Novel hapten for tebuconazole as well as synthetic method and application thereof

A synthetic method and hapten technology applied in the field of hapten to achieve the effect of simple synthetic method, high sensitivity and strong specificity

Active Publication Date: 2017-05-17
HENAN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

So far, there is no report on the enzyme-linked immunoassay

Method used

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  • Novel hapten for tebuconazole as well as synthetic method and application thereof
  • Novel hapten for tebuconazole as well as synthetic method and application thereof
  • Novel hapten for tebuconazole as well as synthetic method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0049] (2) Preparation of immunogen

[0050] Weigh tebuconazole hapten (4.1 mg, 0.01 mmoL) and add it to NN-dimethylformamide (DMF, 1 mL), stir to dissolve, add 1-ethyl-carbodiimide hydrochloride ( EDC, 4.5 mg) and N-hydroxysuccinimide (NHS, 4.4 mg) were reacted at room temperature for 6 hours to obtain liquid A.

[0051] Take hemocyanin (KLH, 20.92 mg / mL, 0.3 mL) and add it to the borate buffer solution (0.2 mol / L, 3.5 mL, pH 8.8), stir and mix well to obtain liquid B. Under an ice bath, add liquid A to liquid B dropwise, stir and react at 4°C for 8-10 hours.

[0052] The reaction solution was dialyzed with phosphate buffer solution (0.01mol / L, pH 7.2) for 3 days, and the dialysate was changed every 6 hours, 8 times. The obtained tebuconazole immunogen is aliquoted, stored frozen and reserved.

[0053] (3) Preparation of the original coating

[0054] Weigh tebuconazole hapten (4.1 mg, 0.01 mmol) and dissolve in NN-dimethylformamide (DMF, 1 mL), then add tributylamine (8 μL) and isob...

Example Embodiment

[0066] Example 1 Synthesis and identification of tebuconazole hapten

[0067] Step 1: Synthesis of intermediate products

[0068] First dissolve tebuconazole (184 mg, 0.6 mmol) in dry dichloromethane (CH 2 Cl 2 , 15 mL), then add triethylamine (C 6 H 15 N, 730 mg, 7.2 mmol). After stirring on an ice bath for 5 minutes, add monoethyl succinate monochloride (C 6 H 9 ClO 3 , 988 mg, 6 mmol), stirred overnight at room temperature, washed with water and saturated brine (15 mL×3 respectively), dried dichloromethane with anhydrous sodium sulfate, filtered, evaporated under reduced pressure and concentrated to dryness to obtain The intermediate product (about 400mg) was directly used in the next step of hydrolysis. The structure of the product was characterized by liquid-mass spectrometry.

[0069] Step 2: Synthesis of Tebuconazole Hapten

[0070] The intermediate product (400 mg, 0.6 mmol) was dissolved in anhydrous methanol-tetrahydrofuran (9 mL: 9 mL) mixture, and an aqueous solution of l...

Example Embodiment

[0073] Example 2 Preparation and identification of tebuconazole antigen

[0074] 1. The tebuconazole hapten is coupled with hemocyanin (KLH) by the active ester method (EDC method) and used as an immunogen. The specific reaction route is as follows:

[0075]

[0076] The tebuconazole hapten is conjugated with chicken ovalbumin (OVA) or bovine serum albumin (BSA) by the mixed acid anhydride method and used as the coating agent. The specific reaction route is as follows:

[0077]

[0078] Description: Among them, Protein is a protein.

[0079] 2. Purification of artificial antigen

[0080] The artificial antigen was desalted and purified by dialysis.

[0081] 3. Identification of artificial antigens

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Abstract

The invention relates to a novel hapten for tebuconazole as well as a synthetic method and application thereof. The structural formula of the novel hapten for tebuconazole is as shown in the specification. The novel hapten for tebuconazole uses the tebuconazole as a raw material. The synthetic route of the novel hapten comprises the following steps: firstly dissolving the tebuconazole into anhydrous dichloromethane, after dissolving, adding triethylamine, stirring under an ice-bath, then adding ethyl succinyl monochloride, stirring in room temperature, staying overnight, washing, drying, filtering, and concentrating to be dry to obtain an intermediate product; dissolving the intermediate product into a mixed solution of absolute methanol and tetrahydrofuran, then adding a water solution of lithium hydroxide monohydrate, stirring in room temperature, staying overnight, adjusting the pH value in an ice bath, adopting a reversed-phase column for purification, concentrating and freeze-drying, and recycling to obtain a colorless oily solid, namely the novel hapten for tebuconazole. According to the method disclosed by the invention, the synthetic method of the novel hapten for tebuconazole is simple, and the tebuconazole antibody prepared by adopting the hapten is high in sensitivity and can be used for establishing an enzyme linked immunosorbent assay for tebuconazole residue detection.

Description

technical field [0001] The invention relates to a hapten, in particular to a novel hapten of tebuconazole and its synthesis method and application. Background technique [0002] Tebuconazole, English name Tebuconazole, is a hydroxyethyl triazole derivative, which is a low-toxic, high-efficiency, broad-spectrum triazole systemic fungicide, and is a sterol demethylation inhibitor. The biosynthesis of ergosterol in the cell membrane of the pathogen makes the pathogen dead due to the inability to form a cell membrane, and can effectively prevent rust and powdery mildew of cereal crops; leaf spot, ring spot and scab of apples and bananas. However, when tebuconazole is widely used for field disease control for a long time, due to its long duration, it has caused serious "3R" (Resistance, Resurgence and Residue) problems; in addition, the study also found that Tebuconazole has certain lethal and teratogenic effects on zebrafish embryo development, and has toxic accumulation and ge...

Claims

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Application Information

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IPC IPC(8): C07D249/08C07K14/795C07K16/44G01N33/535
CPCC07D249/08C07K14/795C07K16/44G01N2430/00
Inventor 陈秀金李兆周王耀高红丽李智丽曹力李道敏胥传来
Owner HENAN UNIV OF SCI & TECH
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