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

A furazolidone and metabolite technology, which is applied in the field of hapten and its preparation, can solve problems such as being unsuitable for on-site monitoring, cumbersome pretreatment process, complicated instruments and equipment, etc., and achieves the effects of good affinity, efficient detection method and low detection cost

Inactive Publication Date: 2013-10-23
BEIJING KWINBON BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently the most common methods used to detect furazolidone metabolites are high performance liquid chromatography (HPLC), liquid chromatography-mass chromatography (LC-MS, LC-MS / MS), etc., due to complex equipment and tedious pretreatment process and High skill requirements for inspectors, not suitable for on-site monitoring and screening of a large number of samples

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1 Synthesis and identification of furazolidone metabolite hapten

[0019] 1. Synthesis of furazolidone metabolite hapten (synthetic route see figure 1 )

[0020] Mix 1.02g of furazolidone metabolite (AOZ) with 20ml N,N-dimethylformamide (DMF) to obtain liquid A; take 2.68-5.36g of terephthalaldehyde and mix 50-100ml DMF to obtain liquid B; room temperature Slowly add liquid A into liquid B dropwise. After the dropwise addition, react at room temperature to 60°C for 2-4 hours, remove the solvent, and purify by column chromatography to obtain a light yellow substance, which is the hapten of the metabolite of furazolidone.

[0021] 2. Identification of furazolidone metabolite hapten

[0022] The synthesized furazolidone metabolite hapten was determined by H NMR spectroscopy, as shown in figure 2 As shown, the peak at 8.0ppm in the spectrum is the signal peak of 4 H introduced on the benzene ring, the peak at 8.3ppm is the signal peak of H on the aldehyde group,...

Embodiment 2

[0023] Example 2 Furazolidone metabolite antigen

[0024] Furazolidone metabolite hapten is coupled with carrier protein to obtain furazolidone metabolite antigen.

[0025] 1. Preparation of immunogen - synthesis of furazolidone metabolite hapten-bovine serum albumin conjugate

[0026] Dissolve 8.5mg of furazolidone metabolite hapten in 1ml of DMF to obtain solution I; take 40mg of bovine serum albumin (BSA) and dissolve it in 9ml of water to obtain solution II; add solution I dropwise to solution II and react at room temperature for 24 hours to obtain solution Ⅲ; Take NaBH 4 Dissolve 20 mg in 0.2ml 0.1mol / L NaOH and add it to solution III, react at 4°C for 2 hours; dialyze with 0.01mol / L PBS at 4°C for 3 days, and change the dialysate 3 times a day to obtain the furazolidone metabolite immunogen; Store at -20°C for later use.

[0027] 2. Preparation of Coating Source—Synthesis of Furazolidone Metabolite Hapten-Ovalbumin Conjugate

[0028] Dissolve 10 mg of furazolidone me...

Embodiment 3

[0031] Embodiment 3 furazolidone metabolite monoclonal antibody

[0032] 1. Preparation of monoclonal antibody to furazolidone metabolite

[0033] Animal immunization: Inject the immunogen into the body of Balb / c mice with an immunization dose of 150 μg / mouse to make them produce polyclonal antibodies.

[0034] Cell fusion and cloning: After the measurement result of mouse serum was higher, the splenocytes were taken and fused with SP2 / 0 myeloma cells at a ratio of 8:1, and the cell supernatant was measured by indirect competitive ELISA, and the positive wells were screened. Positive wells were cloned by limiting dilution until hybridoma cell lines secreting monoclonal antibodies were obtained.

[0035] Cell cryopreservation and recovery: the monoclonal hybridoma cell line of furazolidone metabolites was made into 1×10 6 cells / ml for long-term storage in liquid nitrogen. When recovering, take out the cryopreservation tube, put it into a 37°C water bath to thaw quickly, remo...

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Abstract

The invention discloses a hapten, as well as a preparation method and application thereof, and specifically relates to a furazolidone metabolite hapten. The invention also discloses a preparation method of the hapten and application thereof. A kit quick detection product established based on the furazolidone metabolite hapten is convenient to use, low in detection cost, and efficient, accurate and quick in detection method; the kit quick detection product is capable of detecting a large batch of samples at the same time, and thus suitable for on-site supervision of furazolidone metabolite residues in animal derived food and screening of lots of samples.

Description

technical field [0001] The invention relates to a hapten and its preparation method and application, in particular to a furazolidone metabolite hapten and its preparation method and application. Background technique [0002] Furazolidone (Furazolidone) belongs to nitrofuran drugs, which is a broad-spectrum antibiotic and has certain antibacterial effects on Gram-positive and negative bacteria. Prevention and treatment of chicken blackhead disease. However, studies have shown that nitrofuran drugs and their metabolites have considerable toxicity, teratogenic side effects, and can induce cancer. my country's Ministry of Agriculture document Nongmufa [2002] No. 1 stipulates that the detection limit of furazolidone in food animals is not to be detected. [0003] Since nitrofuran drugs can be metabolized quickly in the body, and the metabolites combined in tissues can persist for a long period of time, it is often necessary to analyze the metabolized products when analyzing the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D263/26C07K14/765C07K14/77C07K14/795C07K14/75C07K14/47C07K1/113C07K16/44G01N33/577
Inventor 万宇平冯才伟罗晓琴孙震陶光灿汪善良冯静杨秀贤朱亮亮刘玉梅崔廷婷石洁
Owner BEIJING KWINBON BIOTECH
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