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

A hybridoma cell line, monoclonal antibody technology, applied in the direction of analytical materials, microbial-based methods, instruments, etc., can solve the problems of expensive equipment, high solvent consumption, etc., and achieve the effect of good detection sensitivity

Pending Publication Date: 2021-06-22
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 s

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Synthesis of hyoscyamine hapten

[0053] Since the small molecule of hyoscyamine is not immunogenic and cannot stimulate the mice to produce an immune response and then produce antibodies, it is necessary to couple hyoscyamine to the protein through protein linkage technology to obtain immunogenicity; in order to better stimulate To generate an immune response in mice, the hyoscyamine hapten was designed, and the derivation process was as follows:

[0054] Weigh hyoscyamine (500mg, 1.73mmol), succinic anhydride (259mg, 2.6mmol), triethanolamine (260mg, 2.6mmol) and dissolve in 10ml of dichloromethane, stir at room temperature for 4h, and terminate the reaction with 1M hydrochloric acid Finally, the pH was adjusted to 5-6, and the mixed solution was removed with dichloromethane to remove impurities. The water layer was purified by preparative high-performance liquid chromatography to obtain a high-purity target substance. After concentration and enrichment, 25...

Embodiment 2

[0055] Example 2: Synthesis of Hyoscyamine Complete Antigen

[0056] Weigh 2.92mg hyoscyamine hapten, 2.6mg N-hydroxysuccinimide (NHS), dissolve in 300μL N,N-dimethylformamide (DMF), stir at room temperature for 5min; then add 4.3mg 1- (3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), stirred at room temperature for 6-8h (referred to as liquid A). Take 10mg KLH, 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 for 12h; then use 0.01M PBS solution Dialyze to remove unreacted small molecular haptens to obtain the complete antigen of hyoscyamine.

Embodiment 3

[0057] Embodiment 3: the synthesis of scopolamine coating former

[0058] Dissolve 2.0 mg of hyoscyamine and 11.20 mg of N,N'-carbonyldiimidazole (CDI) in 300 μL of anhydrous N,N-dimethylformamide (DMF), stir and react at 37°C for 8 hours to obtain hyoscyamine half Antigen solution, i.e. solution A; dilute 5 mg of chicken ovalbumin (OVA) with 1 mL of carbonate buffer solution (CBS) with a concentration of 0.01mol / L to obtain solution B; slowly add solution A to solution B drop by drop Reaction, stirring at room temperature for 12 hours to obtain a reaction solution, which was dialyzed against 0.01 mol / L phosphate buffer saline PBS for 3 days to remove unreacted small molecules to obtain hyoscyamine-coated original.

[0059] The molecular structure of hyoscyamine original drug is shown in the figure:

[0060]

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Abstract

The invention discloses a hybridoma cell strain capable of secreting a scopolamine monoclonal antibody and application of the hybridoma cell strain, and belongs to the field of food safety immunodetection. The hybridoma cell strain Pba capable of secreting the scopolamine monoclonal antibody is preserved in the China General Microbiological Culture Collection Center (CGMCC), is classified and named as a monoclonal cell strain, is preserved on April 23, 2020, and has the preservation number of CGMCC No.19678. A complete antigen of hyoscyamine is used for immunizing a BALB/c mouse, splenocytes of a high-titer and low-IC50 mouse are fused with myeloma cells of the mouse through a PEG method, and a selective culture medium is adopted to screen out hybrid cells after fusion of the two cells; and screening cells by an indirect competitive enzyme-linked immunosorbent assay method, and subcloning for three times to finally obtain the hybridoma cell strain Pba. The monoclonal antibody secreted by the Pba has better detection sensitivity (IC50 value is 1.03 ng/mL) to the hyoscyamine, and can be used for residue detection of the hyoscyamine.

Description

technical field [0001] The invention relates to a hybridoma cell strain secreting scopolamine monoclonal antibody and an application thereof, belonging to the field of food safety immunoassay. Background technique [0002] Hyoscyamine is one of the belladonna alkaloids isolated from the traditional Chinese medicine Tianxianzi and Yangjinhua. Its structure is an ester formed by the condensation of hyoscypolamine and hyoscypolamine. Hyoscyamine is a parasympathetic inhibitor, and its pharmacological action is similar to that of atropine, but it is more toxic and has less clinical application. Hyoscyamine has the function of relieving pain and spasm, and has a good effect on sciatica. Sometimes it is also used to treat epilepsy and seasickness. Mainly used in biochemical research, anticholinergics and gold detection reagents. [0003] At present, the detection methods of scopolamine are mainly instrumental detection, and commonly used methods include high performance liquid c...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/44C07K14/795C07K14/77G01N33/577G01N33/543G01N33/53C12R1/91
CPCC07K16/44C07K14/795C07K14/77C07D451/10G01N33/577G01N33/54346G01N33/5308
Inventor 胥传来郭鑫匡华刘丽强宋珊珊胡拥明
Owner JIANGNAN UNIV
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