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Kit for rapidly detecting amitraz content in crops

A technology of amitraz and a kit, which is applied in the fields of pesticide residue analysis and immunoassay detection, can solve problems such as livestock poisoning and water pollution, and achieve the effect of saving time and reducing operation steps

Inactive Publication Date: 2017-04-26
ZHENJIANG YITE BIOTECH DEV CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in daily production and life, due to unreasonable use, water pollution and livestock poisoning caused by amitraz occur from time to time

Method used

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  • Kit for rapidly detecting amitraz content in crops

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Preparation of kit components

[0044] 1. Antigen synthesis

[0045] a. Dissolve 10 g of carrier bovine serum albumin (BSA) in 35 mL of pH9.6 carbonate buffer, add 10 g of ethylenediamine for activation;

[0046] b. Dissolve 1 g of amitraz in 10 mL of 0.5 M sodium hydroxide solution;

[0047] c. Dissolve 1 g of carbodiimide in 5 mL of pure water, then add it to the amitraz solution and stir at room temperature for 3 hours;

[0048] d. Add the activated carrier protein BSA dropwise to the amitraz solution and stir at 4°C overnight;

[0049] e. The artificial antigen obtained after the reaction was dialyzed against 0.1 M PBS buffer for 5 days, and the buffer was changed 4 times a day; the obtained purified artificial antigen was concentrated by ultrafiltration or lyophilized for storage.

[0050] 2. Preparation of amitraz monoclonal antibody

[0051] a. Animal immunization procedure: BALB / C mice were immunized with amitraz artificial antigen as the immu...

Embodiment 2

[0066] Example 2 The establishment of an enzyme-linked immunosorbent assay kit for the rapid detection of amitraz

[0067] This ELISA kit consists of the following components:

[0068] (1) ELISA plate coated with amitraz antigen;

[0069] (2) Amitraz monoclonal antibody working solution with a protein concentration of 0.5 μg / L;

[0070] (3) Amitraz standard with a concentration of 1 μg / mL;

[0071] (4) Standard diluent: 0.05 mol / L Tris-HCl, pH 8.0, 0.9% NaCl buffer;

[0072] (5) Goat anti-mouse enzyme-labeled secondary antibody working solution labeled with horseradish peroxidase;

[0073] (6) Hydrogen peroxide at the liquid level of substrate chromogenic solution A, and o-phenylenediamine at the liquid level of substrate chromogenic solution B;

[0074] (7) The washing buffer is 0.05 mol / L Tris-HCl, pH 8.0, 0.9% NaCl, 0.04% Tween20;

[0075] (8) The stop solution is 2 mol / L sulfuric acid.

Embodiment 3

[0076] Example 3 Detection of amitraz residues in samples

[0077] 1. Sample pretreatment

[0078] a. Take 1 g of pulverized and representative sample, add 5 mL of acetonitrile;

[0079] b. Thoroughly oscillate and mix for 3-5 minutes, after static layering, take 5 mL supernatant, centrifuge at 4000 r / min for 5 min or filter with quantitative analysis filter paper;

[0080] c. Take 1 mL of the centrifuged supernatant or filtered filtrate, and dry it with nitrogen in a water bath at 50-60 °C;

[0081] d. Add 1 mL of standard diluent and mix well, centrifuge at 4000 r / min for 5 min or filter with quantitative analysis filter paper;

[0082] e. Take the centrifuged supernatant or filtered filtrate for analysis.

[0083] 2. Detection with kit

[0084] Prepare the stock solution of amitraz with a concentration of 1000 ng / mL, carry out doubling dilution, and dilute to 0, 0.3, 0.9, 2.7, 8.1, 24.3 ng / mL 6 concentrations of standard products, according to 100 μL of standard pr...

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Abstract

The present invention provides an enzyme-linked immunoassay kit capable of detecting amitraz in fruits and vegetables, and relates to an immunoassay detection technology. The kit comprises an enzyme label plate coated with amitraz, an amitraz antibody, an amitraz standard substance, a standard substance dilution liquid, an enzyme labeled secondary antibody, a substrate coloring solution, a washing buffer solution, and a terminating solution. The key technology of the present invention is to prepare the monoclonal antibody capable of recognizing amitraz. According to the present invention, the kit uses the high-specificity amitraz monoclonal antibody, and the main reagent is provided in the form of the working liquid, such that the operation steps can be reduced, and the time can be saved.

Description

technical field [0001] The invention belongs to the technical field of pesticide residue analysis and immunoassay detection, and in particular relates to the preparation of a monoclonal antibody capable of recognizing amitraz and its enzyme-linked immunoassay kit. The invention discloses a preparation method of a specific monoclonal antibody, a coating agent and an immunogen and an enzyme-linked immunoassay method. Compared with the prior art, the monoclonal antibody prepared by the invention can recognize amitraz, and the kit and method of the invention have the advantages of simplicity, speed, sensitivity, accuracy and the like. Background technique [0002] Amitraz is a broad-spectrum acaricide with a chemical name of N-methylbis(2,4-dimethylbeniminomethyl)amine, which is widely used in the control of fruit trees, tea, cotton, soybeans, sugar beets and other crops. Kind of pest mites. Both amitraz and its metabolites are carcinogenic and transmissible. Inhalation, inges...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/531G01N33/535G01N33/543G01N21/78G01N21/31
CPCG01N21/31G01N21/78G01N33/531G01N33/535G01N33/543G01N33/577
Inventor 杜霞刘静
Owner ZHENJIANG YITE BIOTECH DEV CO LTD
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