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Preparation methods of nitrofurantoin hapten and nitrofurantoin antigen, and applications of nitrofurantoin hapten and nitrofurantoin antigen in chemiluminescence immunoassay kit

A technology for nitrofurantoin metabolites and haptens, which is applied in the fields of chemical instruments and methods, organic chemistry, immunoglobulin, etc., can solve the problems of non-detection, and achieve the effects of low cost, large amount of processed samples, and simple operation

Inactive Publication Date: 2016-05-25
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

Due to doubts about the safety of furazolidone protein-bound residues, since 1995, the European Union has comprehensively banned the use of furan antibacterial substances, and the detection limit of furan residues in animal-derived foods is not detected

Method used

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  • Preparation methods of nitrofurantoin hapten and nitrofurantoin antigen, and applications of nitrofurantoin hapten and nitrofurantoin antigen in chemiluminescence immunoassay kit
  • Preparation methods of nitrofurantoin hapten and nitrofurantoin antigen, and applications of nitrofurantoin hapten and nitrofurantoin antigen in chemiluminescence immunoassay kit
  • Preparation methods of nitrofurantoin hapten and nitrofurantoin antigen, and applications of nitrofurantoin hapten and nitrofurantoin antigen in chemiluminescence immunoassay kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1, the preparation of nitrofurantoin metabolite hapten

[0031] The preparation method of the nitrofurantoin metabolite hapten, the specific operation steps include:

[0032] ①Put nitrofurantoin metabolite AHD115mg, pyridine 2ml into a 10ml one-mouth bottle, and stir for 5 minutes;

[0033] ② After complete dissolution, cool down to 0°C, add 250 mg of m-carboxybenzenesulfonyl chloride, stir at 0°C for 1 hour, return to room temperature and react overnight;

[0034] ③ After the complete AHD reaction was detected by TLC, the reaction solution was added to 15ml of ice water, stirred for 30min, filtered, washed with water, and dried to obtain 260mg of AHD hapten.

[0035] The structural formula of the hapten is shown in formula 1:

[0036] Formula 1.

Embodiment 2

[0037] Example 2, Preparation of Nitrofurantoin Metabolite Artificial Antigen

[0038] 1. Synthesis of nitrofurantoin metabolite immune antigen

[0039] ① Dissolve 115.7mg of the above-mentioned hapten in 10.7ml of DMF and stir to fully dissolve it. Add EDC221.75mg and NHS191.4mg, react at room temperature for 3h;

[0040] ②Weigh 144.1mg of BSA and dissolve it in 15ml of 0.1M carbonic acid buffer solution (pH=9.6), stir for 10min to fully dissolve;

[0041] ③ Add 3.6ml of the activation solution in step 1 to the protein solution drop by drop in an ice-water bath environment, stir while adding, and react with magnetic stirring (400rpm) at room temperature for 24 hours;

[0042] ④Put the reaction product into a dialysis bag (15cm) rinsed with distilled water, dialyze with 1LPB (1×, pH 7.2) at room temperature (100rpm) for 3 days, and change the liquid 3 times a day (one in the morning, one in the middle, and one in the evening). 9 times, centrifuge the dialysis product at 450...

Embodiment 3

[0049] Embodiment 3, Nitrofurantoin specific antibody preparation

[0050] 1. Preparation of polyclonal antibody against nitrofurantoin metabolites

[0051] The immunogen solution prepared in Example 1 was diluted with PBS buffer solution with pH 7.4 and 0.01M to obtain the immunogen dilution, which was used for the preparation of polyclonal antibodies. New Zealand white rabbits were used as immunized animals.

[0052] The immunization process is as follows:

[0053] First immunization: Mix the immunogen dilution with an equal volume of Freund's complete adjuvant to make an emulsifier, inject it subcutaneously at multiple points on the back of the neck, and the immunization dose is 2.5 mg / cause;

[0054] Booster immunization: 4 weeks after the first immunization, 8 weeks later and 12 weeks later, a booster immunization was carried out each time. The immunogen dilution was mixed and emulsified with an equal volume of Freund's incomplete adjuvant, and injected subcutaneously a...

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Abstract

The present invention discloses a nitrofurantoin metabolite hapten, a corresponding artificial antigen, a monoclonal antibody, and preparation methods and applications of the nitrofurantoin metabolite hapten, the corresponding artificial antigen and the monoclonal antibody. According to the present invention, the nitrofurantoin metabolite hapten and a carrier protein are linked to obtain a nitrofurantoin metabolite antigen, and the nitrofurantoin metabolite antigen can be used for preparation of nitrofurantoin metabolite specific antibody; the preparation method has characteristics of simpleness, feasibility, low cost, and high hapten yield; and the nitrofurantoin metabolite artificial antigen and the nitrofurantoin metabolite specific antibody can be used for preparation of a chemiluminescent enzyme-linked immunosorbent assay kit for detecting nitrofurantoin metabolite residue, and has advantages of simpleness, rapidness, high sample treatment capacity, high sensitivity, strong specificity, and the like.

Description

technical field [0001] The invention relates to a hapten, an antigen and a preparation method thereof, in particular to a preparation method of a nitrofurantoin hapten and an antigen and their application in a chemiluminescence immunoassay kit. Background technique [0002] Nitrofurans are a class of synthetic broad-spectrum antibacterial drugs with a basic structure of 5-nitrofuran ring, mainly including Nitrofurazone, Nitrofurantion, Furazolidone and Furazidone ( Furaltadone). Nitrofuran substances have a killing effect on most Gram-positive bacteria, Gram-negative bacteria, fungi, protozoa and other pathogens, and are widely used in livestock and poultry farming and aquaculture. However, nitrofurans have certain toxicity to livestock and poultry, large doses or long-term continuous administration of nitrofurans can easily cause toxic reactions, manifested as anorexia, diarrhea, gastrointestinal bleeding, peripheral neuritis, excitement, convulsions or Paralysis, etc., c...

Claims

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Application Information

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IPC IPC(8): C07K14/765C07K14/77C07K16/44C07D233/80G01N33/577
Inventor 吴小平王文珺李向梅方沅王世恩苏丽芳杨艳红张桂亮金兴胡蒙蒙陈银辉牛兰兰韩京朋姚琳邢佑尚
Owner 北京维德维康生物技术有限公司
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