Enzyme linked immunosorbent assay kit for detecting zearalenone drug and application thereof

A technology of zearalenone and enzyme-linked immunosorbent reagent, which is used in biological testing, material inspection products, and analysis by chemical reaction of materials, etc., can solve the problems of expensive instruments and cumbersome sample processing.

Active Publication Date: 2013-03-13
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the determination methods of zearalenone mainly include gas chromatography, high performance liquid chromatography, etc. The instrument determination method has high sensitivity, but the sample pretreatment is cumbersome, the instrument is exp

Method used

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  • Enzyme linked immunosorbent assay kit for detecting zearalenone drug and application thereof
  • Enzyme linked immunosorbent assay kit for detecting zearalenone drug and application thereof
  • Enzyme linked immunosorbent assay kit for detecting zearalenone drug and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0057] The preparation of embodiment 1 kit components

[0058] 1. Antigen Synthesis

[0059] a. Synthesis of hapten

[0060] The zearalenone was condensed with hydroxylamine hydrochloride to introduce a new hydroxyl group, and then reacted with succinic anhydride to obtain a hapten with a carboxyl functional group.

[0061] Specific steps for the hapten: 159mg of zearalenone and hydroxylamine hydrochloride were stirred in 5ml of pyridine solution at room temperature for 12-24 hours, and the pyridine and unreacted hydroxylamine were removed by rotary evaporation to obtain a crude product. Add 5ml of tetrahydrofuran and 200μl of pyridine, and stir at room temperature After half an hour, 50 mg of succinic anhydride dissolved in 1 ml of tetrahydrofuran solution was added dropwise, reacted at room temperature for 12-24 hours, the solvent and pyridine were removed by rotary evaporation, and purified by column chromatography to obtain zearalenone hapten.

[0062] b. Immunogen synth...

Embodiment 2

[0095] Example 2 The formation of the ELISA kit for detecting zearalenone

[0096] An enzyme-linked immunosorbent assay kit for detecting zearalenone was set up to include the following components:

[0097] (1) A microtiter plate coated with a zearalenone-coupled antigen;

[0098] (2) Goat anti-mouse anti-antibody labeled with horseradish peroxidase;

[0099] (3) Zearalenone monoclonal antibody working solution;

[0100] (4) 6 bottles of zearalenone standard solution, the concentrations are 0 μg / L, 2 μg / L, 6 μg / L, 20 μg / L, 60 μg / L, 120 μg / L;

[0101] (5) The substrate chromogenic solution is composed of A liquid and B liquid, the substrate chromogenic liquid A liquid is carbamide peroxide, and the substrate chromogenic liquid B liquid tetramethylbenzidine;

[0102] (6) The stop solution is 2mol / L hydrochloric acid;

[0103] (7) The concentrated washing solution is pH 7.2-7.5, containing 0.1-0.2mol / L phosphate buffer solution of 0.8-1.2% Tween-20, 0.3-0.6‰ sodium azide, and...

Embodiment 3

[0104] The detection of zearalenone in the actual sample of embodiment 3

[0105] Sample pretreatment

[0106] Weigh 2.0±0.05g feed sample, add 10ml 60% methanol, shake vigorously for 5min, and centrifuge at room temperature for 5min above 3000g; take 500μl supernatant and add 500μl deionized water to mix evenly; take 20μl for analysis.

[0107] 2. Detection with kit

[0108] Add 20 μl of zearalenone standard solution or sample solution to the microwells of the enzyme-labeled plate coated with zearalenone-conjugated antigen, randomly add 50 μl of enzyme-labeled secondary antibody, and then add zearalenone monoclonal Antibody working solution 80 μl, seal the plate with a cover plate, react in a 25°C incubator for 20 minutes, pour out the liquid in the well, add 250 μl of washing solution in each well, pour out the liquid in the well after 30 seconds, repeat this operation for a total of 5 washes, Finally, use absorbent paper to remove residual water, add substrate chromogenic...

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Abstract

The invention provides an enzyme linked immunosorbent assay kit for detecting zearalenone drugs and an application thereof; the kit comprises an enzyme label plate coated with a coating antigen, an enzyme label, a zearalenone specific antibody working fluid (which is included when the enzyme label plate is coated with the coating antigen and the enzyme label is an enzyme labeled antiantibody, or when the enzyme label plate is coated with an antiantibody and the enzyme label is an enzyme labeled antigen), a zearalenone standard substance solution, a substrate developing solution, a stopping solution, and a concentrated washing liquid. Detection of zearalenone with the kit of the invention comprises the following steps: firstly pretreating a sample, then detecting with the kit, and finally analyzing the detection result. The enzyme linked immunosorbent assay kit provided by the invention can be used for detection of the residual amount of zearalenone in a feed sample, which is simple in operation, low in price, high in sensitivity, suitable for on-site monitoring, and suitable for screening of a large number of samples.

Description

technical field [0001] The invention relates to an enzyme-linked immunoassay detection technology, in particular to an enzyme-linked immunoassay kit for detecting zearalenone and its application. Background technique [0002] Zearalenone (structural formula see figure 1 ) is mainly produced by Fusarium graminearum, which can also be produced by various Fusarium species such as Fusarium pink, Fusarium mongolicum, and Fusarium three-line. Zearalenone exists in many crops such as wheat and soybeans, and corn Chiaralenone is a lactone structure of phenolic dihydroxybenzoic acid, the molecular formula is C 18 h 22 o 50 , it is insoluble in water, carbon disulfide and carbon tetraoxide, soluble in alkaline aqueous solution, ether, benzene, chloroform, methylene chloride, ethyl acetate and acids, slightly soluble in petroleum ether. Zearalenone can not only be produced by mold, but also exists in many higher plants, and it is used as a hormone in plants to regulate the growth o...

Claims

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Application Information

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IPC IPC(8): G01N33/64G01N21/78
Inventor 何方洋韩黎吴鹏罗晓琴赵正苗韩雪琳孙震李勇崔海峰冯静陈勇罗贵昆
Owner BEIJING KWINBON BIOTECH
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