CN105732788A - Preparation method and application of efficient cyhalothrin hapten - Google Patents
Preparation method and application of efficient cyhalothrin hapten Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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Abstract
The invention provides a preparation method and application of efficient cyhalothrin hapten.A synthetic method of the efficient cyhalothrin hapten, provided herein, comprises: subjecting efficient cyhalothrin as a material and succinic anhydride to one-step reaction, and coupling hapten and carrier protein to obtain the efficient cyhalothrin hapten.Experimental results show that the synthetic method of the efficient cyhalothrin hapten is simple, the obtained efficient cyhalothrin hapten is higher in both yield and purity, an antigen hapten and carrier protein coupled herein may be used to immunize an experimental animal to obtain a monoclonal antibody good in specificity and sensitivity, an enzyme linked immunosorbent assay kit and test strip established using the antibody is convenient to use and economical, a detection method is efficient and accurate and available for detecting mass specimens, and the demand for field monitoring of efficient cyhalothrin hapten residue and screening of mass specimens can be met.
Description
Technical field
The present invention relates to the haptenic preparation method of a kind of gamma cyhalothrin residual and application.
Background technology
Gamma cyhalothrin, English name Lambda-Cyhalothrin, it is also called Ai Kening, λ-Grenade (ICI)., belong to pyrethroid, there is the hygienic insecticide of action of contace poison, chemical name: alpha-cyano-3-benzyloxy phenoxy base-3-(2-chloro-3,3, the fluoro-1-acrylic of 3-trichlorine)-2,2-dimethyl cyclopropane carboxylic acid's ester (Z)-(1R, 3R), S-ester and (Z), (1S, 3S), the 1:1 mixture of R-ester.There is action of contace poison, be that the extremely effective one of public health insect is extensively imitated insecticide, have and knock down the advantages such as speed is fast, it is strong to knock down power, and dosage is few.Pathophorous vector pests can be eliminated and prevent and treat various sanitary insect pests.Pyrethroid is known as a new breakthrough of pesticide, is historical 3rd milestone of insecticide.In agricultural production, of many uses.
Gamma cyhalothrin belongs to never poison, headache, dizziness, Nausea and vomiting, both hands can be caused when exposure is big to tremble, whole body is twitched or convulsions, stupor, shock etc., native land man has formulated strict gamma cyhalothrin residue detection standard, to ensure food safety, eliminate potential food safety hidden danger.Such as GB/T5009-2008 " in vegetable food the qualification of organochlorine and the multiple residual of pyrethroid pesticide ", GB/T19649-2006 " in Cereals the mensuration of 475 kinds of pesticide and the flat residual quantity of related chemistry ", SN/T1117-2008 " in import and export food multiple Determining pyrethroid pesticide residues assay method gas chromatography ", existing method for detecting residue is gas chromatography and high performance liquid chromatography, the method depends on large-scale instrument and professional technique testing staff, the detection time is long, need to use organic solvent, environmental pollution can be caused.Seek one quickly, effectively, detection method and means accurately, imperative.Enzyme-linked immune detection method has the advantages that sensitivity is good, degree of accuracy is high, testing cost is low, simple to operate, the detection time is short, can realize that multisample detects simultaneously, plays an important role in food safety quickly detects.
Gamma cyhalothrin is pesticide molecule compound, it is necessary to could produce specific antibody by stimulating animal after being connected with macromolecular substances, thus Enzyme-multiplied immune technique research it is crucial that the preparation of haptenic MOLECULE DESIGN, synthesis and artificial antigen and antibody.Pyrethroid pesticide hapten design mainly has following method with synthesis: 1) using the acid of pyrethroid or alcohol moiety derivatization as hapten；2) active group of pyrethroid ester molecule is changed as hapten.Hapten is exactly connect a spacerarm with certain carbon chain lengths by certain chemical method in the oxide precursor result of non-immunogenicity, and the other end of spacerarm has can with the small-molecule substance of the active group (such as carboxyl, amino, hydroxyl etc.) of carrier protein covalent coupling.Haptenic design is considered as in structure to retain aromatic rings as far as possible.
Both at home and abroad to the more and existing many achievements report of the research of pyrethroid, as Wang Libing et al. has applied for patent of invention " a kind of haptenic synthetic method of cyhalothrin " (patent No.: 201110271124.X), this patent is using hydroxy phenylpropionic acid as raw material, is obtained by reacting hapten through seven steps；China Agricultural University is permitted ship et al. and has been applied for patent of invention " indirect competitive enzyme-linked immunosorbent assay kit of cyhalothrin residual " (patent No.: 200810101544.1), with hydroxy phenylpropionic acid for raw material, through six-step process, acquisition hapten 3-[(±)-(cis)-3-[(Z)-3-chloro-4,4, the fluoro-1-ethyl-2 of 4-tri-, 2-dimethyl] cyclopropyl carbonyl oxygen] phenoxy group] benzenpropanoic acid, above-mentioned two technical scheme, there is reactions steps many, conversion ratio is correlated with relatively low, and the by-product obtained is more, the shortcoming that subsequent purification workload is big.Zhejiang University Cheng Jing is beautiful et al., apply for patent of invention " a kind of high specificity pyrethroid hapten compounds " (patent No.: 200810162533.4), with cypermethrin for raw material, it is adjusted pH extremely acidity, extracts, washes and purification reaction, obtain hapten compound, (RS)-alpha-cyano-3-Phenoxyphenyl (RS)-suitable, trans-2,2-dimethyl-3-carboxyl trimethylene carbonate, this patent is the hapten synthesis route for pyrethroid design, rather than the hapten synthesis for single chrysanthemum ester product.Inspection & Quarantine Technology Center of Fujian Entry-Exit Inspection & Quar, obtain the mandate of patent of invention " pyrethroid pesticide remained method for quick " (patent No.: 200410155382.1), pyrethroid pesticide residual decomposition in detection thing is become containing cyanogen compound by this patent utilization strong base reagent, this invention uses strong acid and strong base reagent, and product is containing cyanogen compound, poisonous and environment is unfriendly.
Li Zhi Ru Youxiu master's thesis, " preparation of pyrethroid pesticide universal antibody and the research of gamma cyhalothrin immunoassay technology ", study with the intermediate-m-phenoxybenzoic acid of pyrethroid pesticide be hapten, chrysanthemumic acid is for primary raw material, it is thus achieved that polyclone universal antibody.
Beijing Kwinbon Biotechnology Co., Ltd. has applied for a patent of invention, " melamine hapten and its preparation method and application " (patent No.: 201110356771.0), this patent reacts with ammeline and succinic anhydrides, prepare melamine hapten, prepare fast detection reagent kit for melamine and test strips, solve the quick detection of tripolycyanamide.
Deficiency for currently available technology scheme, the present invention intends providing a kind of hapten synthesis scheme being specifically designed for gamma cyhalothrin residue detection, antigen is become with carrier protein couplet based on hapten, thus carrying out immunoreation experiment, monoclonal antibody, the advantage with economy, environmental protection, high specificity is obtained through screening.
Summary of the invention
The present invention provides the haptenic synthetic method of key substance prepared by a kind of gamma cyhalothrin antibody, and it is simple that the method has technique, the feature that response rate is high, adopts one-step method to complete, and yield is up to 51.6%；Remain the chemical constitution of gamma cyhalothrin to greatest extent, improve haptenic specificity, and introduce active group-OH, add junction point for antigen preparation.
The present invention is to provide a kind of technical method prepared for the antigen detecting gamma cyhalothrin residual, the hapten being synthesized that gamma cyhalothrin is raw material and succinic anhydrides obtain with carrier protein couplet, have following molecular structural formula:
The haptenic molecular structural formula being synthesized that gamma cyhalothrin is raw material and succinic anhydrides is:
The haptenic synthetic method being synthesized that gamma cyhalothrin is raw material and succinic anhydrides comprises the following steps:
1) with gamma cyhalothrin for raw material, it is dissolved in dichloromethane after drying, passes into nitrogen and protect, obtain solution I；
2) in solution I, succinic anhydrides, stirring and dissolving under room temperature are added；
3) add catalyst aluminum chloride solid and carry out catalytic reaction；
4) TLC lamellae monitoring reaction carries out, until there is no raw material, and stopped reaction；
5) silicagel column purifies, and concentration obtains product, is hapten；
Carrier protein is: bovine serum albumin, Mus serum albumin, rabbit serum proteins, thyroprotein, ovalbumin, hemocyanin or human serum albumin；
Gamma cyhalothrin is that raw material is as follows with the haptenic synthetic route being synthesized of succinic anhydrides:
Reaction temperature is room temperature, and reaction pressure is normal pressure, and the reaction mol ratio of gamma cyhalothrin and succinic anhydrides is 1/1 1/3, and catalyst and gamma cyhalothrin mass ratio are 1%-5%.
The preparation method that the present invention also provides for a kind of gamma cyhalothrin antigen.
1) weigh appropriate gamma cyhalothrin hapten and be dissolved in DMF solution, add EDC and NHS aqueous solution, carry out activation 30 minutes, obtain solution I；
2) carrier protein is dissolved in containing in 30%DMF aqueous solution, obtains solution II；Solution I is joined in solution II, carry out coupling, obtain solution III；
3) with 0.02mol/LPB buffer dialysis solution III, it is thus achieved that gamma cyhalothrin antigen, subpackage also saves backup in-20 DEG C.
Carrier protein can be bovine serum albumin, Mus serum albumin, rabbit serum proteins, thyroprotein, ovalbumin, hemocyanin or human serum albumin.
In sum, the method for the present invention has that technique is simple, workable, yield advantages of higher；Its gained material is gamma cyhalothrin derivant, and this compound is through purification, concentration, Structural Identification, it was demonstrated that for gamma cyhalothrin hapten compound.Based on this compound, coupling can be carried out with carrier protein, prepare antibody, based on this product such as detection such as exploitation enzyme linked immunological kit and colloidal gold test paper card etc..
Accompanying drawing explanation
Fig. 1 gamma cyhalothrin antigenic structure figure.
Fig. 2 gamma cyhalothrin hapten structure chart.
Fig. 3 gamma cyhalothrin hapten synthesis reaction scheme.
Fig. 4 gamma cyhalothrin hapten identifies figure (hydrogen spectrogram).
Fig. 5 gamma cyhalothrin hapten identifies figure (carbon spectrogram).
Detailed description of the invention
The present invention is expanded on further, it should be appreciated that these embodiments are merely to illustrate the present invention, rather than are used for limiting the scope of the present invention below in conjunction with specific embodiment.
Embodiment one: the haptenic synthesis of gamma cyhalothrin and qualification
One, gamma cyhalothrin hapten synthesis
Weigh 0.42g(1mmol respectively) gamma cyhalothrin, 0.10g(1mmol) succinic anhydrides and 0.05g aluminum chloride.
1. by 0.42g gamma cyhalothrin, it is dissolved in dichloromethane after drying, passes into nitrogen and protect, obtain solution I；
2. in solution I, add 0.10g succinic anhydrides, stirring and dissolving under room temperature；
3. add 0.01g aluminum chloride solid and carry out catalytic reaction；
The monitoring reaction of 4.TLC lamellae carries out, until not having raw material or raw material point very shallow, and stopped reaction；
5. silicagel column purifies, and concentration obtains product, is hapten, as in figure 2 it is shown, obtain product 0.205g, and yield 48.8%
。
Two, gamma cyhalothrin hapten is identified
Take above-mentioned Fig. 2 compound, structural analysis is carried out through proton nmr spectra, as shown in Figure 4, in discovery collection of illustrative plates, the peak near 11ppm (mg/L) is the H on hydroxyl, meanwhile, structural analysis is carried out through carbon-13 nmr spectra, as shown in Figure 5, in carbon spectrogram, the peak near 177ppm is the C on carboxyl, and the success of gamma cyhalothrin hapten synthesis is described.
Three, the preparation of gamma cyhalothrin antigen
3.1 immunogenic synthesis
(1) weigh appropriate gamma cyhalothrin hapten 27.5mg and be dissolved in 4mLDMF solution, contain at 2mL and 60mgEDC and 60mgNHS aqueous solution carries out activation 30 minutes, obtain solution I；
(2) carrier protein is dissolved in containing in 5mL30%DMF aqueous solution by 110-264mg, obtains solution II；Solution I is joined in solution II, carry out coupling, obtain solution III；
(3) with 0.02mol/LPB buffer dialysis solution III, dialysing 3 days, every day changes dialysis solution sooner or later, and to remove unreacted small-molecule substance, after having dialysed, it is thus achieved that gamma cyhalothrin antigen, subpackage also saves backup in-20 DEG C.Carrier protein can be bovine serum albumin, Mus serum albumin, rabbit serum proteins, thyroprotein, hemocyanin or human serum albumin.
The synthesis of 3.2 coating antigens
(1) weigh appropriate gamma cyhalothrin hapten 27.5mg and be dissolved in 4mLDMF solution, contain at 2mL and 60mgEDC and 60mgNHS aqueous solution carries out activation 30 minutes, obtain solution I；
(2) ovalbumin is dissolved in containing in 5mL30%DMF aqueous solution by 100-240mg, obtains solution II；Solution I is joined in solution II, carry out coupling, obtain solution III；
(3) with 0.02mol/LPB buffer dialysis solution III, dialysing 3 days, every day changes dialysis solution sooner or later, and to remove unreacted small-molecule substance, after having dialysed, it is thus achieved that gamma cyhalothrin antigen, subpackage also saves backup in-20 DEG C.
The qualification of 3.3 gamma cyhalothrin antigens
By carrier protein, gamma cyhalothrin hapten, gamma cyhalothrin hapten-carrier protein conjugate pH7.4 PB be made into the solution of 0.5mg/ml, return to zero with 0.01mol/LpH7.4PB, it is scanned within the scope of wavelength 260 ~ 750nm with ultraviolet spectrophotometer, draw the absorption curve of carrier protein, gamma cyhalothrin hapten, gamma cyhalothrin hapten-carrier protein conjugate, and calculate its combination ratio.Result shows, there is different absorption curves in three, proving the success of gamma cyhalothrin hapten and carrier protein couplet, the combination of gamma cyhalothrin hapten and bovine serum albumin ratio is for 15-21:1, and the combination of gamma cyhalothrin hapten and oralbumin ratio is for 20-25:1.Molecular structural formula is as shown in Figure 1
。
Four, gamma cyhalothrin monoclonal antibody
4.1 gamma cyhalothrin monoclonal antibody preparation
Animal immune: gamma cyhalothrin hapten and carrier protein couplet thing immunity 8-10 week old Balb/c mice.
Cell fusion and cloning: take the mice spleen cell after immunity, merge under the effect of fusion agent Polyethylene Glycol (PEG) 4000 with SP2/0 myeloma cell, and screening obtains the hybridoma cell strain of energy stably excreting monoclonal antibody.
The monoclonal hybridoma strain of gamma cyhalothrin is obtained through screening.The monoclonal hybridoma strain of gamma cyhalothrin can an unbounded quantity of generation gamma cyhalothrin specific antibody, this antibody specificity is for gamma cyhalothrin, and sensitivity reaches 1 μ g/L.
Cell cryopreservation and recovery: hybridoma frozen stock solution is made 1 × 109The cell suspension of individual/ml, preserves for a long time in liquid nitrogen.Take out cryopreservation tube during recovery, be immediately placed in 37 DEG C of water-bath middling speeds and melt, after centrifugal segregation frozen stock solution, move into and cultivate culture in glassware.
The mensuration of 4.2 antibody titers
The titer measuring antibody with indirect competitive ELISA method is 1: 100000～150000.
Indirect competitive ELISA method: with gamma cyhalothrin hapten-bovine serum albumin conjugate coated elisa plate, add gamma cyhalothrin standard substance working solution solution, monoclonal antibody working solution and ELIAS secondary antibody, 25 DEG C of reaction 30min, pour out liquid in hole, wash 3～5 times with PBST cleaning mixture, pat dry with absorbent paper；Add substrate nitrite ion, after 25 DEG C of reaction 15min, add stop buffer and terminate reaction；Set microplate reader and measure every hole absorbance in wavelength 450nm place.
The specificity of 4.3 monoclonal antibodies
Antibody specificity refers to ability and the comparison with such antigen-analogues ability of its homospecificity antigen combination, and conventional cross reacting rate is as evaluation criterion.Cross reaction is more little, and the specificity of antibody is then more high.
Gamma cyhalothrin, cypermethrin and 3 kinds of medicines of fenvalerate are done serial dilution by this experiment, carry out indirect competitive ELISA respectively with monoclonal antibody, make standard curve, analyze and obtain IC50, then it is calculated as follows cross reacting rate:
Result display cross reacting rate is: gamma cyhalothrin 100%, cypermethrin < 50%, fenvalerate < 10%.Gamma cyhalothrin is had stronger specificity and affinity by antibody of the present invention.
Embodiment two: the haptenic synthesis of gamma cyhalothrin and qualification
One, gamma cyhalothrin hapten synthesis
Weigh 1g(2.4mmol respectively) gamma cyhalothrin, 0.48g(4.8mmol) succinic anhydrides and 0.03g aluminum chloride.
1. by 1g gamma cyhalothrin, it is dissolved in dichloromethane after drying, passes into nitrogen and protect, obtain solution I；
2. in solution I, add 0.48g succinic anhydrides, stirring and dissolving under room temperature；
3. add 0.03g aluminum chloride solid and carry out catalytic reaction；
The monitoring reaction of 4.TLC lamellae carries out, until not having raw material or raw material point very shallow, and stopped reaction；
5. silicagel column purifies, and concentration obtains product, is hapten, as in figure 2 it is shown, obtain product 0.516g, and yield 51.6%
。
Two, gamma cyhalothrin hapten is identified
Take above-mentioned Fig. 2 compound, structural analysis is carried out through proton nmr spectra, as shown in Figure 4, in discovery collection of illustrative plates, the peak near 11ppm (mg/L) is the H on hydroxyl, meanwhile, structural analysis is carried out through carbon-13 nmr spectra, as shown in Figure 5, in carbon spectrogram, the peak near 177ppm is the C on carboxyl, and the success of gamma cyhalothrin hapten synthesis is described.
Three, the preparation of gamma cyhalothrin antigen
3.1 immunogenic synthesis
(1) weigh appropriate gamma cyhalothrin hapten 27.5mg and be dissolved in 4mLDMF solution, contain at 2mL and 60mgEDC and 60mgNHS aqueous solution carries out activation 30 minutes, obtain solution I；
(2) carrier protein is dissolved in containing in 5mL30%DMF aqueous solution by 110-264mg, obtains solution II；Solution I is joined in solution II, carry out coupling, obtain solution III；
(3) with 0.02mol/LPB buffer dialysis solution III, dialysing 3 days, every day changes dialysis solution sooner or later, and to remove unreacted small-molecule substance, after having dialysed, it is thus achieved that gamma cyhalothrin antigen, subpackage also saves backup in-20 DEG C.Carrier protein can be bovine serum albumin, Mus serum albumin, rabbit serum proteins, thyroprotein, hemocyanin or human serum albumin.
The synthesis of 3.2 coating antigens
(1) weigh appropriate gamma cyhalothrin hapten 27.5mg and be dissolved in 4mLDMF solution, contain at 2mL and 60mgEDC and 60mgNHS aqueous solution carries out activation 30 minutes, obtain solution I；
(2) ovalbumin is dissolved in containing in 5mL30%DMF aqueous solution by 100-240mg, obtains solution II；Solution I is joined in solution II, carry out coupling, obtain solution III；
(3) with 0.02mol/LPB buffer dialysis solution III, dialysing 3 days, every day changes dialysis solution sooner or later, and to remove unreacted small-molecule substance, after having dialysed, it is thus achieved that gamma cyhalothrin antigen, subpackage also saves backup in-20 DEG C.
The qualification of 3.3 gamma cyhalothrin antigens
By carrier protein, gamma cyhalothrin hapten, gamma cyhalothrin hapten-carrier protein conjugate pH7.4 PB be made into the solution of 0.5mg/ml, return to zero with 0.01mol/LpH7.4PB, it is scanned within the scope of wavelength 260 ~ 750nm with ultraviolet spectrophotometer, draw the absorption curve of carrier protein, gamma cyhalothrin hapten, gamma cyhalothrin hapten-carrier protein conjugate, and calculate its combination ratio.Result shows, there is different absorption curves in three, proving the success of gamma cyhalothrin hapten and carrier protein couplet, the combination of gamma cyhalothrin hapten and bovine serum albumin ratio is for 15-21:1, and the combination of gamma cyhalothrin hapten and oralbumin ratio is for 20-25:1.Molecular structural formula is as shown in Figure 1:
。
Four, gamma cyhalothrin monoclonal antibody
4.1 gamma cyhalothrin monoclonal antibody preparation
Animal immune: gamma cyhalothrin hapten and carrier protein couplet thing immunity 8-10 week old Balb/c mice.
Cell fusion and cloning: take the mice spleen cell after immunity, merge under the effect of fusion agent Polyethylene Glycol (PEG) 4000 with SP2/0 myeloma cell, and screening obtains the hybridoma cell strain of energy stably excreting monoclonal antibody.
The monoclonal hybridoma strain of gamma cyhalothrin is obtained through screening.The monoclonal hybridoma strain of gamma cyhalothrin can an unbounded quantity of generation gamma cyhalothrin specific antibody, this antibody specificity is for gamma cyhalothrin, and sensitivity reaches 1 μ g/L.
Cell cryopreservation and recovery: hybridoma frozen stock solution is made 1 × 109The cell suspension of individual/ml, preserves for a long time in liquid nitrogen.Take out cryopreservation tube during recovery, be immediately placed in 37 DEG C of water-bath middling speeds and melt, after centrifugal segregation frozen stock solution, move into and cultivate culture in glassware.
The mensuration of 4.2 antibody titers
The titer measuring antibody with indirect competitive ELISA method is 1: 100000～150000.
Indirect competitive ELISA method: with gamma cyhalothrin hapten-bovine serum albumin conjugate coated elisa plate, add gamma cyhalothrin standard substance working solution solution, monoclonal antibody working solution and ELIAS secondary antibody, 25 DEG C of reaction 30min, pour out liquid in hole, wash 3～5 times with PBST cleaning mixture, pat dry with absorbent paper；Add substrate nitrite ion, after 25 DEG C of reaction 15min, add stop buffer and terminate reaction；Set microplate reader and measure every hole absorbance in wavelength 450nm place.
The specificity of 4.3 monoclonal antibodies
Antibody specificity refers to ability and the comparison with such antigen-analogues ability of its homospecificity antigen combination, and conventional cross reacting rate is as evaluation criterion.Cross reaction is more little, and the specificity of antibody is then more high.
Gamma cyhalothrin, cypermethrin and 3 kinds of medicines of fenvalerate are done serial dilution by this experiment, carry out indirect competitive ELISA respectively with monoclonal antibody, make standard curve, analyze and obtain IC50, then it is calculated as follows cross reacting rate:
Result display cross reacting rate is: gamma cyhalothrin 100%, cypermethrin < 50%, fenvalerate < 10%.Gamma cyhalothrin is had stronger specificity and affinity by antibody of the present invention.
Embodiment three: the haptenic synthesis of gamma cyhalothrin and qualification
One, gamma cyhalothrin hapten synthesis
Weigh 2.5g(6mmol respectively) gamma cyhalothrin, 1.8g(18mmol) succinic anhydrides and 0.12g aluminum chloride.
1. by 2.5g gamma cyhalothrin, it is dissolved in dichloromethane after drying, passes into nitrogen and protect, obtain solution I；
2. in solution I, add 1.8g succinic anhydrides, stirring and dissolving under room temperature；
3. add 0.12g aluminum chloride solid and carry out catalytic reaction；
The monitoring reaction of 4.TLC lamellae carries out, until not having raw material or raw material point very shallow, and stopped reaction；
5. silicagel column purifies, and concentration obtains product, is hapten, as in figure 2 it is shown, obtain product 1.26g, and yield 50.4%
。
Two, gamma cyhalothrin hapten is identified
Take above-mentioned Fig. 2 compound, structural analysis is carried out through proton nmr spectra, as shown in Figure 4, in discovery collection of illustrative plates, the peak near 11ppm (mg/L) is the H on hydroxyl, meanwhile, structural analysis is carried out through carbon-13 nmr spectra, as shown in Figure 5, in carbon spectrogram, the peak near 177ppm is the C on carboxyl, and the success of gamma cyhalothrin hapten synthesis is described.
Three, the preparation of gamma cyhalothrin antigen
3.1 immunogenic synthesis
(1) weigh appropriate gamma cyhalothrin hapten 27.5mg and be dissolved in 4mLDMF solution, contain at 2mL and 60mgEDC and 60mgNHS aqueous solution carries out activation 30 minutes, obtain solution I；
(2) carrier protein is dissolved in containing in 5mL30%DMF aqueous solution by 110-264mg, obtains solution II；Solution I is joined in solution II, carry out coupling, obtain solution III；
(3) with 0.02mol/LPB buffer dialysis solution III, dialysing 3 days, every day changes dialysis solution sooner or later, and to remove unreacted small-molecule substance, after having dialysed, it is thus achieved that gamma cyhalothrin antigen, subpackage also saves backup in-20 DEG C.Carrier protein can be bovine serum albumin, Mus serum albumin, rabbit serum proteins, thyroprotein, hemocyanin or human serum albumin.
The synthesis of 3.2 coating antigens
(1) weigh appropriate gamma cyhalothrin hapten 27.5mg and be dissolved in 4mLDMF solution, contain at 2mL and 60mgEDC and 60mgNHS aqueous solution carries out activation 30 minutes, obtain solution I；
(2) ovalbumin is dissolved in containing in 5mL30%DMF aqueous solution by 100-240mg, obtains solution II；Solution I is joined in solution II, carry out coupling, obtain solution III；
(3) with 0.02mol/LPB buffer dialysis solution III, dialysing 3 days, every day changes dialysis solution sooner or later, and to remove unreacted small-molecule substance, after having dialysed, it is thus achieved that gamma cyhalothrin antigen, subpackage also saves backup in-20 DEG C.
The qualification of 3.3 gamma cyhalothrin antigens
By carrier protein, gamma cyhalothrin hapten, gamma cyhalothrin hapten-carrier protein conjugate pH7.4 PB be made into the solution of 0.5mg/ml, return to zero with 0.01mol/LpH7.4PB, it is scanned within the scope of wavelength 260 ~ 750nm with ultraviolet spectrophotometer, draw the absorption curve of carrier protein, gamma cyhalothrin hapten, gamma cyhalothrin hapten-carrier protein conjugate, and calculate its combination ratio.Result shows, there is different absorption curves in three, proving the success of gamma cyhalothrin hapten and carrier protein couplet, the combination of gamma cyhalothrin hapten and bovine serum albumin ratio is for 15-21:1, and the combination of gamma cyhalothrin hapten and oralbumin ratio is for 20-25:1.Molecular structural formula is as shown in Figure 1:
。
Four, gamma cyhalothrin monoclonal antibody
4.1 gamma cyhalothrin monoclonal antibody preparation
Animal immune: gamma cyhalothrin hapten and carrier protein couplet thing immunity 8-10 week old Balb/c mice.
Cell fusion and cloning: take the mice spleen cell after immunity, merge under the effect of fusion agent Polyethylene Glycol (PEG) 4000 with SP2/0 myeloma cell, and screening obtains the hybridoma cell strain of energy stably excreting monoclonal antibody.
The monoclonal hybridoma strain of gamma cyhalothrin is obtained through screening.The monoclonal hybridoma strain of gamma cyhalothrin can an unbounded quantity of generation gamma cyhalothrin specific antibody, this antibody specificity is for gamma cyhalothrin, and sensitivity reaches 1 μ g/L.
Cell cryopreservation and recovery: hybridoma frozen stock solution is made 1 × 109The cell suspension of individual/ml, preserves for a long time in liquid nitrogen.Take out cryopreservation tube during recovery, be immediately placed in 37 DEG C of water-bath middling speeds and melt, after centrifugal segregation frozen stock solution, move into and cultivate culture in glassware.
The mensuration of 4.2 antibody titers
The titer measuring antibody with indirect competitive ELISA method is 1: 100000～150000.
Indirect competitive ELISA method: with gamma cyhalothrin hapten-bovine serum albumin conjugate coated elisa plate, add gamma cyhalothrin standard substance working solution solution, monoclonal antibody working solution and ELIAS secondary antibody, 25 DEG C of reaction 30min, pour out liquid in hole, wash 3～5 times with PBST cleaning mixture, pat dry with absorbent paper；Add substrate nitrite ion, after 25 DEG C of reaction 15min, add stop buffer and terminate reaction；Set microplate reader and measure every hole absorbance in wavelength 450nm place.
The specificity of 4.3 monoclonal antibodies
Antibody specificity refers to ability and the comparison with such antigen-analogues ability of its homospecificity antigen combination, and conventional cross reacting rate is as evaluation criterion.Cross reaction is more little, and the specificity of antibody is then more high.
Gamma cyhalothrin, cypermethrin and 3 kinds of medicines of fenvalerate are done serial dilution by this experiment, carry out indirect competitive ELISA respectively with monoclonal antibody, make standard curve, analyze and obtain IC50, then it is calculated as follows cross reacting rate:
Result display cross reacting rate is: gamma cyhalothrin 100%, cypermethrin < 50%, fenvalerate < 10%.Gamma cyhalothrin is had stronger specificity and affinity by antibody of the present invention.
Claims (4)
1., for detecting an antigen for gamma cyhalothrin residual, it is obtained with carrier protein couplet by the gamma cyhalothrin hapten that to be raw material be synthesized with succinic anhydrides, there is following molecular structural formula:
The haptenic molecular structural formula being synthesized that wherein said gamma cyhalothrin is raw material and succinic anhydrides is:
The haptenic synthetic method being synthesized that described gamma cyhalothrin is raw material and succinic anhydrides comprises the following steps:
1) with gamma cyhalothrin for raw material, it is dissolved in dichloromethane after drying, passes into nitrogen and protect, obtain solution I；
2) in solution I, succinic anhydrides, stirring and dissolving under room temperature are added；
3) add catalyst aluminum chloride solid and carry out catalytic reaction；
4) TLC lamellae monitoring reaction carries out, until there is no raw material, and stopped reaction；
5) silicagel column purifies, and concentration obtains product, is hapten；
Wherein said carrier protein is: bovine serum albumin, Mus serum albumin, rabbit serum proteins, thyroprotein, ovalbumin, hemocyanin or human serum albumin；
Described gamma cyhalothrin is that raw material is as follows with the haptenic synthetic route being synthesized of succinic anhydrides:
。
2. a kind of antigen for detecting gamma cyhalothrin residual according to claims 1, it is characterized in that: the reaction mol ratio of described gamma cyhalothrin and succinic anhydrides is 1/1-1/3, catalyst aluminum chloride solid and gamma cyhalothrin mass ratio are 1%-5%.
3. the preparation method of a kind of antigen for detecting gamma cyhalothrin residual according to claims 1 comprises the following steps:
1) weigh appropriate gamma cyhalothrin hapten and be dissolved in DMF solution, add EDC and NHS aqueous solution, carry out activation 30 minutes, obtain solution I；
2) carrier protein is dissolved in containing in 30%DMF aqueous solution, obtains solution II；Solution I is joined in solution II, carry out coupling, obtain solution III；
3) with 0.02mol/LPB buffer dialysis solution III, it is thus achieved that gamma cyhalothrin antigen, subpackage also saves backup in-20 DEG C.
4. the application of a kind of antigen for detecting gamma cyhalothrin residual according to claims 1 includes: prepare gamma cyhalothrin enzyme linked immunological kit and colloidal gold test paper card.
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| CN107188830A (en) * | 2017-06-20 | 2017-09-22 | 北京勤邦生物技术有限公司 | A kind of pyrethrin pesticide haptens and its preparation method and application |
| CN111812316A (en) * | 2020-06-03 | 2020-10-23 | 北京勤邦生物技术有限公司 | Application of fenpropathrin artificial antigen in enzyme linked immunosorbent assay kit |
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| CN101157611A (en) * | 2007-11-16 | 2008-04-09 | 东华大学 | Polychlorinated biphenyl (PCBs) homologue semiantigen and preparation method thereof |
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| CN107188830A (en) * | 2017-06-20 | 2017-09-22 | 北京勤邦生物技术有限公司 | A kind of pyrethrin pesticide haptens and its preparation method and application |
| CN111812316A (en) * | 2020-06-03 | 2020-10-23 | 北京勤邦生物技术有限公司 | Application of fenpropathrin artificial antigen in enzyme linked immunosorbent assay kit |
| CN111812316B (en) * | 2020-06-03 | 2022-11-18 | 北京勤邦生物技术有限公司 | Application of fenpropathrin artificial antigen in enzyme linked immunosorbent assay kit |
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