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Hybridoma cell strain capable of secreting amitraz monoclonal antibody and application of hybridoma cell strain

A technology of hybridoma cell lines and monoclonal antibodies, which is applied in the fields of analytical materials, biological material analysis, biochemical equipment and methods, etc., can solve the problems of inapplicability to rapid detection of a large number of samples, long detection time, complicated processing, etc., and achieve good results Effects of detection sensitivity and affinity, simplification of synthesis steps, good detection sensitivity and specificity

Inactive Publication Date: 2020-10-13
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 amitraz

Method used

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  • Hybridoma cell strain capable of secreting amitraz monoclonal antibody and application of hybridoma cell strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Synthesis of Amitraz Hapten

[0047] Since the small molecule of amitraz is not immunogenic and cannot stimulate the immune response of mice to produce antibodies, it is necessary to couple amitraz to the protein through protein linkage technology to obtain immunogenicity; protein coupling Active groups commonly used in the technology include amino, carboxyl, hydroxyl, mercapto, etc. Since the molecular structure of amitraz does not contain these active groups, derivatization is required.

[0048] The derivatized amitraz hapten structure of the present invention is as follows:

[0049] .

Embodiment 2

[0050] Embodiment 2: the synthesis of amitraz complete antigen

[0051] Weigh 2.3mg amitraz hapten, 2.2mg N-hydroxysuccinimide (NHS), dissolve in 200μL N,N-dimethylformamide (DMF), stir at room temperature for 10min; then weigh 3.5mg 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), fully dissolved with 100 μL DMF, added to the amitraz hapten solution, stirred at room temperature for 6-8 h (called liquid A). Take 7.5mg KLH, dilute it to 3mg / mL with 0.01M carbonate buffer (CBS) (referred to as solution B), then slowly add solution A to solution B drop by drop, react at room temperature overnight; then use 0.01M PBS The solution was dialyzed to remove the unreacted small molecule hapten to obtain the complete antigen of amitraz, which was identified by ultraviolet absorption scanning method.

Embodiment 3

[0052] Embodiment 3: the synthesis of amitraz coating former

[0053] Dissolve 1.5 mg of amitraz hapten and 2.3 mg of N-hydroxysuccinimide (NHS) in 200 μL of anhydrous N,N-dimethylformamide (DMF), and stir at room temperature for 10 minutes to obtain amitraz hapten Solution: Dissolve 3.8 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in 100 μL of anhydrous DMF and add it to the amitraz hapten solution at room temperature Stir and react for 6-8 hours to obtain solution A; dissolve 10 mg of chicken ovalbumin (OVA) in 3 mL of carbonate buffer solution (CBS) with a concentration of 0.01 mmol / L to obtain solution B; slowly add solution A to the The reaction is carried out in liquid B to obtain a reaction liquid; the reaction liquid is dialyzed with PBS solution to remove unreacted small molecule hapten to obtain the coating source of amitraz.

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Abstract

The invention discloses a hybridoma cell strain capable of secreting an amitraz monoclonal antibody and application of hybridoma cell strain, and belongs to the field of food safety immune detection.The hybridoma cell strain RC capable of secreting the amitraz monoclonal antibody is collected in the China General Microbiological Culture Collection Center (CGMCC), with the collection number of CGMCC No. 19166. The amitraz monoclonal antibody secreted by the strain is used for the analysis and detection of amitraz residues in food safety testing. The amitraz monoclonal antibody cell strain RC can be used for immunoassay detection, and has good detection sensitivity and specificity to amitraz (IC50 value is 0.3 ng / mL, crossover of an amitraz analogue is smaller than 10%, and the crossover rate = (IC50 of IC50 / analogue of amitraz *100%)).

Description

technical field [0001] The invention relates to a hybridoma cell strain secreting amitraz monoclonal antibody and application thereof, which belongs to the field of food safety immune detection. Background technique [0002] Amitraz, also known as mite, is a broad-spectrum formamidine insecticide and acaricide with multiple poisoning mechanisms. It is mainly used to remove mites from fruit trees, vegetables, tea, cotton, soybeans and other crops. Partial Lepidoptera pest eggs are effective, have contact killing and fumigation effects on harmful mites, and can also be used to control acarids in livestock such as cattle and sheep. Because amitraz can effectively prevent bee diseases caused by Divalet mites, and has low toxicity to bees , so it is also widely used to remove mites in hives. Amitraz is moderately toxic to humans and animals, irritating to human skin and mucous membranes, and can inhibit the central nervous system of the human body. There is no specific drug afte...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/44C07K14/795G01N33/577G01N33/53
CPCC07K14/795C07K16/44G01N33/5308G01N33/577G01N2430/10
Inventor 胥传来晁梦佳匡华路倩倩徐丽广刘丽强宋珊珊吴晓玲胡拥明郑乾坤
Owner JIANGNAN UNIV
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