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Enzyme linked immunosorbent assay kit for detecting iprodione and application thereof

An enzyme-linked immunosorbent reagent and isobacterial technology, which is applied in measuring devices, instruments, scientific instruments, etc., can solve the problem of few types of substrates, and achieve the effects of enhanced immunogenicity, high specificity, and simple pretreatment methods.

Active Publication Date: 2016-03-09
ZHENGZHOU TOBACCO RES INST OF CNTC +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most of the detection methods of iprodione are liquid chromatography and gas chromatography, which can detect fewer types of substrates, and there are few related patents and literature reports on the detection method of iprodione in tobacco

Method used

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  • Enzyme linked immunosorbent assay kit for detecting iprodione and application thereof
  • Enzyme linked immunosorbent assay kit for detecting iprodione and application thereof
  • Enzyme linked immunosorbent assay kit for detecting iprodione and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] The preparation of embodiment 1 kit components

[0024] 1. Preparation of iprodione hapten

[0025] Synthesis of compound a: Take 5.0g of 2,6-dichloro-4-nitrophenol and 3.2g of tert-butyl 4-bromobutyrate and stir in acetone, then add 2.6g of anhydrous potassium carbonate as a catalyst and react at 60°C 8h, after the reaction stopped, the solvent was evaporated to dryness, then extracted with water and ethyl acetate, dried with anhydrous sodium sulfate, concentrated, passed through a 200-300 mesh silica gel column, separated and purified by chromatography to obtain 5.23g of compound a, with a yield of 87% . 1HNMR (CDCl3, 300MHZ) δ: 7.945(1H,d,J=0.000),4.144(2H,t,J=7.500),7.945(1H,d,J=0.000),1.973(2H,tt,J=7.500 ,J=7.367),2.359(2H,t,J=7.367),1.414(3H),1.414(3H),1.414(3H,s).

[0026] Synthesis of compound b: add 3.1g of zinc powder and 1mL of glacial acetic acid to 20mL of water, react at 90°C for 30min, then add 5.2g of compound a in ethanol solution 80mL, react at 60°C...

Embodiment 2

[0054] Embodiment 2 detects the formation of the ELISA kit of iprodione

[0055] An enzyme-linked immunosorbent assay kit for detecting iprodione was set up to include the following components:

[0056] (1) ELISA plate coated with iprodione-conjugated antigen;

[0057] (2) 6 bottles of iprodione standard solution, the concentrations are 0μg / L, 4μg / L, 12μg / L, 36μg / L, 108μg / L, 324μg / L;

[0058] (3) Goat anti-mouse anti-antibody labeled with horseradish peroxidase;

[0059] (4) Iprodione-specific antibody;

[0060] (5) The substrate chromogenic solution is composed of liquid A and liquid B, liquid A is carbamide peroxide, and liquid B is tetramethylbenzidine;

[0061] (6) The stop solution is 2mol / L sulfuric acid;

[0062] (7) The washing solution has a pH value of 7.4, contains 0.5%~1.0% Tween-20, 0.01‰~0.03‰ sodium azide preservative, and 0.1~0.3mol / L phosphate buffer solution, and the percentages are by weight volume percentage;

[0063] (8) The complex solution is a pho...

Embodiment 3

[0064] The detection of iprodione in embodiment 3 tobacco leaves

[0065] 1. Sample pretreatment

[0066] Weigh 1.0±0.05g of tobacco leaf sample into a 50mL polystyrene centrifuge tube, add 10mL of tobacco leaf extract (measure 100ml of methanol, add 100mL of deionized water, and mix well); use a homogenizer to fully break it; Filter the broken sample with a filter membrane; pipette 200mL of the filtrate and add 800mL of complex solution, mix well; take 50mL for analysis.

[0067] 2. Detection with kit

[0068]Add 50 μL of the standard / sample to the corresponding microwell, then add 50 μL / well of the antibody working solution, shake gently to mix, cover the plate with a cover film and place it in a dark environment at 25°C for 30 minutes. Carefully uncover the cover plate membrane, shake off the liquid in the well, wash fully with 250 μL / well of washing working solution for 4-5 times, with an interval of 10 seconds between each time, and pat dry with absorbent paper. Add 10...

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Abstract

The invention provides an enzyme linked immunosorbent assay kit for detecting iprodione and application thereof. The enzyme linked immunosorbent assay kit comprises an elisa plate coated with a coating antigen, an iprodione standard substance solution, an enzyme-labeled secondary antibody, an iprodione specific antibody, a substrate color developing solution, a stop solution, a scrubbing solution and a redissolved solution. The coating antigen is an iprodione coupling antigen, and the enzyme-labeled secondary antibody is an enzyme-labeled goat-anti-mouse antibody. The invention further discloses a method using the enzyme linked immunosorbent assay kit for detecting iprodione. The enzyme linked immunosorbent assay kit can be used for detecting the content of iprodione in tobacco leaf samples, and is easy to operate, low in cost, high in sensitivity, capable of achieving on-site monitoring and suitable for screening a great number of samples.

Description

technical field [0001] The invention relates to an enzyme-linked immunosorbent detection technology, in particular to an enzyme-linked immunosorbent assay kit for detecting iprodione and its application, which can qualitatively and quantitatively detect the residual amount of iprodione in tobacco leaves. Background technique [0002] Iprodione, also known as diphenhydramine, has the molecular formula C 13 h 13 Cl 2 N 3 o 3 , belonging 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. Iprodione pesticide is a testing item with national limit requirements...

Claims

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Application Information

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IPC IPC(8): G01N33/535
CPCG01N33/535
Inventor 陈黎范子彦杨昌松冯静潘立宁胡斌唐纲岭刘惠民鲁亚辉
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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