CN102336831A - Anti-sildenafil specific antibody - Google Patents
Anti-sildenafil specific antibody Download PDFInfo
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- CN102336831A CN102336831A CN201010237964XA CN201010237964A CN102336831A CN 102336831 A CN102336831 A CN 102336831A CN 201010237964X A CN201010237964X A CN 201010237964XA CN 201010237964 A CN201010237964 A CN 201010237964A CN 102336831 A CN102336831 A CN 102336831A
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Abstract
The invention provides a preparation method of a sildenafil artificial antigen and antibody, and relates to a preparation method of an artificial hapten, antigen and antibody maximizing the retention of a sildenafil structure. A sildenafil hapten prepared by a chemical synthesis method and a carrier KLH protein are connected and synthesized into an artificial antigen; and an antibody is prepared by the following steps: immunizing animals with the artificial antigen, taking blood, separating out antiserum and purifying. By designing and synthesizing the hapten and artificial antigen having a sildenafil structure, the invention highlights the molecule-specific antigenic determinant, overcomes difficulties in chemical synthesis, and produces the high-affinity antibody through animal immunization inducement. Compared with other similar methods, the invention has the characteristics of specificity, sensitivity, accuracy, high speed, convenience, low cost and the like; and the designed and synthesized hapten lays a foundation for the preparation of the antibody having favorable specificity.
Description
Technical field
The compound that the present invention relates to select a kind of maximum possible to comprise the Virga original structure is as the Virga haptin, and haptin is processed antigen and then produced antibody; And this type of haptin, antigenic synthetic and preparation method for antibody.The invention belongs to biological technical field.
Background technology
Along with global economic integration and food trade internationalization, food safety has become the global challenge public health problem important with the whole world.The Along with people's growth in the living standard, People more and more is paid close attention to the health of oneself, and functional food also receives people's favor day by day.But illegal interpolation drug matters was very outstanding in the functional food in the last few years; Illegal interpolation Virga in " kidney tonifying, establishing-Yang " type functional food; Not only caused great harm to the patient; Also brought loss economically, badly influenced consumption confidence and China food in the international market the competitive power of China people for food.Therefore; Quickening is developed the food safety Study on rapid detection technique; Structuring food prods quality and safety security system to quickening China's food circulation area now for changing, improve the competitive power of food in the world market, has important practical significance and far-reaching strategic importance.
The Virga that illegally is added in the functional food has bigger spinoff, and human body is caused bigger harm.Since Virga in functional food in additive capacity trace very all, instrumental method detects has a lot of restrictions and defective, therefore set up a kind of economy, reliable, special, responsive, method is significant fast and effectively.This problem is prepared the Virga haptin according to the Virga structure; Carry out obtaining behind the immunity test Virga antibody of high specific; Optimization through test conditions; The Virga enzyme-linked immune detection method that a kind of sample pre-treatments is simple, accuracy is high has been set up in the research of sample-pretreating method, for the development of Virga test kit is from now on laid a good foundation.
Summary of the invention
The present invention designs and has synthesized small molecules target analytes haptin; And with the carrier protein coupling; Prepare effective artificial antigen, immune animal prepares small molecules analyte specific antibody, utilizes the specificity immunology reaction of antigen-antibody and is prone to the amplification of the affinity tag of identification to be detected; Thereby ultramicron small molecules target compound in the detection sample of qualitative, quantitative can be used for sample and measures.The key of this technical study is the preparation of haptenic molecular designing, synthetic and holoantigen and antibody.
The present invention is that to reach technical scheme that above purpose designs be the synthetic molecules structural formula Virga haptin that is shown in the following figure
, then haptin being connected synthetic artificial antigen with carrier proteins, immune animal obtains antibody.
(1) (47.5mg 0.1mmol) joins 60% sodium hydride (5.2mg, 0.13mmol are dispersed in the MO) at N, in the suspension-s of dinethylformamide (1.5mL) (nitrogen protection) to Virga.Behind the stirring at room 1h, dripping bromine ETHYLE ACETATE (19mg, N 0.11mmo), dinethylformamide (0.8mL) solution.Behind the stirring at room 15min, continue to stir 16h in 60 ℃ of oil baths.N is divided exactly in decompression, and dinethylformamide behind the adding 1mL water, is used the dichloromethane extraction product in the resistates.United extraction liquid after the water washing, is told organic layer, anhydrous magnesium sulfate drying.The filtering siccative, after filtrate decompression concentrated, the resistates column chromatography for separation must be than pure products.
(2) above-mentioned substituted acetic acid methyl esters sample is dissolved in the 1mL THF, adds lithium hydroxide aqueous solution under the stirring at room, stirring at room 5-6h, sample point completely dissolve in thin layer plate.After vacuum rotary steam fell THF, methylene dichloride extracted impurity earlier.After transferring pH value of solution 2-3 with the hydrochloric acid of 5mol/L, the substituted acetic acid that makes with dichloromethane extraction.United extraction liquid, washing back anhydrous magnesium sulfate drying.
(3) artificial antigen is synthetic: take by weighing 423.2mg Virga haptin and 126.7mg N-hydroxy-succinamide (NHS) and be dissolved in the 20mL anhydrous tetrahydro furan (THF), ice bath stirs 0.5h down.Add and be dissolved with 227.0mg N, the 5mLTHF solution of N-NSC 57182 (DCC).Ice bath continues down to stir 1h, rises to room temperature naturally, stirred overnight.Revolve steaming and obtain product, product is dissolved in 1mL N-N N (DMF) solution.This solution is dropwise added (10mg KLH is dissolved in the 2mL 130mmol/L sodium bicarbonate buffer liquid, and pH 8.1) in the albumen KLH solution that 2mL concentration is 5mg/mL in ice bath, stir, react 5h under the room temperature; Accurately take by weighing 4.7mg EDC again and add in the reaction solution, stir, 4 ℃ of reactions are spent the night; With reaction solution dialysis three days in 4 ℃, the phosphate buffer soln (PBS) of pH 7.4, accurately measure the volume of protein conjugate solution then then, measure concentration and add sodium azide; Packing ,-20 ℃ of preservations.
(4) preparation of antibody and purifying
Immune animal is selected 2 of Japan large ear rabbits, and at 3 months monthly ages, about 1.5 kilograms of body weight are numbered respectively No. 1, No. 2.Immunity is taked subcutaneous and intramuscular injection is carried out jointly.
Immune programme for children: initial immunity: for improving antigenic immunity, adopt by Freund's complete adjuvant emulsive immunogen, dosage is 1mg, is dissolved in the NaCl of 1mL 0.9%, mixes with the Freund's complete adjuvant of 1mL and carries out emulsification.Booster immunization: carried out booster immunization in the 14th day and 28 days behind the initial immunity, dosage is 0.5mg, is dissolved in the NaCl of 1mL 0.9%, mixes with the Freund's incomplete adjuvant of 1mL and carries out emulsification.Later on whenever once, be total to immunity 6 times at a distance from 28 days booster immunizations.Since the 2nd booster immunization, each immunity after 10 days animal pilot production blood is carried out serum titer mensuration and avidity is measured.Adopt the femoral artery blood-collecting method that animal is adopted whole blood after the last immunity, after centrifugal treating, collect whole serum, take the immunoaffinity chromatography method to carry out antibody purification, store for future use in 4 ℃ behind the sodium azide of gained antibody interpolation 0.1% (W/V).
The step of serum titer detection method:
1) the Virga coating antigen for preparing is dissolved in pH 9.6Na
2CO
3-NaHCO
3Damping fluid in; With Virga coating antigen dilution be 1.0 μ g/100 μ L as coating buffer, the every hole of 96 hole microwell plates adds 100 μ L, room temperature is placed and is spent the night or 37 ℃ of constant temperature incubations 2～3 hours; With PBST is phosphate buffered saline buffer 0.05% (V/V), Tween20 washing lotion washing three times;
2) every hole adds 200 μ L 1%KLH/PBS confining liquids, seals 1 hour, with washing lotion washing four times;
3) the Virga antiserum(antisera) with various extension rates joins in the micropore separately, the parallel application of sample in three holes, concussion mixing 5～10min, room temperature reaction 1～2 hour;
4) wash microwell plate 3～4 times with washing lotion, liquid in the hole is got rid of, buckle with thieving paper and do, add 100 liters of goat-anti rabbit ELIAS secondary antibodies to every micropore, 37 ℃ were reacted 30 minutes;
5) wash microwell plate 3～4 times with washing lotion, liquid in the hole is got rid of, buckle with thieving paper and do, add the mixed solution of substrate A and substrate B, the proportioning of the mixed solution of this substrate A and substrate B is 14.6: 0.45, and reaction is 0.5 hour under the room temperature;
6) in each micropore, add stop buffer with termination reaction, read absorbance with ELIASA, the serum dilution that according to light absorption value is at 1 o'clock is drawn from 24 days to the 120th day Virga antibody titer curve of immunity for tiring.
Advantage of the present invention and positively effect are:
1. the present invention has farthest kept the chemical structure of Virga; Be the new compound of initiating both at home and abroad; Immunizing antigen with this haptin preparation goes the immune animal maximum possible to keep the molecular structure of original Virga, and this has the antibody of high degree of specificity that assurance is provided for obtaining to Virga.
2. the present invention has characteristics such as special, sensitive, accurate, quick, cheapness, design, the synthetic haptin lays a good foundation for the good antibody of preparation specificity.
3. through verification experimental verification, above-mentioned haptin, its compound method is simple, and used main raw material such as Virga, and cheap, the easy acquisition of THF all can be buied in general chemical reagents corporation.
4. the present invention is through the above-mentioned haptin of verification experimental verification, its simple synthetic method, and combined coefficient is high, and reactions step is few, has improved the controllability of reaction; In addition, extraction, the purification process of synthetic product are easy, are easy to popularize.
Embodiment
Below in conjunction with accompanying drawing, the embodiment of the invention is further specified; Following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
Embodiment 1:
1. haptenic synthetic
The present invention selects the haptin of Virga, and molecular structural formula is:
Concrete preparation method is: (47.5mg 0.1mmol) joins 60% sodium hydride (5.2mg, 0.13mmol are dispersed in the MO) at N, in the suspension-s of dinethylformamide (1.5mL) (nitrogen protection) to Virga.Behind the stirring at room 1h, dripping bromine ETHYLE ACETATE (19mg, N 0.11mmo), dinethylformamide (0.8mL) solution.Behind the stirring at room 15min, continue to stir 16h in 60 ℃ of oil baths.N is divided exactly in decompression, and dinethylformamide behind the adding 1mL water, is used the dichloromethane extraction product in the resistates.United extraction liquid after the water washing, is told organic layer, anhydrous magnesium sulfate drying.The filtering siccative, after filtrate decompression concentrated, the resistates column chromatography for separation must be than pure products.
2. hydrolysis
Above-mentioned substituted acetic acid methyl esters sample is dissolved in the 1mL THF, adds lithium hydroxide aqueous solution under the stirring at room, stirring at room 5-6h, sample point completely dissolve in thin layer plate.After vacuum rotary steam fell THF, methylene dichloride extracted impurity earlier.After transferring pH value of solution 2-3 with the hydrochloric acid of 5mol/L, the substituted acetic acid that makes with dichloromethane extraction.United extraction liquid, washing back anhydrous magnesium sulfate drying is the Virga haptin.
3, artificial antigen is synthetic
Take by weighing 423.2mg Virga haptin and 126.7mg N-hydroxy-succinamide (NHS) and be dissolved in the 20mL anhydrous tetrahydro furan (THF), ice bath stirs 0.5h down.Add and be dissolved with 227.0mgN, the 5mL THF solution of N-NSC 57182 (DCC).Ice bath continues down to stir 1h, rises to room temperature naturally, stirred overnight.Revolve steaming and obtain product, product is dissolved in 1mLN-N N (DMF) solution.This solution is dropwise added (10mg KLH is dissolved in the 2mL 130mmol/L sodium bicarbonate buffer liquid, and pH 8.1) in the albumen KLH solution that 2mL concentration is 5mg/mL in ice bath, stir, react 5h under the room temperature; Accurately take by weighing 4.7mg EDC again and add in the reaction solution, stir, 4 ℃ of reactions are spent the night; With reaction solution dialysis three days in 4 ℃, the phosphate buffer soln (PBS) of pH 7.4, accurately measure the volume of protein conjugate solution then then, measure concentration and add sodium azide; Packing ,-20 ℃ of preservations.
4, envelope antigen is synthetic
The Virga haptin of experiment gained combines to process envelope antigen with OVA, be used for coated elisa plate.Its specific practice is synthetic with artificial antigen.
5, the preparation of antibody and purifying:
Immune animal is selected 2 of Japan large ear rabbits, and at 3 months monthly ages, about 1.5 kilograms of body weight are raised in the standard test Animal House, observe continuously 4 days, confirms to carry out immunity after physical appearance normally.Immunity is taked subcutaneous and intramuscular injection is carried out jointly.
Initial immunity: taking by weighing dosage is 1mg, is dissolved in the NaCl of 1mL 0.9%, mixes with the Freund's complete adjuvant of 1mL and carries out emulsification, and emulsion is used for immunity.
Booster immunization: carried out booster immunization in the 14th day and 28 days behind the initial immunity, take by weighing 0.5mg, be dissolved in the NaCl of 1mL 0.9%, mix with the Freund's incomplete adjuvant of 1mL and carry out emulsification, emulsion carries out immunity.Every at a distance from 28 days booster immunizations once since the 4th immunity, immunity is 6 times altogether.Since the 2nd booster immunization, each immunity is carried out serum titer to animal pilot production blood after 10 days and is measured.Adopt the femoral artery blood-collecting method that animal is adopted whole blood after the last immunity; After centrifugal treating, collect whole serum; Take the proteinA-SepharoseCL-4B immune affinity chromatographic column to carry out antibody purification, store for future use in 4 ℃ behind the sodium azide of gained antibody interpolation 0.1% (W/V).
6, the step of serum titer detection method:
Encapsulate: the Virga coating antigen for preparing is dissolved in pH 9.6Na
2CO
3-NaHCO
3Damping fluid in; With Virga coating antigen dilution be 1.0 μ g/100 μ L as coating buffer, the every hole of 96 hole microwell plates adds 100 μ L, room temperature is placed and is spent the night or 37 ℃ of constant temperature incubations 2～3 hours; With PBST is phosphate buffered saline buffer 0.05% (V/V), Tween20 washing lotion washing three times;
Sealing: every hole adds 200 μ L, 1% bovine serum albumins (BSA)/PBS confining liquid, seals 1 hour, with washing lotion washing four times;
Application of sample: the Virga antiserum(antisera) of various extension rates is joined in the micropore separately, the parallel application of sample in three holes, concussion mixing 5～10min, room temperature reaction 1～2 hour;
Add ELIAS secondary antibody: wash microwell plate 3～4 times with washing lotion, liquid in the hole is got rid of, buckle with thieving paper and do, add 100 microlitre goat-anti rabbit ELIAS secondary antibodies to every micropore, 37 ℃ were reacted 30 minutes;
Add substrate: wash microwell plate 3～4 times with washing lotion, liquid in the hole is got rid of, buckle with thieving paper and do, chromogenic substrate uses TMB (TMB).Every hole adds 150 microlitre TMB-hydrogen peroxide solutions (the 5mg TMB is dissolved in the 1ml substrate buffer solution), and reaction is 0.5 hour under the room temperature;
Stop: in each micropore, add stop buffer with termination reaction, read absorbance with ELIASA, the serum dilution that according to light absorption value is at 1 o'clock is for tiring.
Claims (4)
1. the artificial antigen of Virga and antibody is characterized in that using molecular formula to do
Haptin be connected synthetic artificial antigen with KLH albumen, is connected with horseradish peroxidase and synthesizes enzyme-labelled antigen, wherein artificial antigen passes through immune new zealand white rabbit again, gets blood, isolates the antiserum(antisera) purifying and makes antibody;
2. the preparation method of the described Virga artificial antigen of claim 1 is characterized in that it being to make with following step:
1) the 47.5mg Virga is joined the N that 1.5mL contains 60% sodium hydride, also use nitrogen protection in the suspension-s of dinethylformamide, behind the stirring at room 1h, drip the N that contains the 19mg METHYL BROMOACETATE; Dinethylformamide 0.8mL behind the stirring at room 15min, continues to stir 16h in 60 ℃ of oil baths, N is divided exactly in decompression; Dinethylformamide adds 1mL water in the resistates, use the dichloromethane extraction product, united extraction liquid; After the water washing, tell organic layer, anhydrous magnesium sulfate drying, filtering siccative; After filtrate decompression concentrated, the resistates column chromatography for separation obtained the substituted acetic acid methyl esters and connects the arm product;
2) above-mentioned substituted acetic acid methyl esters sample is dissolved in the 1mL THF, adds lithium hydroxide aqueous solution under the stirring at room, stirring at room 5-6h; Sample point completely dissolve in thin layer plate, after vacuum rotary steam fell THF, methylene dichloride extracted impurity earlier; Behind the hydrochloric acid accent pH value of solution 2-3 with 5mol/L; With the substituted acetic acid that dichloromethane extraction makes, united extraction liquid, washing back anhydrous magnesium sulfate drying can obtain the Virga haptin;
3) take by weighing 423.2mg Virga haptin and 126.7mg N-hydroxy-succinamide (NHS) and be dissolved in the 20mL anhydrous tetrahydro furan (THF), ice bath stirs 0.5h down, adds to be dissolved with 227.0mgN; The 5mL THF solution of N-NSC 57182 (DCC), ice bath continue down to stir 1h, rise to room temperature naturally; Stirred overnight; Revolve steaming and obtain product, product is dissolved in 1mL N-N N (DMF) solution, this solution is dropwise added concentration in ice bath be 5mg mL
-12mL KLH solution in, stir, react 5h under the room temperature, accurately take by weighing 4.7mg EDC again and add in the reaction solution, stir, 4 ℃ of reactions are spent the night, and with reaction solution dialysis three days in 4 ℃, the phosphate buffer soln of pH 7.4, can obtain artificial antigen then;
3. the preparation method of the described Virga enzyme-labelled antigen of claim 1 is characterized in that it being to make with following step:
Claim accurately that respectively claim 2 gained Virga haptin 16.975mg is dissolved in the 1mL N-N dimethyl formamide solution; This solution is dropwise added in ice bath in the HRP solution that 2mL concentration is 5mg/mL; Stir; React 5h under the room temperature, 4 ℃ of reactions are spent the night, and then reaction solution dialysis in 4 ℃, the phosphate buffer soln of pH 7.4 can be obtained the enzyme-labelled antigen of Virga in three days;
4. the preparation method of the described Virga antibody of claim 1 is characterized in that it being to make with following step:
Immune animal is selected female new zealand white rabbit for use; Immunization method adopts subcutaneous and intramuscular injection, carries out four times booster immunization after just exempting from, booster immunization respectively at initial immunity after 2 weeks, 4 weeks and 6 week back immunity three times; After this carried out the 5th immunity at interval in one month; Immunity was got blood by the auricular vein of rabbit in back 9 days, the detection of tiring, and specific practice is:
Initial immunity: get 1mg and be dissolved in the solution that 0.9% NaCl solution and Freund's complete adjuvant equal-volume prepared, carry out animal immune according to the said method synthetic of claim 2 artificial antigen,
Booster immunization: be dissolved in the solution that 0.9% NaCl solution and Freund's incomplete adjuvant equal-volume are prepared with the above-mentioned artificial antigen of 0.5mg, carry out animal immune,
Antibody purification: the periodic monitor animal's antibody is tired; When antibody reaches suitable tiring to certain envelope antigen, gather blood, and centrifugal acquisition antiserum(antisera); Use G-Sepharose albumen affinity column antagonistic Serum to carry out purifying, obtain the specific antibody of anti-Virga.
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Cited By (6)
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| CN104502554A (en) * | 2014-12-17 | 2015-04-08 | 郭杰标 | Immunoassay method for nano-colloidal gold marker of tadalafil and analogs thereof |
| CN105353095A (en) * | 2015-11-16 | 2016-02-24 | 华南农业大学 | Sildenafil and its analogue immunodetection method |
| CN108409744A (en) * | 2018-03-23 | 2018-08-17 | 广东工业大学 | Silaenafil haptens, artificial antigen and preparation method thereof |
| CN108752345A (en) * | 2018-03-23 | 2018-11-06 | 广东工业大学 | Silaenafil haptens, artificial antigen and preparation method thereof |
| CN110357890A (en) * | 2019-06-20 | 2019-10-22 | 华南农业大学 | A kind of colloidal gold strip and its preparation method and application detecting silaenafil class drug |
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2010
- 2010-07-27 CN CN201010237964XA patent/CN102336831A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104502554A (en) * | 2014-12-17 | 2015-04-08 | 郭杰标 | Immunoassay method for nano-colloidal gold marker of tadalafil and analogs thereof |
| CN105353095A (en) * | 2015-11-16 | 2016-02-24 | 华南农业大学 | Sildenafil and its analogue immunodetection method |
| CN108409744A (en) * | 2018-03-23 | 2018-08-17 | 广东工业大学 | Silaenafil haptens, artificial antigen and preparation method thereof |
| CN108752345A (en) * | 2018-03-23 | 2018-11-06 | 广东工业大学 | Silaenafil haptens, artificial antigen and preparation method thereof |
| CN110357890A (en) * | 2019-06-20 | 2019-10-22 | 华南农业大学 | A kind of colloidal gold strip and its preparation method and application detecting silaenafil class drug |
| CN110357890B (en) * | 2019-06-20 | 2021-10-29 | 华南农业大学 | Colloidal gold test strip for detecting sildenafil drugs and preparation method and application thereof |
| CN111273007A (en) * | 2020-03-13 | 2020-06-12 | 内江师范学院 | Kit and detection method for rapidly detecting fish Edwardsiella ictaluri |
| CN111273007B (en) * | 2020-03-13 | 2023-08-01 | 内江师范学院 | Kit for rapidly detecting Edwardsiella ictaluri and detection method |
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