CN108689985A - A kind of preparation method and application of safrole haptens and antigen - Google Patents
A kind of preparation method and application of safrole haptens and antigen Download PDFInfo
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- CN108689985A CN108689985A CN201810539737.9A CN201810539737A CN108689985A CN 108689985 A CN108689985 A CN 108689985A CN 201810539737 A CN201810539737 A CN 201810539737A CN 108689985 A CN108689985 A CN 108689985A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D317/00—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms
- C07D317/08—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3
- C07D317/44—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems
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- C07D317/48—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring
- C07D317/50—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to atoms of the carbocyclic ring
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
A kind of preparation method and application of safrole haptens and antigen, it is characterised in that:The safrole haptens is obtained by the reaction by 5- (the bromo- 2- propylene -1- bases of 2-) -1,3- benzodioxolanes and alanine;The safrole antigen is obtained with carrier protein couplet by safrole haptens.Antigen prepared by the present invention shows the safrole antigenic determinant of specificity, and safrole monoclonal antibody can be prepared by immune animal.The antibody specificity height of generation, high sensitivity, enzyme linked immunological kit, colloidal gold strip and the time-resolved fluoroimmunoassay chromatograph test strip that can be used for preparing detection safrole, to realize the quick detection of safrole in essence spice for cigarette, tobacco and tobacco product, food.
Description
Technical field
The present invention relates to a kind of preparation method and applications of safrole haptens and antigen.Belong to immunochemical technique neck
Domain.
Background technology
Safrole is also known as safrole, safrole, 1-ally-3,4-methy-lene dioxy benzene, and colourless or yellowish liquid is not soluble in water, be soluble in chloroform, ether and
Other non-polar organic solvents have camphorwood smell, are naturally occurring in the plants such as Chinese cassia tree, nutmeg, black pepper and purple perilla, are to be permitted
The main component of such as yellow camphor tree essential oil of more edible natural essential oils, aniseed essential oil and camphorated oil.Safrole class compound includes mainly Huang
Camphor tree element, isosafrole, dihydrosafrole etc..Due to its comfortable smell, sassafras oil is once in food, articles for washing and cosmetics
It is middle to be used as flavoring agent and deodorant tune.But Recent study shows that safrole can form safrole-deoxyribose core in hepatic tissue
Acid adduct has human body certain toxic side effect, easy modificator gene mutation and hepatic injury or even liver cancer, is digestive system, blood
The strong carcinogen of liquid system, urinary system.Therefore, countries in the world are forbidden using safrole as fragrance and food additives at present
Directly use.Due to the toxic side effect of safrole class compound, safrole class compound analysis accurate, quickly, easy is established
Detection method, for safeties important roles such as assessment food, cosmetics, tobaccos.
Currently, safrole mainly passes through direx process, ultrasonic extraction method, Simultaneous distillation, steam distillation
Deng extraction, in conjunction with gas chromatography(GC), gas chromatography-mass spectrography(GC-MS), gas chromatography tandem mass spectrometry method(GC-MS/
MS), high performance liquid chromatography(HPLC), ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)Detection.But due to this
A little analysis methods need the testing staff of expensive large-scale instrument and equipments and profession, pretreatment process it is complicated, it is cumbersome, detect
It is of high cost, analyze speed is slow, it is difficult to meet the needs of safrole content rapid screening in field monitoring and great amount of samples.Based on anti-
The immunoassay method of original antibody specific recognition can be with the safrole content in qualitative and quantitative detection sample.This analysis method
It is of less demanding to instrument and equipment, fast and convenient, the general pretreatment without carrying out complexity to sample, high sensitivity, high specificity,
It is of less demanding to the professional technique of user of service, it is easy universal and promotes, the needs of quickly analysis detection can be met, be particularly suitable for
The quick analysis of scene screening and a large amount of samples.Immunoassay provides a new analysis for safrole research and detects approach.
Immunoassay has become a brand-new field of analysis and research at present, and American Chemical Society is by immunoassay and gas-chromatography, liquid phase
Chromatography is classified as three big mainstays of analysis jointly.The invention belongs to micromolecular compound immunochemistries and retention analysis technology to lead
Domain is related to organic synthesis, immunochemistry and biochemistry etc., by immunology, immunochemistry basic principle and biotechnology hand
Section, design, synthesized micromolecule target analytes haptens, and be coupled with carrier protein, prepare effective artificial antigen.It prepares
Antigen can be exempted from by the way that animal preparation is immunized to the antibody of small molecule analyte specific recognition using the specificity of antigen-antibody
Epidemiology reacts and the amplification of the marker of easily detected identification, ultramicron small molecule object in quantitative detection sample.
The MOLECULE DESIGN of haptens is the committed step for generating specific antibody and establishing immunoassay method with synthesis.Artificial antigen
It prepares, including binding site, combination, carrier and haptens and the difference in any structure of analyte substance of interest, it is all
Topological sex character including such as molecular size, shape, ingredient, configuration, conformation, polarity, cloud density, all may great shadow
Ring the property of corresponding antibodies.About the preparation method of safrole haptens and antigen, there is not been reported at present.
Invention content
The purpose of the present invention is based on above-mentioned prior art situation and provides a kind of system of safrole haptens and antigen
Preparation Method and its application.
The purpose of the present invention is achieved through the following technical solutions:
A kind of preparation method of safrole haptens is by 5- (the bromo- 2- propylene -1- bases of 2-) -1,3- benzodioxolanes and ammonia
Base propionic acid is obtained by the reaction, and molecular structure is:
。
It is as follows:
5- (the bromo- 2- propylene -1- bases of 2-) -1,3- benzodioxolanes, 1.0 g is taken, anhydrous dimethyl sulphoxide is added(DMSO) 50 mL
Dissolving, stirring clarification, adds 0.48 g of potassium hydroxide, adds 0.20 g of anhydrous sodium iodide, after being vigorously stirred, add alanine 0.41
G, 100 DEG C of reaction 4h.Stop reaction, add 50 mL of water, add 1mol/L salt acid for adjusting pH value to 6, add 80 mL of ethyl acetate, extracts
Three times, merge organic phase, revolving is evaporated, upper silicagel column, is 1 with volume ratio:5 ethyl acetate/petroleum ether elution separation, obtains
Haptens product propionic acid safrole 0.91g.
The safrole haptens can be used for making the antigen system raw material of animal immune.
A kind of preparation method of safrole antigen is obtained with carrier protein couplet by the safrole haptens.It is described
Carrier protein is bovine serum albumin(BSA), ovalbumin, keyhole limpet hemocyanin or human serum albumins.
It is as follows:
The preparation of immunizing antigen:9 mg of safrole haptens is taken, 0.3 mL of n,N-Dimethylformamide is added to dissolve, clarification adds carbon
Change diimine(EDC)8.3 mg stir 20 min, add 7 mg of N- succinimides, continue to stir 30 min, obtain haptens work
Change liquid A liquid;Take bovine serum albumin(BSA)(BSA)50 mg add 0.8% sodium-chloride water solution, 4 mL to dissolve, obtain B liquid;A liquid is slow
It being added drop-wise in B liquid, 4 DEG C of 4 h of stirring, 0.02 mol/L PB buffer solutions dialysis purification 3 days obtains safrole-BSA immunizing antigens,
Packing, -20 DEG C save backup.
The preparation of envelope antigen:6 mg of safrole haptens is taken, adds 0.2 mL of dimethyl sulfoxide (DMSO) to dissolve, adds 20 μ of triethylamine
L, 18 μ L of chlorination iso-butyl formate, mixing, 4 DEG C of 30 min of reaction obtain haptens activating solution A liquid;Take ovalbumin(OVA)
50 mg add 0.8% sodium-chloride water solution, 4 mL to dissolve, obtain B liquid;A liquid is slowly dropped in B liquid, 4 DEG C stirring 4 h, 0.02
Mol/L PB buffer solutions dialysis purification 3 days obtains safrole-OVA envelope antigens, and packing, -20 DEG C save backup.
The monoclonal antibody obtained using the safrole antigen-immunized animal, can be used for establishing enzyme linked immunosorbent assay (ELISA)
Method and immune chromatography test paper rapid test method, to realize safrole in essence spice for cigarette, tobacco and tobacco product, food
Quick detection.
The safrole haptens synthesized in the present invention had not only remained the chemical constitution of safrole to the greatest extent, but also had properly
The linking arm of length, the antigen prepared with the haptens show the safrole antigenic determinant of specificity so that filter out height
The safrole monoclonal antibody of specificity is possibly realized.The antibody specificity height of generation, high sensitivity can be used for preparing detection yellow
Enzyme linked immunological kit, colloidal gold strip and the time-resolved fluoroimmunoassay chromatograph test strip of camphor tree element, to realize cigarette perfume
The quick detection of safrole in smart fragrance, tobacco and tobacco product, food.
Description of the drawings
Fig. 1:Safrole hapten synthesis route map.
Specific implementation mode
With reference to specific embodiment, the present invention is further explained.It should be understood that these embodiments are merely to illustrate this
Invention, and be not used to limit the scope of the invention.
The preparation of 1 safrole haptens of embodiment
1, the synthesis of safrole haptens
5- (the bromo- 2- propylene -1- bases of 2-) -1,3- benzodioxolanes, 1.0 g is taken, 50 mL of anhydrous DMSO is added to dissolve, stirring is clear
Clearly, add potassium hydroxide 0.48g, add 0.20 g of anhydrous sodium iodide, after being vigorously stirred, add 0.41 g of alanine, 100 DEG C of reactions 4
h.Stop reaction, add 50 mL of water, adds 1mol/L salt acid for adjusting pH value to 6,80 mL of ethyl acetate, extraction is added three times, to be associated with
Machine phase, revolving are evaporated, upper silicagel column, ethyl acetate/petroleum ether(V/v, 1/5)Elution separation, it is yellow to obtain haptens product propionic acid
Camphor tree element 0.91g, yield 87.37%.
2, the identification of safrole haptens
Above-mentioned haptens is taken to be identified through nuclear magnetic resonance spectroscopy,1H-NMR(CDCl3, 300MHz)δ:11.0(1H, -COOH),
6.90(1H, ArH, J=6.864), 6.68(1H, ArH), 6.76(1H, ArH), 6.07(2H, s, -CH2-), 4.16
(2H, s, -CH2-), 3.75(1H, dd, =CH2), 3.12(2H, t, -CH2-), 2.49(2H, t, -CH2-), 2.0
(1H, s, -NH-).
Chemical shift δ=11.0 are the resonance absorbing peak of carboxyl hydrogen in collection of illustrative plates, and δ=3.12,2.49 are sub- on spacerarm
The resonance absorbing peak of methyl hydrogen, the presence at these peaks, it was demonstrated that spacerarm is coupled successfully, and safrole haptens structure is correct.
The preparation of 2 safrole antigen of embodiment
1, the synthesis of safrole immunogene
Safrole haptens and bovine serum albumin(BSA)(BSA)Coupling obtains immunizing antigen.
9 mg of safrole haptens is taken, 0.3 mL of n,N-Dimethylformamide is added to dissolve, clarification adds carbodiimides
(EDC)8.3 mg stir 20 min, add 7 mg of N- succinimides, continue to stir 30 min, obtain haptens activating solution A liquid;
Take bovine serum albumin(BSA)(BSA)50 mg add 0.8% sodium-chloride water solution, 4 mL to dissolve, obtain B liquid;A liquid is slowly dropped to B
In liquid, 4 DEG C of 4 h of stirring, 0.02 mol/L PB buffer solutions dialysis purification 3 days obtains safrole-BSA immunogenes, dispenses, -20
It DEG C saves backup.
2, the synthesis of safrole coating antigen
Safrole haptens and ovalbumin(OVA)Coupling obtains envelope antigen.
6 mg of safrole haptens is taken, adds 0.2 mL of dimethyl sulfoxide (DMSO) to dissolve, adds 20 μ L of triethylamine, chlorination isobutyl formate
18 μ L of ester, mixing, 4 DEG C of 30 min of reaction obtain haptens activating solution A liquid;Take ovalbumin(OVA)50 mg add 0.8% chlorine
Change sodium water solution 4 mL dissolvings, obtains B liquid;A liquid is slowly dropped in B liquid, 4 DEG C of 4 h of stirring, 0.02 mol/L PB bufferings
Liquid dialysis purification 3 days obtains safrole-OVA envelope antigens, and packing, -20 DEG C save backup.
3, the identification of safrole antigen
In the ratio of synthesis safrole coupled antigen reaction haptens used, carrier protein and coupled product, carry out ultraviolet(200
~ 400 nm)Sweep measuring, by comparing three respectively the absorbance value of 260 nm and 280 nm calculate its combine than.Coupling
The maximum absorption band of object safrole hapten-carrier albumen with safrole haptens, carrier protein maximum absorption band compared with send out
Apparent variation has been given birth to, has shown that the synthesis of safrole hapten-carrier albumen is successful.It is computed, the knot of haptens and BSA
Composition and division in a proportion is 15:1, and the combination ratio of OVA is 10:1.
The preparation of 3 safrole monoclonal antibody of embodiment
1, animal immune
In the immunogene injection Balb/c Mice Bodies that step 1 is obtained, immunizing dose is 150 μ g/, its is made to generate antiserum.
2, cell fusion and cloning
It takes the Balb/c mouse boosting cells for generating specific antibody to be merged with myeloma cell SP20, is exempted from using indirect competitive enzyme-linked
Epidemic disease analysis method measures cell supernatant, screens positive hole.Cloning is carried out to positive hole using limiting dilution assay, obtains and builds
The hybridoma cell strain of vertical production monoclonal antibody.
3, cell cryopreservation and recovery
It takes the hybridoma in exponential phase that cell suspension is made with frozen stock solution, is sub-packed in cryopreservation tube, it is long in liquid nitrogen
Phase preserves.Cryopreservation tube is taken out when recovery, 37 DEG C of water-bath middling speeds is immediately placed in and melts, and after centrifugation removal frozen stock solution, is moved into culture bottle
Culture.
4, the preparation and purification of monoclonal antibody
Using method is induced in vivo, by Balb/c mouse(8 week old)Intraperitoneal injection sterilizing paraffin oil, pneumoretroperitoneum injection hybridization in 7 ~ 14 days
Oncocyte acquires ascites after 7 ~ 10 days.Ascites purifying is carried out through octanoic acid-saturated ammonium sulfate method, purity is reflected through SDS-PAGE electrophoresis
It is fixed, bottle packing, -20 DEG C of preservations.
5, the measurement of antibody titer
The potency that antibody is measured with indirect competitive ELISA method is 1:(50000~100000).
Indirect competitive ELISA method:With safrole haptens-OVA conjugate coated elisa plates, safrole standard items are added
The sheep anti mouse antiantibody solution of solution, safrole monoclonal antibody solution and horseradish peroxidase-labeled, 25 DEG C of reactions 30
Min pours out liquid in hole, is washed 3 ~ 5 times with cleaning solution, is patted dry with blotting paper;Substrate developing solution, 25 DEG C of 15 min of reaction are added
Afterwards, terminate liquid is added and terminates reaction;Setting microplate reader is measured at 450 nm of wavelength per hole absorbance value.
6, the measurement of monoclonal antibody specificity
The ability and the comparison with such antigen-analogues ability that antibody specificity refers to its homospecificity antigen binding, often
Use cross reacting rate as evaluation criterion.Cross reaction is smaller, and the specificity of antibody is then higher.
Yellow camphor tree analog is serially diluted by this experiment, carries out indirect competitive ELISA with monoclonal antibody respectively, makes mark
Directrix curve, analysis obtain IC50, cross reacting rate is then calculated as follows:
The cross reacting rate of the antibody and safrole analog the results are shown in Table 1.
1 cross reacting rate of table is tested
| Compound | Cross reacting rate(%) |
| Safrole | 100 |
| Isosafrole | 13.2 |
| Dihydrosafrole | 6.7 |
| Cumarin | < 1 |
| Vanillic aldehyde | < 1 |
| Damascenone | < 1 |
As it can be seen from table 1 safrole monoclonal antibody and isosafrole, dihydrosafrole, cumarin, vanillic aldehyde, Tujue's alkene
The equal no cross reaction of ketone.
Claims (8)
1. a kind of preparation method of safrole haptens, it is characterised in that:It is by 5- (the bromo- 2- propylene -1- bases of 2-) -1,3- benzos
Dioxolanes is obtained by the reaction with alanine, and molecular structure is:
。
2. the preparation method of safrole haptens as described in claim 1, it is characterised in that:The specific steps of the preparation method
It is as follows:
5- (the bromo- 2- propylene -1- bases of 2-) -1,3- benzodioxolanes, 1.0 g is taken, anhydrous dimethyl sulphoxide is added(DMSO) 50 mL
Dissolving, stirring clarification, adds 0.48 g of potassium hydroxide, adds 0.20 g of anhydrous sodium iodide, after being vigorously stirred, add alanine 0.41
G, 100 DEG C of reaction 4h, stops reaction, adds 50 mL of water, add 1mol/L salt acid for adjusting pH value to 6, add 80 mL of ethyl acetate, extract
Three times, merge organic phase, revolving is evaporated, upper silicagel column, is 1 with volume ratio:5 ethyl acetate/petroleum ether elution separation, obtains
Haptens product propionic acid safrole 0.91g.
3. the application of safrole haptens prepared by method as described in claim 1, it is characterised in that:The safrole haptens
It can be used for making the antigen system raw material of animal immune.
4. a kind of preparation method of safrole antigen, it is characterised in that:It is the safrole haptens made from claim 1 and load
Body protein is coupled to obtain.
5. the preparation method of safrole antigen as claimed in claim 4, it is characterised in that:The carrier protein is that ox blood is pure
Albumen, ovalbumin, keyhole limpet hemocyanin or human serum albumins.
6. the preparation method of safrole antigen as claimed in claim 4, which is characterized in that be as follows:Take safrole
9 mg of haptens adds 0.3 mL of n,N-Dimethylformamide to dissolve, and clarification adds 8.3 mg of carbodiimides, stirs 20 min, adds
7 mg of N- succinimides continues to stir 30 min, obtains haptens activating solution A liquid;50 mg of bovine serum albumin(BSA) is taken, is added
0.8% sodium-chloride water solution, 4 mL dissolvings, obtain B liquid;A liquid is slowly dropped in B liquid, 4 DEG C of stirrings 4 h, 0.02 mol/L
PB buffer solutions dialysis purification 3 days obtains safrole antigen, and packing, -20 DEG C save backup.
7. the preparation method of safrole antigen as claimed in claim 4, which is characterized in that be as follows:Take safrole
6 mg of haptens adds 0.2 mL of dimethyl sulfoxide (DMSO) to dissolve, adds 20 μ L of triethylamine, 18 μ L of chlorination iso-butyl formate, mixing, 4 DEG C
30 min are reacted, haptens activating solution A liquid is obtained;50 mg of ovalbumin is taken, adds 0.8% sodium-chloride water solution, 4 mL to dissolve, obtains
To B liquid;A liquid is slowly dropped in B liquid, 4 DEG C of 4 h of stirring, 0.02 mol/L PB buffer solutions dialysis purification 3 days obtains safrole
Antigen, packing, -20 DEG C save backup.
8. the application of safrole antigen prepared by method as claimed in claim 4, it is characterised in that:It is immune using safrole antigen
The monoclonal antibody that animal obtains can be used for preparing enzyme linked immunological kit, colloidal gold strip and the time of detection safrole
Resolved fluorometric immuno-chromatographic test paper strip, to realize the quick of safrole in essence spice for cigarette, tobacco and tobacco product, food
Detection.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111825562A (en) * | 2020-06-04 | 2020-10-27 | 北京勤邦生物技术有限公司 | Chlordan hapten, artificial antigen and antibody, and preparation method and application thereof |
| CN114539205A (en) * | 2022-03-18 | 2022-05-27 | 华南农业大学 | Safrole hapten, artificial antigen, antibody and preparation method and application thereof |
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| CN114539205A (en) * | 2022-03-18 | 2022-05-27 | 华南农业大学 | Safrole hapten, artificial antigen, antibody and preparation method and application thereof |
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