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A hybridoma cell line secreting gamimycin monoclonal antibody and its application

A hybridoma cell line and monoclonal antibody technology, which is applied to the analysis of materials, instruments, peptides, etc., can solve the problems of long detection time, complicated processing, and inapplicability to rapid detection of a large number of samples, and achieve good detection sensitivity and specificity Effect

Active Publication Date: 2021-11-12
JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods have shortcomings such as complicated sample pretreatment and long detection time, and are not suitable for rapid detection of a large number of samples. In order to protect the interests of consumers, it is necessary to establish an efficient and rapid detection method for gamithromycin

Method used

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  • A hybridoma cell line secreting gamimycin monoclonal antibody and its application
  • A hybridoma cell line secreting gamimycin monoclonal antibody and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Example 1: Synthesis of Gamithromycin Hapten

[0045] Since the small molecule of gaminomycin is not immunogenic and cannot stimulate the immune response of mice to produce antibodies, it is necessary to couple gaminomycin to the protein through protein linkage technology to obtain immunogenicity; protein Active groups commonly used in coupling techniques include amino, carboxyl, hydroxyl, mercapto, etc. Since the molecular structure of gamithromycin does not contain amino, carboxyl, and contains many hydroxyl groups, its analogues were found to derive carboxyl groups.

[0046] The derivatized gamithromycin hapten structure of the present invention is as follows:

[0047] .

Embodiment 2

[0048] Example 2: Synthesis of Gamimycin Complete Antigen

[0049] Weigh 4.0mg gamithromycin hapten (GAM-COOH), 3.1mg N-hydroxysuccinimide (NHS), dissolve in 300μL N,N-dimethylformamide (DMF), stir at room temperature for 10min ; Then weigh 4.9 mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), fully dissolve it with 100 μL DMF, add it to the GAM-COOH solution, and stir at room temperature Reaction 4-6h (called A solution). Take 6mg KLH, dilute it to 3mg / mL with 0.01M carbonate buffer solution (CBS) (called solution B), then slowly add solution A to solution B drop by drop, react at room temperature overnight; then use 0.01M PBS solution Dialyze to remove the unreacted small molecule hapten to obtain the complete antigen GAM-COOH-KLH, which is identified by the ultraviolet absorption scanning method.

Embodiment 3

[0050] Embodiment 3: the synthesis of gamimycin coating former

[0051] Dissolve 4.2mg gamithromycin hapten (GAM-COOH) and 2.3mg N-hydroxysuccinimide (NHS) in 300μL anhydrous N,N-dimethylformamide (DMF), and stir at room temperature for 10min , to obtain gamimycin hapten (GAM-COOH) solution; 3.8 mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was dissolved in 100 μL anhydrous DMF Finally, add it to the GAM-COOH solution, stir at room temperature and react for 4-6 hours to obtain liquid A; dilute 6 mg of chicken ovalbumin (OVA) with 1 mL of carbonate buffer solution (CBS) with a concentration of 0.01 mmol / L to obtain Solution B; slowly add solution A to solution B dropwise for reaction to obtain a reaction solution; dialyze the reaction solution with PBS solution to remove unreacted small molecule haptens to obtain the coating original (GAM-COOH-OVA).

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Abstract

The invention discloses a hybridoma cell line secreting gamimycin monoclonal antibody and its application, belonging to the field of food safety immunoassay. The hybridoma cell line ABC11 secreting gamimycin monoclonal antibody of the present invention has been deposited in the General Microbiology Center CGMCC of the China Microbiological Culture Collection Management Committee, with a storage date of November 28, 2019, and a storage number of CGMCC No.19167. The gamimycin monoclonal antibody secreted by the strain is used for the analysis and detection of gamimycin residues in food safety testing. The garamimycin monoclonal antibody cell line obtained by the present invention can be used for immunoassay detection, and it has better detection sensitivity and specificity (IC 50 The value is 0.82ng / mL, less than 10% crossover to gamithromycin analogues, crossover rate=(IC of gamithromycin 50 / analogue IC 50 )×100%).

Description

technical field [0001] The invention relates to a hybridoma cell strain secreting gamimycin monoclonal antibody and an application thereof, belonging to the field of food safety immunoassay. Background technique [0002] Gamithromycin (Gamithromycin) is a new type of animal-specific semi-synthetic macrolide veterinary antibiotics. It plays a role of antibacterial and bactericidal by inhibiting bacterial RNA-dependent protein synthesis. It has a broad antibacterial spectrum, It has the advantages of strong antibacterial activity, low residue, safety and high efficiency. In foreign countries, gamithromycin has been widely used in the prevention and treatment of respiratory diseases in non-lactating cows and pigs. In China, Gamithromycin Injection was marketed domestically through the registration of imported veterinary drugs by the Ministry of Agriculture and Rural Affairs in 2017. It is used for the treatment and prevention of major pathogenic bacteria such as Mannella hemol...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/44C07K14/795G01N33/577G01N33/53
CPCC07K14/795C07K16/44G01N33/5308G01N33/577
Inventor 胥传来路倩倩匡华徐丽广刘丽强宋珊珊吴晓玲胥欣欣胡拥明郑乾坤
Owner JIANGNAN UNIV
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