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An enzyme linked immuno-detection kit suitable for furazolidone retention analysis and application

An enzyme-linked immunosorbent assay and furazolidone technology, applied in the field of immunochemical analysis, can solve problems such as no patent reports on furazolidone ELISA kits

Inactive Publication Date: 2006-02-08
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, studies on furazolidone ELISA methods have been carried out abroad (Cooper KM, Elliott C T, Kennedy D G. Detection of 3-amino-2-oxazolidinone (AOZ), a tissue-bound metabolite of thenitrofuran furazolidone, in prawn tissue by enzyme immunoassay. Food Additives and Contaminants, 2004, 21 (9) 841-848), but there is no patent report of furazolidone ELISA kit at home and abroad

Method used

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  • An enzyme linked immuno-detection kit suitable for furazolidone retention analysis and application
  • An enzyme linked immuno-detection kit suitable for furazolidone retention analysis and application
  • An enzyme linked immuno-detection kit suitable for furazolidone retention analysis and application

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Experimental program
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Effect test

Embodiment 1

[0021] Embodiment 1, the preparation of antigen, antibody

[0022] 1.1 Synthesis of artificial antigen (immunogen, coater)

[0023] Add 10 mL of distilled water and 3.0 g of p-aldehyde benzoic acid into a 100 mL beaker with a magnetic stirrer device, slowly add N, N-dimethylformamide (DMF) dropwise until the p-aldehyde benzoic acid is completely dissolved, and add AOZ while stirring 1.0 g, filtered after reacting for 2 hours, washed with water three times to obtain 3-(4-carboxybenzylidene)-amino-2-oxazolidinone (abbreviated as CPAOZ, the same below) as the reactant of AOZ and p-formylbenzoic acid. Dissolve 23.4 mg of CPAOZ in 2 mL of DMF, add 27.5 mg of dicyclohexylcarbodiimide (DCC) and 14.4 mg of N-hydroxysuccinimide (NHS) while stirring, and react with magnetic stirring at 4°C overnight. After centrifugation, the supernatant is liquid A. Weigh 170 mg of bovine serum albumin (BSA) and dissolve it in 10 mL of 0.1 mol / L PBS (pH 8.0), add 1 mL of DMF, stir and dissolve to pre...

Embodiment 2

[0027] Embodiment 2, establishment of furazolidone indirect competitive detection method

[0028] 2.1 Determination of the optimal coating concentration of antigen and the optimal working concentration of antibody

[0029] Determined by the square matrix titration test, the point where the absorbance value is about 1.0 and the difference between the left and right adjacent absorbance values ​​is the largest is taken as the reference point. Operation steps: Coat the first row of the 96-well ELISA plate with 2000 μg / L of the coating agent, and the second to eighth rows are sequentially coated with 1000, 500, 250, 120, 60, 30, 15 μg / L of the original Be original. Overnight at 4°C, block with 1% ovalbumin at 37°C for 1 hour, wash twice, pat dry, add 100 μL to the 1st to 9th column of the microtiter plate in sequence, the dilution factor is 10000, 20000, 40000, 80000, 160000, For 320000, 640000, 1280000, 2560000 3-amino-2-oxazolidinone antibodies, add sample diluent to the 10th c...

Embodiment 3

[0039] Example 3, the preparation of the enzyme-linked immunoassay kit for furazolidone residue analysis of the present invention

[0040] 3.1 Composition of the enzyme-linked immunoassay kit of the present invention

[0041] The kit of the present invention is mainly composed of a box body (1), an ELISA plate (2), 6 bottles of AOZ standard solution (3), horseradish peroxidase (HRP)-labeled goat anti-rabbit antibody (4), AOZ antibody Solution (5), Substrate Color Development Solution A (6), Substrate Color Development Solution B (7), Stop Solution (8), Wash Solution (9), Sample Diluent Solution (10) and Foam Holder (11) composition.

[0042] 3.2 Preparation of reagents

[0043] Prepared by conventional methods: where coating diluent: Na 2 CO 3 1.5g, NaHCO 3 2.9g, Na 2 N 3 0.2g, add double distilled water to 1000mL, adjust to pH9.6; blocking solution: ovalbumin 0.1g dissolved in pH7.4PBS 100mL; washing solution: NaCl 8.0g, KH 2 PO 4 0.2g, Na 2 HPO 4 12H 2 O 2.9g, KC...

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Abstract

The invention discloses an enzyme immune detecting agent box suit for analyzing the surplus furazolidone and its application in the field of chemo immunology analyzing technology. The agent box is mainly formed by a 3-amido-2-AOZ peculiarity antibody, a AOZ standard solution which comprises AOZ and the ferment standard plate of the egg albumin complex. The sample releases AOZ after alcaine hydrolysis, then the benzaldehyde derivatizes over the night, MAX column purifies, it adopts indirect contest ELISA method to detect the animal structure such as liver and the AOZ surplus in muscle. The core technology comprises: artificial antibody synthesis, antibody preparing, ELISA method establishing, and ELISA agent box adjustment.

Description

technical field [0001] The invention relates to an enzyme-linked immunoassay kit suitable for the analysis of furazolidone residues, which is suitable for determining the residues of furazolidone residue marker 3-amino-2-oxazolidinone (abbreviated as AOZ, the same below) in animal foods. The invention belongs to the technical field of immunochemical analysis. Background technique [0002] Furazolidone, also known as furazolidone, belongs to nitrofuran drugs and is a synthetic broad-spectrum antibacterial drug. It was once widely used as a growth-promoting agent in livestock, poultry, and aquaculture. Toxicology studies have found that furazolidone is carcinogenic and mutagenic. The European Union listed furazolidone as a banned drug in 1995, and then the United States, Japan, Australia and other countries all canceled the use of furazolidone in livestock and poultry production. The "List of Veterinary Drugs and Other Compounds Banned for Fo...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N33/531
Inventor 袁宗辉常超王玉莲赵春保王帅兵高爱中陈冬梅陶燕飞伍金娥彭大鹏
Owner HUAZHONG AGRI UNIV
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