Preparation method and application of a kind of iprodione hapten and antigen

A technology of iprodione and hapten, which is applied in the field of preparation of iprodione hapten and antigen, can solve the problems of poor timeliness, cumbersome processing, and time-consuming, and achieve rapid detection, high sensitivity, and high antibody specificity

Active Publication Date: 2021-09-03
ZHENGZHOU TOBACCO RES INST OF CNTC +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 a kind of iprodione hapten and antigen
  • Preparation method and application of a kind of iprodione hapten and antigen
  • Preparation method and application of a kind of iprodione hapten and antigen

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Experimental program
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Embodiment 1

[0029] The preparation of embodiment 1 iprodione hapten

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

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

[0032] 2) Add the above white solid into 150mL 6% sodium hydroxide aqueous solution, heat to 90°C for 3h under stirring, cool to room temperature, extract a small amount of unreacted raw materials and by-products with ethyl acetate, and use 4N Adjust the pH value to 2 with hydrochloric acid, and filter to obtain a white solid

[0033]...

Embodiment 2

[0040] The preparation of embodiment 2 iprodione antigen

[0041] 1. Synthesis of Iprodione Immunogen

[0042] Iprodione hapten is coupled 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 hours to obtain hapten activation solution A; 50mg of bovine serum albumin (BSA) was fully dissolved in 3.8mL of phosphate buffered saline (PBS) to obtain solution B, and solution A was added dropwise to solution B, stirred at room temperature for 5 hours, and dialyzed against 0.01mol / L PBS at 4°C for 3 hours. day, 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] The iprodione hapten was coupled with ovalbumin (OVA) to obtain the coating source.

[0046] Take 7.0 mg of iprodione...

Embodiment 3

[0055] The preparation of embodiment 3 iprodione monoclonal antibody

[0056] 1. Obtaining hybridoma cells

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

[0058] 2) Booster immunization twice: starting 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 immunogen solution without any adjuvant, and then executed three days later Mice, whose spleen w...

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Abstract

A preparation method and application of an iprodione hapten and an antigen. The iprodione hapten is produced by reacting 3,5-dichlorophenylisocyanate and glycine ethyl ester hydrochloride to produce hydrolysis, and then undergoes a cyclization reaction to obtain 3- (3,5‑dichlorophenyl)‑2,4‑imidazolidinedione, reacted with 6‑isocyanatocaproic acid methyl ester obtained by reaction of 6‑aminocaproic acid methyl ester hydrochloride with triphosgene Generate 6‑(3‑(3,5‑dichlorophenyl)‑2,4‑dioximidazolidinyl‑1‑carboxamido) methyl hexanoate, which is finally obtained by hydrolysis under acidic conditions; iprodione antigen It is obtained by coupling iprodione hapten with carrier protein. The antigen prepared by the invention presents specific iprodione epitope, which makes it possible to screen highly specific iprodione monoclonal antibody. The antibody produced has high specificity and high sensitivity, and can be used for enzyme-linked immunosorbent assay and rapid assay with 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 (Iprodione) belongs to dicarboximides, is a broad-spectrum contact type protective fungicide, widely used in tobacco, fruit trees, 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 2mg / kg, the maximum residue limit in tomato, apple and pear is 5mg / kg, and in grape , The maximum ...

Claims

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Application Information

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Patent Type & Authority Patents(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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