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Nitrofurantoin metabolite hapten as well as preparation method and application thereof

A technology for nitrofurantoin metabolites and haptens, applied in chemical instruments and methods, fibrinogen, animal/human peptides, etc., can solve the problems of cumbersome pretreatment process, complex equipment, unsuitable for on-site monitoring, etc., and achieve detection methods High efficiency, low detection cost, and good affinity

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

AI Technical Summary

Problems solved by technology

Currently the most commonly used methods for detecting nitrofurantoin metabolites are high performance liquid chromatography (HPLC), liquid chromatography-mass chromatography (LC-MS, LC-MS / MS), etc. 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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  • Nitrofurantoin metabolite hapten as well as preparation method and application thereof
  • Nitrofurantoin metabolite hapten as well as preparation method and application thereof
  • Nitrofurantoin 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 nitrofurantoin metabolite hapten

[0019] One, the synthesis of nitrofurantoin metabolite hapten (synthetic route such as figure 1 )

[0020] Mix 1.15g of nitrofurantoin metabolite (AHD) 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 nitrofurantoin metabolite hapten.

[0021] 2. Identification of nitrofurantoin metabolite hapten

[0022] The synthesized nitrofurantoin metabolite hapten was determined by H NMR spectroscopy, as shown in figure 2 As shown, the peak at about 8.0ppm in the spectrum is the signal peak of the 4 H introduced on the benzene ring, the peak at about 8.4ppm is the signal peak...

Embodiment 2

[0023] Example 2 Nitrofurantoin metabolite antigen

[0024] The nitrofurantoin metabolite hapten is coupled with a carrier protein to obtain the nitrofurantoin metabolite antigen.

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

[0026] Dissolve 12 mg of nitrofurantoin metabolite hapten in 1 ml of DMF to obtain solution I; take 40 mg of bovine serum albumin (BSA) and dissolve it in 6 ml 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 ;Dissolve 14mg of NaBH4 with 0.2ml 0.1mol / L NaOH and add it to solution III, react at 4°C for 2h; dialyze with 0.01mol / L PBS at 4°C for 3d, change the dialysate 3 times a day, and obtain the nitrofurantoin metabolite immunogen; , stored at -20°C for later use.

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

[0028] Dissolve 10 mg...

Embodiment 3

[0031] Example 3 Nitrofurantoin metabolite monoclonal antibody

[0032] 1. Preparation of monoclonal antibody against nitrofurantoin metabolites

[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 nitrofurantoin 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 quic...

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Abstract

The invention discloses a nitrofurantoin metabolite hapten. The nitrofurantoin metabolite hapten is characterized in that the molecular structural formula is shown in formula (1) in the description). The invention further provides a synthetic method of the nitrofurantoin metabolite hapten and an application of the nitrofurantoin metabolite hapten used as a raw material of antigen systems in animal immunization. An antibody prepared from the nitrofurantoin metabolite hapten disclosed by the invention is used in an ELISA (enzyme-linked immuno sorbent assay) method for detecting nitrofurantoin metabolites, the use is convenient, and the detection cost is low; and the detection method is efficient, accurate and fast, can be used for simultaneously detecting large-batch samples and is suitable for on-site supervision of residual nitrofurantoin metabolites in animal-origin foods and screening of the large-batch samples.

Description

technical field [0001] The invention relates to a hapten and its preparation method and application, in particular to a nitrofurantoin metabolite hapten and its preparation method and application. Background technique [0002] Nitrofurantoin, belonging to nitrofuran drugs, is a synthetic antibacterial drug, often used as a broad-spectrum antibiotic to prevent and treat gastrointestinal diseases caused by Salmonella and Escherichia in pigs, cattle, poultry and bees . However, studies in recent years have shown that nitrofuran drugs and their metabolites are highly toxic, have teratogenic side effects, and can induce cancer. Feed use. [0003] Due to the thermal and chemical instability of nitrofuran drugs, their metabolites are often used as residue markers. Currently the most commonly used methods for detecting nitrofurantoin metabolites are high performance liquid chromatography (HPLC), liquid chromatography-mass chromatography (LC-MS, LC-MS / MS), etc. High skill require...

Claims

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Application Information

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IPC IPC(8): C07D233/80C07K14/765C07K14/435C07K14/77C07K14/795C07K14/75C07K16/44G01N33/577
Inventor 何方洋万宇平陶光灿吴鹏孙震冯静朱亮亮蒲小容胡德专扶胜崔廷婷张荃
Owner BEIJING KWINBON BIOTECH
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