 mg of carbendazim hapten, dissolve it in 1 mL of DMF, add 0.21 mL of isopropyl ...

Embodiment 3

[0045] Example 3 Preparation of carbendazim monoclonal antibody

[0046] 1. Obtaining hybridoma cells

[0047] 1) The first immunization: fully emulsify the carbendazim hapten-BSA conjugate (immunogen) with 3 times Freund's complete adjuvant, and subcutaneously inject Balb / c mice aged 8-10 weeks at an immunization dose of 150 μg / piece;

[0048] 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;

[0049] 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 myel...

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Abstract

The invention discloses a preparation method and application of a carbendazim hapten and antigen. The method is characterized in that the carbendazim hapten is prepared from 2-aminobenzimidazole and butanedioic anhydride which are reacted; the carbendazim antigen is obtained by coupling the carbendazim hapten and carrier protein. The antigen prepared through the method presents a specific carbendazim antigenic determinantantigen epitope, so that screening out a high-specificity carbendazim 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 determination method, so that carbendazim in tobacco and food is fast detected.

Description

technical field [0001] The invention relates to a preparation method and application of carbendazim hapten and antigen. The invention belongs to the technical field of pesticide immunochemistry. Background technique [0002] Carbendazim (Carbendazim) is a benzimidazole fungicide. It is a good broad-spectrum and systemic fungicide. It is effective against most pathogenic bacteria in Ascomycetes, Basidiomycetes and half-knowledge fungi. It is widely used in Disease control of crops, Chinese herbal medicine, tobacco, etc. Carbendazim has stable chemical properties and can be absorbed by seeds, roots and leaves of plants. It has a long residual effect period and has certain toxicity to humans and animals. For different crops, my country has formulated the maximum residue limit standard of carbendazim, among which, the maximum residue limit of carbendazim in grains is between 0.05 - 2 mg / kg, and the maximum residue limit in vegetables is 0.02 - 5mg / kg. kg, the maximum residue l...

Claims

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Application Information

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IPC IPC(8): C07D235/30C07K14/435C07K14/765C07K14/77C07K14/795G01N33/532G01N33/535G01N33/558G01N33/577
CPCC07D235/30C07K14/435C07K14/765C07K14/77C07K14/795C07K19/00G01N33/532G01N33/535G01N33/558G01N33/577
Inventor 陈黎范子彦刘惠民唐纲岭樊美娟崔华鹏潘立宁鲁亚辉冯才伟吴小胜
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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