CN101161681B - Method for preparing double-flumethasone artificial antigen - Google Patents

Abstract

A preparation method of artificial antigen of diflucortolone is provided, which belongs the biochemistry field. The present invention adopts diflucortolone as the raw material, and utilizes the reaction between uccinic anhydride and diflucortolone to produce diflucortolone 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 diflucortolone, i.e. diflucortolone bovine serum protein. The present invention synthesizes the artificial antigen of diflucortolone 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

A kind of preparation method of double-flumethasone artificial antigen

Technical field

A kind of preparation method of double-flumethasone artificial antigen belongs to biological chemical field.

Background technology

(Flumethasone FLM) belongs to the synthetic glucocoricoid to diflucortolone, and chemical name is 6 α, 9 α-two pregna-fluorides-16 Alpha-Methyl-11 β, 17 α, 21-trihydroxy--1,4-diene-3,20 diketone.Have antianaphylaxis, antiphlogistic effect, it has cumulative toxicity, and in the forbidden drugs list is listed this medicine by many countries, especially must not detect in beef product and the milk.The current residual detection method of diflucortolone 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 diflucortolone 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 diflucortolone immune analysis method, be necessary to provide a kind of preparation method of effective double-flumethasone artificial antigen.

Summary of the invention

The preparation method who the purpose of this invention is to provide a kind of double-flumethasone 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 double-flumethasone artificial antigen is a raw material with the diflucortolone, utilizes the reaction of Succinic anhydried and diflucortolone to generate the diflucortolone 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 diflucortolone is diflucortolone-bovine serum albumin.Its reaction equation is:

Diflucortolone diflucortolone-21-succinic acid half-ester

Diflucortolone-bovine serum albumin

Processing step is:

(1) preparation of artificial semiantigen:

Diflucortolone and Succinic anhydried batching mol ratio are 1: 3, take by weighing diflucortolone 0.4mmol and Succinic anhydried 1.2mmol places the 50mL round-bottomed flask, add the 2.5mL anhydrous pyridine,, reactant is changed in 4 ℃ of frozen water that 10mL contains 10% hydrochloric acid at 60 ℃ of following heated and stirred 6h, 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 diflucortolone 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 (NHS), 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;

Phosphate buffered saline buffer (PBS): preparation 0.2mol/L disodium phosphate soln: Sodium phosphate dibasic 71.64g adds water to 1000mL.The sodium dihydrogen phosphate of preparation 0.2mol/L: SODIUM PHOSPHATE, MONOBASIC 35.61g adds water to 1000mL.The disodium phosphate soln 94.7mL of 0.2mol/L is mixed the phosphate buffered saline buffer that is pH8.0 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: diflucortolone-bovine serum albumin with the ultrapure water of 2 * 2L.

(3) evaluation of double-flumethasone 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, 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 distinguished

Mensuration is by two kinds of molecular conecentrations of link coupled. and 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 3.20mgmL ^-1

Coupling ratio is measured: preparation diflucortolone concentration is 50 μ gmL ^-120% ethanolic soln, by UV scanning as can be known the maximum absorption wavelength of diflucortolone be 241nm.

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 241nm place, be blank with the deionized water, measure light absorption value.With FLM, FLM-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 _FLM-BSA-ε _BAS)/ε _FLM (2)

The present invention calculates crosslinking rate and is about 17.

Beneficial effect of the present invention: the present invention has synthesized the artificial antigen of diflucortolone, 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 liquid chromatogram of Fig. 1 diflucortolone artificial semiantigen.

The mass spectrum of Fig. 2 diflucortolone artificial semiantigen.

UV scanning figure before and after the preparation of Fig. 3 double-flumethasone artificial antigen

Embodiment

(1) preparation of artificial semiantigen:

Take by weighing 0.16g diflucortolone and 0.12g Succinic anhydried in the 50mL round-bottomed flask, add the 2.5mL anhydrous pyridine, mixture is at 60 ℃ of following heated and stirred 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 1 μ L 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 3/4 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 45.93mg (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.

Phosphate buffered saline buffer: 0.2mol/L Sodium phosphate dibasic liquid: Sodium phosphate dibasic 71.64g adds water to 1000mL.The SODIUM PHOSPHATE, MONOBASIC liquid of joining 0.2mol/L: SODIUM PHOSPHATE, MONOBASIC 35.61g adds water to 1000mL.The Sodium phosphate dibasic liquid of 94.7mL is mixed the phosphate buffered saline buffer that is pH 8.0 with the SODIUM PHOSPHATE, MONOBASIC liquid 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: diflucortolone-bovine serum albumin.

(3) evaluation of double-flumethasone 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 3.20mgmL ^-1

Coupling ratio is measured: preparation diflucortolone concentration is 50 μ gmL ^-120% ethanolic soln, by UV scanning as can be known the maximum absorption wavelength of diflucortolone be 241nm.

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 241nm place, be blank with the deionized water, measure light absorption value.With FLM, FLM-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 _FLM-BSA-ε _BAS)/ε _FLM (2)

The present invention calculates crosslinking rate and is about 17.

Claims (1)

1. the preparation method of a double-flumethasone artificial antigen is characterized in that with the diflucortolone being raw material, utilizes the reaction of Succinic anhydried and diflucortolone to generate the diflucortolone succinic acid half-ester, obtains artificial semiantigen; Utilize active ester method to make described artificial semiantigen change the active ester intermediate into, make it then to combine with bovine serum albumin, the artificial antigen of preparation diflucortolone is diflucortolone-bovine serum albumin; Step is:

(1) preparation of artificial semiantigen:

Diflucortolone and Succinic anhydried batching mol ratio are 1: 3, take by weighing diflucortolone 0.4mmol and Succinic anhydried 1.2mmol places the 50mL round-bottomed flask, add the 2.5mL anhydrous pyridine,, reactant is changed in 4 ℃ of frozen water that 10mL contains 10% hydrochloric acid at 60 ℃ of following heated and stirred 6h, 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 described artificial semiantigen 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-layer plate 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 above-mentioned artificial semiantigen of 0.09mmol, 0.09mmol N-hydroxy-succinamide, 0.099mmol N, the N-dicyclohexylcarbodiimide 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;

Phosphate buffered saline buffer: the disodium phosphate soln 94.7mL of 0.2mol/L is mixed the phosphate buffered saline buffer that is pH8.0 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: diflucortolone-bovine serum albumin with the ultrapure water of 2 * 2L.

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