CN102336830A - Anti-nandrolone specific antibody - Google Patents
Anti-nandrolone specific antibody Download PDFInfo
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- CN102336830A CN102336830A CN2010102379565A CN201010237956A CN102336830A CN 102336830 A CN102336830 A CN 102336830A CN 2010102379565 A CN2010102379565 A CN 2010102379565A CN 201010237956 A CN201010237956 A CN 201010237956A CN 102336830 A CN102336830 A CN 102336830A
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Abstract
The invention provides a preparation method of a nandrolone artificial antigen and antibody, and relates to a preparation method of an artificial hapten, antigen and antibody maximizing the retention of a nandrolone structure. A nandrolone hapten prepared by a chemical synthesis method and a carrier BSA 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 nandrolone 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 nandrolone original structure is as the nandrolone 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 adulteration is serious day by day in the consequent functional food, and human consumer's physical and mental health in serious harm.In recent years, security issues become increasingly urgent for functional food, also proposed new requirement for adulterant quality inspection survey technology in the food, and the investigator is actively seeking to satisfy the food rapid detection and the analytical procedure of the following aspects characteristics: highly sensitive; High specific; The wide scope of application can be measured types of functionality food; Low testing cost; Be prone to commercialization.Immunodetection is the most widely used Fast Detection Technique that meets this requirement at present.
Nandrolone has stronger toxic side effect, can cause cancer, hepatitis and dizzy.Malice admixture nandrolone very easily works the mischief to human body in the functional food, and is therefore significant to the detection of nandrolone in the functional food.Since 17 β-NT in functional food interpolation all very the trace; The general more complicated of composition of while functional food; The matrix influence is difficult to eliminate; The conventional sense method is difficult to reach ideal and detects effect, therefore presses for a kind of economy, reliable, special, responsive, the method fast and effectively set up.
Malice adulteration to functional food; This paper is prepared the highly sensitive of adulterant nandrolone, the antibody of high specificity; Set up simply, nandrolone SPE-enzyme-linked immune detection method fast, support for the functional food false distinguishing provides a kind of reliable technique.
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 the technical scheme that above purpose designs be the nandrolone haptin that the synthetic molecules structural formula is shown in the following figure
, then haptin being connected synthetic artificial antigen with carrier proteins, immune animal obtains antibody.
(1) accurately take by weighing the 0.5488mg nandrolone and be dissolved in the 13.5mL anhydrous tetrahydro furan, add the 0.4g Succinic anhydried, 0.55mL triethylamine and 0.243mg DMAP, 60 ℃ of reactions are spent the night, and outstanding the steaming extremely of solvent done.
(2) with residue obtained with the organic solvent ethyl acetate/petroleum ether (1/1, v/v) after the dissolving, cross the chromatography column purifying.Through optimizing, finally select ethyl acetate/petroleum ether (1/1, v/v) be developping agent.Obtain white crystal after the filtrating that obtains revolved steaming, be 17 β-NT haptin.
(3) artificial antigen is synthetic: accurately take by weighing above-mentioned gained 17 β-NT haptin 0.025mmol (9.36mg) and 9.3mg EDC respectively, be dissolved in 1mL N-N N (DMF) solution.This solution is dropwise added (10mg BSA is dissolved in the 2mL 130mmol/L sodium bicarbonate buffer liquid, and pH 8.1) in the protein B SA 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), the preparation of coating antigen
Be connected with OVA with the nandrolone haptin and promptly process envelope antigen, be used to encapsulate reaction.
(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 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 nandrolone coating antigen for preparing is dissolved in pH 9.6 Na
2CO
3-NaHCO
3Damping fluid in; With nandrolone 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 nandrolone 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 microlitre 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 nandrolone 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 nandrolone; 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 nandrolone, and this has the antibody of high degree of specificity that assurance is provided for obtaining to nandrolone.
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 nandrolone, 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 nandrolone, and molecular structural formula is:
Concrete preparation method is: accurately takes by weighing the 0.5488mg nandrolone and is dissolved in the 13.5mL anhydrous tetrahydro furan, add the 0.4g Succinic anhydried, and 0.55mL triethylamine and 0.243mg DMAP, 60 ℃ of reactions are spent the night, and outstanding the steaming extremely of solvent done.
2. purifying
With residue obtained with the organic solvent ethyl acetate/petroleum ether (1/1, v/v) after the dissolving, cross the chromatography column purifying.Through optimizing, finally select ethyl acetate/petroleum ether (1/1, v/v) be developping agent.Obtain white crystal after the filtrating that obtains revolved steaming, be 17 β-NT haptin.
3, artificial antigen is synthetic
Accurately take by weighing above-mentioned gained 17 β-NT haptin 0.025mmol (9.36mg) and 9.3mg EDC respectively, be dissolved in 1mL N-N N (DMF) solution.This solution is dropwise added (10mg BSA is dissolved in the 2mL 130mmol/L sodium bicarbonate buffer liquid, and pH 8.1) in the protein B SA 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 nandrolone 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).
Claims (4)
1. the artificial antigen of nandrolone and antibody is characterized in that using molecular formula to do
17 β-NT haptin be connected synthetic artificial antigen with BSA 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 nandrolone artificial antigen of claim 1 is characterized in that it being to make with following step:
(1) the haptenic synthesis step of nandrolone
Accurately take by weighing the 0.5488mg nandrolone and be dissolved in the 13.5mL anhydrous tetrahydro furan, add the 0.4g Succinic anhydried, 0.55mL triethylamine and 0.243mg DMAP, 60 ℃ of reactions are spent the night, and outstanding the steaming extremely of solvent done;
(2) haptenic purifying
With residue obtained with the organic solvent ethyl acetate/petroleum ether (1/1, v/v) after the dissolving, cross the chromatography column purifying.Through optimizing, finally select ethyl acetate/petroleum ether (1/1, v/v) be developping agent.Obtain white crystal after the filtrating that obtains revolved steaming, be 17 β-NT haptin;
(3) artificial antigen is synthetic
Accurately take by weighing above-mentioned gained 17 β-NT haptin 0.025mmol (9.36mg) and 9.3mg EDC respectively, be 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;
3. the preparation method of the described nandrolone enzyme-labelled antigen of claim 1 is characterized in that it being to make with following step:
17 β of right 2 gained-NT haptin combines to process enzyme-labelled antigen with HRP.Method is identical with the preparation method of immunizing antigen: accurately take by weighing above-mentioned gained 17 β-NT haptin 0.025mmol (9.36mg) and 9.3mg EDC respectively, be dissolved in 1mLN-N N (DMF) solution.This solution is dropwise added (10mg HRP is dissolved in the 2mL 130mmol/L sodium bicarbonate buffer liquid, and pH 8.1) in the HRP 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; Then with reaction solution dialysis three days in 4 ℃, the phosphate buffer soln (PBS) of pH7.4; Accurately measure the volume of enzyme-labelled antigen solution then, measure concentration and add Thiomersalate, 4 ℃ of preservations;
4. the preparation method of the described nandrolone 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-nandrolone.
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| CN2010102379565A CN102336830A (en) | 2010-07-27 | 2010-07-27 | Anti-nandrolone specific antibody |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104849476A (en) * | 2015-05-22 | 2015-08-19 | 天津科技大学 | Enzyme-labeled antigen for protein anabolic hormone immunoassay and analysis method |
| CN110128529A (en) * | 2019-04-11 | 2019-08-16 | 中国农业科学院兰州畜牧与兽药研究所 | A kind of synthetic method of nortestosterone artificial antigen |
| CN113563292A (en) * | 2021-06-18 | 2021-10-29 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Henbane hapten, artificial antigen, preparation methods of henbane hapten and artificial antigen, antibody and application of henbane hapten and artificial antigen |
-
2010
- 2010-07-27 CN CN2010102379565A patent/CN102336830A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104849476A (en) * | 2015-05-22 | 2015-08-19 | 天津科技大学 | Enzyme-labeled antigen for protein anabolic hormone immunoassay and analysis method |
| CN110128529A (en) * | 2019-04-11 | 2019-08-16 | 中国农业科学院兰州畜牧与兽药研究所 | A kind of synthetic method of nortestosterone artificial antigen |
| CN110128529B (en) * | 2019-04-11 | 2023-07-07 | 中国农业科学院兰州畜牧与兽药研究所 | Synthetic method of nortestosterone artificial antigen |
| CN113563292A (en) * | 2021-06-18 | 2021-10-29 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Henbane hapten, artificial antigen, preparation methods of henbane hapten and artificial antigen, antibody and application of henbane hapten and artificial antigen |
| CN113563292B (en) * | 2021-06-18 | 2023-02-28 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Henbane hapten, artificial antigen, preparation methods of henbane hapten and artificial antigen, antibody and application of henbane hapten and artificial antigen |
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