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Enzyme-linked immunosorbent assay kit for detecting carbendazim and its application

A technology of immunological reagents and carbendazim enzymes, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., to achieve the effects of high accuracy, low detection limit and simple structure

Active Publication Date: 2017-06-30
ZHENGZHOU TOBACCO RES INST OF CNTC +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no simple and fast detection method

Method used

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  • Enzyme-linked immunosorbent assay kit for detecting carbendazim and its application
  • Enzyme-linked immunosorbent assay kit for detecting carbendazim and its application
  • Enzyme-linked immunosorbent assay kit for detecting carbendazim and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1 Preparation of kit components

[0029] 1. Preparation of carbendazim hapten

[0030] The carbendazim raw material drug is subjected to nitration reaction, and a nitro group is introduced on the benzene ring, and a hapten product with an aromatic amine is obtained after reduction.

[0031] Add 20 mL of trifluoroacetic acid and 2 mL of trifluoroacetic anhydride, take an ice-water bath, lower to 0°C, add 0.5 g of ammonium nitrate, stir for 1 h, add 1.0 g of carbendazim in trifluoroacetic acid solution, continue stirring, and react for 2 h . Stop the reaction, neutralize to neutral with dilute sodium hydroxide solution, extract with dichloromethane, wash with water, evaporate to dryness, wash and crystallize with ether to obtain 0.76 g of compound a with a yield of 67%. 1H NMR(CDCl3, 300MHZ)δ: 8.31 (1H, dd, J=1.616, J=1.239), 7.69 ( 1H, dd, J=8.716, J=1.616), 7.64 (1H, dd, J=8.716, J =1.239), 3.85(3H, s).

[0032] 0.7 g of compound a was dissolved in ethano...

Embodiment 2

[0056] Example 2 Establishment of an enzyme-linked immunosorbent assay kit for detecting carbendazim

[0057] An enzyme-linked immunosorbent assay kit for detecting carbendazim was constructed to include the following components:

[0058] (1) Enzyme plate coated with carbendazim-conjugated antigen;

[0059] (2) 6 bottles of carbendazim standard solution, the concentrations are 0 µg / L, 0.1 µg / L, 0.3 µg / L, 0.9 µg / L, 2.7 µg / L, 8.1 µg / L;

[0060] (3) Carbendazim anti-antibody labeled with horseradish peroxidase;

[0061] (4) The substrate chromogenic solution is composed of liquid A and liquid B, liquid A is carbamide peroxide, and liquid B is tetramethylbenzidine;

[0062] (5) The stop solution is 2 mol / L sulfuric acid;

[0063] (6) The washing liquid has a pH value of 7.4, contains 0.5%~1.0% Tween-20, 0.01‰~0.03‰ sodium azide preservative, and 0.1~0.3 mol / L phosphate buffer;

[0064] (7) The complex solution is a phosphate buffer solution with a pH value of 7.0 and 0.02 mol / ...

Embodiment 3

[0065] Example 3 Detection of carbendazim in tobacco leaves

[0066] 1. Sample pretreatment

[0067] Weigh 1.0±0.05 g of tobacco leaf sample into a 50 ml polystyrene centrifuge tube, add 10 mL of tobacco leaf extract, and thoroughly crush it with a homogenizer; filter the crushed sample with a filter membrane; pipette the filtrate for 50 Add 700 mL of deionized water to 1 mL, and mix well; then pipette 50 mL of the above liquid and add 950 mL of reconstitution working solution, and mix well; take 50 mL for analysis.

[0068] 2. Detection with kit

[0069] Add 50 mL of standard solution / sample to the corresponding microwell, then add 50 mL / well of antibody working solution, shake and mix gently, cover the plate with a cover film and place it in a light-proof environment at 25°C for 30 min. Carefully peel off the cover film, dry the liquid in the wells, wash fully with 250 mL / well of washing working solution for 4-5 times with an interval of 10 s, and pat dry with absorbent pa...

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Abstract

The invention provides a carbendazim detection ELISA (enzyme linked immunosorbent assay) kit which includes an ELISA plate coated with a coating antigen, a carbendazim standard solution, a carbendazim antibody, an enzyme-labeled secondary antibody, a substrate coloring liquid, a terminating liquid, a washing liquid and a complex solution, the coating antigen is a carbendazim coupling antigen, and the enzyme-labeled secondary antibody is an enzyme labeled carbendazim antibody. The invention also discloses a carbendazim detection method using the ELISA kit, and the method includes first, pretreatment of a sample, then detection using the ELISA kit and final detection result analyzing. The ELISA kit can be used to detect the content of carbendazim in a tobacco leaf sample, and has the advantages of being simple in operation, low in cost, high in sensitivity, capable of monitoring on site and suitable for screening a large 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 carbendazim and its application, which can qualitatively and quantitatively detect the residual amount of carbendazim in tobacco leaves. Background technique [0002] my country is a big country that produces and uses chemical pesticides. The harm and impact of long-term use of pesticides on the ecological environment and human health has aroused people's great concern. Carbendazim [carbendazim, N-(2-benzopyrazolyl) carbamate methyl ester] is a benzopyrazole, which is a good broad-spectrum, systemic fungicide for Ascomycetes, Basidiomycetes and Most pathogenic bacteria in half-known fungi are effective, and are widely used in the disease prevention and control of crops and Chinese herbal medicine. Carbendazim has stable chemical properties and can be absorbed by seeds, roots and leaves of plants. It has ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577
CPCG01N33/577
Inventor 范子彦陈黎鲁亚辉罗晓琴潘立宁胡斌唐纲岭刘惠民赵正苗
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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