CN101643506A - Preparation of monoclonal antibody of specific combined sibutramine and demethylated sibutramine - Google Patents
Preparation of monoclonal antibody of specific combined sibutramine and demethylated sibutramine Download PDFInfo
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- CN101643506A CN101643506A CN200910115825A CN200910115825A CN101643506A CN 101643506 A CN101643506 A CN 101643506A CN 200910115825 A CN200910115825 A CN 200910115825A CN 200910115825 A CN200910115825 A CN 200910115825A CN 101643506 A CN101643506 A CN 101643506A
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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
The invention belongs to the immunochemical technique field, be specifically related to the preparation method of a kind of demethylated sibutramine artificial antigen, and preparation can specific combined sibutramine and the monoclonal antibody of demethylated sibutramine.
Background technology
Sibutramine (sibutramine) is serotonin and NRI its chemistry by name (±) N-{1-(1-(4-chloro-phenyl-) the cyclobutyl)-3-methyl butyl of Knoll company in listing in 1997 }-N, N dimethylamine.The main mechanism of sibutramine interior metabolism product is to suppress the re-uptake of norepinephrine, serotonin and Dopamine HCL, increase the concentration of synaptic cleft norepinephrine, serotonin and Dopamine HCL, act on nervus centralis, accelerate metabolism and depress appetite, increase full sense, promote intravital energy expenditure, blood fat reducing level and cholesterol levels are used for the treatment of obesity clinically.
U.S. food Drug Administration (US-FDA) is in this medicine of official approval in November, 1997 special-purpose medicaments as treatment of obesity, and regulation must be used under physician guidance.Chinese food Drug Administration (SFDA) is after in July, 2000, approval was gone on the market as two kind new medicines with it, and same regulation is pressed the prescription drugs management.Under physician guidance, do not take sibutramine, can produce a series of untoward reactions and side effect, mainly contain the untoward reaction of Digestive tract, cardiovascular systems, neural psychiatric system.
In addition, take sibutramine and also can produce some rare untoward reactions, mainly contain renal impairment, influence, fash, alopecia or the like sexual function.The clinician should be specifically noted that the observation to its untoward reaction when using sibutramine, reduce drug combination, forbids using in teenage and elderly population, to guarantee medication person's safety.
But the Western medicine Sibutramine hydrochloride but illegally mixes in the manufacturer of some Chinese medicine slimming health product in product.Mix pseudo-malpractice for hitting this false making effectively, ensure the people's drug safety, necessary foundation is reliably at the detection method of illegally mixing the Western medicine Sibutramine hydrochloride.At present, the detection method of sibutramine and demethylated sibutramine mainly contains: tlc, high performance liquid chromatography, high performance liquid chromatography-mass spectrometry method or the like, but these method apparatus expensive, working cost height, complicated operating process are not suitable in department of basic unit and extensively carry out.And for the analog of sibutramine medicine, its moving law and known drug on chromatographic column differs widely, and causes omission easily.
Summary of the invention
The objective of the invention is to use monoclonal antibody technique, the monoclonal antibody of preparation specific combined sibutramine and demethylated sibutramine.
The present invention be specifically related to a kind of make the sibutramine drug effect must group by complete reservation and be exposed to the sibutramine method for preparing artificial antigen of carrier proteins outside, and, use hybridoma technology screening, evaluation to obtain the monoclonal antibody of the necessary group of specific recognition sibutramine drug effect based on this artificial immunizing antigen, detection antigen.
Use hybridoma technology screening, identify and obtain the monoclonal antibody that specific recognition sibutramine drug effect must group, must based on the sibutramine pharmacophoric group by complete reservation and be exposed to the carrier proteins outside artificial immunization antigen, detect antigen.So, in the process of synthetic artificial antigen, must select for use the sibutramine drug effect to carry out the chemistry connection in group site in addition.Small-molecule drug is connected on the carrier proteins, will utilizes groups such as amino on the small-molecule drug, carboxyl, sulfydryl usually.But, in the structure beyond the necessary group of sibutramine drug effect, do not have these groups.Select suitable connection site, satisfy the requirement of synthetic qualified artificial antigen.
The present invention is achieved by the following technical solutions.
1, demethylated sibutramine artificial immunization is antigenic synthetic.
1. the preparation of glycosylation BSA: the molar ratio according to glucose and lactose and bovine serum albumin is 20-25 all: 1 mixes, regulate pH to 9.0-9.5,40-42 ℃ of shaking table reaction 2 hours, reducing sugar all was incorporated on the amino by forming the Schieve bond, finishes the modification to carrier proteins.
2. modificationization bovine serum albumin, glutaraldehyde, demethylated sibutramine with 1: (25-30): (80-90) molar ratio mixes, regulate pH to 9.0-9.5,40-42 ℃ was reacted 6-8 hour, demethylated sibutramine is connected on the carrier proteins by bifunctional reagent.4 ℃ of fully dialysis in 0.01M PBS (pH7.2-7.4) solution then.Finish the antigenic preparation of demethylated sibutramine artificial immunization.
2, the demethylated sibutramine manual detection is antigenic synthetic.
Ovalbumin (OVA), glutaraldehyde, methyl sibutramine sibutramine with 1: (15-20): (60-75) molar ratio mixes, regulate pH to 9.0-9.5,40-42 ℃ was reacted 6-8 hour, the methyl sibutramine is connected on the carrier proteins by bifunctional reagent.4 ℃ of fully dialysis in 0.01M PBS (pH7.2-7.4) solution then.Finish the antigenic preparation of demethylated sibutramine manual detection.
3,, use sibutramine artificial immunization antigen immune mouse according to the prior art of pertinent literature report.
4, according to the prior art of pertinent literature report, screen anti-sibutramine monoclonal antibody with the cytogamy hybridoma technology.
5, the structure specificity of monoclonal antibody is identified:
1. use sibutramine analog demethylated sibutramine, suppress the ELISA method with competition each strain specific combined sibutramine monoclonal antibody is carried out the cross reaction detection;
2. differentiating the monoclonal antibody that cross reacting rate is big, is exactly can specific combined sibutramine and the monoclonal antibody of analog.
Demethylated sibutramine of the present invention is synthetic by prior art.
The present invention is highly sensitive, high specificity, convenient and swift, simple to operate, with low cost, need not the equipment by costliness, is well suited for promoting in detection department of the vast basic unit of China.
Description of drawings
Accompanying drawing 1 is the demethylated sibutramine synthetic route chart.
Accompanying drawing 2 is demethylated sibutramine artificial immunization antigen and manual detection antigen synthetic route chart.
Embodiment
The present invention will be further described by following examples.
Embodiment one.The demethylated sibutramine synthetic method.
1. the preparation of 1-(4-chloro-phenyl-) cyclobutyronitrile: with 65g 4-chlorobenzene acetonitrile and 85g 1, the 3-dibromopropane is dissolved in the 120mL ether, fully stirs down in 20 ℃ this solution is slowly splashed in the 300mL methyl-sulphoxide suspension that contains 95g potassium hydroxide (fine powder).Stir 1h, reactant is cooled to 15 ℃, drip frozen water 200mL, keep temperature to be no more than 20 ℃ simultaneously.In reaction mixture, add the 250mL ether, filter with sintered filter funnel.Wash filter residue with ether, merging filtrate and washings divide water-yielding stratum with separating funnel, use 250m extracted with diethyl ether three times, combined ether layer and evaporate to dryness.The resistates underpressure distillation gets colorless oil.2. 1-(1-(4-chloro-phenyl-) cyclobutyl)-3-methylbutylamine (demethylated sibutramine) preparation: 50g 1-(4-chloro-phenyl-) cyclobutyronitrile is dissolved in the 180mL dry toluene, this solution is splashed in the isobutyl bromide magnesium solution, ether is removed by distillation.Mixture stirs 18h at 90 ℃.Cooling is added to reactant in the suspension of the hot sodium borohydride of 30g/1000mL Virahol slightly, the mixture heating up backflow 6h that obtains, solvent evaporated behind the room temperature placement 18h.Resistates adds 1000mL water mixing, places 30min, uses 300mL ethyl acetate extraction 3 times.Branch ester output layer divides water-yielding stratum to regulate pH to 8.0 with yellow soda ash with the extraction of pH4.0 aqueous hydrochloric acid, uses 200mL ethyl acetate extraction 2 times, merges ester layer evaporate to dryness and just obtains 1-(1-(4-chloro-phenyl-) cyclobutyl)-3-methylbutylamine (demethylated sibutramine).
Embodiment two: demethylated sibutramine artificial immunization is antigenic synthetic.
1. the preparation of glycosylation BSA: according to glucose and lactose and the molar ratio of bovine serum albumin all is to mix at 20: 1, regulate pH to 9.0 with the 0.1M sodium carbonate solution, 42 ℃ of shaking table reactions 2 hours, reducing sugar all is incorporated on the amino by forming the Schieve bond, finishes the modification to carrier proteins.
2. prepare demethylated sibutramine artificial immunization antigen by the glutaraldehyde single stage method: modificationization bovine serum albumin, glutaraldehyde, demethylated sibutramine are mixed with 1: 25: 80 molar ratio, regulate pH to 9.0 with the 0.1M sodium carbonate solution, 40 ℃ were reacted 6 hours, demethylated sibutramine are connected on the carrier proteins by bifunctional reagent.Then to 0.05M PBS (pH7.2) solution 4 ℃ the dialysis 3 days, during change liquid 10 times.Finish the antigenic preparation of demethylated sibutramine artificial immunization.
Embodiment three: the demethylated sibutramine manual detection is antigenic synthetic.
Ovalbumin (OVA), glutaraldehyde, methyl sibutramine sibutramine are mixed with 1: 15: 60 molar ratio, regulate pH to 9.0 with the 0.1M sodium carbonate solution, 40 ℃ were reacted 6 hours, the methyl sibutramine are connected on the carrier proteins by bifunctional reagent.Then to 0.05M PBS (pH7.2) solution 4 ℃ the dialysis 3 days, during change liquid 10 times.Finish the antigenic preparation of demethylated sibutramine manual detection.
Embodiment five: use sibutramine artificial immunization antigen immune mouse.
1. after bent person of good sense worker's immunizing antigen of cloth and the emulsification of equivalent Freund's complete adjuvant, adopt subcutaneous multi-point injection (100 μ g/0.2mL only) mode that the female BALB/c mouse in 44 ages in week is carried out subcutaneous immunity to the west of; 2. after just exempting from for 4 weeks, get sibutramine artificial immunization antigen and the emulsification of equivalent Freund's incomplete adjuvant, mouse is adopted subcutaneous injection booster immunization (100 μ g/ only); Per three all booster immunizations are 1 time later on; 3. merge first three day, adopt the tail vein injection sibutramine artificial immunization antigen aqueous solution (50 μ g/ only).The cytogamy experiment is carried out in preparation;
Embodiment six: screen anti-sibutramine monoclonal antibody with the cytogamy hybridoma technology.
1. with 1 * 10
8Splenocyte and 2 * 10
7Myeloma cell SP2/0 is mixed in the 50mL fusion pipe, adds the DMEM substratum to 30mL.The centrifugal 7min of 1000rpm is with supernatant exhaustion as far as possible; 2. on palm, touch the fusion pipe bottom, make sedimentation cell loose evenly, put preheating in 40 ℃ of water-baths.Added 50%PEG (pH 8.0) 1mL that is preheated to 40 ℃ with the 1mL suction pipe in 1 minute, the limit edged shakes mixing gently.Added the incomplete DMEM substratum that 25mL is preheated to 37 ℃ with the 5mL suction pipe in 90 seconds, room temperature leaves standstill 10min.Centrifugal 7 minutes of 800rpm abandons supernatant; 3. add 5mL HAT perfect medium, the pressure-vaccum sedimentation cell suspends and mixing it gently, adds the HAT substratum then to 90mL.Divide to be filled to and merge the 96 porocyte culture plates of cultivating the day before yesterday that contain feeder cell, every hole 100 μ L; Culture plate is put 37 ℃ of 5%CO
2Cultivate in the incubator; 4. be cultured to the 5th day, with the HT perfect medium 1/2HAT nutrient solution in the hole that swaps out.Be cultured to the 7th day, with HT substratum all nutrient solutions that swap out in the hole; 5. observe the hybridoma growing state every day, when treating that hybridoma grows to 10% above hole floorage, the sucking-off nutrient solution carries out antibody test; Suppress the ELISA method with competition, filter out the hybridoma of the anti-sibutramine monoclonal antibody of secretion.
Embodiment seven.The structure specificity of monoclonal antibody is differentiated.
1. use the bent name of sibutramine analog demethyl west cloth, with competition inhibition ELISA method each strain specific combined sibutramine monoclonal antibody is carried out cross reaction and detect;
2. differentiating the monoclonal antibody that cross reacting rate is big, is exactly can specific combined sibutramine and the monoclonal antibody of analog.
Claims (1)
1, the MONOCLONAL ANTIBODIES SPECIFIC FOR of specific combined sibutramine and demethylated sibutramine, comprise that antigenic synthetic, the immune mouse of antigenic synthetic, the manual detection of artificial immunization, the screening of monoclonal antibody, the structure specificity of monoclonal antibody identify, it is characterized in that artificial immunization is antigenic to synthesize:
1. the preparation of glycosylation BSA: the molar ratio according to glucose and lactose and bovine serum albumin all is 20-25: 1 mixes, and regulates pH to 9.0-9.5, reacts 2 hours at 40-42 ℃ of shaking table;
2. modificationization bovine serum albumin, glutaraldehyde, demethylated sibutramine with 1: (25-30): (80-90) molar ratio mixes, regulate pH to 9.0-9.5,40-42 ℃ was reacted 6-8 hour, 4 ℃ of fully dialysis in pH7.2-7.4,0.01M PBS solution then;
Manual detection is antigenic to synthesize: ovalbumin, glutaraldehyde, methyl sibutramine sibutramine with 1: (15-20): (60-75) molar ratio mixes, regulate pH to 9.0-9.5,40-42 ℃ was reacted 6-8 hour, then at pH7.2-7.4, and 4 ℃ of fully dialysis in the 0.01M PBS solution.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103267842A (en) * | 2013-05-15 | 2013-08-28 | 郭杰标 | Immune colloidal gold method for detecting diclofenac illegally added in Chinese patent medicament |
| CN109022367A (en) * | 2018-08-16 | 2018-12-18 | 江南大学 | The anti-sibutramine monoclonal antibody specific hybridoma cell strain of one plant of secretion and its application |
-
2009
- 2009-08-27 CN CN200910115825A patent/CN101643506A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103267842A (en) * | 2013-05-15 | 2013-08-28 | 郭杰标 | Immune colloidal gold method for detecting diclofenac illegally added in Chinese patent medicament |
| CN103267842B (en) * | 2013-05-15 | 2016-03-02 | 郭杰标 | Detect the immune colloidal gold method of the illegal interpolation Diclofenac of Chinese patent drug |
| CN109022367A (en) * | 2018-08-16 | 2018-12-18 | 江南大学 | The anti-sibutramine monoclonal antibody specific hybridoma cell strain of one plant of secretion and its application |
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Open date: 20100210 |