Mouse monoclonal antibody cell strain for resisting amoxicillin and ampicillin

A technology of ampicillin mice and monoclonal antibodies, which is applied in chemical instruments and methods, biochemical equipment and methods, instruments, etc., and can solve the problems of high cost, poor timeliness, and high technical requirements

Inactive Publication Date: 2011-07-13
PEOPLES REPUBLIC OF CHINA BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these two methods are sensitive and accurate, they have long detection time, poor timeliness, high cost, high technical requirements, and expensive instruments. They are not suitable for the screening and determination of a large number of samples, and cannot meet the requirements of food safety management for fast and efficient detection methods. requirements

Method used

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  • Mouse monoclonal antibody cell strain for resisting amoxicillin and ampicillin
  • Mouse monoclonal antibody cell strain for resisting amoxicillin and ampicillin
  • Mouse monoclonal antibody cell strain for resisting amoxicillin and ampicillin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Example 1: Antigen cross-linking

[0015] 1. Immunogen synthesis:

[0016] Carbodiimide hydrochloride (EDCoHCl) two-step method: Weigh 10-20mg of amoxicillin, 50-150mg of EDCoHC, 5-20mg of NHS (N-hydroxysuccinimide) into a one-mouth bottle, add Fully dissolve in 1-4mL DMF (dimethylformamide), put into the rotor of a magnetic stirrer, seal and avoid light, and react at room temperature for 10-20hrs to obtain amoxicillin reaction solution, which is called A solution. Then weigh 10-50 mg of KLH (keyhole limpet hemocyanin) and fully dissolve it in 1-8 mL of PBS (phosphate buffered saline), called solution B. Then add solution A to solution B dropwise, seal it away from light, and stir overnight at 2-8°C. After the above reaction is completed, the reaction solution is transferred to a dialysis bag pretreated with PBS for 10-32 hrs, and dialyzed with PBS for 12-72 hrs in a refrigerator at 2-8°C with stirring, and the medium is changed several times in between. The dialyzed...

Embodiment 2

[0019] Example 2: Preparation of Monoclonal Antibody

[0020] 1. Immunization of mice and titer determination

[0021] Use amoxicillin-KLH cross-linked product as immunization antigen, immunize Balb / c pure line mice, take antigen at 30-150μg / mouse, mix with Freund's complete adjuvant in equal amount, after fully emulsification, mouse neck Multiple subcutaneous injections on the back. The second immunization was carried out 3 weeks after the first immunization, with the same dose as the first one, emulsified with Freund's incomplete adjuvant, and injected subcutaneously at multiple points on the back of the mouse. Three weeks after the second immunization, the same dose of antigen was dissolved in saline and injected intraperitoneally. 5-15 days after the end of immunization, blood was collected from the tail of the mice, the serum was separated, and the titer of antiserum was determined by indirect ELISA.

[0022] Method for determining antibody titer by indirect ELISA: dil...

Embodiment 3

[0043] Example 3: Production and affinity purification of monoclonal antibodies

[0044] 1. Production of monoclonal antibody

[0045] To ensure the purity of the antibody, the hybridoma cells were cultured in a serum-free medium (Hyclone product, catalog number SH30800). Cells were cultured routinely (cell density was 5-10×10 5 When subcultured, the inoculation density was 1-2×10 5 ), after expanding to 1 L, the culture medium was not changed until about 10 days later, all the hybridoma cells died, and the supernatant was collected by centrifugation, which contained highly specific antibodies.

[0046] 2. Protein G affinity purification

[0047] The Protein G chromatography column (Bio-Rad, Bio-Rad) used for antibody purification was pre-equilibrated with 3-5 column bed volume equilibration buffer (10 mM PBS, pH7.4). The collected supernatant was filtered with a disposable 0.22 μm filter and loaded onto the Protein G chromatography column through a peristaltic pump. Coll...

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Abstract

The invention relates to a mouse monoclonal antibody cell strain for resisting both amoxicillin and ampicillin, belonging to the technical field of B cell hybridoma. The mouse monoclonal antibody cell strain for resisting the amoxicillin and the ampicillin has the preservation number of CGMCC NO.2674. The monoclonal antibody aiming at the amoxicillin has 100 percent of cross reaction with the ampicillin which also belongs to the penicillin antibiotics, and has no cross reaction with cefazolin of cephalosporin antibiotics. The invention can be used for rapidly detecting the residual condition of the amoxicillin and the ampicillin in animal-sourced food, and can be further used for developing an immunity detection product rapidly and accurately detecting the amoxicillin and the ampicillin in the animal-sourced food.

Description

technical field [0001] The invention relates to a mouse monoclonal antibody capable of simultaneously resisting amoxicillin and ampicillin, and belongs to the technical field of B cell hybridoma. Background technique [0002] Antibiotics are widely used in the prevention and treatment of various microbial infectious diseases in poultry and livestock. However, antibiotics have serious side effects. The harm of long-term use of antibiotics is mainly to cause bacterial resistance, which will cause the spread and spread of drug-resistant strains, and even make a specific tissue cell resistant. Excessive abuse of antibiotics causes antibiotics to remain in animal products, and eventually accumulate in the human body in various ways, resulting in a large number of drug-resistant strains in the human body, losing resistance to certain diseases, causing allergies, anaphylaxis, and ultimately affecting the human body. Severely toxic to organs. Amoxicillin and ampicillin belong to t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577C07K16/44C12N5/20
Inventor 王金花高启祥卢晓宇刘晨丁海勤史秋磊徐超一张朝晖李小林韩深云环
Owner PEOPLES REPUBLIC OF CHINA BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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