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Preparation of monoclonal antibody of specific combined sibutramine and demethylated sibutramine

A technology of desmethylsibutramine and monoclonal antibody, applied in the field of immunochemistry, can solve the problems of high operation cost, complicated operation process, expensive equipment and the like, and achieve the effects of low cost, simple operation and high sensitivity

Inactive Publication Date: 2010-02-10
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the detection methods of sibutramine and demethyl sibutramine mainly include: thin layer chromatography, high performance liquid chromatography, high performance liquid chromatography-mass spectrometry, etc., but these methods are expensive in equipment and operating costs High, complex operation process, not suitable for widespread implementation in grassroots departments
Moreover, for the structural analogs of sibutramine drugs, their operation rules on the chromatographic column are quite different from those of known drugs, which may easily lead to missed detection

Method used

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  • Preparation of monoclonal antibody of specific combined sibutramine and demethylated sibutramine
  • Preparation of monoclonal antibody of specific combined sibutramine and demethylated sibutramine

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specific Embodiment approach 2

[0027] Specific embodiment two: Synthesis of desmethylsibutramine artificial immune antigen.

[0028] ①Preparation of glycosylated BSA: Mix glucose, lactose and bovine serum albumin at a molar ratio of 20:1, adjust the pH to 9.0 with 0.1M sodium carbonate solution, react on a shaker at 42°C for 2 hours, and reduce sugar completely Modification of the carrier protein is accomplished by forming a Schieve bond to the amino group.

[0029] ②Preparation of demethylsibutramine artificial immune antigen by glutaraldehyde one-step method: mix modified bovine serum albumin, glutaraldehyde and demethyl sibutramine in a molar ratio of 1:25:80, and use Adjust the pH to 9.0 with 0.1M sodium carbonate solution, and react at 40°C for 6 hours, so that desmethylsibutramine is connected to the carrier protein through the bifunctional reagent. Then, the 0.05M PBS (pH7.2) solution was dialyzed at 4°C for 3 days, during which the solution was changed 10 times. Complete the preparation of desmeth...

specific Embodiment approach 3

[0030] Specific embodiment three: Desmethylsibutramine artificially detects the synthesis of antigens.

[0031] Mix ovalbumin (OVA), glutaraldehyde, and sibutramine-methyl sibutramine in a molar ratio of 1:15:60, adjust the pH to 9.0 with 0.1M sodium carbonate solution, and react at 40°C for 6 hours. The methyl sibutramine is linked to the carrier protein through the bifunctional reagent. Then, the 0.05M PBS (pH7.2) solution was dialyzed at 4°C for 3 days, during which the solution was changed 10 times. Complete the preparation of desmethylsibutramine artificial detection antigen.

specific Embodiment approach 5

[0032] Embodiment 5: Using sibutramine to immunize mice with artificial immunization antigen.

[0033] ①After emulsifying sibutramine artificial immunization antigen with an equal amount of complete Freund's adjuvant, subcutaneously immunized four 4-week-old female BALB / c mice by subcutaneous multi-point injection (100 μg / 0.2 mL per mouse); ②After 4 weeks of primary immunization, the sibutramine artificial immunization antigen was emulsified with the same amount of Freund's incomplete adjuvant, and the mice were boosted by subcutaneous injection (100 μg / mouse); after that, boosted immunization once every three weeks;③ Three days before fusion, sibutramine artificial immunization antigen aqueous solution (50 μg / rat) was injected into the tail vein. Prepare for cell fusion experiments;

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Abstract

The invention discloses preparation of monoclonal antibody of specific combined sibutramine and demethylated sibutramine, comprising the following steps of: synthesizing immune antigen; detecting antigen synthesis; immunizing mouse; screening monoclonal antibody; and identifying the structural specificity of the monoclonal antibody. The synthesis of the immune antigen comprises the following stepsof: (1) mixing glucose, lactose and bovine serum albumin according to mole ratio being 20-25:1, adjusting pH value to 9.0-9.5, and table reacting for 2 hours under the temperature of 40-42 DEG C; and(2) mixing the modified bovine serum albumin, glutaraldehyde and demethylated sibutramine according to mol ratio being 1:(25-30): (80-90), adjusting pH value to be 9.0-9.5, reacting for 6-8 hours under the temperature of 40-42 DEG C, and completely dialyzing in 0.01 M PBS solution with pH value being 7.2-7.4 under the temperature of 4 DEG C. The detection of the antigen synthesis comprises the following steps of: mixing the ovalbumin, the glutaraldehyde and the methyl sibutramine with mol ration being 1: (15-20): (60-75), adjusting pH value to be 9.0-9.5, reacting for 6-8 hours under the temperature of 40-42 DEG C, and completely dialyzing in 0.01 M PBS solution with pH value being 7.2-7.4 under the temperature of 4 DEG C. The preparation has high sensitivity, strong specificity, convenience, simple operation, low cost, and no expensive equipment.

Description

technical field [0001] The invention belongs to the technical field of immunochemistry, and in particular relates to a method for preparing an artificial antigen of desmethyl sibutramine, and prepares a monoclonal antibody capable of specifically binding to sibutramine and desmethyl sibutramine. Background technique [0002] Sibutramine (sibutramine) is a serotonin and norepinephrine reuptake inhibitor listed by Knoll in 1997. Its chemical name is (±)N-{1-[1-(4-chlorophenyl) ring butyl]-3-methylbutyl}-N,N-dimethylamine. The main mechanism of action of sibutramine metabolites is to inhibit the reuptake of norepinephrine, 5-hydroxytryptamine and dopamine, increase the concentration of norepinephrine, 5-hydroxytryptamine and dopamine in the synaptic cleft, act on the central nervous system, and accelerate Metabolism and suppress appetite, increase satiety, promote energy consumption in the body, reduce blood lipid levels and cholesterol levels, clinically used for the treatmen...

Claims

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Application Information

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IPC IPC(8): C07K14/765C07K14/795C07K1/113C12N15/06C12P21/08
Inventor 许杨郭杰标何庆华李燕萍黄志兵
Owner NANCHANG UNIV
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