CN103145831A - Synthesis method of specific salbutamol artificial antigen - Google Patents
Synthesis method of specific salbutamol artificial antigen Download PDFInfo
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Abstract
The invention discloses a synthesis method of a specific salbutamol artificial antigen, belonging to the technical field of biological chemical engineering. The synthesis method disclosed by the invention comprises the following steps of: activating salbutamol through a formaldehyde solution, and connecting 6-aminocaproic acid in the ortho-position of a phenolic hydroxyl group to obtain a salbutamol hapten; and coupling a carboxyl group on the salbutamol hapten with an amino group on a carrier protein to obtain the salbutamol artificial antigen. The synthesis method disclosed by the invention can make up for the insufficiencies and defects of the existing salbutamol antigen synthesis technologies, the salbutamol artificial antigen with high specificity is obtained, the specificity of a produced antibody is high, and the sensitivity is high; and experimental results show that the antiserum titre of an animal immunized by using the salbutamol artificial antigen disclosed by the invention can achieve 80000, the detection limit is 0.5ng/mL, and the half-inhibitory concentration IC50 is 5ng/mL. The antigen or the antibody disclosed by the invention can be used for establishing an enzyme-linked immunosorbent analytical method and a colloidal gold test strip rapid assay method so as to rapidly detect the residues of the salbutamol in a food and further realize broad application prospects.
Description
Technical field
A kind of synthetic method of high specific salbutamol artificial antigen belongs to technical field of biochemical industry.
Background technology
Salbutamol (salbutamol) is a kind of fugitive beta 2 adrenoreceptor agonists, as anti-asthmatic.Add micro-salbutamol in animal feed, can increase the cutability of livestock and meat change rate, minimizing fat, but its toxicity is far above the Ractopamine hydrochloride with identical function.10 of September in 2002 forbid within Chinese territory using salbutamol in feed and animal drinking water.The salbutamol structural formula is suc as formula (1).
Formula (1)
China mainly contains high performance liquid chromatography (HPLC), Liquid Chromatography/Mass Spectrometry (LC/MS), enzyme-linked immunosorbent assay (Enzyme-linked immunosorbent assay, ELISA), colloidal gold strip method etc. to the detection method of salbutamol at present.Instrument analytical method exists sample must dilute through multistep, filter, extract, the shortcoming that preparation is complicated, loaded down with trivial details.Although instrument analytical method is the conclusive evidence method that salbutamol detects, due to its complex operation, and long Sample pretreatment process, cause testing cost high, cycle is long, can't satisfy sample rapid screening in enormous quantities, and the requirement of field quick detection.ELISA and colloidal gold strip are owned by France in immuno analytical method, have higher sensitivity and specificity, and the purity requirement to sample during detection is not high and easy and simple to handle, are applicable to the field quick detection of great amount of samples.
The key factor that affects immune analysis method is specific antigen and antibody.Traditional salbutamol artificial antigen is generally by the hydroxyl coupling on succinyl oxide and salbutamol molecule, derive carboxyl again with albumen coupling.Due to three hydroxyls being arranged on salbutamol, can participate in reaction, therefore the carboxy derivatives that uses the succinyl oxide method to obtain is a mixture, and the final antibody that obtains by immune animal is for this mixture, and the specificity of antibody and sensitivity meeting are greatly affected.
The active hydrogen at the present invention phenolic hydroxyl group ortho position from the salbutamol molecule is set about, the access 6-aminocaprolc acid, obtain the salbutamol carboxy derivatives of single structure, this derivative is the salbutamol haptens, and salbutamol haptens and carrier protein couplet are formed salbutamol artificial antigen at last.
Summary of the invention
Purpose of the present invention: for deficiency and the defective of existing salbutamol antigen synthetic technology and corresponding antibodies, a kind of novel salbutamol haptens and complete antigen synthetic method are provided, make the salbutamol monoclonal antibody of preparation high specific become possibility.
Technical scheme of the present invention:
Salbutamol hapten compound provided by the invention has molecular structure shown in Compound I.
Compound (1)
Salbutamol artificial antigen compound provided by the invention has molecular structure shown in compound ii.
Compound ii
The synthetic method of one species specificity salbutamol artificial antigen, take salbutamol as raw material, salbutamol and the synthetic salbutamol haptens of 6-aminocaprolc acid reaction are Compound I, Compound I activates by 1-ethyl-3-(3-dimethylaminopropyl) phosphinylidyne diimine EDC and N-hydroxy-succinamide NHS or activates by tri-n-butylamine and isobutyl chlorocarbonate, making salbutamol artificial antigen with carrier protein couplet again is compound ii, and synthetic route is as shown in reaction formula (1):
Reaction formula (1)
Synthesis technique is:
(1) the haptenic preparation of salbutamol
Salbutamol adds dissolve with ethanol, at room temperature, drips the formaldehyde solution reaction (salbutamol: the mol ratio of formaldehyde is 1:1) of mass concentration 37%, reaction 30min, then add and the equimolar 6-aminocaprolc acid of salbutamol, stirring reaction 6h.With reaction solution pH regulator to 2～3, add ethyl acetate extraction, 37 ℃ of rotary evaporations of organic phase are done, and obtain the salbutamol haptens, i.e. Compound I.
(2) preparation of salbutamol artificial antigen
Carboxyl on haptens and the amino on carrier proteins are carried out coupling obtain artificial antigen, with the artificial antigen dialysis, then carry out ultraviolet and identify (Fig. 1).
(A) with Compound I N, dinethylformamide (DMF) dissolving, Compound I and N-hydroxy-succinamide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) phosphinylidyne diimine (EDC) is at 4 ℃ of lucifuge stirring reaction 1h, Compound I: N-hydroxy-succinamide (NHS): the mol ratio of 1-ethyl-3-(3-dimethylaminopropyl) phosphinylidyne diimine (EDC) is 1:1.5:2, at room temperature reaction 12h, get the salbutamol haptens solution of activation again;
Get bovine serum albumin, the mol ratio of Compound I and bovine serum albumin is 80:1, bovine serum albumin is dissolved with 0.1M pH9.6 carbonate buffer solution, bovine serum albumin concentration after dissolving is greater than 3mg/mL, and the volume ratio of carbonate buffer solution and DMF (DMF) is 5:1; The salbutamol haptens solution of activation is added drop-wise at a slow speed in bovine serum albumin solution, reacts 24h under room temperature, with PBS damping fluid dialysis 2 days, during change PBS damping fluid 4 times, namely obtain salbutamol artificial antigen, i.e. compound ii;
Or (B) with Compound I N, dinethylformamide (DMF) dissolving, 0 ℃ of reaction 1h of Compound I and tri-n-butylamine and isobutyl chlorocarbonate, Compound I: tri-n-butylamine: the mol ratio of isobutyl chlorocarbonate is 1:1.2:1.2, gets the salbutamol haptens solution of activation;
Get bovine serum albumin, the mol ratio of Compound I and bovine serum albumin is 80:1, bovine serum albumin is dissolved with 0.1M pH9.6 carbonate buffer solution, 0 ℃ of precooling 30min, bovine serum albumin concentration after dissolving is greater than 3mg/mL, and the volume ratio of carbonate buffer solution and DMF (DMF) is 5:1; Under 0 ℃ of condition, the haptens solution that activates is added drop-wise at a slow speed in bovine serum albumin solution, react 1h under 0 ℃ of condition, then react 24h under room temperature, with PBS damping fluid dialysis 2 days, during change PBS damping fluid 4 times, namely obtain salbutamol artificial antigen, i.e. compound ii.
Described salbutamol, 6-aminocaprolc acid purity is all greater than 95%.
Described carrier proteins is: the bovine serum albumin cBSA of bovine serum albumin BSA, cationization, keyhole limpet hemocyanin KLH, hemocyanin LPH, chicken ovalbumin OVA or human serum albumin HSA a kind of.
The application in preparation salbutamol antibody of above-mentioned salbutamol haptens or artificial antigen compound also belongs to protection scope of the present invention.
The salbutamol antibody that above-mentioned salbutamol artificial antigen compound immune animal obtains also belongs to protection scope of the present invention, and described antibody is polyclonal antibody and/or monoclonal antibody.
The application of above-mentioned salbutamol haptens or artificial antigen compound or the antibody salbutamolum residue in detecting food also belongs to the scope of protection of the invention.
Beneficial effect of the present invention: the present invention is novel salbutamol complete antigen synthetic method, and the specific salbutamol antigenic determinant that complete antigen presents makes the salbutamol monoclonal antibody that filters out high specific become possibility.
Experimental result shows, the antiserum titre that obtains with antigen-immunized animal of the present invention can reach 80000, detects to be limited to 0.5ng/mL, 503nhibiting concentration IC
50Be 5ng/mL.The antibodies specific that produces is high, highly sensitive.Antigen of the present invention or antibody can be used for setting up ELISA adsorption analysis method and Colloidal gold stripes, thereby are used for the salbutamolum residue of rapid detection food.
Description of drawings
Fig. 1 salbutamol artificial antigen ultraviolet spectrogram.
Embodiment
The experimental technique that uses in following example if no special instructions, is ordinary method.
The material that uses in following example, reagent etc. if no special instructions, all can obtain from commercial channels.
The haptenic preparation of salbutamol
Salbutamol 200mg(0.836mmol), add the 15mL dissolve with ethanol, under room temperature, drip 37% formaldehyde solution 99 μ L, stirring reaction 30min, then add 6-aminocaprolc acid 110mg(0.836mmol), continue stirring reaction 6h.Slowly drip 6M HCl in reaction solution, regulate pH to 2～3, add the 10mL ethyl acetate extraction 3 times, collect organic phase, 37 ℃ of rotary evaporations are done, and obtain the salbutamol haptens.
The preparation of embodiment 2, salbutamol artificial antigen
Get 25mg(0.0678mmol) the salbutamol haptens, add 2mL N, dinethylformamide (DMF) dissolving, add respectively again N-hydroxy-succinamide (NHS), the mol ratio of 1-ethyl-3-(3-dimethylaminopropyl) phosphinylidyne diimine (EDC) (haptens, NHS, EDC) is 1:1.5:2), under 4 ℃, the lucifuge mixing, stirring reaction 60min, then at room temperature reaction 12h.Get 56mg(0.00084mmol) bovine serum albumin (mol ratio of haptens and bovine serum albumin is 80:1), add 10mL0.1M pH9.6 carbonate buffer solution.The haptens solution of activation is added drop-wise at a slow speed in bovine serum albumin solution, reacts 24h under room temperature.With PBS damping fluid dialysis 2 days, during change PBS damping fluid 4 times, namely obtain salbutamol artificial antigen.
The preparation of embodiment 3, salbutamol artificial antigen
Get 25mg(0.0678m mol) the salbutamol haptens, add 2mL DMF (DMF) dissolving, 0 ℃ of precooling 30min.Under 0 ℃, add respectively tri-n-butylamine, isobutyl chlorocarbonate (mol ratio of haptens, tri-n-butylamine, isobutyl chlorocarbonate is 1:1.2:1.2), 0 ℃ of reaction 1h.Get 56mg(0.00084mmol) bovine serum albumin (mol ratio of haptens and bovine serum albumin is 80:1), add 10mL0.1M pH9.6 carbonate buffer solution, 0 ℃ of precooling 30min.Under 0 ℃ of condition, the haptens solution that activates is added drop-wise at a slow speed in bovine serum albumin solution, react 1h under 0 ℃ of condition, then react 24h under room temperature.With PBS damping fluid dialysis 2 days, during change PBS damping fluid 4 times, namely obtain salbutamol artificial antigen.
Embodiment 4, the sero-fast preparation of salbutamol
The salbutamol artificial antigen that makes take embodiment 2 is selected 6-8 age in week as immunogen, and female BALB/C mice is immune animal, adopts freund's adjuvant to carry out immunity, 5 of immune mouses.The freund's adjuvant immunization method is: head exempts to get appropriate immunogen and mixes with the equal-volume Freund's complete adjuvant, and emulsification is good by the subcutaneous multi-point injection immunity of nape section, at interval of 3 all booster immunizations once.
Embodiment 5, the sero-fast mensuration of salbutamol
One, adopt indirect ELISA method to detect serum titer, concrete operation step is as follows:
(1) coated: with the salbutamol artificial antigen of gained in embodiment 3 with 0.05M pH9.6 carbonate buffer solution since 10 μ g/mL doubling dilutions, 100 μ L/ holes, 37 ℃ are reacted 2h.
(2) washing: solution in plate is inclined, dry, and wash 3 times with washings, each 3min.
(3) sealing: after patting dry, add 200 μ L/ hole confining liquids, 37 ℃ of reaction 2h.The washing post-drying is standby.
(4) application of sample: embodiment 4 gained salbutamol antiserum(antisera)s are begun doubling dilution from 1:1000, and join in each dilution coated hole, 100 μ L/ holes, 37 ℃ of reaction 1h; Fully after the washing, add the HRP-sheep anti-mouse igg of 1:3000 dilution, 100ul/ hole, 37 ℃ of reaction 1h.
(5) colour developing: enzyme plate is taken out, and fully after washing, every hole adds the TMB nitrite ion of 100 μ L, 37 ℃ of lucifuge reaction 15min.
(6) stop and measure: every hole adds 100 μ L stop buffers with termination reaction, then measures the OD in each hole with microplate reader
450Value.
(7) interpretation as a result: with OD
450Value is tired more than or equal to the ELISA that the highly diluted multiple of 2.1 times of (being P/N 〉=2.1) corresponding serum of negative control hole is serum.
Two, lowest detectable limit, half suppress and specific detection
Concrete operation step is as follows:
(1) determine the working concentration of coating antigen and antibody with above-mentioned indirect ELISA square formation volumetry, with OD
450Value corresponding antigen and antibody concentration when 1.5 left and right are the suitableeest working concentration.
(2) coated: coating antigen is diluted to the suitableeest working concentration with coated damping fluid, 100 μ L/ holes, 37 ℃ are reacted 2h.
(3) washing and sealing: method operates with above-mentioned indirect elisa method.
(4) preparation salbutamol standardized solution: the salbutamol standard substance are become the mother liquor of 1mg/mL with the PBS solution preparation of 0.01mol/L, pH7.4, then, before application of sample, then become to need concentration with the PBS solution doubling dilution of 0.01mol/L, pH7.4.
(5) application of sample: every hole adds each concentration standard product of salbutamol of 50 μ L doubling dilutions, and then adds the antiserum(antisera) of the suitable extension rate of 50 μ L/ Kongzuis, 37 ℃ of reaction 1h.Fully after the washing, add the HRP-sheep anti-mouse igg of 1:3000 dilution, 100 μ L/ holes, 37 ℃ of reaction 1h.
(6) color reaction: enzyme plate is taken out, and fully after washing, every hole adds the TMB nitrite ion of 100 μ L, 37 ℃ of lucifuge reaction 15min.
(7) stop and measure: every hole adds 100 μ L stop buffers with termination reaction, then measures the OD in each hole with microplate reader
450Value.
(8) data processing: take the logarithm of each concentration of salbutamol as X-coordinate, take OD value corresponding to each concentration of salbutamol as ordinate zou, drawing standard curve, calculation of half inhibitory concentration (IC
50, i.e. OD
450Corresponding standard substance concentration when value drops to 50% from A0 corresponding to zero standard solution), thus judge whether antiserum(antisera) has specificity to salbutamol.
(9) standard substance of salbutamol are changed into Ractopamine hydrochloride, special sieve of horse-ride step, clenbuterol and measure as stated above IC
50Value, and calculate cross reacting rate.
Cross reacting rate (%)=IC
50(salbutamol)/IC
50(analogue)
3 repetitions, results averaged are established in experiment.
Result shows, four exempt from after, mouse resisting anteserum is tired and can be reached 80000, detects to be limited to 0.5ng/mL, salbutamol IC
50Be 5ng/mL, the cross reacting rate of each analogue is all less than 0.1.
Claims (6)
1. the synthetic method of a species specificity salbutamol artificial antigen, it is characterized in that take salbutamol as raw material, salbutamol and the synthetic salbutamol haptens of 6-aminocaprolc acid reaction are Compound I, Compound I activates by 1-ethyl-3-(3-dimethylaminopropyl) phosphinylidyne diimine EDC and N-hydroxy-succinamide NHS or activates by tri-n-butylamine and isobutyl chlorocarbonate, carrying out coupling with carrier proteins again, to make salbutamol artificial antigen be compound ii, and synthetic route is as shown in reaction formula:
(1) the haptenic preparation of salbutamol
Salbutamol adds dissolve with ethanol, at room temperature, drips the formaldehyde solution reaction of mass concentration 37%, and salbutamol: the mol ratio of formaldehyde is 1:1, reaction 30min, then add and the equimolar 6-aminocaprolc acid of salbutamol, stirring reaction 6h; With reaction solution pH regulator to 2～3, add ethyl acetate extraction, 37 ℃ of rotary evaporations of organic phase are done, and obtain the salbutamol haptens, i.e. Compound I;
(2) preparation of salbutamol artificial antigen
(A) with Compound I N, dinethylformamide DMF dissolving, Compound I and N-hydroxy-succinamide NHS and 1-ethyl-3-(3-dimethylaminopropyl) phosphinylidyne diimine EDC is at 4 ℃ of lucifuge stirring reaction 1h, Compound I: the mol ratio of N-hydroxy-succinamide NHS:1-ethyl-3-(3-dimethylaminopropyl) phosphinylidyne diimine EDC is 1:1.5:2, at room temperature reaction 12h, get the salbutamol haptens solution of activation again;
Get bovine serum albumin, the mol ratio of Compound I and bovine serum albumin is 80:1, bovine serum albumin is dissolved with 0.1M pH9.6 carbonate buffer solution, bovine serum albumin concentration after dissolving is greater than 3mg/mL, and the volume ratio of carbonate buffer solution and DMF DMF is 5:1; The salbutamol haptens solution of activation is added drop-wise at a slow speed in bovine serum albumin solution, reacts 24h under room temperature, with PBS damping fluid dialysis 2 days, during change PBS damping fluid 4 times, namely obtain salbutamol artificial antigen, i.e. compound ii;
Or (B) with Compound I N, dinethylformamide DMF dissolving, 0 ℃ of reaction 1h of Compound I and tri-n-butylamine and isobutyl chlorocarbonate, Compound I: tri-n-butylamine: the mol ratio of isobutyl chlorocarbonate is 1:1.2:1.2, gets the salbutamol haptens solution of activation;
Get bovine serum albumin, the mol ratio of Compound I and bovine serum albumin is 80:1, bovine serum albumin is dissolved with 0.1M pH9.6 carbonate buffer solution, 0 ℃ of precooling 30min, bovine serum albumin concentration after dissolving is greater than 3mg/mL, and the volume ratio of carbonate buffer solution and DMF DMF is 5:1; Under 0 ℃ of condition, the haptens solution that activates is added drop-wise at a slow speed in bovine serum albumin solution, react 1h under 0 ℃ of condition, then react 24h under room temperature, with PBS damping fluid dialysis 2 days, during change PBS damping fluid 4 times, namely obtain salbutamol artificial antigen, i.e. compound ii.
2. the synthetic method of specificity salbutamol artificial antigen according to claim 1 is characterized in that: described carrier proteins is bovine serum albumin cBSA, keyhole limpet hemocyanin KLH, hemocyanin LPH, chicken ovalbumin OVA or human serum albumin HSA a kind of of bovine serum albumin BSA, cationization.
3. the synthetic method of specificity salbutamol artificial antigen according to claim 1, it is characterized in that: described salbutamol haptens is Compound I, and its molecular structural formula is:
Compound I.
4. the synthetic method of specificity salbutamol artificial antigen according to claim 1, it is characterized in that: described salbutamol artificial antigen is compound ii, its molecular structural formula is:
Compound ii.
5. with the application of the synthetic salbutamol artificial antigen of the described method of claim 1, it is characterized in that preparing salbutamol antibody, described antibody is polyclonal antibody and/or monoclonal antibody.
6. with the application of the synthetic salbutamol artificial antigen of the described method of claim 1, it is characterized in that detecting the salbutamolum residue in food.
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| CN104031142A (en) * | 2014-06-10 | 2014-09-10 | 江南大学 | Synthesis method of highly specific folic acid complete antigen and application of folic acid complete antigen |
| CN104610079A (en) * | 2014-10-13 | 2015-05-13 | 湖州海创生物科技有限公司 | Salbutamol hapten derivative, and artificial salbutamol antigen and preparation method and application thereof |
| CN105315166A (en) * | 2014-06-27 | 2016-02-10 | 北京维德维康生物技术有限公司 | Preparation methods of salbutamol hapten and salbutamol antigen, and application thereof in quantum dot immunofluorescence kit |
| CN106916221A (en) * | 2017-04-20 | 2017-07-04 | 山西大学 | A kind of method for synthesizing Terbutaline and bovine serum albumin(BSA) conjugate |
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| CN106916221A (en) * | 2017-04-20 | 2017-07-04 | 山西大学 | A kind of method for synthesizing Terbutaline and bovine serum albumin(BSA) conjugate |
| CN109651503A (en) * | 2018-11-28 | 2019-04-19 | 天津国际生物医药联合研究院 | A kind of preparation method and application of artificial antigen |
| CN110004117A (en) * | 2019-04-11 | 2019-07-12 | 中抗生物医药(杭州)有限公司 | Secrete hybridoma cell strain, monoclonal antibody and the application of salbutamol monoclonal antibody |
| CN110627726A (en) * | 2019-09-23 | 2019-12-31 | 北京勤邦生物技术有限公司 | Prochloraz hapten, artificial antigen and antibody, and preparation method and application thereof |
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| CN112625119A (en) * | 2020-11-25 | 2021-04-09 | 杭州隆基生物技术有限公司 | Estrone antigen and preparation method thereof |
| CN113444166A (en) * | 2021-06-24 | 2021-09-28 | 深圳市疾病预防控制中心（深圳市卫生检验中心、深圳市预防医学研究所） | Carprofen artificial antigen, antibody, and synthetic method and application thereof |
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Application publication date: 20130612 |