CN104280541A - Beta-epinephrine receptor stimulant multi-cluster antigens and wide-spectrum specific antibodies and preparation thereof - Google Patents
Beta-epinephrine receptor stimulant multi-cluster antigens and wide-spectrum specific antibodies and preparation thereof Download PDFInfo
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Abstract
The invention discloses multi-cluster antigens and antibodies which are used for detecting multiple residues of a beta-epinephrine receptor stimulant and preparation methods of the antigens and the antibodies. The invention provides eight beta-stimulant multi-cluster antigens which are respectively clenbuterol-ractopamine-bovine serum albumin, clenbuterol-salbutamol-bovine serum albumin, salbutamol-ractopamine-bovine serum albumin, clenbuterol-salbutamol-ractopamine-bovine serum albumin, clenbuterol-ractopamine-ovalbumin, clenbuterol-salbutamol-ovalbumin, salbutamol-ractopamine-ovalbumin and clenbuterol-salbutamol-ractopamine-ovalbumin. Four wide-spectrum specific antibodies for detecting multiple residues of the beta-epinephrine receptor stimulant are prepared by adopting the multi-cluster antigens by animal immunization.
Description
Technical field
The invention belongs to immunochemistry and analysis technical field, be specifically related to a kind of receptor,β activator (hereinafter referred to as beta-2-agonists) polygen and wide spectrum specific antibody and preparation thereof.
Background technology
Receptor,β activator, is called for short beta-2-agonists, and be the chemically residual hazardous material that in animal derived food, recall rate is the highest, harm is the most serious, wherein foremost is exactly Clenbuterol (clenbuterol, CL), is commonly called as " clenbuterol hydrochloride ".The eighties in last century, the scientific research personnel of Zhi An company of the U.S. surprisingly finds, " clenbuterol hydrochloride " can strengthen animal tallow and decompose, and promotes protein synthesis, greatly shortens meat market periods, significantly improve carcass lean meat percentage, feed return rate and economic benefit.Promote rapidly all over the world subsequently, the later stage eighties, import China into and feed manufacturing emterprise in many areas and aquaculture are promoted the use of, become animal husbandry " adjuvant " the most widely very soon.
" clenbuterol hydrochloride ", except Clenbuterol, also comprises the compound that more than 20 structures and characteristics such as salbutamol (salbutamol, SAL), Ractopamine (ractopamine, RAC) is similar.This compounds enters selectively acting after in body and, in adenyl cyclase, causes human body to produce low sensitivity phenomenon, and asthma incidence and occurring degree raise; Also can cause endocrine disturbance, bring out chromosome aberration and malignant tumour, can causing death time serious.Therefore China and in the world most countries all expressly forbid that " clenbuterol hydrochloride " is as feed addictive.
At present, the detection method of beta-2-agonists class residue of veterinary drug mainly comprises high performance liquid chromatography (HPLC), Liquid Chromatography-Mass Spectrometry (LC-MS), GC-MS(gas chromatography-mass spectrography) (GC-MS), euzymelinked immunosorbent assay (ELISA) (ELISA) and fluorescent immune method (FIA) etc.
Chromatography is the main detection method of beta-2-agonists residue of veterinary drug, but chromatography testing cost is high, complicated operation, a large amount of negative sample duplicate detection; And euzymelinked immunosorbent assay (ELISA) has the advantages such as high specificity, sample pre-treatments be simple, but the feature of high specificity also determines its fatal defects: a kind of enzyme linked immunological kit or test strips can only detect a kind of residue of veterinary drug simultaneously, and other similar residue of veterinary drug cannot be detected, Here it is easily there is undetected reason (" the two remittance clenbuterol hydrochloride " event as caused a sensation throughout the country for 2011); In addition, along with the appearance of the multiple substitute of beta-2-agonists, if detected completely, a sample needs to use multiple testing product, substantially increases testing cost and working time, loses the meaning of rapid screening.
Many residual immunoassays can detect a class medicament residue simultaneously, the advantage of its low cost, high flux, rapid field is that existing chromatography and specific immunity method are incomparable, be one of gordian technique of following field of detection of food safety, cause the attention of people gradually.
Summary of the invention
The object of the present invention is to provide a kind of receptor,β activator polygen and wide spectrum specific antibody and preparation method thereof.
The present invention is by the following technical solutions:
A kind of receptor,β activator polygen, it is characterized in that, described polygen is the conjugate of two or more receptor,β activator and carrier protein, described carrier protein is bovine serum albumin(BSA) or ovalbumin, and described receptor,β activator is selected from Clenbuterol, salbutamol and Ractopamine.
Polygen of the present invention is on carrier protein, the receptor,β activator haptens that coupling is two or more successively obtains, haptens is selected from Clenbuterol, salbutamol and Ractopamine, wherein Clenbuterol haptens adopts heavy nitrogen coupling, and salbutamol and Ractopamine adopt mixed anhydride method coupling.
Polygen of the present invention comprises eight kinds of beta-2-agonists polygens, be specially Clenbuterol-Ractopamine-bovine serum albumin(BSA) (CL-RAC-BSA), Clenbuterol-salbutamol-bovine serum albumin(BSA) (CL-SAL-BSA), salbutamol-Ractopamine-bovine serum albumin(BSA) (SAL-RAC-BSA), Clenbuterol-salbutamol-Ractopamine-bovine serum albumin(BSA) (CL-SAL-RAC-BSA), Clenbuterol-Ractopamine-ovalbumin (CL-RAC-OVA), Clenbuterol-salbutamol-ovalbumin (CL-SAL-OVA), salbutamol-Ractopamine-ovalbumin (SAL-RAC-OVA) and Clenbuterol-salbutamol-Ractopamine-ovalbumin (CL-SAL-RAC-OVA).
Wherein, Clenbuterol-salbutamol-bovine serum albumin(BSA) (CL-SAL-BSA) or Clenbuterol-salbutamol-ovalbumin (CL-SAL-OVA) are for haptens with Clenbuterol and salbutamol, with bovine serum albumin(BSA) or ovalbumin for carrier, prepare according to the following steps:
1) on bovine serum albumin(BSA) or ovalbumin with heavy nitrogen coupling Clenbuterol haptens, then carry out purifying:
By 0.2mmol clenobuterol hydrochloride in being dissolved in 1 ~ 5ml watery hydrochloric acid, the sodium nitrite solution slowly adding 0.2 ~ 2mol/L becomes purple to starch potassium iodide paper, after continuing reaction 1 ~ 3h, reactant liquor is slowly joined 5 ~ 10ml to contain in the sodium carbonate buffer of 5 ~ 10mg/ml carrier protein, then continue reaction 3 ~ 5h.By above-mentioned reactant liquor phosphate buffer solution dialysis 3 ~ 6 times, freeze drying can obtain Clenbuterol-carrier protein couplet thing freeze-dried powder.
2) in step 1) product on mixed anhydride method coupling salbutamol haptens, then carry out purifying:
0.2mmol salbutamol sulfate is dissolved in 5 ~ 10ml absolute ethyl alcohol, adds 0.2mmol glutaric anhydride, under room temperature, react 3 ~ 5h, centrifugal.Lower floor's solid is dissolved in 5 ~ 10ml DMF, adds 0.2mmol tri-n-butylamine and 0.2mmol isobutyl chlorocarbonate, continue reaction 1 ~ 3h.Above-mentioned reactant liquor is slowly joined 5 ~ 10ml to contain in the phosphate buffer solution of 5 ~ 10mg/ml Clenbuterol-carrier protein couplet thing, room temperature reaction spends the night.By above-mentioned reactant liquor phosphate buffer solution dialysis 3 ~ 6 times, freeze drying can obtain Clenbuterol-salbutamol-carrier protein couplet thing freeze-dried powder.
Clenbuterol-Ractopamine-bovine serum albumin(BSA) (CL-RAC-BSA) or Clenbuterol-Ractopamine-ovalbumin (CL-RAC-OVA) are for haptens with Clenbuterol and Ractopamine, with bovine serum albumin(BSA) or ovalbumin for carrier, prepare according to the following steps:
1) on bovine serum albumin(BSA) or ovalbumin with mixed anhydride method coupling Ractopamine haptens, then carry out purifying:
0.2mmol glutaric anhydride is dissolved in 5ml pyridine, adds 0.2mmol ractopamine hydrochloric, room temperature reaction 10 ~ 20h, solvent evaporated, is dissolved in 5 ~ 10ml DMF by gained grease, add 0.2mmol tri-n-butylamine and 0.2mmol isobutyl chlorocarbonate, continue reaction 1 ~ 3h.Above-mentioned reactant liquor is slowly joined 5 ~ 10ml contain in the phosphate buffer solution of 5 ~ 10mg/ml carrier protein, room temperature reaction spends the night.By above-mentioned reactant liquor phosphate buffer solution dialysis 3 ~ 6 times, freeze drying can obtain Ractopamine-carrier protein couplet thing freeze-dried powder.
2) in step 1) product on heavy nitrogen coupling Clenbuterol haptens, then carry out purifying:
By 0.2mmol clenobuterol hydrochloride in being dissolved in 1 ~ 5ml watery hydrochloric acid, the sodium nitrite solution slowly adding 0.2 ~ 2mol/L becomes purple to starch potassium iodide paper, after continuing reaction 1 ~ 3h, reactant liquor is slowly joined 5 ~ 10ml to contain in the sodium carbonate buffer of 5 ~ 10mg/ml Ractopamine-carrier protein couplet thing, then continue reaction 3 ~ 5h.By above-mentioned reactant liquor phosphate buffer solution dialysis 3 ~ 6 times, freeze drying Clenbuterol-Ractopamine-carrier protein couplet thing freeze-dried powder.
Salbutamol-Ractopamine-bovine serum albumin(BSA) (SAL-RAC-BSA) or salbutamol-Ractopamine-ovalbumin (SAL-RAC-OVA) are for haptens with salbutamol and Ractopamine, with bovine serum albumin(BSA) or ovalbumin for carrier, prepare according to the following steps:
1) on bovine serum albumin(BSA) or ovalbumin with mixed anhydride method coupling salbutamol haptens, then carry out purifying:
0.2mmol salbutamol sulfate is dissolved in 5 ~ 10ml absolute ethyl alcohol, adds 0.2 mmol glutaric anhydride, under room temperature, react 3 ~ 5h, centrifugal.Lower floor's solid is dissolved in 5 ~ 10ml DMF, adds 0.2mmol tri-n-butylamine and 0.2mmol isobutyl chlorocarbonate, continue reaction 1 ~ 3h.Above-mentioned reactant liquor is slowly joined 5 ~ 10ml contain in the phosphate buffer solution of 5 ~ 10mg/ml carrier protein, room temperature reaction spends the night.By above-mentioned reactant liquor phosphate buffer solution dialysis 3 ~ 6 times, freeze drying can obtain salbutamol-carrier protein couplet thing freeze-dried powder.
2) in step 1) product on continue with mixed anhydride method coupling Ractopamine haptens, then carry out purifying:
0.2mmol glutaric anhydride is dissolved in 5ml pyridine, add 0.2 mmol ractopamine hydrochloric, room temperature reaction 10 ~ 20h, solvent evaporated, gained grease is dissolved in 5 ~ 10ml N, dinethylformamide, adds 0.2mmol tri-n-butylamine and 0.2mmol isobutyl chlorocarbonate, continues reaction 1 ~ 3h.Above-mentioned reactant liquor is slowly joined 5 ~ 10ml to contain in the phosphate buffer solution of 5 ~ 10mg/ml salbutamol-carrier protein couplet thing, room temperature reaction spends the night.By above-mentioned reactant liquor phosphate buffer solution dialysis 3 ~ 6 times, freeze drying can obtain salbutamol-Ractopamine-carrier protein couplet thing freeze-dried powder.
Clenbuterol-salbutamol-Ractopamine-bovine serum albumin(BSA) (CL-SAL-RAC-BSA) or Clenbuterol-salbutamol-Ractopamine-ovalbumin (CL-SAL-RAC-OVA) are for haptens with Clenbuterol, salbutamol and Ractopamine, with bovine serum albumin(BSA) or ovalbumin for carrier, can according to the following steps in any one prepares:
1) on Clenbuterol-Ractopamine-carrier protein couplet thing with mixed anhydride method coupling salbutamol, then carry out purifying:
0.2mmol salbutamol sulfate is dissolved in 5 ~ 10ml absolute ethyl alcohol, adds 0.2mmol glutaric anhydride, under room temperature, react 3 ~ 5h, centrifugal.Lower floor's solid is dissolved in 5 ~ 10ml DMF, adds 0.2mmol tri-n-butylamine and 0.2mmol isobutyl chlorocarbonate, continue reaction 1 ~ 3h.Above-mentioned reactant liquor is slowly joined 5 ~ 10ml to contain in the phosphate buffer solution of 5 ~ 10mg/ml Clenbuterol-Ractopamine-carrier protein couplet thing, room temperature reaction spends the night.By above-mentioned reactant liquor phosphate buffer solution dialysis 3 ~ 6 times, freeze drying can obtain Clenbuterol-salbutamol-Ractopamine-carrier protein couplet thing freeze-dried powder.
2) on Clenbuterol-salbutamol-carrier protein couplet thing with mixed anhydride method coupling Ractopamine, then carry out purifying:
0.2mmol glutaric anhydride is dissolved in 5ml pyridine, add 0.2mmol ractopamine hydrochloric, room temperature reaction 10 ~ 20h, solvent evaporated, gained grease is dissolved in 5 ~ 10ml N, dinethylformamide, adds 0.2mmol tri-n-butylamine and 0.2 mmol isobutyl chlorocarbonate, continues reaction 1 ~ 3h.Above-mentioned reactant liquor is slowly joined 5 ~ 10ml to contain in the phosphate buffer solution of 5 ~ 10mg/ml Clenbuterol-salbutamol-carrier protein couplet thing, room temperature reaction spends the night.By above-mentioned reactant liquor phosphate buffer solution dialysis 3 ~ 6 times, freeze drying can obtain Clenbuterol-salbutamol-Ractopamine-carrier protein couplet thing freeze-dried powder.
3) on salbutamol-Ractopamine-carrier protein couplet thing with heavy nitrogen coupling Clenbuterol, then carry out purifying:
By 0.2mmol clenobuterol hydrochloride in being dissolved in 1 ~ 5ml watery hydrochloric acid, the sodium nitrite solution slowly adding 0.2 ~ 2mol/L becomes purple to starch potassium iodide paper, continue reaction 1 ~ 3h, reactant liquor is slowly joined 5 ~ 10ml to contain in the phosphate buffer solution of 5 ~ 10mg/ml salbutamol-Ractopamine-carrier protein couplet thing, room temperature reaction spends the night.By above-mentioned reactant liquor phosphate buffer solution dialysis 3 ~ 6 times, freeze drying can obtain Clenbuterol-salbutamol-Ractopamine-carrier protein couplet thing freeze-dried powder.
Present invention also offers the receptor,β activator wide spectrum specific antibody that described polygen is obtained; Particularly four kinds of beta-2-agonists wide spectrum specific antibody, described wide spectrum specific antibody is respectively and is obtained by animal immune by receptor,β activator polygen CL-RAC-BSA, CL-SAL-BSA, SAL-RAC-BSA or CL-SAL-RAC-BSA.
Beneficial effect of the present invention: the present invention has successfully synthesized eight kinds of beta-2-agonists polygens, and prepare beta-2-agonists wide spectrum specific antibody based on animal immune method.Described beta-2-agonists polygen can be used for constructing immune-electrochemistry sensor, based on beta-2-agonists wide spectrum specific antibody to the cross-immune reaction of beta-2-agonists, the detection of multiple beta-2-agonists to comprising Clenbuterol, salbutamol, Ractopamine, his woods of special step, Mabuterol and Tulobuterol can be realized.Because the beta-2-agonists wide spectrum specific antibody adopted all has higher cross reacting rate to multiple beta-2-agonists, thus can realize the common detection to beta-2-agonists, and many residual immunoassays has quick, efficient, highly sensitive advantage.
Describe the present invention below in conjunction with specific embodiment.Protection scope of the present invention is not limited with embodiment, but is limited by claim.
Accompanying drawing explanation
Fig. 1 is the ultraviolet-visible spectrogram of CL, BSA and CL-BSA conjugate.
Fig. 2 is the ultraviolet-visible spectrogram of SAL, CL-BSA conjugate and CL-SAL-BSA conjugate.
Fig. 3 is the ultraviolet-visible spectrogram of SAL, BSA and SAL-BSA conjugate.
Fig. 4 is the ultraviolet-visible spectrogram of RAC, SAL-BSA conjugate and SAL-RAC-BSA conjugate.
Fig. 5 is the ultraviolet-visible absorption spectroscopy figure of RAC, OVA and RAC-OVA conjugate.
Fig. 6 is the ultraviolet-visible absorption spectroscopy figure of CL, RAC-OVA conjugate and CL-RAC-OVA conjugate.
Fig. 7 is the ultraviolet-visible absorption spectroscopy figure of SAL, CL-RAC-OVA conjugate and CL-SAL-RAC-OVA conjugate.
Embodiment
The preparation of embodiment 1CL-SAL-BSA conjugate
1) on BSA with heavy nitrogen coupling CL haptens, then carry out purifying:
By 0.2mmol clenobuterol hydrochloride in being dissolved in 1 ~ 5ml watery hydrochloric acid, the sodium nitrite solution slowly adding 0.2 ~ 2mol/L becomes purple to starch potassium iodide paper, after continuing reaction 1 ~ 3h, reactant liquor is slowly joined 5 ~ 10ml to contain in the sodium carbonate buffer of 5 ~ 10mg/ml BSA, then continue reaction 3 ~ 5h.By above-mentioned reactant liquor phosphate buffer solution dialysis 3 ~ 6 times, freeze drying can obtain CL-BSA conjugate freeze-dried powder.Ultraviolet-visible absorption spectroscopy figure (see accompanying drawing 1) according to CL, BSA and CL-BSA conjugate can be calculated coupling ratio, BSA: CL=1: 28.
2) in step 1) product on mixed anhydride method coupling SAL haptens, then carry out purifying:
0.2mmol salbutamol sulfate is dissolved in 5 ~ 10ml absolute ethyl alcohol, adds 0.2mmol glutaric anhydride, under room temperature, react 3 ~ 5h, centrifugal.Lower floor's solid is dissolved in 5 ~ 10ml DMF, adds 0.2mmol tri-n-butylamine and 0.2mmol isobutyl chlorocarbonate, continue reaction 1 ~ 3h.Above-mentioned reactant liquor is slowly joined 5 ~ 10ml contain in the phosphate buffer solution of 5 ~ 10mg/mlCL-BSA conjugate, room temperature reaction spends the night.By above-mentioned reactant liquor phosphate buffer solution dialysis 3 ~ 6 times, freeze drying can obtain CL-SAL-BSA conjugate freeze-dried powder.Ultraviolet-visible absorption spectroscopy figure (see accompanying drawing 2) according to SAL, CL-BSA conjugate and CL-SAL-BSA conjugate can be calculated coupling ratio, BSA: CL: SAL=1: 28: 17.
The preparation of embodiment 2SAL-RAC-BSA conjugate
1) on BSA with mixed anhydride method coupling SAL haptens, then carry out purifying:
0.2 mmol salbutamol sulfate is dissolved in 5 ~ 10ml absolute ethyl alcohol, adds 0.2mmol glutaric anhydride, under room temperature, react 3 ~ 5h, centrifugal.Lower floor's solid is dissolved in 5 ~ 10ml DMF, adds 0.2mmol tri-n-butylamine and 0.2mmol isobutyl chlorocarbonate, continue reaction 1 ~ 3h.Above-mentioned reactant liquor is slowly joined 5 ~ 10ml to be contained in the phosphate buffer solution of 5 ~ 10mg/mlBSA, and room temperature reaction spends the night.By above-mentioned reactant liquor phosphate buffer solution dialysis 3 ~ 6 times, freeze drying can obtain SAL-BSA conjugate freeze-dried powder.Ultraviolet-visible absorption spectroscopy figure (see accompanying drawing 3) according to SAL, BSA and SAL-BSA conjugate can be calculated coupling ratio, BSA: SAL=1: 8.5.
2) in step 1) product on continue with mixed anhydride method coupling RAC haptens, then carry out purifying:
0.2mmol glutaric anhydride is dissolved in 5ml pyridine, adds 0.2mmol ractopamine hydrochloric, room temperature reaction 10 ~ 20h, solvent evaporated, is dissolved in 5 ~ 10ml DMF by gained grease, add 0.2mmol tri-n-butylamine and 0.2mmol isobutyl chlorocarbonate, continue reaction 1 ~ 3h.Above-mentioned reactant liquor is slowly joined 5 ~ 10ml contain in the phosphate buffer solution of 5 ~ 10mg/mlSAL-BSA conjugate, room temperature reaction spends the night.By above-mentioned reactant liquor phosphate buffer solution dialysis 3 ~ 6 times, freeze drying can obtain SAL-RAC-BSA conjugate freeze-dried powder.Ultraviolet-visible absorption spectroscopy figure (see accompanying drawing 4) according to RAC, SAL-BSA conjugate and SAL-RAC-BSA conjugate can be calculated coupling ratio, BSA: SAL:RAC=1: 8.5: 30.
The preparation of embodiment 3CL-RAC-OVA conjugate
1) on OVA with mixed anhydride method coupling RAC haptens, then carry out purifying:
0.2mmol glutaric anhydride is dissolved in 5ml pyridine, adds 0.2mmol ractopamine hydrochloric, room temperature reaction 10 ~ 20h, solvent evaporated, is dissolved in 5 ~ 10ml DMF by gained grease, add 0.2mmol tri-n-butylamine and 0.2mmol isobutyl chlorocarbonate, continue reaction 1 ~ 3h.Above-mentioned reactant liquor is slowly joined 5 ~ 10ml to be contained in the phosphate buffer solution of 5 ~ 10mg/mlOVA, and room temperature reaction spends the night.By above-mentioned reactant liquor phosphate buffer solution dialysis 3 ~ 6 times, freeze drying can obtain RAC-OVA conjugate freeze-dried powder.Ultraviolet-visible absorption spectroscopy figure (see accompanying drawing 5) according to RAC, OVA and RAC-OVA conjugate can be calculated coupling ratio, OVA:RAC=1: 20.
2) in step 1) product on heavy nitrogen coupling CL haptens, then carry out purifying:
By 0.2mmol clenobuterol hydrochloride in being dissolved in 1 ~ 5ml watery hydrochloric acid, the sodium nitrite solution slowly adding 0.2 ~ 2mol/L becomes purple to starch potassium iodide paper, after continuing reaction 1 ~ 3h, reactant liquor is slowly joined 5 ~ 10ml to contain in the sodium carbonate buffer of 5 ~ 10mg/ml RAC-OVA conjugate, then continue reaction 3 ~ 5h.By above-mentioned reactant liquor phosphate buffer solution dialysis 3 ~ 6 times, freeze drying CL-RAC-OVA conjugate freeze-dried powder.Ultraviolet-visible absorption spectroscopy figure (see accompanying drawing 6) according to CL, RAC-OVA conjugate and CL-RAC-OVA conjugate can be calculated coupling ratio, OVA: RAC: CL=1: 20: 16.
The preparation of embodiment 4CL-SAL-RAC-OVA conjugate
0.2mmol salbutamol sulfate is dissolved in 5 ~ 10ml absolute ethyl alcohol, adds 0.2mmol glutaric anhydride, under room temperature, react 3 ~ 5h, centrifugal.Lower floor's solid is dissolved in 5 ~ 10ml DMF, adds 0.2mmol tri-n-butylamine and 0.2mmol isobutyl chlorocarbonate, continue reaction 1 ~ 3h.Reactant liquor is slowly joined 5 ~ 10ml to contain in the phosphate buffer solution of 5 ~ 10mg/mlCL-RAC-OVA conjugate, room temperature reaction spends the night.By above-mentioned reactant liquor phosphate buffer solution dialysis 3 ~ 6 times, freeze drying can obtain CL-SAL-RAC-OVA conjugate freeze-dried powder.Ultraviolet-visible absorption spectroscopy figure (see accompanying drawing 7) according to SAL, CL-RAC-OVA conjugate and CL-SAL-RAC-OVA conjugate can be calculated coupling ratio, OVA:RAC: CL: SAL=1: 20: 16: 21.
CL-RAC-BSA, CL-SAL-OVA, SAL-RAC-OVA and CL-SAL-RAC-BSA conjugate can be prepared respectively according to the method similar to embodiment 1-4.
The preparation of the anti-CL-RAC-BSA of embodiment 5, anti-CL-SAL-BSA, anti-SAL-RAC-BSA and anti-CL-SAL-RAC-BSA antibody
Be about the healthy White Rabbit of 2kg as immunogen immune body weight with the conjugate (one in CL-RAC-BSA, CL-SAL-BSA, SAL-RAC-BSA or CL-SAL-RAC-BSA) synthesized.During first immunisation, 0.25mg immunogene to be mixed with equivalent Freund's complete adjuvant and fully emulsified, dorsal sc multi-point injection.Carry out emulsification with same dose of immunogens and equivalent incomplete Freund's adjuvant after two weeks and carry out booster immunization, every two weeks booster immunizations once, are strengthened three times altogether.After last immune 10 days, jugular vein blood collection is carried out to White Rabbit, 30min is left standstill under being placed on 4 DEG C of environment, carry out purifying by ammonium sulfate multi stage precipitation method again, anti-CL-RAC-BSA, anti-CL-SAL-BSA, anti-SAL-RAC-BSA and anti-CL-SAL-RAC-BSA polyclonal antibody can be obtained.
Embodiment 6 antibody titer measures
By after purifying the different multiple of serum-dilution, resist CL-RAC-BSA, anti-CL-SAL-BSA, anti-SAL-RAC-BSA and anti-CL-SAL-RAC-BSA antibody respectively with indirect elisa method and carry out titration, result is as follows:
CL-SAL-BSA is sero-fast 1: 6400, the CL-RAC-BSA that tires that to be the antiserum titre of 1: 1600, CL-SAL-RAC-BSA be is sero-fast to tire be the antiserum titre of 1: 12800, SAL-RAC-BSA is 1: 3200.
Claims (7)
1. a receptor,β activator polygen, it is characterized in that, described polygen is the conjugate of two or more receptor,β activator and carrier protein, described carrier protein is bovine serum albumin(BSA) or ovalbumin, and described receptor,β activator is selected from Clenbuterol, salbutamol and Ractopamine.
2. receptor,β activator polygen according to claim 1, it is characterized in that: be Clenbuterol-salbutamol-bovine serum albumin(BSA) or Clenbuterol-salbutamol-ovalbumin conjugate, with Clenbuterol and salbutamol for haptens, with bovine serum albumin(BSA) or ovalbumin for carrier, prepare according to the following steps:
1) on bovine serum albumin(BSA) or ovalbumin with heavy nitrogen coupling Clenbuterol haptens, then carry out purifying:
By 0.2mmol clenobuterol hydrochloride in being dissolved in 1 ~ 5ml watery hydrochloric acid, the sodium nitrite solution adding 0.2 ~ 2mol/L becomes purple to starch potassium iodide paper, after continuing reaction 1 ~ 3h, reactant liquor is joined 5 ~ 10ml to contain in the sodium carbonate buffer of 5 ~ 10mg/ml carrier protein, then continue reaction 3 ~ 5h; By above-mentioned reactant liquor phosphate buffer solution dialysis 3 ~ 6 times, freeze drying can obtain Clenbuterol-carrier protein couplet thing freeze-dried powder;
2) in step 1) product on mixed anhydride method coupling salbutamol haptens, then carry out purifying:
0.2mmol salbutamol sulfate is dissolved in 5 ~ 10ml absolute ethyl alcohol, adds 0.2mmol glutaric anhydride, under room temperature, react 3 ~ 5h, centrifugal, lower floor's solid is dissolved in 5 ~ 10ml DMF, add 0.2mmol tri-n-butylamine and 0.2mmol isobutyl chlorocarbonate, continue reaction 1 ~ 3h; Above-mentioned reactant liquor is joined 5 ~ 10ml to contain in the phosphate buffer solution of 5 ~ 10mg/ml Clenbuterol-carrier protein couplet thing, room temperature reaction spends the night; By above-mentioned reactant liquor phosphate buffer solution dialysis 3 ~ 6 times, freeze drying can obtain Clenbuterol-salbutamol-carrier protein couplet thing freeze-dried powder.
3. receptor,β activator polygen according to claim 1, it is characterized in that: be Clenbuterol-Ractopamine-bovine serum albumin(BSA) or Clenbuterol-Ractopamine-ovalbumin conjugate, with Clenbuterol and Ractopamine for haptens, with bovine serum albumin(BSA) or ovalbumin for carrier, prepare according to the following steps:
1) on bovine serum albumin(BSA) or ovalbumin with mixed anhydride method coupling Ractopamine haptens, then carry out purifying:
0.2mmol glutaric anhydride is dissolved in 5ml pyridine, adds 0.2mmol ractopamine hydrochloric, room temperature reaction 10 ~ 20h, solvent evaporated, is dissolved in 5 ~ 10ml DMF by gained grease, add 0.2mmol tri-n-butylamine and 0.2mmol isobutyl chlorocarbonate, continue reaction 1 ~ 3h; Above-mentioned reactant liquor is joined 5 ~ 10ml contain in the phosphate buffer solution of 5 ~ 10mg/ml carrier protein, room temperature reaction spends the night; By above-mentioned reactant liquor phosphate buffer solution dialysis 3 ~ 6 times, freeze drying can obtain Ractopamine-carrier protein couplet thing freeze-dried powder;
2) in step 1) product on heavy nitrogen coupling Clenbuterol haptens, then carry out purifying:
By 0.2mmol clenobuterol hydrochloride in being dissolved in 1 ~ 5ml watery hydrochloric acid, the sodium nitrite solution adding 0.2 ~ 2mol/L becomes purple to starch potassium iodide paper, after continuing reaction 1 ~ 3h, reactant liquor is joined 5 ~ 10ml to contain in the sodium carbonate buffer of 5 ~ 10mg/ml Ractopamine-carrier protein couplet thing, then continue reaction 3 ~ 5h; By above-mentioned reactant liquor phosphate buffer solution dialysis 3 ~ 6 times, freeze drying Clenbuterol-Ractopamine-carrier protein couplet thing freeze-dried powder.
4. receptor,β activator polygen according to claim 1, it is characterized in that: be salbutamol-Ractopamine-bovine serum albumin(BSA) or salbutamol-Ractopamine-ovalbumin conjugate, with salbutamol and Ractopamine for haptens, with bovine serum albumin(BSA) or ovalbumin for carrier, prepare according to the following steps:
1) on bovine serum albumin(BSA) or ovalbumin with mixed anhydride method coupling salbutamol haptens, then carry out purifying:
0.2mmol salbutamol sulfate is dissolved in 5 ~ 10ml absolute ethyl alcohol, adds 0.2mmol glutaric anhydride, under room temperature, react 3 ~ 5h, centrifugal, lower floor's solid is dissolved in 5 ~ 10ml DMF, add 0.2mmol tri-n-butylamine and 0.2mmol isobutyl chlorocarbonate, continue reaction 1 ~ 3h; Above-mentioned reactant liquor is joined 5 ~ 10ml contain in the phosphate buffer solution of 5 ~ 10mg/ml carrier protein, room temperature reaction spends the night; By above-mentioned reactant liquor phosphate buffer solution dialysis 3 ~ 6 times, freeze drying can obtain salbutamol-carrier protein couplet thing freeze-dried powder;
2) in step 1) product on continue with mixed anhydride method coupling Ractopamine haptens, then carry out purifying:
0.2mmol glutaric anhydride is dissolved in 5ml pyridine, adds 0.2mmol ractopamine hydrochloric, room temperature reaction 10 ~ 20h, solvent evaporated, is dissolved in 5 ~ 10ml DMF by gained grease, add 0.2mmol tri-n-butylamine and 0.2mmol isobutyl chlorocarbonate, continue reaction 1 ~ 3h; Above-mentioned reactant liquor is joined 5 ~ 10ml to contain in the phosphate buffer solution of 5 ~ 10mg/ml salbutamol-carrier protein couplet thing, room temperature reaction spends the night; By above-mentioned reactant liquor phosphate buffer solution dialysis 3 ~ 6 times, freeze drying can obtain salbutamol-Ractopamine-carrier protein couplet thing freeze-dried powder.
5. receptor,β activator polygen according to claim 1, it is characterized in that: be Clenbuterol-salbutamol-Ractopamine-bovine serum albumin(BSA) or Clenbuterol-salbutamol-Ractopamine-ovalbumin conjugate, with Clenbuterol, salbutamol and Ractopamine for haptens, with bovine serum albumin(BSA) or ovalbumin for carrier, prepare by any one method following:
1) on Clenbuterol-Ractopamine-carrier protein couplet thing with mixed anhydride method coupling salbutamol, then carry out purifying:
0.2mmol salbutamol sulfate is dissolved in 5 ~ 10ml absolute ethyl alcohol, adds 0.2mmol glutaric anhydride, under room temperature, react 3 ~ 5h, centrifugal, lower floor's solid is dissolved in 5 ~ 10ml DMF, add 0.2mmol tri-n-butylamine and 0.2mmol isobutyl chlorocarbonate, continue reaction 1 ~ 3h; Above-mentioned reactant liquor is joined 5 ~ 10ml to contain in the phosphate buffer solution of 5 ~ 10mg/ml Clenbuterol-Ractopamine-carrier protein couplet thing, room temperature reaction spends the night; By above-mentioned reactant liquor phosphate buffer solution dialysis 3 ~ 6 times, freeze drying can obtain Clenbuterol-salbutamol-Ractopamine-carrier protein couplet thing freeze-dried powder;
2) on Clenbuterol-salbutamol-carrier protein couplet thing with mixed anhydride method coupling Ractopamine, then carry out purifying:
0.2mmol glutaric anhydride is dissolved in 5ml pyridine, adds 0.2mmol ractopamine hydrochloric, room temperature reaction 10 ~ 20h, solvent evaporated, is dissolved in 5 ~ 10ml DMF by gained grease, add 0.2mmol tri-n-butylamine and 0.2mmol isobutyl chlorocarbonate, continue reaction 1 ~ 3h; Above-mentioned reactant liquor is joined 5 ~ 10ml to contain in the phosphate buffer solution of 5 ~ 10mg/ml Clenbuterol-salbutamol-carrier protein couplet thing, room temperature reaction spends the night; By above-mentioned reactant liquor phosphate buffer solution dialysis 3 ~ 6 times, freeze drying can obtain Clenbuterol-salbutamol-Ractopamine-carrier protein couplet thing freeze-dried powder;
3) on salbutamol-Ractopamine-carrier protein couplet thing with heavy nitrogen coupling Clenbuterol, then carry out purifying:
By 0.2mmol clenobuterol hydrochloride in being dissolved in 1 ~ 5ml watery hydrochloric acid, the sodium nitrite solution slowly adding 0.2 ~ 2mol/L becomes purple to starch potassium iodide paper, continue reaction 1 ~ 3h, reactant liquor is joined 5 ~ 10ml to contain in the phosphate buffer solution of 5 ~ 10mg/ml salbutamol-Ractopamine-carrier protein couplet thing, room temperature reaction spends the night; By above-mentioned reactant liquor phosphate buffer solution dialysis 3 ~ 6 times, freeze drying can obtain Clenbuterol-salbutamol-Ractopamine-carrier protein couplet thing freeze-dried powder.
6. the receptor,β activator wide spectrum specific antibody that polygen according to claim 1 is obtained.
7. receptor,β activator wide spectrum specific antibody according to claim 6, it is characterized in that, described wide spectrum specific antibody is obtained by animal immune by receptor,β activator polygen CL-RAC-BSA, CL-SAL-BSA, SAL-RAC-BSA or CL-SAL-RAC-BSA.
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