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Preparation method and application of iprodione hapten and antigen

A technology for isobacteride and hapten, which is applied in the field of preparation of isobacteride hapten and antigen, can solve the problems of inability to carry out large-scale detection on site, difficult to popularize, and cumbersome to handle, and achieves rapid detection, good affinity, and crossover. The effect of low response rate

Active Publication Date: 2019-01-25
ZHENGZHOU TOBACCO RES INST OF CNTC +2
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  • 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 iprodione hapten and antigen
  • Preparation method and application of iprodione hapten and antigen
  • Preparation method and application of iprodione hapten and antigen

Examples

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Embodiment 1

[0029]Example 1 Preparation of iprodione hapten

[0030] 1. Synthesis of iprodione hapten (synthetic route see figure 1 )

[0031] 1) Add 8.37 g (60 mmol) ethyl glycine hydrochloride and 16.5 mL (120 mmol) triethylamine into 100 mL dichloromethane, add 10 g (53 mmol) 3,5-dichloromethane dropwise at 0°C Dichloromethane solution of phenylisocyanate, stirred at room temperature overnight, filtered to remove insoluble solids, the filtrate was washed with 2 N hydrochloric acid, saturated sodium bicarbonate solution, and saturated brine, and the organic layer was dried with anhydrous magnesium sulfate. After distilling off the solvent, a white Solid ethyl 2-(3-(1,5-dichlorophenylureido))acetate.

[0032] 2) Add the above white solid into 150 mL of 6% sodium hydroxide aqueous solution, heat to 90°C for 3 hours under stirring, cool to room temperature, extract a small amount of unreacted raw materials and by-products with ethyl acetate, and use the water phase at 0°C The pH was adj...

Embodiment 2

[0040] Example 2 Preparation of iprodione antigen

[0041] 1. Synthesis of Iprodione Immunogen

[0042] Iprodione hapten is conjugated with bovine serum albumin (BSA) to obtain immunogen.

[0043] Take 9.0 mg of iprodione hapten, dissolve it in 1.0 mL of dimethylformamide (DMF), add 0.18 mL of isobutyl chloroformate, add 0.3 mL of pyridine, and stir at room temperature for 5 h to obtain hapten activation solution A; Take 50 mg of bovine serum albumin (BSA) and fully dissolve it in 3.8 mL of phosphate buffered saline (PBS) to obtain liquid B. Add liquid A to liquid B dropwise, stir at room temperature for 5 h, and add 0.01 mol / L PBS at 4 °C for 3 days to remove unreacted small molecular substances to obtain iprodione-BSA immunogen; store at -20 °C for future use.

[0044] 2. Synthesis of iprodione coating agent

[0045] Iprodione hapten was coupled with ovalbumin (OVA) to obtain the coating source.

[0046] Take 7.0 mg of iprodione hapten, dissolve it in 1.0 mL of DMF, add ...

Embodiment 3

[0055] Example 3 Preparation of iprodione monoclonal antibody

[0056] 1. Obtaining hybridoma cells

[0057] 1) First immunization: fully emulsify the iprodione hapten-BSA conjugate (immunogen) with the same amount of complete Freund's adjuvant, and subcutaneously inject 6-week-old Balb / c mice at an immunization dose of 150 μg / Only;

[0058] 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;

[0059] 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 myelom...

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Abstract

The invention provides a preparation method and application of iprodione hapten and antigen. The iprodione hapten is obtained through the following steps: taking 3,5-dichlorophenyl isocyanate and ethyl glycinate hydrochloride to react to generate ethyl 2-(3-(1,5-dichlorophenylcarbamido)) acetate, hydrolyzing to obtain 2-(3-(1,5-dichlorophenylcarbamido))acetic acid, carrying out ring-forming reaction to obtain 3-(3,5-dichlorophenyl)-2,4-imidazolidindione, reacting with methyl 6-isocyanatocaproate generated by reaction of methyl 6-aminohexanoate hydrochloride and triphosgene, so as to generate methyl 6-(3-(3,5-dichlorophenyl)-2,4-dioxyiminazolealkyl-1-formylamino)caproate; finally, hydrolyzing under an acidic condition. The iprodione antigen is obtained by coupling the iprodione hapten and carrier protein. The antigen prepared by the invention has a specific iprodione antigenic determinant, so that a high-specificity iprodione monoclonal antibody is possibly screened. A generated antibody has high specificity and high sensitivity and can be used for enzyme-linked immunization and rapid determination of test paper.

Description

technical field [0001] The invention relates to a preparation method and application of iprodione hapten and antigen. The invention belongs to the technical field of pesticide immunochemistry. Background technique [0002] Iprodione belongs to dicarboximides, and is a broad-spectrum contact-type protective fungicide, widely used in tobacco, fruit tree, vegetable disease control and fruit storage and preservation. Iprodione can be absorbed systemically through roots, and can effectively control fungi that are resistant to benzimidazole systemic fungicides. Its main control targets are diseases caused by Botrytis, Alternaria, and Sclerotinia, such as Botrytis cinerea, early blight, black spot, and Sclerotinia. For different crops, my country has formulated the maximum residue limit standard of iprodione, among which the maximum residue limit in rapeseed and cucumber is 2 mg / kg, and the maximum residue limit in tomato, apple and pear is 5 mg / kg. The maximum residue limit in g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D233/80C07K14/435C07K14/765C07K14/77C07K14/795G01N33/532G01N33/535G01N33/558G01N33/577
CPCC07D233/80C07K14/435C07K14/765C07K14/77C07K14/795C07K19/00G01N33/532G01N33/535G01N33/558G01N33/577
Inventor 陈黎范子彦刘惠民唐纲岭樊美娟崔华鹏颜权平万宇平申梁吴小胜
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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