CN101161680A - Method for preparing beta-corlan artificial antigen - Google Patents
Method for preparing beta-corlan artificial antigen Download PDFInfo
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- CN101161680A CN101161680A CNA2007101352436A CN200710135243A CN101161680A CN 101161680 A CN101161680 A CN 101161680A CN A2007101352436 A CNA2007101352436 A CN A2007101352436A CN 200710135243 A CN200710135243 A CN 200710135243A CN 101161680 A CN101161680 A CN 101161680A
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Abstract
A preparation method of artificial antigen of betamethasone is provided, which belongs the biochemistry field. The present invention adopts betamethasone as the raw material, and utilizes the reaction between uccinic anhydride and betamethasone to produce betamethasone succinate and obtain the artificial semi-antigen, then the active ester method is used to transform the semi-antigen into a intermediate body of active ester, which is combined with bovine serum to prepare the artificial antigen of betamethasone, i.e. betamethasone bovine serum protein. The present invention synthesizes the artificial antigen of betamethasone antigen, and the prepared product can be used for researches on immunization method with glucocorticoid has simplified and effective synthetic procedures, as well as feasibility to be applied to immune analysis, thereby providing a convenient path for the further research by the people, and being capable of satisfying the domestic needs on the study on the object.
Description
Technical field
A kind of preparation method of beta-corlan artificial antigen belongs to biological chemical field.
Background technology:
(Betamethasone BET) belongs to the synthetic glucocoricoid to Betamethasone Valerate, and chemical name is 16 Alpha-Methyls-11 β, 17 α, 21-trihydroxy--9 α-pregna-fluoride-1,4-diene-3,20-diketone.Have the glycometabolic effect of antianaphylaxis, anti-inflammatory and influence, often be used to treat the ketoacidosis of inflammatory reaction, immunological disease, ox of domestic animal and sheep pregnancy induced hypertension syndrome etc.Betamethasone Valerate also is often used as growth stimulant, thereby the feed intake that increases domestic animal makes it reach the purpose of weightening finish.Yet, proving that through toxicology test this medicine has to mutagenicity, cumulative toxicity, the ADI value is 0.000015mg/kgbw/day.If with being intended to add this medicine in the feed, the medicine of accumulating enters human body by food chain, will cause huge harm.In the forbidden drugs list is listed this type of medicine by many for this reason countries.The current residual detection method of Betamethasone Valerate commonly used in the world has: thin-layer chromatography (TLC), liquid chromatography (LC), gas-chromatography (GC), gas-matter coupling method (GC-MS), liquid-matter coupling method (LC-MS) etc., but these instrument analytical methods not only need expensive plant and instrument, also than higher, need through complicated sample pre-treatment just can carry out the requirement of sample.The external research of having carried out already the Betamethasone Valerate immune analysis method, but domestic still blank out in this respect at present.In order to strengthen the supervision of domestic meat product and to ensure people health, be necessary to launch research to the Betamethasone Valerate immune analysis method, be necessary to provide a kind of preparation method of effective beta-corlan artificial antigen.
Summary of the invention:
The preparation method who the purpose of this invention is to provide a kind of beta-corlan artificial antigen, prepared product can be used for the research of glucocorticoids immunization method, for people's research from now on provides approach easily.
Technical scheme of the present invention: a kind of preparation method of beta-corlan artificial antigen is a raw material with the Betamethasone Valerate, utilizes the reaction of Succinic anhydried and Betamethasone Valerate to generate the Betamethasone Valerate succinic acid half-ester, obtains artificial semiantigen; Utilize active ester method to make haptens change the active ester intermediate into, make it then to combine with bovine serum albumin, the artificial antigen of preparation Betamethasone Valerate is Betamethasone Valerate-bovine serum albumin.Its reaction equation is:
Betamethasone Valerate Betamethasone Valerate-21-succinic acid half-ester
Betamethasone Valerate-bovine serum albumin
Processing step is:
(1) preparation of artificial semiantigen:
Betamethasone Valerate and Succinic anhydried batching mol ratio are 1: 3, take by weighing Betamethasone Valerate 0.8mmol and Succinic anhydried 2.4mmol in the 50mL round-bottomed flask, add the 5mL anhydrous pyridine, mixture is at 60 ℃ of following heated and stirred reaction 6h, and reactant is changed in 4 ℃ of frozen water that 20mL contains 10% hydrochloric acid, the adularescent precipitation is separated out, centrifugal pyridine and the excessive Succinic anhydried removed, filter cake is with deionized water wash for several times, and is centrifugal, obtain the Betamethasone Valerate haptens after the loft drier drying, place 4 ℃ of preservations;
The making of silica gel thin-layer plate: take by weighing silica gel 12g, be dissolved in the sodium cellulose glycolate solution of 40mL 0.5% mass concentration, fully stir into pasty state with glass stick, ultra-sonic oscillation 1 minute, be uniformly coated on the sheet glass, after the In Shade seasoning, the baking oven of putting into 105 ℃ activates 1h, and it is standby to put into loft drier at last;
Thin layer chromatography detects: 1cm place, silica gel thin sheet lower end makes a sea line, draw reaction solution 1 μ L point on line, point sample dries up after finishing, and thin plate is put into chromatography cylinder, and chromatographic solution is a methyl alcohol: chloroform: the ammoniacal liquor volume ratio is 21: 78: 1, be expanded to thin plate 3.5-4cm place, get plate, dry up solvent, thin plate is placed uv analyzer, observing at the 254nm wavelength, is that 0.21 place observes the colour developing point as reaction end at Rf;
(2) preparation of artificial antigen:
Preparation A liquid: take by weighing the 0.09mmol haptens, 0.09mmol N-hydroxy-succinamide, 0.099mmolN, the N-dicyclohexylcarbodiimide is dissolved in the dioxane of 1.8mL in the 20mL beaker, stirs under the room temperature and spends the night, abandon precipitation after centrifugal, clear liquid is the active ester intermediate, is A liquid;
Phosphate buffered saline buffer: the disodium phosphate soln 94.7mL of 0.2mol/L is mixed the phosphate buffered saline buffer that is pH 8.0 with the sodium dihydrogen phosphate 5.3mL of 0.2mol/L;
Preparation B liquid: the bovine serum albumin that takes by weighing 120.6mg is dissolved in the phosphate buffered saline buffer of pH 8.0 of 9mL, and 4 ℃ of low temperature stir half an hour, and this liquid is B liquid;
Under 4 ℃ of low temperature stir, A liquid dropwise is added drop-wise in the B liquid, after all dripping off, mixed solution stirs and returned to room temperature in 1 hour, promptly obtains the artificial antigen mixed solution;
Dialysis tubing pre-treatment: get the dialysis tubing of 10cm, in boiling water, boil 10min, use 60 ℃ deionized water rinsing 3min again, be kept in 4 ℃ of deionized waters standby;
The artificial antigen mixed solution is moved in the dialysis tubing, dialysed 3 days, use lyophilization that the liquid in the dialysis tubing is made powder at last, promptly obtain artificial antigen: Betamethasone Valerate-bovine serum albumin with the ultrapure water of 2 * 2L.
(3) evaluation of beta-corlan artificial antigen
Estimate in the conjugate by the method for the ratio of two kinds of molecules of link coupled (coupling ratio), though kind is a lot, but all be to be set up by the principle of two kinds of molecule contents of link coupled (or relative content) according to detecting in the conjugate. spectrophotometry is to utilize material that the principle that absorption and its concentration of light is proportionlity is measured respectively by two kinds of molecular conecentrations of link coupled. in macromole and small molecules conjugate, two kinds of molecules all have different separately ultraviolet scanning spectrums, and show the character of spectrogram superposition.
The conjugate determination of protein concentration: compound concentration is 0,40,60,80,100,120,160,200 μ gmL
-1Bovine serum albumen solution 1.5mL, add 5mL coomassie brilliant blue staining liquid, mixing immediately, warm 5 minutes of 30 ℃ of water-baths, each concentration is made parallel sample. survey light absorption value, the relation curve of drafting protein concentration and light absorption value at 595nm place.Antigenic solution is diluted by a certain percentage, measure the light absorption value of antigenic solution at the 595nm place, obtain the corresponding proteins concentration of antigenic solution from curve.The protein concentration that this experimental calculation gets antigenic solution is 4.57mgmL
-1
Coupling ratio is measured: preparation Betamethasone Valerate concentration is 50 μ gmL
-120% ethanolic soln, by UV scanning as can be known the maximum absorption wavelength of Betamethasone Valerate be 240nm.
Prepare 200 μ gmL
-1The aqueous solution of bovine serum albumin is diluted to about 200 μ gmL with coupled product with deionized water
-1, survey light absorption value at the 240nm place, be blank with the deionized water, measure light absorption value.With BET, BET-BSA, the mass concentration of BET is converted to volumetric molar concentration according to its relative molecular mass, calculates separately molar extinction coefficient according to formula (1) then:
Wherein, A is an absorbancy, and C is a volumetric molar concentration.
Again according to formula (2) estimation crosslinking rate:
M
DEX∶M
BAS＝(ε
BET-BSA-ε
BAS)/ε
BET (2)
The present invention calculates crosslinking rate and is about 23.
Beneficial effect of the present invention: the present invention has synthesized the artificial antigen of Betamethasone Valerate, prepared product can be used for the research of glucocorticoids immunization method, synthesis step is succinct, effectively, can be used in the middle of the immunoassay fully, for people's research later on provides approach easily, can satisfy domestic needs to its research.
Description of drawings
The high-efficient liquid phase chromatogram of Fig. 1 Betamethasone Valerate artificial semiantigen.
The mass spectrum of Fig. 2 Betamethasone Valerate artificial semiantigen.
UV scanning figure before and after the preparation of Fig. 3 beta-corlan artificial antigen
Embodiment
(1) preparation of artificial semiantigen:
Take by weighing 0.32g Betamethasone Valerate and 0.24g Succinic anhydried in the 50mL round-bottomed flask, add the 5mL anhydrous pyridine, mixture is at 60 ℃ of following heated and stirred reaction 6h, reactant is changed in the frozen water (4 ℃) that contains 10% hydrochloric acid (v/v), the adularescent precipitation is separated out, and centrifugal pyridine and the excessive Succinic anhydried of going out, deionized water wash are for several times, centrifugal, preserve after the loft drier drying.
Reaction end monitoring: the making of silica gel thin-layer plate, take by weighing silica gel 12g, be dissolved in the sodium cellulose glycolate solution of 40mL 0.5% mass concentration, fully stir into pasty state with glass stick, ultra-sonic oscillation 1 minute are uniformly coated on the sheet glass, after the In Shade seasoning, the baking oven of putting into 105 ℃ activates 1h, and it is standby to put into loft drier at last.
Thin layer chromatography detects: 1cm place, silica gel thin sheet lower end makes a sea line, draws reaction solution 1ul point on line, and point sample dries up after finishing, thin plate is put into chromatography cylinder, and chromatographic solution is a methyl alcohol: chloroform: ammoniacal liquor, volume ratio are 21: 78: 1, be expanded to thin plate 3.5-4cm place, get plate, dry up solvent.Thin plate being placed uv analyzer, observe at the 254nm wavelength, is that 0.21 place observes the colour developing point as reaction end at Rf.
(2) preparation of artificial antigen:
Preparation A liquid: take by weighing 44.31mg (0.09mmol) haptens, 10.35mg (0.09mmol) N-hydroxy-succinamide (NHS), 20.4mg (0.099mmol) N, N-dicyclohexylcarbodiimide (DCC) is in the 20mL beaker, be dissolved in the dioxane of 1.8mL, stir under the room temperature and spend the night, abandon precipitation after centrifugal, clear liquid is the active ester intermediate, is A liquid.
Phosphoric acid salt (PBS) damping fluid: the 0.2mol/L Sodium phosphate dibasic: Sodium phosphate dibasic 71.64g adds water to 1000ml.0.2mol/L SODIUM PHOSPHATE, MONOBASIC: SODIUM PHOSPHATE, MONOBASIC 35.61g adds water to 1000ml.The Sodium phosphate dibasic of 94.7ml is mixed the phosphate buffered saline buffer that is ph8.0 with the SODIUM PHOSPHATE, MONOBASIC of 5.3ml.
Preparation B liquid: the BSA that takes by weighing 120.6mg is dissolved in the phosphate buffered saline buffer of PH 8.0 of 9ml, and low temperature stirs half an hour, and this liquid is B liquid.
Under low temperature (4 ℃) stirs, A liquid dropwise is added drop-wise to B liquid, after all dripping off, mixed solution stirs and returned to room temperature in 1 hour, promptly obtains the artificial antigen mixed solution.
Dialysis tubing pre-treatment: get the dialysis tubing of 10cm, in boiling water, boil 10min, use 60 ℃ deionized water rinsing 3min again, be kept in 4 ℃ of deionized waters standby.
The artificial antigen mixed solution is moved in the dialysis tubing, dialysed 3 days with the ultrapure water of 2 * 2L.Use lyophilization that the liquid in the dialysis tubing is made powder at last, promptly obtain artificial antigen: Betamethasone Valerate-bovine serum albumin.
(3) evaluation of beta-corlan artificial antigen
Estimate in the conjugate by the method for the ratio of two kinds of molecules of link coupled (coupling ratio), though kind is a lot, but all be to be set up by the principle of two kinds of molecule contents of link coupled (or relative content) according to detecting in the conjugate. spectrophotometry is to utilize material that the principle that absorption and its concentration of light is proportionlity is measured respectively by two kinds of molecular conecentrations of link coupled. in macromole and small molecules conjugate, two kinds of molecules all have different separately ultraviolet scanning spectrums, and show the character of spectrogram superposition.
The conjugate determination of protein concentration: compound concentration is 0,40,60,80,100,120,160,200 μ gmL
-1Bovine serum albumen solution 1.5mL, add 5mL coomassie brilliant blue staining liquid, mixing immediately, warm 5 minutes of 30 ℃ of water-baths, each concentration is made parallel sample. survey light absorption value, the relation curve of drafting protein concentration and light absorption value at 595nm place.Antigenic solution is diluted by a certain percentage, measure the light absorption value of antigenic solution at the 595nm place, obtain the corresponding proteins concentration of antigenic solution from curve.The protein concentration that the present invention calculates antigenic solution is 4.57mgmL
-1
Coupling ratio is measured: preparation Betamethasone Valerate concentration is 50 μ gmL
120% ethanolic soln, by UV scanning as can be known the maximum absorption wavelength of Betamethasone Valerate be 240nm.
Prepare 200 μ gmL
-1The aqueous solution of bovine serum albumin is diluted to about 200 μ gmL with coupled product with deionized water
-1, survey light absorption value at the 240nm place, be blank with the deionized water, measure light absorption value.With BET, BET-BSA, the mass concentration of BAS is converted to volumetric molar concentration according to its relative molecular mass, calculates separately molar extinction coefficient according to formula (1) then:
Wherein, A is an absorbancy, and C is a volumetric molar concentration.
Again according to formula (2) estimation crosslinking rate:
M
DEX∶M
BAS＝(ε
BET-BSA-ε
BAS)/ε
BET (2)
The present invention calculates crosslinking rate and is about 23.
Claims (1)
1. the preparation method of a beta-corlan artificial antigen is characterized in that with the Betamethasone Valerate being raw material, utilizes the reaction of Succinic anhydried and Betamethasone Valerate to generate the Betamethasone Valerate succinic acid half-ester, obtains artificial semiantigen; Utilize active ester method to make haptens change the active ester intermediate into, make it then to combine with bovine serum albumin, the artificial antigen of preparation Betamethasone Valerate is Betamethasone Valerate-bovine serum albumin; Step is:
(1) preparation of artificial semiantigen:
Betamethasone Valerate and Succinic anhydried batching mol ratio are 1: 3, take by weighing Betamethasone Valerate 0.8mmol and Succinic anhydried 2.4mmol in the 50mL round-bottomed flask, add the 5mL anhydrous pyridine, mixture is at 60 ℃ of following heated and stirred reaction 6h, and reactant is changed in 4 ℃ of frozen water that 20mL contains 10% hydrochloric acid, the adularescent precipitation is separated out, centrifugal pyridine and the excessive Succinic anhydried removed, filter cake is with deionized water wash for several times, and is centrifugal, obtain the Betamethasone Valerate haptens after the loft drier drying, place 4 ℃ of preservations;
The making of silica gel thin-layer plate: take by weighing silica gel 12g, be dissolved in the sodium cellulose glycolate solution of 40mL 0.5% mass concentration, fully stir into pasty state with glass stick, ultra-sonic oscillation 1 minute, be uniformly coated on the sheet glass, after the In Shade seasoning, the baking oven of putting into 105 ℃ activates 1h, and it is standby to put into loft drier at last;
Thin layer chromatography detects: 1cm place, silica gel thin sheet lower end makes a sea line, draw reaction solution 1 μ L point on line, point sample dries up after finishing, and thin plate is put into chromatography cylinder, and chromatographic solution is a methyl alcohol: chloroform: the ammoniacal liquor volume ratio is 21: 78: 1, be expanded to thin plate 3.5-4cm place, get plate, dry up solvent, thin plate is placed uv analyzer, observing at the 254nm wavelength, is that 0.21 place observes the colour developing point as reaction end at Rf;
(2) preparation of artificial antigen:
Preparation A liquid: take by weighing the 0.09mmol haptens, 0.09mmol N-hydroxy-succinamide, 0.099mmolN, the N-dicyclohexylcarbodiimide is dissolved in the dioxane of 1.8mL in the 20mL beaker, stirs under the room temperature and spends the night, abandon precipitation after centrifugal, clear liquid is the active ester intermediate, is A liquid;
Phosphate buffered saline buffer: the disodium phosphate soln 94.7mL of 0.2mol/L is mixed the phosphate buffered saline buffer that is pH 8.0 with the sodium dihydrogen phosphate 5.3mL of 0.2mol/L;
Preparation B liquid: the bovine serum albumin that takes by weighing 120.6mg is dissolved in the phosphate buffered saline buffer of pH 8.0 of 9mL, and 4 ℃ of low temperature stir half an hour, and this liquid is B liquid;
Under 4 ℃ of low temperature stir, A liquid dropwise is added drop-wise in the B liquid, after all dripping off, mixed solution stirs and returned to room temperature in 1 hour, promptly obtains the artificial antigen mixed solution;
Dialysis tubing pre-treatment: get the dialysis tubing of 10cm, in boiling water, boil 10min, use 60 ℃ deionized water rinsing 3min again, be kept in 4 ℃ of deionized waters standby;
The artificial antigen mixed solution is moved in the dialysis tubing, dialysed 3 days, use lyophilization that the liquid in the dialysis tubing is made powder at last, promptly obtain artificial antigen: Betamethasone Valerate-bovine serum albumin with the ultrapure water of 2 * 2L.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007101352436A CN101161680A (en) | 2007-11-01 | 2007-11-01 | Method for preparing beta-corlan artificial antigen |
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|---|---|---|---|
| CNA2007101352436A CN101161680A (en) | 2007-11-01 | 2007-11-01 | Method for preparing beta-corlan artificial antigen |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103940999A (en) * | 2013-01-19 | 2014-07-23 | 北京勤邦生物技术有限公司 | A dipstick used for testing betamethasone and application thereof |
| CN115637258A (en) * | 2022-10-20 | 2023-01-24 | 江南大学 | Clobetasol propionate artificial antigen, monoclonal antibody, hybridoma cell strain and application thereof |
-
2007
- 2007-11-01 CN CNA2007101352436A patent/CN101161680A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103940999A (en) * | 2013-01-19 | 2014-07-23 | 北京勤邦生物技术有限公司 | A dipstick used for testing betamethasone and application thereof |
| CN103940999B (en) * | 2013-01-19 | 2016-06-01 | 北京勤邦生物技术有限公司 | A kind of test strip detecting Betamethasone Valerate and its preparation method and application |
| CN115637258A (en) * | 2022-10-20 | 2023-01-24 | 江南大学 | Clobetasol propionate artificial antigen, monoclonal antibody, hybridoma cell strain and application thereof |
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Open date: 20080416 |