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A hybridoma cell line secreting anti-bisamide compound monoclonal antibody and its application

A hybridoma cell line and monoclonal antibody technology, applied in the field of immunochemistry, can solve the problems of consumption, expensive equipment, high solvent, etc.

Active Publication Date: 2022-04-12
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Despite the high sensitivity and specificity of these chromatography-based methods, there are some disadvantages such as the need for thorough sample cleanup, high solvent consumption, expensive equipment, and skilled technicians

Method used

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  • A hybridoma cell line secreting anti-bisamide compound monoclonal antibody and its application
  • A hybridoma cell line secreting anti-bisamide compound monoclonal antibody and its application
  • A hybridoma cell line secreting anti-bisamide compound monoclonal antibody and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1: Synthesis of Hapten (Hapten 1)

[0061] Since small molecules are not immunogenic and cannot stimulate mice to produce an immune response to produce antibodies, it is necessary to couple small molecules to proteins through protein linkage technology to obtain immunogenicity; commonly used in protein coupling techniques Active groups include amino, carboxyl, hydroxyl, mercapto, etc. In order to obtain monoclonal antibodies that can specifically recognize cyclobromide, cyantraniliprole, chlorantraniliprole, and tetrachlorantraniliprole, it is necessary to design and derive a For a good hapten, the specific derivatization steps are as follows.

[0062] Step 1: Synthesis of tert-butyl 4-(2-amino-3-bromo-5-chlorobenzamido)butyrate

[0063]

[0064] Add 1.0g (4.0mmol) 2-amino-3-bromo-5-chlorobenzoic acid, 10ml toluene and 0.80mL (12.0mmol) thionyl chloride to the reaction flask, reflux for 3 hours, remove the solvent, add 5mL tetrahydrofuran and wait Use; drop...

Embodiment 2

[0068] Example 2: Synthesis of Complete Antigen

[0069] Weigh 2.8mg hapten (Hapten 1), 1.6mg N-hydroxysuccinimide (NHS), dissolve in 300μL N,N-dimethylformamide (DMF), stir at room temperature for 10min; then weigh 2.6mg 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), fully dissolved with 100μL DMF, added to the hapten solution, stirred at room temperature for 6-8h (called Liquid A). Take 10mg of BSA, dilute it to 5mg / 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 solution Dialyzed to remove unreacted small molecular hapten to obtain the complete antigen, which was identified by UV absorption scanning method.

Embodiment 3

[0070] Embodiment 3: the synthesis of coating original

[0071]In order to improve the sensitivity, a new coating hapten (Hapten2) structure is designed as follows:

[0072]

[0073] The synthesis method of hapten (Hapten2) is similar to that of hapten (Hapten1). The specific steps for the synthesis of the coating agent are as follows: dissolve 3.6 mg hapten (Hapten2) and 2.3 mg N-hydroxysuccinimide (NHS) in 300 μL of anhydrous N,N-dimethylformamide (DMF), and stir the reaction at room temperature After 10 min, the hapten solution was obtained; 3.8 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was dissolved in 100 μL of anhydrous DMF, and added to the hapten solution , stirred at room temperature and reacted for 6-8 hours to obtain liquid A; dilute 10 mg of chicken ovalbumin (OVA) with 1 mL of carbonate buffer solution (CBS) with a concentration of 0.01mol / L to obtain liquid B; Slowly add it into liquid B for reaction to obtain a reaction liquid;...

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Abstract

The invention provides a hybridoma cell line secreting anti-bisamide compound monoclonal antibody and application thereof, belonging to the technical field of immunochemistry. The deposit number of the hybridoma cell line is: CGMCC No.22322. The present invention mixes and emulsifies the complete antigen with Freund's adjuvant, and subcutaneously immunizes mice to obtain high-titer, low-IC 50 Mouse splenocytes were fused with mouse myeloma cells by the PEG method, and the hybrid cells after the fusion of the two cells were screened out using selective medium; the cells were then screened by indirect competitive enzyme-linked immunosorbent method and subcloned three times, and finally obtained A monoclonal antibody hybridoma cell line. The monoclonal antibody secreted by this cell line has good detection sensitivity to cyclic bromantraniliprole, cyantraniliprole, chlorantraniliprole, and tetrachlorantraniliprole, and can be used for the detection of these four pesticide residues in food.

Description

technical field [0001] The invention belongs to the technical field of immunochemistry, in particular to a hybridoma cell strain secreting anti-bisamide compound monoclonal antibody and application thereof. Background technique [0002] Cyclaniliprole, cyantraniliprole, chlorantraniliprole, and tetrachlorantraniliprole are all bisamide insecticides. Bisamide insecticides are ryanodine receptor activators, which have excellent control effects on lepidopteran pests on rice and other crops, are safe to non-target organisms, and are incompatible with other existing insecticides with other modes of action. cross resistance. At present, 4 varieties are commercialized, namely flubendiamide, which was launched in 2006, chlorantraniliprole, which was launched in 2007, cyantraniliprole, which was launched in 2013, and tetrachlorantraniliprole, which was launched in 2014. [0003] Chlorantraniliprole was discovered by DuPont with flubendiamide as the lead compound after in-depth pilo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C12N15/06C07K16/44G01N33/577G01N33/53
CPCC07K16/44C12N5/163G01N33/577G01N33/5308C07K2317/33G01N2430/10
Inventor 胥传来郭鑫匡华刘丽强宋珊珊胡拥明吴爱红徐丽广郝昌龙孙茂忠马伟
Owner JIANGNAN UNIV
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