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Anti-clorprenaline monoclonal antibody hybridoma cell strain and application thereof

A hybridoma cell line and monoclonal antibody technology, which is applied in the field of food safety immunology detection, can solve the problems of clorprenaline residue, high cost, cumbersome operation, etc., and achieve good detection sensitivity and specificity

Inactive Publication Date: 2018-09-11
JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the culprit in the first illegal addition of "clenbuterol" to veterinary drugs uncovered in my country in 2013 was also clorprenaline hydrochloride, which was added to 6 kinds of medicines for treating colds in animals, including "30% Tilmicosin Injection". In veterinary drugs, clorprenaline remains in animals
[0003] The methods for detecting clorpronaline at present mainly include liquid chromatography (HPLC), liquid chromatography tandem mass spectrometry (LC-MS / MS), enzyme-linked immunosorbent assay, methods such as immunoaffinity chromatographic column and electrochemical sensor, However, these methods have the disadvantages of cumbersome operation, time-consuming, and expensive costs, and cannot realize the rapid detection of a large number of samples. Therefore, it is of great significance to establish a fast and simple detection method.

Method used

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  • Anti-clorprenaline monoclonal antibody hybridoma cell strain and application thereof
  • Anti-clorprenaline monoclonal antibody hybridoma cell strain and application thereof
  • Anti-clorprenaline monoclonal antibody hybridoma cell strain and application thereof

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Experimental program
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Embodiment 1

[0025] The preparation of embodiment 1 hybridoma cell line 4G1

[0026] 1. Hapten Synthesis

[0027] Weigh compound 1 (20g, 121.9mmol) dissolved in alcohol (150mL) in 4mL H2SO4 and stir overnight at 80°C, put the compound in an ice bath and extract with organic waste gas (EA extraction), the organic layer was washed with brine, washed with Na 2 SO 4 Drying and evaporation gave compound 2 in a yield of 98.4%. Compound 2 (23g, 119.8mmol) was added into 230mL dichloromethane (DCM) solution containing 19mL ethoxychloric acid and 76g aluminum chloride, and stirred at room temperature for 4 hours. The compound was ice-bathed and extracted with DCM, the organic layer was washed with brine, washed with Na 2 SO 4 Drying and evaporation afforded compound 3 as a yellow oil with a yield of 53.6%. To compound 3 (15 g, 64.1 mmol) in methanol (150 mL) was added NBS (12 g, 67.3 mmol) in portions and stirred at 60 °C for 1 hour, 200 mL of water was poured into the mixture and extracted wi...

Embodiment 2

[0040] The application of embodiment 2 antibody

[0041] The monoclonal antibody prepared by the hybridoma cell line 4G1 was applied to the ELISA addition recovery test of clorprenaline through the in vivo ascites, and the specific steps were as follows:

[0042] (1) Coating: The coated original CLP-OVA was diluted with 0.05M pH9.6 carbonate buffer solution starting from 2 μg / mL, 100ul / well, and reacted at 37°C for 2h.

[0043] (2) Washing: Pour off the solution in the plate, spin dry, and wash 3 times with washing solution, 3 minutes each time.

[0044] (3) Blocking: After patting dry, add 200ul / well blocking solution, and react at 37°C for 2h. Wash and tumble dry for later use.

[0045] (4) Adding samples: Dilute the antiserum starting from 1:1000, and add it to the coated wells of each dilution, 100ul / well, and react at 37°C for 30min; after fully washing, add 1:3000 diluted HRP - Goat anti-mouse IgG, 100ul / well, react at 37°C for 30min.

[0046] (5) Color development: ...

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Abstract

The invention discloses an anti-clorprenaline monoclonal antibody hybridoma cell strain and application thereof, and belongs to the field of food safety immunodetection. A method is characterized in that after the clorprenaline complete antigen is subjected to mixed emulsification with the same quantity of a Freund's adjuvant, the BALB / c mouse immunization is performed through back subcutaneous injection. In the first immunization, the complete Freund's adjuvant is used; then, in-complete Freund's adjuvants are used. The high-valence low-IC50 mouse splenocytes are fused with mouse myeloma cells by a PEG method; through indirect competition ELISA screening and three times of subclone, a hybridoma cell strain is obtained. The monoclonal antibody secreted by the cell strain has good specificity and detection sensitivity; the IC50 value is 0.435ng / ml; the strain can be used for detecting clorprenaline residue in food.

Description

technical field [0001] The invention relates to an anti-chlorprenaline monoclonal antibody hybridoma cell line and an application thereof, belonging to the field of food safety immunology detection. Background technique [0002] beta 2 - receptor agonists (β 2 -agonist) can obviously promote the synthesis of protein and the decomposition of fat in animals, so it is often used as an animal feed additive to achieve the purpose of increasing the lean meat rate of animals. But beta 2 - Receptor agonists are slowly metabolized in animals and have cumulative toxicity, and the edible part of animals enters the human body through the food chain, thereby causing harm to human health. Chlorprenaline, whose chemical name is 1-(2-chlorophenyl)-2-isopropylamino-ethanol, is also a beta 2 - Receptor agonists are commonly used clinically to treat asthma-like diseases. But when a certain dose is reached, it and other beta 2 -Receptor agonists have nutrient redistribution effects in ani...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/44G01N33/577G01N33/558C07C229/34C07C227/08C07C227/16C07C227/18C07C67/08C07C69/612C07C67/343C07C67/307C07C69/738
CPCC07C67/08C07C67/307C07C67/343C07C227/08C07C227/16C07C227/18C07K16/44G01N33/558G01N33/577G01N2430/00C07C69/612C07C69/738C07C229/34
Inventor 匡华蒋薇胥传来徐丽广马伟刘丽强吴晓玲宋珊珊胡拥明
Owner JIANGNAN UNIV
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