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Kit for rapidly detecting content of milneb in crops

A Dyson ring and kit technology, which is applied in the field of monoclonal antibody preparation and enzyme-linked immunoassay kit, specific monoclonal antibody, and enzyme-linked immunoassay, can solve the tedious pretreatment steps, long analysis time, detection The problem of large sample volume is required, so as to save time and reduce operation steps

Inactive Publication Date: 2016-01-27
JIANGSU WISE SCI & TECH DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, HPLC is the main method for the determination of Dyson ring. Due to the shortcomings of this method, such as long analysis time, large sample volume required for detection, and cumbersome pretreatment steps, it is not suitable for the determination of a large number of samples.

Method used

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  • Kit for rapidly detecting content of milneb in crops

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The preparation of embodiment 1 kit components

[0044] 1. Antigen synthesis

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

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

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

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

[0049] e. The artificial antigen obtained after the reaction was dialyzed against 0.1M 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 Dysencycline monoclonal antibody

[0051] a. Animal immunization procedure: use Dysencycline artificial antigen as the immunogen t...

Embodiment 2

[0066] Embodiment 2 is used for the formation of the enzyme-linked immunosorbent assay kit of rapid detection of Dyson ring

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

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

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

[0070] (3) The concentration is 1 μg / mL Dyson ring standard;

[0071] (4) Standard diluent: 0.05mol / LTris-HCl, pH8.0, 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 0.05mol / LTris-HCl, pH8.0, 0.9%NaCl, 0.04%Tween20;

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

Embodiment 3

[0076] The detection of Dyson ring residue in the sample of embodiment 3

[0077] 1. Sample pretreatment

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

[0079] b. Fully oscillate and mix for 3-5 minutes, after standing for stratification, take 5mL supernatant and centrifuge at 4000r / min for 5min 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 1mL of standard diluent and mix well, centrifuge at 4000r / min for 5min 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 1000ng / mL Disen ring mother solution, perform double dilution, and dilute to 0, 0.3, 0.9, 2.7, 8.1, 24.3ng / mL 6 concentrations of standard products, according to 100 μL standard or sample to be tested and...

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Abstract

The invention provides an enzyme-linked immunosorbent assay kit capable of detecting milneb in fruits and vegetables, and relates to the immunoassay detection technology. The kit includes an ELISA plate coated with milneb, a milneb antibody, a milneb standard, a standard diluent, an enzyme-labelled secondary antibody, a substrate developing liquid, a washing buffer liquid and a stopping liquid. The key technology comprises that a monoclonal antibody which can identify milneb is prepared. The kit adopts the high-specificity milneb monoclonal antibody, a main reagent is provided in a form of a working liquid, the operation steps can be reduced, and the time is 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 Dysencycline and an 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 present invention can recognize Dysencycline, and the kit and method of the present invention have the advantages of simplicity, rapidity, sensitivity, accuracy and the like. Background technique [0002] Daisenhuan is mainly used for the prevention and control of various diseases on vegetables and fruit trees, and it is also effective in the prevention and treatment of downy mildew and wheat rust on melons and cabbage. Ethylenethiourea, the degradation product of Dysencycline in the environ...

Claims

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Application Information

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IPC IPC(8): G01N33/577
Inventor 洪霞戴蔚蔚杜霞
Owner JIANGSU WISE SCI & TECH DEV
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