CN101029067A - Synthesis of ivermectin artificial antigen - Google Patents
Synthesis of ivermectin artificial antigen Download PDFInfo
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- CN101029067A CN101029067A CN 200610041918 CN200610041918A CN101029067A CN 101029067 A CN101029067 A CN 101029067A CN 200610041918 CN200610041918 CN 200610041918 CN 200610041918 A CN200610041918 A CN 200610041918A CN 101029067 A CN101029067 A CN 101029067A
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Abstract
A process for synthesizing the artificial antigen of ivermectin includes synthesizing the semi-antigen 4'-0-(mono)-succinyl IVM and synthesizing the artificial antigen by N-hydroxy succinylimide ester method.
Description
Technical field
The invention belongs to the drug residue detection technique of biological technical field.
Background technology
Ivermectin (Ivermectin is designated hereinafter simply as IVM) is the semi-synthetic macrolide antibiotics from actinomyces Steptomyoesavermitilis, is the dihydro reduzate (22,23-dihydro AVM) of Avrmectin.IVM belongs to the medicine that toxicity is big, the off-drug period is grown in the residue of veterinary drug source of pollution, elimination is slow in animal body, the residence time is longer.IVM and meta-bolites thereof in animal body delay or accumulate after, the circulation of its residual component enters the human body and the ecosystem, producing other has the effect of obvious harm to human body, also may have teratogenesis, carcinogenic, mutagenesis (three cause) effect.
The domestic and international at present research at IVM mainly concentrates on aspect the veterinary drug pharmacological action, reports seldom for the research that determination of plasma concentration method, pharmacokinetics and drug residue are analyzed, and the analytical procedure of employing is also mostly to be high performance liquid chromatography (HPLC) assay methods.The using dosage of IVM very little (0.2～0.3 μ g/kg), water-soluble extremely low, organizing the Chinese traditional medicine maximum residue limit(MRL) is 0.015～0.02 μ g/kg, thereby very difficult to the residual detection of IVM.At present, the residual conventional method of analysis of IVM is still the high performance liquid chromatography (HPLC) based on the fluorescence derivation detection of (1980) such as Tolan, and analytic process is very loaded down with trivial details, and the operational condition harshness has seriously limited its operability in practice.It is easy that enzyme immunoassay (ELISA) method detects residue of veterinary drug, reliably, sensitivity, but domesticly do not see enzyme linked immunosorbent detection IVM report, in similar medicine, Li Junsuo (journal of animal science and veterinary medicine, 1996,27 (6): 531-538) Avrmectin Antibody Preparation and ELISA method for detecting residue thereof are studied; In existing research, veterinary drug, agricultural chemicals artificial immunogen synthetic vectors are used bovine serum albumin more.He Jihong, Shen Jianzhong (Chinese animal doctor's magazine, 2005,1:15-17) grade is studied preparation and the evaluation of having carried out the doractin artificial antigen, and the two used artificial immunogen synthetic vectors is bovine serum albumin.Because bovine serum albumin three-dimensional arrangement complexity, haptens is connected with carrier proteins, can produce the antibody at the carrier proteins position behind the immune animal, and sero-fast specificity is reduced, and homogeneity is poor, and detection sensitivity is low in the elisa assay in later stage.
Summary of the invention
In view of the present situation of the residual detection of at present domestic and international IVM, the invention provides the new synthetic method of a kind of ivermectin artificial antigen, provide research technique for further studying IVM enzyme linked immunological retention analysis method.
The present invention makes the haptens of Simulation with I VM feature structure, and artificial antigen is made in the free amine group coupling of itself and carrier proteins.The preparation artificial antigen obtains antiserum(antisera) behind the injection animal, obtains specific polyclonal antibody, is the key step and the core technology of enzyme linked immunological (ELISA) retention analysis.
Secondly, the present invention selects for use poly-l-lysine (PLL) to prepare the ivermectin artificial antigen as coupling carrier, and poly-l-lysine is linear polypeptide, and is simple in structure, immunogenicity is poor, understands the antibody that produces at the carrier proteins position after having overcome haptens and carrier proteins being connected immune animal.The poly-lysine free amine group is many simultaneously, is 10 times of bovine serum albumin approximately.But, do not see successfully report in domestic farming, the Artificial Antigens for Veterinary Drugs preparation because coupling condition requires comparatively strictness.
Based on above-mentioned, purpose of the present invention realizes by following steps:
(1) haptens 4, and " it is poly-that O-(list)-succinyl-IVM synthetic is divided into three steps
The first step: with TERT-BUTYL DIMETHYL CHLORO SILANE (t-BuMe
2SiCl) with the C of IVM
5-OH selective protection;
Get 1.0g IVM 6ml N, dinethylformamide (DMF) dissolving under the mechanical stirring, adds 0.47g imidazoles and 0.52g t-BuMe
2SiCl (with 1ml DMF predissolve).Behind 25 ℃ of stirring 2.5h, reaction solution mixes with the ethyl acetate of 100ml, and the 50ml distilled water wash is collected ethyl acetate layer, washes concentrating under reduced pressure ester layer repeatedly 3 times; Resistates redissolves with the 1ml methylene dichloride.Through silicagel column GF
254(200-400 order) separated product, elutriant is CH
2CI
2: (100: 2, v/v), TLC monitored reaction to THF, collects R
fIt is 0.47 component.Concentrating under reduced pressure after the vacuum-drying, gets white powder.It is 95.2% that HPLC analyzes main component content, identifies its molecular weight by mass spectrum.
Reaction formula is: IVM+t-BuMe
2SiCl → 5-t-BuMe
2SiO-IVM+HCl
Second step: at IVM C
4" the OH position makes up spacer molecule and active group
Get 0.5g 5-O-t-BuMe
2Si IVM is dissolved in the methylene dichloride of 10ml, adds 0.28g4-Dimethylamino pyridine (DAMP) successively, 0.5ml triethylamine and 0.90g succinyl oxide, and under the magnetic agitation condition, backflow 2.5h, reaction solution becomes chocolate.Remove methylene dichloride in the reaction solution by pressure reducing mode, residue is washed 3-5 time repeatedly with 3.6% hydrochloric acid 20ml and distilled water 50ml washing ether layer with the heavy molten after-filtration of 100ml ether, filtrate, and making pH is neutrality.Collect ether layer evaporate to dryness, redissolve, with TLC method separated product (GF with the 1ml methylene dichloride
254, developping agent is CH
2Cl
2: THF: CH
3OH 95: 5: 5 v/v/v), collects R
fBe 0.5 component,, collect elutriant with methyl alcohol drip washing, concentrating under reduced pressure, vacuum-drying, pale yellow powder.It is 94.3% that HPLC analyzes main component content; Mass spectrum is identified its molecular weight.
Reaction formula is: 5-t-BuMe
2SiO-IVM+C
4H
4O
3→ 5-t-BuMe
2SiO-4 " O-(list) succinyl-IVM
The 3rd step: go protection, obtain C
4" O-(list) succinyl-IVM
Get 5-t-BuMe
2" O-(list) succinyl-IVM 0.3g 17ml dissolve with methanol; add 0.2g p-methyl benzenesulfonic acid (being dissolved in the methyl alcohol of 10ml in advance) under 25 ℃ of magnetic agitation behind 25 ℃ of magnetic agitation 25min, adds the ethyl acetate of 100ml; behind the mixing to SiO-4, with the 2%NaHCO of 50ml
3Washing is again with distilled water wash 3-5 time, to pH neutrality.Collect ester layer concentrating under reduced pressure, with methylene dichloride 1ml redissolution residue, with TLC method separated product (GF
254, developping agent is CH
2Cl
2: THF: HA
C90: 9.7: 0.3, v/v/v), collect mobility (R
f) be 0.2 component, with ethyl acetate drip washing, collect elutriant, concentrating under reduced pressure, vacuum-drying, pale yellow powder.It is 93.6% that HPLC analyzes main component content; Mass spectrum is identified its molecular weight.
Reaction formula is: 5-t-BuMe
2SiO-4 " O-(list) succinyl-IVM+C
7H
8O
3S → 4 " O-succinyl-IVM
(2) artificial antigen is synthetic
Adopt the synthetic artificial antigen of N-hydroxy-succinamide ester method (NHS)
The first step: get 10.0mg4 " O-(list) succinyl-IVM (artificial semiantigen of preparation) puts in the 5ml round-bottomed flask, adds 1.0mL DMF dissolving, adds 2.5mgNHS and 4.5mgEDCHCL more successively, after the mixing, magnetic agitation 10h at room temperature.
Second step: in the 15ml triangular flask, add the 5.4ml damping fluid and be borate (0.2mol/L, pH9.0), 0.6ml N, dinethylformamide (DMF), behind the mixing, (PLL) is dissolved in wherein with the 25mg poly-lysine.
The 3rd step: the first step reaction solution is dropwise added in 0.5h in the solution of second step preparation, after stirring 1h under 25 ℃, go to 4 ℃ and stir 6h down.
The 4th step: the 3rd step product is packed in the dialysis tubing (is 3500 by molecular weight), and dialyzate is the PBS of 0.01mol/L, and pH7.2 changed 1 dialyzate every 12 hours, dialysed 72 hours.Take out dialysis tubing, put into the beaker that fills polyethylene glycol 6000, the moisture content of dialysis tubing is blotted.
The 5th step: with Sephacryl S-200 purifying, utilize ultra-violet absorption spectrum that proteic connection of haptens and carrying agent identified that its binding ratio is 1: 29 as calculated, obtain purer IVM artificial antigen, frozen down at-20 ℃.
The beneficial effect of advantage of the present invention and generation is:
1. ivermectin is a classic insect repellent in the Avrmectin family, is able to widespread use in livestock industry is produced, and its method for detecting residue is seen in the HPLC method that mostly is of report.Rapid screening technology during the enzyme immunoassay technology is analyzed as residue of veterinary drug, it is the main flow direction that ensures animal food safety, the most commonly used is ELISA method and test strip technology, but the domestic enzyme immunoassay of still not seeing the IVM retention analysis, tracing it to its cause may be the molecular structure more complicated of IVM, and it is carried out artificial reconstructed and prepare artificial antigen existing great challenge.The present invention has successfully synthesized the artificial antigen of IVM with regard to the constructional feature of IVM, has prepared the polyclonal antibody at IVM, lays a good foundation for further studying IVM enzyme linked immunological retention analysis method;
2. be usually used in Artificial Antigens for Veterinary Drugs synthetic carrier protein and comprise bovine serum albumin (BSA), ovalbumin (OVA), human serum protein (HSA) and keyhole limpet hemocyanin (KLH), wherein commonly used with bovine serum albumin again.Because BSA physics and chemistry is stable, economy is easy to get, and the intramolecularly free amino group is many, with hapten conjugation rate height, and has under different pH values and the ionic strength and containing under some organic solvent state and can both keep bigger solubleness.In recent years, all there is the document introduction to do carrier (poly L Methionin commonly used) both at home and abroad with the polypeptide of synthetic, autoimmunization originality is very poor but can increase haptenic immunity, help body and produce the higher antibody of specificity, and have than BSA more freedom amino (with the free amino group number of the poly-lysine of the suitable molecular weight of BSA be 10 times of the contained number of BSA approximately), can improve carrier protein and haptenic coupling rate greatly.Doing the report that carrier prepares Artificial Antigens for Veterinary Drugs with poly-lysine does not find as yet, the present invention is by repeatedly testing coupling condition, success is that carrier has synthesized 4 with poly-lysine PLL (MW3～80,000), and " O-(list) succinyl-IVM-PLL; as artificial immunogen has created condition for further studying IVM enzyme linked immunological retention analysis method.
Embodiment is referring to summary of the invention.
Claims (1)
1, the new synthetic method of a kind of ivermectin artificial antigen, its feature comprises haptens 4 " the synthetic and synthetic artificial antigen of employing N-hydroxy-succinamide ester method of O-(list)-succinyl-IVM:
1) haptens 4 '-O-(list)-succinyl-IVM synthetic
The first step: with the C of TERT-BUTYL DIMETHYL CHLORO SILANE with succinyl-IVM
5-OH selective protection
Get 1.0g IVM 6ml N, dinethylformamide (DMF) dissolving under the mechanical stirring, adds 0.47g imidazoles and 0.52g t-BuMe
2SiCl, with 1ml DMF predissolve, behind 25 ℃ of stirring 2.5h, reaction solution mixes with the ethyl acetate of 100ml, and the 50ml distilled water wash is collected ethyl acetate layer, washes concentrating under reduced pressure ester layer repeatedly 3 times; Resistates redissolves with the 1ml methylene dichloride; Through 200-400 order silicagel column GF
254Separated product, elutriant are CH
2Cl
2: THF, its volume ratio 100: 2, TLC monitors reaction, collects R
fIt is 0.47 component; Concentrating under reduced pressure after the vacuum-drying, gets white powder;
Reaction formula is: IVM+t-BuMe
2SiCl → 5-t-BuMe
2SiO-IVM+HCl
Second step: at IVM C
4" the OH position makes up spacer molecule and active group
Get 0.5g 5-t-BuMe
2SiO-IVM is dissolved in the methylene dichloride of 10ml, adds 0.28g 4-Dimethylamino pyridine successively, 0.5ml triethylamine and 0.90g succinyl oxide, and under the magnetic agitation condition, backflow 2.5h, reaction solution becomes chocolate.Remove methylene dichloride in the reaction solution by pressure reducing mode, residue is washed 3-5 time repeatedly with 3.6% hydrochloric acid 20ml and distilled water 50ml washing ether layer with the heavy molten after-filtration of 100ml ether, filtrate, and making pH is neutrality; Collect ether layer evaporate to dryness, redissolve, use GF with the 1ml methylene dichloride
254TLC method separated product, developping agent are CH
2Cl
2: THF: CH
3OH, R is collected in its volume ratio position 95: 5: 5
fBe 0.5 component,, collect elutriant with methyl alcohol drip washing, concentrating under reduced pressure, vacuum-drying, pale yellow powder;
Reaction formula is: 5-t-BuMe
2SiO-IVM+C
4H
4O
3→ 5-t-BuMe
2SiO-4 " O-(list) succinyl-IVM
The 3rd step: go protection, obtain C
4" O-(list) succinyl-IVM
Get 5-r-BuMe
2" the 0.2g p-methyl benzenesulfonic acid that adds the 10ml dissolve with methanol under O-(list) the succinyl-IVM 0.3g 17ml dissolve with methanol, 25 ℃ of magnetic agitation behind 25 ℃ of magnetic agitation 25min, adds the ethyl acetate of 100ml to SiO-4, behind the mixing, with the 2%NaHCO of 50ml
3Washing is again with distilled water wash 3-5 time, to pH neutrality; Collect ester layer concentrating under reduced pressure, with methylene dichloride 1ml redissolution residue, with GF
254TLC method separated product, developping agent are CH
2Cl
2: THF: HA
C, its volume ratio is 90: 9.7: 0.3, collects mobility (R
f) be 0.2 component, with ethyl acetate drip washing, collect elutriant, concentrating under reduced pressure, vacuum-drying, pale yellow powder;
Reaction formula is: 5-t-BuMe
2SiO-4 " O-(list) succinyl-IVM+C
7H
8O
3S → 4 " O-succinyl-IVM
(2) artificial antigen is synthetic
Adopt the synthetic artificial antigen of N-hydroxy-succinamide ester method
The first step: the artificial semiantigen 4 of getting 10.0mg preparation " O-(list) succinyl-IVM puts in the 5ml round-bottomed flask, adds 1.0ml DMF and dissolves, and adds 2.5mgNHS and 4.5mg EDCHCL more successively, after the mixing, magnetic agitation 10h at room temperature;
Second step: adding 5.4ml 0.2mol/L pH9.0 damping fluid in the 15ml triangular flask is borate, 0.6ml DMF, and behind the mixing, (PLL) is dissolved in wherein with the 25mg poly-lysine;
The 3rd step: the first step reaction solution is dropwise added in 0.5h in the solution of second step preparation, after stirring 1h under 25 ℃, go to 4 ℃ and stir 6h down;
The 4th step: the 3rd step product is packed in molecular weight is 3500 dialysis tubing, and dialyzate is the PBS of 0.01mol/L, and pH7.2 changed 1 dialyzate every 12 hours, dialysed 72 hours; Take out dialysis tubing, put into the beaker that fills polyethylene glycol 6000, the moisture content of dialysis tubing is blotted;
The 5th step: with Sephacryl S-200 purifying, utilize ultra-violet absorption spectrum that haptens and being connected of carrier proteins are identified that its binding ratio is 1: 29 as calculated, obtain purer IVM artificial antigen, frozen down at-20 ℃.
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| CN103048445A (en) * | 2012-12-14 | 2013-04-17 | 广东工业大学 | Preparation method of bisphenol A-coated antigen with polylysine as carrier and application thereof |
| CN103048445B (en) * | 2012-12-14 | 2015-02-04 | 广东工业大学 | Preparation method of bisphenol A-coated antigen with polylysine as carrier and application thereof |
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| CN107446030A (en) * | 2017-09-21 | 2017-12-08 | 福建省农业科学院农业生物资源研究所 | A kind of Bt Pesticidal toxins of ivermectin coupling and its application |
| CN107417774A (en) * | 2017-09-21 | 2017-12-01 | 福建省农业科学院农业生物资源研究所 | A kind of Bt Pesticidal toxins of ivermectin coupling and its application |
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| CN107417774B (en) * | 2017-09-21 | 2021-04-09 | 福建省农业科学院农业生物资源研究所 | Ivermectin-coupled Bt insecticidal toxin and application thereof |
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| CN109541216A (en) * | 2018-10-26 | 2019-03-29 | 北京勤邦生物技术有限公司 | Detect enzyme linked immunological kit and its application of ivermectin and avermectin |
| CN109541216B (en) * | 2018-10-26 | 2022-10-21 | 北京勤邦生物技术有限公司 | Enzyme linked immunosorbent assay kit for detecting ivermectin and abamectin and application thereof |
| CN109748946A (en) * | 2019-01-22 | 2019-05-14 | 谢金兵 | A kind of synthesis and application of protein nano particle |
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