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Detection kit and method of furazolidone metabolin

A technology for furazolidone and metabolites, which is applied in the field of furazolidone metabolite detection kits, can solve the problems that the sensitivity of banned drugs cannot meet the requirements, is not suitable for rapid detection, and the instrument is expensive, and achieves reduction of subjectivity, simple and fast operation, and high performance. Effects of Specificity and Sensitivity

Inactive Publication Date: 2010-08-18
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, AOZ determination methods mainly include liquid chromatography / ultraviolet (LC / UV) analysis, liquid chromatography / mass spectrometry (LC / MS), liquid chromatography tandem mass spectrometry (LC / MS / MS), but these methods The required instruments are expensive and the experimental process is cumbersome, so it is not suitable for on-site rapid detection
At present, microbial methods and enzyme-linked immunoassays are widely used in the rapid detection of drug residues. However, these two methods often fail to meet the sensitivity requirements for the detection of banned drugs.
The rapid screening method represented by chemiluminescence immunoassay (Chemiluminescence Immunoassay, CLIA) is the mainstream technology of immunology, but there is no report on the detection of furazolidone metabolite AOZ by chemiluminescence immunoassay (CLIA) at home and abroad.

Method used

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  • Detection kit and method of furazolidone metabolin
  • Detection kit and method of furazolidone metabolin
  • Detection kit and method of furazolidone metabolin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Preparation of monoclonal antibodies

[0035] (1) Preparation of immune antigen

[0036] The furazolidone metabolite AOZ reacts with p-aldehyde benzoic acid to prepare 4-CPAOZ. Then 4-CPAOZ is coupled with bovine serum albumin to obtain 4-CPAOZ-BSA conjugate, which is the immune antigen.

[0037] (2) Monoclonal antibody preparation

[0038] 1) Animal immunization: immunize mice with the above-mentioned synthetic immune antigen, and mix the immune antigen with the same amount of Freund's complete adjuvant to make an emulsifier during the first immunization, and then subcutaneously inject multiple points on the back of the neck, taking the same at 2-3 weeks apart The dose of immune antigen plus the equal amount of Freund's incomplete adjuvant is mixed and emulsified, and the booster is once. After the fourth immunization, the abdominal cavity is boosted once, and the spleen cells are taken 3 days later.

[0039] 2) Cell fusion and cloning: Take spleen cells of immunized...

Embodiment 2

[0042] Example 2 Preparation of the detection plate and reagents in the furazolidone metabolite AOZ detection kit

[0043] In this example 2, the furazolidone metabolite AOZ detection kit is equipped with a detection plate, detection target 4-NPAOZ standard solution, AOZ derivatized hapten monoclonal antibody, enzyme-labeled secondary antibody, substrate reaction solution, derivatizing agent, The sample diluent and 10× concentrated washing solution are prepared as follows:

[0044] (1) Preparation of AOZ microwell detection plate for furazolidone metabolism

[0045] Use 0.2M carbonate buffer of pH 9.6 as coating solution, dilute furazolidone metabolite derivatized hapten and ovalbumin conjugate (4-CPAOZ-OVA) to 200ng / ml, add 100μl / well In the chemiluminescence well plate, coat overnight at 4°C. Spin dry, add 1% gelatin, pH 7.4 phosphate buffer at 37°C for 2 hours at 200μl / well, wash with 1% sodium azide, pH 7.4 phosphate washing solution 3 times, spin dry, and pack Store in a bag ...

Embodiment 3

[0056] Example 3 Using the kit of the present invention to detect furazolidone metabolite AOZ

[0057] (1) Sample pretreatment

[0058] Pretreatment of animal tissues (chickens, pigs), honey, eggs, fish, and shrimp samples:

[0059] ①Take 1g sample into a centrifuge tube, add 4ml water, and fully break it under a homogenizer;

[0060] ②Then add 0.5ml of 1M HCl, 25μl of 25mM p-nitrobenzaldehyde, vortex for 1 minute and derivatize at 37°C for 16 hours (or derivatize at 55°C for 4 hours);

[0061] ③Cool to room temperature, add 0.1mol / LNa 2 HPO 4 5mL, 1mol / L NaOH 0.4mL, shake for 1min, add ethyl acetate 5mL, shake for 1min, centrifuge at 5000r / min for 6min;

[0062] ④ Take 2.5mL of ethyl acetate layer and blow dry with nitrogen, add 1mL of n-hexane to dissolve, and add 1mL of sample diluent, shake for 1min, centrifuge at 5000r / min for 10min;

[0063] ⑤Remove the lower clear liquid and pay attention to the neutral pH value, dilute it with the sample diluent for later use.

[0064] (2) The test...

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Abstract

The invention discloses a chemiluminescence detection kit of furazolidone metabolin, comprising a detection plate and a reagent, wherein the reagent comprises an antibody and a detection target standard solution, wherein the detection target is a p-nitrobenzene derivative 4-NPAOZ of furazolidone metabolin 3-amino-2-oxazolidone. The detection kit of the furazolidone metabolin not only can realize the fast and mass detection, but also has very high specificity and sensitivity, and achieves the detection sensitivity of 0.005 ppb.

Description

Technical field [0001] The invention relates to the technical field of bioengineering, in particular to a furazolidone metabolite detection kit and a detection method thereof. Background technique [0002] AOZ (chemical name: 3-amino-2-oxazolidinone) is the main metabolic residue of furazolidone (Furanzolidone, FZ) in the organism. Hydroxyethyl hydrazine, a decomposition product produced by gastric acid, is a highly toxic substance that is harmful to humans. Risk of cancer. [0003] Furazolidone, also known as furazolidone, is the most representative of nitrofuran drugs (furazolidone, furantoin, nitrofurantoin, nitrofurazone). It is a synthetic antibacterial drug, which is yellow or crystalline powder. It has a molecular weight of 225.16 and a melting point of 245-258°C. It decomposes when exposed to alkali, and the color gradually darkens under strong light. The first discovery of the antibacterial activity of 5-nitrofuran was in the biosynthesis and microbiology experiments con...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53G01N33/577G01N21/76
Inventor 王权李健陈沁徐仙陈永军蒋蔚赵新宇顾惠明
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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