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
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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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