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A kind of preparation method and application of thiabendazole hapten

A thiabendazole and thiabendazole enzyme technology is applied in the fields of antigen, monoclonal antibody preparation, and thiabendazole hapten, which can solve the problems of complex and expensive instruments, cumbersome sample processing process, unsuitable for sample screening, etc. The effect of large sample volume, simple and feasible preparation method and low cost

Active Publication Date: 2017-11-28
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The current TBZ detection method is mostly chromatography, but it requires complex and expensive instruments, and the sample pretreatment process is cumbersome, which is not suitable for the screening of a large number of samples on site

Method used

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  • A kind of preparation method and application of thiabendazole hapten
  • A kind of preparation method and application of thiabendazole hapten
  • A kind of preparation method and application of thiabendazole hapten

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1, preparation and identification of thiabendazole hapten

[0032] 1. Preparation of thiabendazole hapten

[0033] Mix 220 mg of 5-hydroxythiabendazole, 500 mg of phthalic anhydride and 2 mL of pyridine in 20 mL of dimethyl sulfoxide, stir and react at 80°C for 10 hours, evaporate the solvent, and purify by column chromatography The product shown in formula 1, i.e. monothiabendazole phthalate was obtained.

[0034] 2. Identification of thiabendazole hapten

[0035] The thiabendazole hapten prepared in step 1 was identified by NMR.

[0036] NMR see figure 2 . The spectrum showed two groups of aromatic ring signal peaks increased at about 8.0 ppm and carboxyl signal peaks increased at about 13.3 ppm, indicating that the hapten was successfully synthesized.

Embodiment 2

[0037] Embodiment 2, preparation and identification of thiabendazole artificial antigen

[0038] 1. Synthesis of thiabendazole immune antigen (TBZ-BSA)

[0039] (1) Take 10 mg of the product shown in Formula 1 and dissolve it in 1 mL of dimethylformamide;

[0040] (2) Take 50 μL of isobutyl chloroformate and stir at 4°C for 24 h to obtain reaction solution A;

[0041] (3) Weigh 50 mg of bovine serum albumin (BSA), add 3.8 mL of carbonate buffer solution with pH 9.0, fully dissolve, add reaction solution A drop by drop to the protein solution, and stir at room temperature for 24 h;

[0042] (4) Dialyze with 0.01mol / L phosphate buffer at 4°C for 3 days, and change the dialysate 3 times a day to remove unreacted small molecular substances;

[0043] (5) Aliquot and store at -20°C for later use.

[0044] 2. Synthesis of thiabendazole-coated antigen (TBZ-OVA)

[0045] (1) Take 10 mg of the product shown in Formula 1 and dissolve it in 1 mL of dimethylformamide;

[0046] (2) Ta...

Embodiment 3

[0052] Example 3, Preparation and Specific Identification of Enzyme-labeled Monoclonal Antibody

[0053] One, the preparation of thiabendizumab

[0054]1. Use 100 μg of the immunogen (TBZ-BSA) prepared above, dissolve the immunogen in normal saline and mix it with Freund’s complete adjuvant in an equal volume, and inject subcutaneously on the back of the neck to immunize Balb / c females aged 6-8 weeks. On the 7th, 14th, and 28th day after the initial immunization, mix the immunogen and Freund's incomplete adjuvant in equal volumes, and each additionally immunize once, and 3 days before the fusion, 100 μg of the immune complex / rat, without Freund's adjuvant Add another immunization.

[0055] 2. Proceed according to the conventional method, take the splenocytes of the immunized mice and mix them with the mouse myeloma cells (SP2 / 0) in the logarithmic growth phase, and then slowly add the preheated fusion agent (PEG4000) within 45s for fusion, Suspend evenly with HAT medium, the...

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Abstract

The invention discloses a thiabendazole hapten, and corresponding artificial antigen and monoclonal antibody. The invention also discloses the preparation methods and applications of the thiabendazole hapten and the corresponding artificial antigen and monoclonal antibody. The thiabendazole hapten is a product represented by the formula 1. When the product represented by the formula 1 is connected with carrier protein, thiabendazole antigen can be obtained. The thiabendazole antigen can be used for preparing thiabendazole specific antibody. The preparation method provided by the invention is simple and feasible, and has low cost and high hapten yield. The thiabendazole artificial antigen can be used for producing specific antibody aiming at thiabendazole through immunizing animals, and can be used for preparing enzyme-linked immunosorbent assay kits used for detecting thiabendazole residue. The kit is simple and fast, and has various advantages such as large sample processing amount, high sensitivity, high specificity, and the like.

Description

technical field [0001] The invention belongs to the technical field of food safety detection, and in particular relates to a preparation method of thiabendazole hapten, antigen and monoclonal antibody and an application thereof. Background technique [0002] Thiabendazole English name TBZ (Thiabendazole), alias: tekedo; 10 h 7 N 3 S, molecular weight: 201.25, the original drug is white powder, melting point: 304-305°C, sublimation at 310°C. Solubility at room temperature: acetone 28g / L, methanol 9.3g / L, dimethyl sulfoxide 80g / L, dimethylformamide 39.0g / L, benzene 2.3g / L, chloroform 0.8g / L. Stable in neutral, acidic and alkaline aqueous solutions. [0003] Thiabendazole belongs to benzimidazoles (BMZs). It is a broad-spectrum, high-efficiency, and low-toxicity anthelmintic. function and is still widely used today. Thiabendazole can cause loss of appetite, nausea, vomiting, abdominal pain, diarrhea, dizziness, headache, drowsiness and yellow vision. Announcement No. 235...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D417/04C07K14/765C07K16/44G01N33/543
CPCC07D417/04C07K1/1077C07K14/765C07K16/44C07K19/00
Inventor 万宇平罗晓琴吴鹏韩京朋崔海峰蒲小容李海静刘春雪
Owner BEIJING KWINBON BIOTECH
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