CN102372776A - Synthesis method of II pyrethroid artificial antigen - Google Patents
Synthesis method of II pyrethroid artificial antigen Download PDFInfo
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- CN102372776A CN102372776A CN2011102711432A CN201110271143A CN102372776A CN 102372776 A CN102372776 A CN 102372776A CN 2011102711432 A CN2011102711432 A CN 2011102711432A CN 201110271143 A CN201110271143 A CN 201110271143A CN 102372776 A CN102372776 A CN 102372776A
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Abstract
The invention provides a synthesis method of an II pyrethroid artificial antigen, belonging to the technical field of biochemical engineering. The synthesis method provided by the invention comprises the steps of: with cyphenothrin as a raw material, oxidizing the cyphenothrin by adopting potassium permanganate and sodium periodate to obtain an artificial hapten; connecting the artificial hapten with a carrier protein BSA by adopting an active ester method, and estimating the coupling ratio of immunogen by adopting an ultraviolet spectrophotometry. The method is used for synthesizing II pyrethroid artificial antigen, has simple and effective synthesis steps, can be completely applied in immunological analysis, provides a novel artificial antigen for successive research of people, and meets the research demand at home.
Description
Technical field
The compound method of a kind of artificial antigen of II class pyrethroid pesticide belongs to technical field of biochemical industry.
Background technology
II class pyrethroid pesticide is one type of important synthetic pesticide, has the characteristics of wide spectrum, efficient, low toxicity, low residue, is widely used in the pest control in fields such as agricultural, forestry, animal cultivation and public health.Along with a large amount of uses of II class pyrethroid pesticide, pesticide residue have not only caused environmental pollution, but also jeopardize human health.Research shows: pyrethrin has potential genetoxic, genotoxicity and immunotoxicity; Therefore; Developed country has proposed the proneness limit index to technology barriers to pesticide residue in the agricultural-food, thereby makes the agricultural residual problem in the agricultural and animal products cause global concern.The method for detecting residue of pyrethroid is more at present, like HPLC (HPLC), gc-electron capture method (GC-ECD) and gas chromatography-mass spectrum (GC-MS) etc.For these chromatographic detection methods, although it is highly sensitive, accuracy good, the pre-treatment purification techniques step of sample is various, cause length consuming time, and instrument is expensive, needs the professional to operate, and is not suitable for the requirement of field quick detection especially.Eighties of last century is since the eighties; Along with the application of immunological technique in Detecting Pesticide; Enzyme immunoassay (ELISA) technical measurement pesticide residue have developed into one of important method of the many residue detection of pyrethrin at present, and it is strong to have specificity, simple to operate; Fast, the sensitive advantage, and one of gordian technique of enzyme linked immunological is immunogenic design and synthetic.Up to the present; Domestic report about the how residual detection of II class pyrethroid does not still have the haptin synthetic immunogen to its whole common structures, and the preparation that the haptin that contains the whole common structures of II class pyrethroid is used for detecting the complete antigen of II class pyrethroid has been synthesized in design for this reason.
Summary of the invention
The compound method that the purpose of this invention is to provide a kind of general artificial antigen.Prepared product is used for the immune analysis method research of II class pyrethroid pesticide, for from now on research provides a kind of novel artificial antigen.
Technical scheme of the present invention: the compound method of a kind of II class pyrethroid artificial antigen; With the cyphenothrin is raw material; Carry out oxidation through potassium permanganate and sodium periodate; Obtain the haptin of II class pyrethroid pesticide, with active ester method with itself and carrier proteins BSA coupling, with the coupling ratio of ultraviolet spectrophotometry and SDS-PAGE method estimation conjugate.Its reaction equation is:
Its synthesis step is:
(1) the haptenic preparation of II class pyrethroid
Take by weighing 1.20g (3.2mmol) cyphenothrin in the tool plug triangular flask of 100mL, add the trimethyl carbinol of 25.0mL and the water of 17.0mL again, mixing; Take by weighing the NaIO of 3.48mg (0.22mmol) then
4, 42.57mg (3.6mmol) KMnO
4And 9010mg (85mmol) Na
2CO
3Add respectively in the above-mentioned triangular flask.Add phase-transfer catalyst methyl trioctylphosphine ammonium chloride at last, normal temperature reaction down spends the night.After reaction finishes, carry out acidifying with 6N hydrochloric acid, with ethyl acetate extraction three times, the merging organic phase; Wash organic phase with hypo solution then, then use anhydrous magnesium sulfate drying, concentrate; Column chromatography purification, with quality than sherwood oil: ETHYLE ACETATE: acetic acid=69.9:30:0.1, collect R
f=0.36 component concentrates, and obtains flaxen oily matter and is II class pyrethroid haptin.
(2) II class pyrethroid artificial antigen preparation
Take by weighing the haptin of 50.0mg (0.027mmol); 7.8mg (0.04mmol) 1-ethyl-carbodiimide hydrochloride (EDC); 0.66mg (0.0054mmol) lutidine (DMAP) and 4.1mg (0.04mmol) triethylamine are put into reaction flask A; Add 300 μ LN, after dinethylformamide (DMF) dissolving, obtain A liquid; In reaction flask B, add 9.32mg (0.081 mmol) N-hydroxy succinic acid imines (NHS) and 100 μ L N then, dinethylformamide (DMF) obtains B liquid after the dissolving.B liquid dropwise is added drop-wise in the A liquid, and reaction is 12 hours under the room temperature, obtains C liquid.The BSA that takes by weighing 18mg (0.27 μ mol) is dissolved in the borate buffer solution of 4mL pH 8.4, gets protein soln.Under ice bath, C liquid dropwise is added drop-wise in the protein soln, reacted 6 hours.After reaction finishes, centrifugal, get supernatant, obtain II class pyrethroid artificial antigen mixed solution.
Dialysis tubing pre-treatment: dialysis tubing pre-treatment: the dialysis tubing of clip 9.0cm, boil 6-8min, use deionized water rinsing again 5 times, be kept in 4 ℃ of deionized waters subsequent use;
II class pyrethroid artificial antigen mixed solution moved into handled in the dialysis tubing, with 0.01M pH 7.4 phosphate buffered saline buffer (PBS solution) dialysis 72 hours, changed a dialyzate in 6-8 hour, during replacing dialyzate 6-9 time.At last II class pyrethroid artificial antigen mixed solution is divided in the centrifuge tube of 1.0mL, is kept at-20 ℃, subsequent use.
(3) evaluation of II class pyrethroid pesticide artificial antigen
Coupling ratio is measured: adopt ultraviolet spectrophotometry that the coupling ratio of II class pyrethroid artificial antigen is measured;
Antibody titer is measured: adopt the enzyme plate method to measure.
Beneficial effect of the present invention: the present invention successfully synthesizes the artificial antigen of II class pyrethroid; Synthesis step is simple; Effectively; Can be used for fully in the middle of the immunoassay, for people's research later on provides approach easily, the polyclonal antibody that this method produces can satisfy domestic needs to its research.
Description of drawings
The haptenic liquid chromatogram of Fig. 1-a.1-b haptin mass spectrum.Explain: the molecular weight of 6.52min peak tie substance is 365, is the haptenic peak of II class pyrethroid.
Fig. 2 II class pyrethroid artificial semiantigen nuclear-magnetism is identified figure, H
1NMR (CDCl
3): δ; 1.0-1.5 (6H, s, (CH
3)
2C), 2.0-2.5 (2H, s, CO (CH)
2), 6.3-6.5 (1H, s, CNCH), 7.5-7.0 (9H, s, ArH).
The UV scanning figure of Fig. 3 II class pyrethroid immunogen Hapten-BSA, 1, BSA: bovine serum albumin; 2, Hapten-BSA: the conjugate of haptin and bovine serum albumin; 3, the haptin of Hapten:II class pyrethroid.
The UV scanning figure of Fig. 4 II class pyrethroid coating antigen Hapten-OVA, OVA: chicken egg white; 2, Hapten-OVA: the conjugate of haptin and chicken egg white; 3, the haptin of Hapten:II class pyrethroid.
The II class pyrethroid standard that Fig. 5 utilizes indirect ELISA method to record suppresses curve.Fenpropathrin: Fenvalerate; Cypermethrin: PP-383; Deltametnrin: Deltamethrin; Fenvalerate: fenvalerate.
Embodiment
(1) haptenic preparation
Take by weighing 1.20g (3.2mmol) cyphenothrin in the tool plug triangular flask of 100mL, add the trimethyl carbinol of 25.0mL and the water of 17.0mL again, mixing; Take by weighing the NaIO of 3.48mg (0.22mmol) then
4, 42.57mg (3.6mmol) KMnO
4And 9010mg (85mmol) Na
2CO
3Add respectively in the above-mentioned triangular flask.Add phase-transfer catalyst methyl trioctylphosphine ammonium chloride at last, normal temperature reaction down spends the night.After reaction finishes, carry out acidifying with 6N hydrochloric acid, with ethyl acetate extraction three times, the merging organic phase; Wash organic phase with hypo solution then, then use anhydrous magnesium sulfate drying, concentrate; Column chromatography purification is with sherwood oil: ETHYLE ACETATE: acetic acid=69.9:30:0.1, the component of collecting Rf=0.36; Concentrate, obtain flaxen oily matter, be the haptin of II class pyrethroid.
(2) complete antigen preparation
Take by weighing the haptin of 0.027mmol (50.0mg); 0.04mmol (7.8mg) 1-ethyl-carbodiimide hydrochloride (EDC); 0.0054mmol (0.66mg) lutidine (DMAP) and 0.04mmol (4.1mg) triethylamine are put into reaction flask A; Add 300 μ L N, after dinethylformamide (DMF) dissolving, obtain A liquid; In reaction flask B, add 0.081 mmol (9.32mg) N-hydroxy succinic acid imines (NHS) and 100 μ L N then, dinethylformamide (DMF) obtains B liquid after the dissolving.B liquid dropwise is added drop-wise in the A liquid, and reaction is 12 hours under the room temperature, obtains C liquid.The BSA that takes by weighing 18mg (0.27 μ mol) is dissolved in the borate buffer solution of 4mL pH 8.4, gets protein soln.Under ice bath, C liquid dropwise is added drop-wise in the protein soln, reacted 6 hours.After reaction finishes, centrifugal, get supernatant, obtain the mixed antigen liquid of II class pyrethroid pesticide.
Dialysis tubing pre-treatment: dialysis tubing pre-treatment: the dialysis tubing of clip 9.0cm, boil 6-8min, use deionized water rinsing again 5 times, be kept in 4 ℃ of deionized waters subsequent use;
The artificial antigen mixed solution is moved in the dialysis tubing, with 0.01M pH 7.4 phosphate buffered saline buffer (PBS solution) dialysis 72 hours, changed a dialyzate in 6-8 hour, during replacing dialyzate 7-9 time.At last immunogen is divided in the centrifuge tube of 1.0mL, is kept at-20 ℃, subsequent use.
(3) evaluation of II class pyrethroid pesticide artificial antigen
Coupling ratio is measured: by the method for the ratio of two kinds of molecules of link coupled (coupling ratio), this patent adopts ultraviolet spectrophotometry that the coupling ratio of complete antigen is estimated in the estimation conjugate.
Principle: be to utilize material that the principle that absorption and its concentration of light is proportionlity is measured respectively by two kinds of molecular conecentrations of link coupled; In macromole and small molecules conjugate; Two kinds of molecules all have different separately ultraviolet scanning spectrums, and show the character of spectrogram superposition.
The Xylene Brilliant Cyanine G method is measured the protein concentration of conjugate: adopt the bovine serum albumin standardized solution of ultrapure water preparation 1mg/mL, and then its concentration is diluted successively is 0,40; 60,80,100 μ g/mL; The diluent that in enzyme plate, adds the bovine serum albumin of 48 μ L respectively then adds 240 μ L coomassie brilliant blue staining liquid, mixings immediately; Measure behind the 5min, each concentration is done 3 parallel appearance. and survey light absorption value at the 595nm place, draw the relation curve of protein concentration and light absorption value.Antigenic solution is diluted by a certain percentage, measure the light absorption value of antigenic solution at the 595nm place, obtain the corresponding proteins concentration 3.0mg/mL of antigenic solution from curve.
Molar absorption coefficient ε: preparation haptin concentration is 0; 10; 20% methanol solution of 20,30 μ g/mL can know that through UV scanning haptenic maximum absorption wavelength is 278nm; Survey light absorption value at the 278nm place, each concentration is done 3 parallel appearance. and molar absorptivity is calculated as: ε=light absorption value/volumetric molar concentration.
Coupling ratio estimation: prepare 20% methanol solution of 200 μ g/mL bovine serum albumins, coupled product is diluted to 200 μ g/mL with 20% methyl alcohol, survey light absorption value at the 278nm place; With 20% methyl alcohol is blank; The light absorption value of measuring is A1, A2, and coupling ratio r is: r=((A2-A1)/ε)/(200*10
-3/ 66200) wherein ε is molar absorptivity (L/mol), and 66200 is the molecular weight of bovine serum albumin, 200 * 10
-3Be bovine serum albumin concentration (μ g/mL).Calculate coupling ratio r=21:1.
Antibody titer is measured: adopt the enzyme plate method to measure.
(1) sero-fast preparation
1) laboratory animal: the healthy new zealand white rabbit that to select 22 monthly ages, body weight be 1.5-2 kg is a laboratory animal.
2) antigen configuration: immunogen is diluted with saline water, be made into the solution of 2 mg/mL.
3) emulsification: with complete with equivalent or the incomplete freund adjuvant of above-mentioned solution with mixing paddling process with its emulsification, until an emulsion is splashed in the water, is not scattered float on the water surface till.
4) immunization method: initial immunity reagent that emulsification is good is multi-point injection in the rabbit back, and 1 mg/ only.Immunity behind the initial immunity is called booster immunization, and booster immunization is used freund 's incomplete adjuvant emulsification, and booster immunization is intramuscular injection, per two all booster immunizations once, dosage is identical with initial immunity.
5) blood sampling: from ear edge vein exploitating blood, adopt the indirect competitive enzyme-linked immunosorbent method to measure antiserum titre behind 4 booster immunizations.Wait to tire reach requirement after, adopt the ear vein bloodletting acquisition antiserum(antisera) that combines with the heart bloodletting, be collected in 50 mL and sterilize in the plastic centrifuge tube.
6) preparation of serum: place under 37 ℃ 2h; Change refrigerator overnight again over to, centrifugal (5000rpm 10min), obtains antiserum(antisera), preserves down for 4 ℃, and is subsequent use.
(2) antibody titer determination step:
1) coating antigen is encapsulated 96 hole enzyme plates with encapsulating damping fluid as serial dilution, 100 μ L/ holes are in 4 ℃ of refrigerator overnight.Take out enzyme plate and be back to room temperature next day, and 200 μ L PBST solution are injected in every hole, and vibration 3 min firmly get rid of washings on the shaking table, do at the thieving paper arsis, wash 3 times.Following washing methods is identical.
2) behind the thorough washing, with sealing damping fluid sealase target, take out behind incubation 2 h in 37 ℃ of incubation casees in 200 μ L/ holes, dries for use.
3) positive serum serial dilution correspondence is joined preceding 6 of enzyme plate and be listed as, the 7th row add negative serum, and 50 μ L/ holes are hatched the 30min after scouring, clapped and do for 37 ℃.
4) every hole adds 100 μ L, and the goat anti-rabbit igg of the HRP mark of 1:3000 dilution is hatched the 30min after scouring, clapped and do for 37 ℃.
5) every hole adds 100 μ L colour developing liquid (TMB and substrate solution ratio are 1:5), and the 37 ℃ of reactions in dark place, 15 min take out every hole, back and add 50 μ L stop buffers (sulfuric acid of 2 mol/L), measure light absorption value A with ELIASA
450
(3) determination step of antibody susceptibility:
1) encapsulate: the coating antigen with setting concentration encapsulates enzyme-linked reaction plate, 100 μ L/holes, and 4 ℃ are spent the night.
2) washing: with PBST washing reaction plate three times, 3min at every turn, 200 μ L/holes dry Sptting plate then.
3) sealing: adopt sealing buffered soln to seal, 200 μ L/holes, 37 ℃ of sealing 2h.
4) washing: with 2)
5) competition: with PBST standard substance are diluted to 0,0.15,0.31,0.625,1.25,2.5,5.0,10 μ g/mL series concentration, other establishes a PBST blank, 50 μ L/holes.Every then hole adds the antiserum(antisera) of 4000 times of 50 μ L dilutions, in 37 ℃ of incubation 30min.
6) washing: with 2)
7) add ELIAS secondary antibody (goat-anti rabbit HRP-IgG, 1:3000), 100 μ L/holes, 37 C react 30min.
8) washing: with 2)
9) colour developing: add substrate TMB100 μ L/hole, colour developing 15min.
10) stop: add stop buffer 50 μ L/holes.
11) measure: detect OD with ELIASA
450Nm.
The mensuration that table 1 II class pyrethroid rabbit anteserum is tired
Remarks: after No. 1 on behalf of No. 1 rabbit five, serum exempt from, ear vein blood sampling is measured tiring and suppresses.Hap-ova has represented the conjugate of haptin and chicken egg white (OVA).Tiring of serum: when the light absorption value (OD value) of positive serum more than or equal to 2.1 times of negative control hole (P/N >=2.1), light absorption value is greater than 0.2 simultaneously, this moment, the extension rate of serum was tiring of serum.Can get tiring of serum by last table is 32000.
Claims (1)
1. the compound method of an II class pyrethroid artificial antigen is characterized in that with the cyphenothrin being raw material, adopts potassium permanganate and sodium periodate to carry out oxidation, obtains artificial semiantigen; Adopt active ester method that haptin is linked to each other with carrier proteins BSA, estimate immunogenic coupling ratio with ultraviolet spectrophotometry and SDS-PAGE method; Step is:
(1) the haptenic preparation of II class pyrethroid
Take by weighing the 1.20g cyphenothrin in the tool plug triangular flask of 100mL, add the trimethyl carbinol of 25.0mL and the water of 17.0mL again, mixing; Take by weighing the NaIO of 3.48mg then
4, 42.57mg KMnO
4With 9010mg Na
2CO
3Add respectively in the above-mentioned triangular flask; Add phase-transfer catalyst methyl trioctylphosphine ammonium chloride at last, normal temperature reaction down spends the night; After reaction finishes, carry out acidifying with 6N hydrochloric acid, with ethyl acetate extraction three times, the merging organic phase; Wash organic phase with hypo solution then, then use anhydrous magnesium sulfate drying, concentrate; Column chromatography purification, elutriant are the mixed solvent with mass ratio Shi You Mi ︰ Yi Suan Yi Zhi ︰ acetic acid=69.9 ︰, 30 ︰ 0.1, collect the component of Rf=0.36; Concentrate, obtain flaxen oily matter, be II class pyrethroid haptin;
(2) II class pyrethroid artificial antigen preparation
Take by weighing the haptin of 50.0mg, 7.8mg 1-ethyl-carbodiimide hydrochloride EDC, 0.66mg lutidine DMAP and 4.1mg triethylamine are put into reaction flask A, add 300 μ L N again, after the dinethylformamide DMF dissolving, obtain A liquid; In reaction flask B, add 9.32mgN-hydroxy succinic acid imines NHS and 100 μ L N then, dinethylformamide DMF obtains B liquid after the dissolving; B liquid dropwise is added drop-wise in the A liquid, reacts 12h under the room temperature, obtain C liquid; The BSA that takes by weighing 18mg is dissolved in the borate buffer solution of 4mL pH 8.4, gets protein soln; Under ice bath, C liquid dropwise is added drop-wise in the protein soln reaction 6h; After reaction finishes, centrifugal, get supernatant, obtain II class pyrethroid artificial antigen mixed solution;
The dialysis tubing pre-treatment: the dialysis tubing of clip 9.0cm, boil 6-8min, use deionized water rinsing again 5 times, be kept in 4 ℃ of deionized waters subsequent use;
II class pyrethroid artificial antigen mixed solution is moved in the dialysis tubing; With 0.01M pH 7.4 phosphate buffered saline buffer dialysis 72h; 6-8h changes a dialyzate, during change dialyzate 7-9 time, at last II class pyrethroid artificial antigen is divided in the centrifuge tube of 1.0mL; Be kept at-20 ℃, subsequent use;
(3) evaluation of II class pyrethroid artificial antigen
Coupling ratio is measured: adopt ultraviolet spectrophotometry that the coupling ratio of II class pyrethroid artificial antigen is measured;
Antibody titer is measured: adopt the enzyme plate method to measure.
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| CN107449900A (en) * | 2017-06-07 | 2017-12-08 | 广东产品质量监督检验研究院 | Carbamate and chrysanthemum esters multi-residue determination antigen, antibody and preparation method and application |
| CN111735951A (en) * | 2020-06-03 | 2020-10-02 | 北京勤邦生物技术有限公司 | Test strip for detecting fenpropathrin and application thereof |
| 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 |
| CN115611774A (en) * | 2022-09-26 | 2023-01-17 | 华南农业大学 | Fenpropathrin hapten, artificial antigen and fenpropathrin detection method |
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| CN107449900A (en) * | 2017-06-07 | 2017-12-08 | 广东产品质量监督检验研究院 | Carbamate and chrysanthemum esters multi-residue determination antigen, antibody and preparation method and application |
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| 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 |
| CN111735951B (en) * | 2020-06-03 | 2022-11-18 | 北京勤邦生物技术有限公司 | Test strip for detecting fenpropathrin and application thereof |
| CN115611774A (en) * | 2022-09-26 | 2023-01-17 | 华南农业大学 | Fenpropathrin hapten, artificial antigen and fenpropathrin detection method |
| CN115611774B (en) * | 2022-09-26 | 2024-04-12 | 华南农业大学 | Fenpropathrin hapten, artificial antigen and fenpropathrin detection method |
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