Furazolidone metabolite hapten and antigen as well as preparation method and application thereof

A furazolidone and metabolite technology, which is applied in the field of furazolidone metabolite hapten, can solve the problems of expensive instruments, high equipment requirements, and high operator requirements, and achieves great economic value and social significance, high purity and yield, and simple detection. Effect

Inactive Publication Date: 2010-02-10
北京维德维康生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The current methods for detecting AOZ residues include LC-UV, LC-MS and LC-MS-MS. This type of technology requires high equipment requireme...

Method used

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  • Furazolidone metabolite hapten and antigen as well as preparation method and application thereof
  • Furazolidone metabolite hapten and antigen as well as preparation method and application thereof
  • Furazolidone metabolite hapten and antigen as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1, Preparation and identification of furazolidone metabolite hapten

[0038] 1. Preparation of furazolidone metabolite hapten

[0039] 3-amino-2-oxazolidinone: 5-hydroxy-2-nitro-benzaldehyde: succinic anhydride = 1:3.8:8.3 (molar ratio).

[0040] 1. Preparation of solution A and solution B

[0041] Solution A: 25 mg of 3-amino-2-oxazolidinone was dissolved in 1 mL of distilled water, and set aside.

[0042] Solution B: 155mg of 5-hydroxy-2-nitro-benzaldehyde, dissolved in 4mL of ethanol, set aside.

[0043] 2. At room temperature (20-30°C), gradually add solution A to solution B with continuous stirring, and react for 7 hours to obtain solution C.

[0044] 3. The solution C was centrifuged at 5000 rpm for 10 minutes, the precipitate was taken out, washed twice with a small amount of absolute ethanol, and dried in a drying oven at 60°C to obtain a yellow powder.

[0045] 4. Transfer the product of step 3 to a 20 mL round bottom flask, add 200 mg of succinic a...

Embodiment 2

[0052] Example 2, Preparation and Identification of Furazolidone Metabolite Artificial Antigen

[0053] 1. Preparation of Furazolidone Metabolite Artificial Antigen

[0054]1. Dissolve the furazolidone metabolite hapten (compound shown in formula (I)) prepared in Example 1 in N, N-dimethylformamide (DMF), then add N, N'-dicyclohexylcarbodisulfide Imine (DCC) and N-hydroxysuccinimide (NHS), stirred at room temperature (20-30°C) for 9 hours, overnight at 4°C, centrifuged to separate the supernatant; furazolidone metabolite hapten: DCC: NHS: DMF =1:1:1:4 (molar ratio).

[0055] 2. Add the supernatant to the phosphate buffer (0.01 mol / L, pH 7.4) in which the carrier protein (BSA) is dissolved, and continue to stir for 5 hours to obtain the furazolidone metabolite artificial antigen.

[0056] 3. Put the solution in step 2 into a dialysis bag, dialyze with phosphate buffer (0.01 mol / L, pH 7.4) at 4°C for 72 hours, and change the water 10 times.

[0057] 4. Pass the dialysate thro...

Embodiment 3

[0060] Embodiment 3, preparation and specific identification of antiserum

[0061] 1. Preparation of antiserum from immunized animals

[0062] Five New Zealand white rabbits were used as immunized animals, and the artificial antigen of furazolidone metabolite (prepared in Example 2) was used as the immunogen, and the immunizing dose for each immunization was 1 mg / kg. The immunization method is as follows: at the first immunization, fully emulsify the immunogen with an equal volume of Freund's complete adjuvant, inject it subcutaneously at multiple points on the back of the neck, and take it at intervals of 3-4 weeks. Mix the immunogen with Freund's incomplete adjuvant to make an emulsifier Booster immunization once, a total of 7 times of immunization, the last time without adjuvant.

[0063] From the second booster immunization, blood was collected from the rabbit’s ear vein on the 8th day after each immunization. After the blood was collected, it was placed in a refrigerator...

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Abstract

The invention discloses furazolidone metabolite hapten and antigen as well as a preparation method and application thereof. The furazolidone metabolite hapten provided by the invention is the compoundshown in the formula (I), and the furazolidone metabolite antigen can be obtained by connecting the compound shown in the formula (I) and carrier protein. The furazolidone metabolite antigen can be applied to preparing the specific antibody of the furazolidone metabolite. The method for preparing the compound is simple in operation and the compound is high in purity and yield. The specific antibody with high affinity for the furazolidone metabolite is generated after the compound of the invention is used for immunizing animals, and enzyme-linked immunosorbent assay established by using the antibody can be used for detecting the residual furazolidone metabolite in the animal derived foods in a rapid, sensitive and simple way. The invention has great economic value and social meaning.

Description

technical field [0001] The invention relates to a furazolidone metabolite hapten, an antigen, a preparation method and application thereof. Background technique [0002] Furazolidone, also known as furazolidone, belongs to nitrofuran drugs. It is a synthetic antibacterial drug with stable efficacy. It can inhibit or kill a variety of Gram-positive and negative bacteria, and is effective against certain protozoa and fungi It has a certain effect, and it is often used clinically to prevent intestinal infection in livestock and poultry. According to data reports, furazolidone has strong teratogenic, carcinogenic, mutagenic and other toxic and side effects, and its residue in animal food directly threatens human health. In 1995, the European Union banned the use of furazolidone in food animals, and then the United States, Japan, Australia, and China all canceled the use of furazolidone in livestock and poultry production, but incidents of furazolidone residues exceeding the sta...

Claims

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Application Information

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IPC IPC(8): C07D263/26C07K14/765C07K14/77C07K1/113C07K16/44G01N33/566G01N33/531
Inventor 王战辉江海洋吴小平王亚辉张静阮永磊黄思扬徐飞
Owner 北京维德维康生物技术有限公司
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