CN103601807A - Preparation method of monoclonal antibody with oxime carbamate pesticide butocarboxim resistant property - Google Patents
Preparation method of monoclonal antibody with oxime carbamate pesticide butocarboxim resistant property Download PDFInfo
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- WDQAHRJGGIAFLZ-GQCTYLIASA-N CC(/C(/C)=N/O)SC Chemical compound CC(/C(/C)=N/O)SC WDQAHRJGGIAFLZ-GQCTYLIASA-N 0.000 description 1
- BTWLYQZCZZOJJV-UHFFFAOYSA-N CC(C)=NOC(Cl)=O Chemical compound CC(C)=NOC(Cl)=O BTWLYQZCZZOJJV-UHFFFAOYSA-N 0.000 description 1
- MCRHVDXCHVOUSH-UHFFFAOYSA-N CC(C)=NOC(NCCCC(O)=O)=O Chemical compound CC(C)=NOC(NCCCC(O)=O)=O MCRHVDXCHVOUSH-UHFFFAOYSA-N 0.000 description 1
- LXZOLYDIANYOIR-RJFKTOBHSA-N CC1[C@@H](C/C(/C)=N/OC(Cl)=O)C1 Chemical compound CC1[C@@H](C/C(/C)=N/OC(Cl)=O)C1 LXZOLYDIANYOIR-RJFKTOBHSA-N 0.000 description 1
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Abstract
The invention relates to a preparation method of a monoclonal antibody with an oxime carbamate pesticide butocarboxim resistant property, and belongs to the biological technical field. The invention discloses a preparation method of a monoclonal antibody which is dedicated for specifically identifying oxime carbamate pesticide butocarboxim and an application of the antibody in the high-sensitive and rapid detection of butocarboxim residual in agricultural products and agricultural production environment. A butocarboxim semi-antigen and a carrier protein are coupled through a lively ester method so as to form a complete antigen, the complete antigen is used to immunize a Balb/C rat, hybridoma cells are prepared from spleen cells and Sp2/0 myeloma cells of the immunized rat through a hybridoma technique, so that a monoclonal antibody which can stably secrete an anti-butocarboxim component is obtained. The preparation technique of the antibody is feasible, the whole antigen preparation process does not need special instrument or equipment, and the mass production of the antibody is easy.
Description
(1) technical field
The present invention is the preparation of oxime carbamate class agricultural chemicals butocarboxim monoclonal antibody, belongs to biological technical field.Be exclusively used in the monoclonal antibody preparation of specific recognition carboxylamine oxime lipid agricultural chemicals butocarboxim, and the residual high-sensitivity rapid detection of butocarboxim in agricultural-food and agriculture production environment, be specially adapted to batch samples and detect and field monitoring.
(2) background technology
Along with scientific and technical progress, agricultural chemicals has become the Main Means of prevention and elimination of disease and pests, to agriculture stable yields, volume increase, plays a part very important.Since the seventies in last century, because organochlorine pesticide is limited the use of or forbids, attend by anti-organic phosphorous insecticide insect species increasing simultaneously, the consumption of carbamate chemicals for agriculture is increased year by year.Carbamate chemicals for agriculture has become one of pesticide species that China's usage quantity is larger at present, be widely used in the pest control on the cash crop such as grain, vegetables, fruit, main products has dimethacarb, carbaryl, aldicarb, carbofuran, meta-tolyl-N-methylcarbamate (MTMC), fenobucarb, butocarboxim etc.Although this class agricultural chemicals has the advantages such as insecticidal spectrum is wide, drug effect is fast, but along with the consumption of carbamate chemicals for agriculture increases year by year, it is also increasing to the negative impact of human and environment, and this just makes carbamate pesticide residue analyzing and testing receive increasing concern.
At present, the residue detection of carbamate chemicals for agriculture adopts the methods such as tlc, vapor-phase chromatography, high performance liquid chromatography more.The detectability of these methods is low, highly sensitive, existing relevant technical bid will definitely be for reference, but due to the instrument of needs costliness, special operator, and sample pre-treatments is complicated, cost is high, the time is long, therefore can not meet better fast and convenient Site Detection requirement.
The immunologic detection method of amino formate has advantages of fast, cheap, easy, special, can be portable and carry out field monitoring.Overcome the shortcoming of traditional detection method.By the haptens of the amino methylcarbamoyl oximino ester pesticides of chemosynthesis, obtain complete antigen with carrier protein couplet, preparation is for the specific monoclonal antibody of carboxylamine oxime lipid agricultural chemicals butocarboxim in environment and agricultural-food.Set up butocarboxim immunological detection method, the completing of the method, the gordian technique such as will solve that haptens is synthetic, prepared by coupling, monoclonal antibody, sets up the immunology Fast Detection Technique of butocarboxim.This patent is not only food safety detection, and is that the entry and exit of agricultural products in China etc. detect.The water area monitoring of environmental monitoring department provides a kind of new technique means and detection method.The Sustainable Healthy Development of agricultural products in China, solution food-safety problem are had important practical significance and important society, economic worth.The immunology detection technology of butocarboxim still belongs to blank on international and domestic, has not yet to see the research report of these class methods.
(3) summary of the invention
The preparation method who the object of this invention is to provide butocarboxim monoclonal antibody, by the artificial semiantigen of chemosynthesis butocarboxim, prepare complete antigen, immunity Balb/C mouse, the butocarboxim monoclonal antibody of preparing high specificity by hybridoma technology, and for agricultural-food and the residual high-sensitivity rapid detection of agriculture production environment butocarboxim.
(4) technical scheme
The preparation method of butocarboxim monoclonal antibody, comprising:
1. haptens preparation:
1.1H1's is synthetic
1g propionaldoxime and 1.88g Succinic anhydried add triethylamine 2.15ml in 20ml THF (anhydrous) N2 environment, reflux 24h, after acidifying, ethyl acetate extracts to obtain 2.6g product, by ethyl acetate and 1: 1 heating for dissolving of sherwood oil, take advantage of then recrystallization of heat filtering, obtain 1.74g product.1HNMR(300MH，CDC13)δ：2.97(s，3H)，3.11(s，3H)，4.00(s，4H)。
1.2H2's is synthetic
2.95g triphosgene is dissolved in 20ml methylene dichloride together with 8ml pyridine, with 20ml methylene dichloride, dissolve 3.3mmol acetoxime, under-15 ℃ of conditions, slowly this solution is dripped in the mixed solution of triphosgene and pyridine, after dropping, slowly rise to room temperature, reaction 5h, filters, with cold wash three times, dry with anhydrous Na 2SO4.
Take product acyl chlorides 3.275g (about 12.1mmo1), dissolve in 4m11,4-dioxane, puts 5 ℃ of refrigerators cooling, obtains A liquid; Take β-Beta Alanine 1.989g (about 22.3mmol), dissolve into 4ml4mol/LNaOH solution, put 5 ℃ of refrigerators cooling, obtain B liquid; Separately get 3ml4mol/L NaOH solution, put 5 ℃ of refrigerators cooling, obtain C liquid.A liquid and C liquid are divided into 5 equal portions, and at interval of respectively adding 1 part in 5 minutes clockwise B liquid, whole reaction is put in frozen water, under magnetic agitation, carries out simultaneously.Application of sample all completes and starts to clock, and finishes reaction after 2 hours.With concentrated hydrochloric acid, regulating the pH value of the whole liquid of reaction is 4, ethyl acetate for target product (50mL * 3) extraction; Ethyl acetate with rare HCl washing for several times, extracts with 1mol/LNaHCO3 solution (50mL * 2) mutually; Water is used concentrated hydrochloric acid acidifying again, and ethyl acetate extracts again; Anhydrous Na 2SO4 is dry, and gains underpressure distillation is concentrated into 13.8ml, in concentrated solution, adds gradually 31ml normal hexane, and white crystals thing is separated out, and filters.Crystallisate obtains 3.898g through cryodrying; Through at 5 ℃ of toluene and deionized waters, wash, weigh and obtain Hapten1 compared with sterling 1.871g after cryodrying, productive rate is 48%.1H?NMR(300MHz，DMSO)δ：2.22(2H，t，J＝7.2Hz)，1.67(m，2H)，3.07(m，2H)，1.98(s，3H)，1.97(s，3H)。
1.3H3's is synthetic
1.3.13-first sulfydryl-2-butanone oxime 1.
3-first sulfydryl-2-butanone (0.2g, 1.69mmol) is dissolved in the pyridine of 2ml, adds oxammonium hydrochloride (0.36g, 5.13mmol), stirs 24h.Stopped reaction, with 3mol/L salt acid for adjusting pH value, is extracted with ethyl acetate (10ml * 3) under ice bath, and anhydrous sodium sulfate drying, filters, and is spin-dried for.Obtain colorless oil 1. (0.2,88.9%).1H?NMR(300MHz，CDC13)δ：3.43(m，2H)，1.97(s，3H)，1.93(s，3H)，1.36(d，3H，J＝7.2Hz)。
1.3.20-[N-(2-oxyethyl group-2-oxoethyl) carboxamide]-3-first sulfydryl Diacetylmonoxime is 2.
Product 1. (0.2g, 1.50mmol) is dissolved in the methylene dichloride of I0ml with 0.1ml triethylamine, drips wherein the mixing solutions of the methylene dichloride of isocyanide ethyl acetoacetic acid ethyl ester (0.2g, 1.55mmol) and 4ml.Under normal temperature, stir 1h, be then warming up to 40-45 ℃ and react again 1h.Cooling, wash (20ml x3) with water.Organic layer anhydrous sodium sulfate drying, filters, and concentrated white oily matter is (0.20g, 50.9%) 2..1H?NMR(300MHz，CDC13)δ：4.14(m，2H)，3.97(m，2H)，3.41(m，1H)，1.98(s，3H)，1.91(s，3H)，1.32(d，3H，J＝6.9Hz)，1.20(t，3H，J＝6.9Hz)。
1.3.3O-(N-acetic acid carboxamide)-3-first sulfydryl Diacetylmonoxime H3
Product 2. (0.20g, 0.76mmol) joins in the aqueous solution of sodium carbonate (0.58g sodium carbonate, 18ml water), adds methyl alcohol to material dissolution.At 55 ℃, stir half hour.Be cooled to normal temperature, organic phase is spin-dried for.Water layer uses 3mol/L salt acid for adjusting pH to 3-4.Ethyl acetate extraction (20ml * 3), anhydrous sodium sulfate drying, filters, and concentrates to obtain crude product.Through column chromatography separated [eluent: ethyl acetate: sherwood oil (1: 2, v/v)], obtain faint yellow oily matter H3 (0.16g, 89.9%).1H?NMR(300MHz，CDC13)δ：4.11(d，2H，J＝5.1Hz)，3.48(m，1H)，2.06(s，3H)，1.97(s，3H)，1.38(d，3H，J＝6.9Hz)。
1.4H4's is synthetic
1.4.13-first sulfydryl-2-butanone oxime 3.
3-first sulfydryl-2-butanone (0.2g, 1.69mmol) is dissolved in the pyridine of 2ml, adds oxammonium hydrochloride (0.36g, 5.13mmol), stirs 24h.Stopped reaction, with 3mol/L salt acid for adjusting pH value, is extracted with ethyl acetate (10ml * 3) under ice bath, and anhydrous sodium sulfate drying, filters, and is spin-dried for.Obtain colorless oil 3. (0.2g, 85.9%).1H?NMR(300MHz，CDC13)δ：3.43(m，1H)，1.97(s，3H)，1.93(s，3H)，1.36(d，3H，J＝7.2Hz)。
1.4.2O-[N-(4-ketobutyric acid)]-3-first sulfydryl Diacetylmonoxime H4
Product is (0.2g, 1.50mmol) 3., and Succinic anhydried (0.15g, 1.50mmol) is dissolved in the tetrahydrofuran (THF) of 5ml, and adding 1ml triethylamine is catalyzer.Stirring at normal temperature 24h.After reaction finishes, be spin-dried for solvent, add ethyl acetate dilution, use 3mol/L salt acid for adjusting pH value to 3-4.Be extracted with ethyl acetate (10ml * 3), anhydrous sodium sulfate drying, filters, and is spin-dried for.Through the separated [eluent: ethyl acetate: sherwood oil (1: 2, v/v)), obtains faint yellow oily matter H4 (0.2g, 57.3%) of column chromatography.1H?NMR(300MHz，CDC13)δ：3.60(m，1H)，2.75(s，4H)，2.02(s，3H)，2.00(s，3H)，1.38(d，3H，J＝7.2Hz)。
1.5H5's is synthetic
1.5.13-[(1-methyl-2-oxopropyl)], mercapto-propionate 4.
The chloro-2-butanone of 3-(1.06g, 10mmol), 3-mercapto-propionate (1.22g, 10mmol), is dissolved in 11ml methylene dichloride, adds 1ml triethylamine.Ice bath stirs 30min, removes ice bath, under normal temperature, stirs and spends the night, and has insolubles in process always.After reaction finishes, add 2.5ml water, insolubles dissolves.Separatory, gets organic layer.Water 10ml extraction once again.Get organic layer, anhydrous sodium sulfate drying, filters, and concentrates to obtain crude product.Through chromatographic column separated [eluent: ethyl acetate: sherwood oil (1: 4, v/v)], obtain colorless oil 4. (0.68g, 35.8%).1H?NMR(300MHz，CDC13)δ：3.70(s，3H)，3.55(m，1H)，2.72(m，2H)，2.59(m，2H)，2.29(s，3H)，1.40(d，3H，J＝3.9Hz)。
1.5.23-1 (1-methyl-2-isonitroso propyl group)] mercapto-propionate is 5.
Product is (0.68g, 3.58mmol) 4., is dissolved in the pyridine of 3.5ml, adds oxammonium hydrochloride (0.74g, 10.7mmol) to stir 24h.Stopped reaction, with 3mol/L salt acid for adjusting pH value, is extracted with ethyl acetate (10ml * 3) under ice bath, and anhydrous sodium sulfate drying, filters, and is spin-dried for.Obtain colorless oil 5. (0.57g, 78%).1H?NMR(300MHz，CDCl3)δ：3.69(s，3H)，3.60(m，1H)，2.71(m，2H)，2.60(m，2H)，1.93(s，3H)，1.35(d，3H，J＝7.2Hz)。
1.5.3O-sulfydryl Diacetylmonoxime 6. for (N-ethyl carboxamide)-3-(3-methoxyl group-3-oxopropyl)
Product is (0.57g, 2.78mmol) 5., and ethyl isocyanate (0.21g, 3.02mmol) is dissolved in 5ml ether, adds 3ml triethylamine, stirs 12h.After reaction finishes, water: ether (20ml: 10ml) extractive reaction liquid, water layer is used ethyl acetate (20mlx2) washing again, and organic layer anhydrous sodium sulfate drying filters, and concentrates to obtain crude product.Through column chromatography separated [eluent: ethyl acetate: sherwood oil (1: 5, v/v)], obtain colorless oil 6. (0.51g, 67.1%).1HNMR(300MHz，CDCl3)δ：3.69(s，3H)，3.60(m，1H)，3.34(m，2H)，2.71(m，2H)，2.68(m，2H)，2.05(s，3H)，1.38(d，3H，J＝7.2Hz)，1.21(t3H，J＝7.2Hz)。
1.5.4O-(N-ethyl carboxamide)-3-(2-propyloic) sulfydryl Diacetylmonoxime H5
Product 6. (0.51g, 1.84mmol) joins in the aqueous solution of sodium carbonate (1.43g sodium carbonate, 36ml water), adds methyl alcohol to material dissolution.At 75 ℃, stir one hour.Be cooled to normal temperature, organic phase is spin-dried for.Water layer uses 3mol/L salt acid for adjusting pH to 3-4.Ethyl acetate extraction (20mlx3), anhydrous sodium sulfate drying, filters, and concentrates to obtain thick product.Through chromatographic column separated [eluent: ethyl acetate: sherwood oil (1: 2, v/v)], obtain faint yellow oily matter H5 (0.28g, 58.1%).1HNMR(300MHz，CDCl3)δ：3.63(m，1H)，(m，2H)，2.66(m，4H)，2.06(s，3H)，1.38(d，3H，J＝7.2Hz)，1.20(t，3H，J＝7.2Hz)。
2. antigen preparation:
The preparation of immunizing antigen: take haptens 0.25mmol and 0.25mmol NHS, dissolve with 1.5ml DMF solution.Take 0.25mmol DCC and be dissolved in 1ml DMF, this solution is slowly added in the solution of above-mentioned haptens and NHS.Under magnetic agitation, room temperature sealed reaction is 7 hours.Reacting whole liquid, to put 4 ℃ of refrigerators cooling more than 2 hours, through 12000rpm centrifugal 5 minutes, gets the active ester liquid of supernatant and join very slowly in the BSA albumen of 3ml15mg/ml, reaction stirring at room 4 hours under magnetic agitation.After question response completes, reaction solution is placed in dialysis tubing, in the PB of 0.2mol/L pH6.8 damping fluid, 4 ℃ are stirred dialysis, within every four hours, change dialyzate one time, dialyse altogether 60 hours.After dialysis finishes, the white emulsion (HA-BSA) in dialysis tubing being taken out to packing is stored in-20 ℃ of refrigerators.
Synthesizing of envelope antigen: method, with the coupling of immunizing antigen, is about to BSA and changes OVA into, obtains H-OVA.
3. mouse immune:
Using the dH1-BSA preparing as immunogen immune 6-8 age in week female Balb/C mouse, the immunogen consumption 200 μ g of each every mouse, immunity is five times altogether, immunity for the first time adopts by isopyknic immunogen and Freund's complete adjuvant emulsification, abdominal injection.Immune interval is after three weeks for the first time, and 2 weeks, five each immune intervals of for the second time to the, adopt isopyknic immunogen and Freund's incomplete adjuvant emulsification;
From for the third time, after each immunity one week, the specificity of indirect non-competing ELISA method detection serum antibody for caudal vein blood sampling:
(1) coated: with coated damping fluid (0.05mol/L, pH9.6), envelope antigen (dHl-OVA) is diluted to 1000 times and add enzyme plate, every hole 50 μ l, hatch 2h in 37 ℃ of incubators;
(2) wash plate: with washings PBST (0.05% polysorbas20,0.01mol/L, pH7.4), wash 5 times, thieving paper pats dry;
(3) sealing: every hole adds 100 μ L0.1% gelatin, hatches 1.5h for 37 ℃;
(4) wash plate: with (2);
(5) add primary antibodie: every hole adds 50 μ L through the mice serum to be checked of PBST dilution, with the serum of immune mouse not, make negative control, hatch 1h for 37 ℃;
(6) wash plate: with (2);
(7) add ELIAS secondary antibody body: every hole adds 50 μ L through the horseradish peroxidase-goat anti-mouse igg of 1: 20000 times of PBST dilution, hatches 1h for 37 ℃;
(8) wash plate: with (2);
(9) colour developing: every hole adds the freshly prepared nitrite ion of 50 μ L, 37 ℃ of incubation 10min;
(10) stop: every hole adds the H of 25 μ L2mol/L
2s0
4solution;
(11) absorbance measurement: each hole light absorption value of measuring 450nm wavelength place by microplate reader;
(12) result judgement: return to zero with blank; Using without immune serum as negative control, if treat gaging hole OD
450value is more than or equal to 2.1 times of negative hole values and is decided to be the positive.
4. cytogamy
By positive value screening Balb/C mouse, the high Balb/C mouse of positive value is directly used dH1-BSA immunity, after three days, get spleen, the Sp2/0 myeloma cell of splenocyte and logarithmic phase prepares hybridoma by hybridoma technology, 37 ℃, 5% CO2gas incubator was cultivated after 10-12 days, by detection, merged hole supernatant screening hybridoma;
5. hybridoma screening:
Coating antigen dH1-OVA coated elisa plate with 1000 times of dilutions, 1% gelatin sealing, get the culture supernatant of hybridoma, adopt non-competing indirect elisa method tentatively to screen that (as mentioned above, wherein primary antibodie is hybridoma supernatant to method, with the positive contrast of serum of immune mouse, with the negative contrast of myeloma cell's supernatant), choose the cell hole being positive, cell conditioned medium is retained for next step and is detected, if the hole of being negative is still negative after repeating once, can give up.
The cell hole being positive for previous step adopts indirect competitive ELISA method further to confirm, the concrete operations of indirect competitive ELISA method are as follows:
(1) coated: with coated damping fluid (0.05mol/L, pH9.6), envelope antigen (dH1-OVA) is diluted to 1000 times and add enzyme plate, every hole 50 μ l, hatch 2h in 37 ℃ of incubators;
(2) wash plate: with washings PBST (0.05% polysorbas20,0.01mol/L, pH7.4), wash 5 times, thieving paper pats dry;
(3) sealing: every hole adds 120 μ L0.1% gelatin, hatches 1.5h for 37 ℃;
(4) wash plate: with (2);
(5) add agricultural chemicals and cell conditioned medium mixture: it is pesticidal solutions (PBST dilution) the 25 μ L of 100 μ g/mL that every hole adds concentration, then adds cell conditioned medium 25 μ L, hatches 1h for 37 ℃, PBST washing 5 times, and be arranged in parallel positive control and negative control;
(6) wash plate: with (2);
(7) add ELIAS secondary antibody body: every hole adds 50 μ L through the horseradish peroxidase-goat anti-mouse igg of 1: 20000 times of PBST dilution, hatches 1h for 37 ℃;
(8) wash plate: with (2);
(9) colour developing: every hole adds the freshly prepared nitrite ion of 50 μ L, 37 ℃ of incubation 10min;
(10) stop: every hole adds the H of 25 μ L2mol/L
2sO
4solution;
(11) absorbance measurement: each hole light absorption value of measuring 450nm wavelength place by microplate reader;
The hole that agricultural chemicals butocarboxim is occurred to obvious competitive inhibition reaction is the positive hole that produces anti-carboxylamine oxime lipid agricultural chemicals butocarboxim antibody, subclone is carried out by limiting dilution assay in positive hole, and the supernatant of positive hole secretion is prepared butocarboxim monoclonal antibody.
The hybridoma cell strain obtaining is preserved in Chinese Typical Representative culture collection center (China, Wuhan, Wuhan University), deposit number on December 25th, 2012: CCTCC NO:C2012190, Classification And Nomenclature: hybridoma cell strain 1C9D8.
Beneficial effect advantage of the present invention and positively effect show:
(1) antibody is pioneering: this antibody, by synthetic dH1-BSA artificial antigen immunity Blab/C, obtains the monoclonal antibody of specific recognition butocarboxim by hybridoma technology, belong in the world at home pioneering.
(2) good stability of antigen and antibody:: the synthetic butocarboxim antigen of this method has good stability, and-20 ℃ of refrigerators at least can be deposited and within 5 years, not affect its immunogenicity.Cryopreservation of Hybridoma Cells at least keeps 3 years in liquid nitrogen, and the stability of antibody also can be relatively good, and antibody-20 of purifying ℃ refrigerator at least can be deposited 2 years.
(3) haptens type is unique: carbamate chemicals for agriculture butocarboxim is chain structure, there is no complicated molecular structure, and in the preparation process of antibody, receives good effect, has good sensitivity and specificity.
Embodiment
The detection of embodiment 1 butocarboxim standard substance (Dr.Ehrensorfer, Germany)
Adopt indirect competitive ELISA method, detection curve is carried out to desk study.Method is as follows:
Coated: with coated damping fluid (0.05mol/L, pH9.6), envelope antigen (H5-OVA) is diluted to 1000 times and add enzyme plate, every hole 50 μ l, hatch 2h in 37 ℃ of incubators;
(2) wash plate: with washings PBST (0.05% polysorbas20,0.01mol/L, pH7.4), wash 5 times, thieving paper pats dry;
(3) sealing: every hole adds 120 μ L1% gelatin, hatches 1.5h for 37 ℃;
(4) wash plate: with (2);
(5) add agricultural chemicals and mixtures of antibodies: with 3 times of stepwise dilution butocarboxim standardized solution of PBS phosphoric acid buffer, every hole adds 25 μ L, then adds the antibody-solutions 25 μ L that dilute certain multiple, hatches 1h for 37 ℃, PBST washing 5 times, and be arranged in parallel positive control and negative control;
(6) wash plate: with (2);
(7) add ELIAS secondary antibody body: every hole adds 50 μ L through the horseradish peroxidase-goat anti-mouse igg (Wuhan doctor's moral company) of 1: 20000 times of PBST dilution, hatches 1h for 37 ℃;
(8) wash plate: with (2);
(9) colour developing: every hole adds the freshly prepared nitrite ion of 50 μ L, 37 ℃ of incubation 10min;
(10) stop: every hole adds the H of 25 μ L2mol/L
2sO
4solution;
By microplate reader (Thermo CO.), measure each hole light absorption value at 450nm wavelength place, and calculate inhibiting rate.The IC of antibody
50=0.837 μ g/mL.Result shows, the antibody capable of preparation is identified butocarboxim preferably.
In embodiment 2 apple samples, detect butocarboxim residual quantity
H5-OVA coated elisa plate 50 μ L/ holes, 8000 times of butocarboxim antibody dilutions are as working concentration, 1% gelatin is closure, PBS phosphoric acid buffer pH value is 7.5,, ionic strength is that 0.4M, organic solvent are methyl alcohol, 50 μ L/ holes in enzyme plate.
Testing sample is prepared:
Apple sample is bought from Formocarbam supermarket, Nanjing, takes the sample of 5g homogenate in advance, adds standard substance (butocarboxim) to the final concentration of 60 μ g/g, mixes, the standing 1h of room temperature, adds the methyl alcohol of 10mL, fully vibration, 4000rpm, centrifugal 5min, supernatant liquor moves in 25mL volumetric flask.Precipitation rejoins 10mL methyl alcohol, fully vibration, and 4000rpm, centrifugal 5min, supernatant liquor moves in 25mL volumetric flask, and is settled to 25mL with optimum working buffer liquid (not containing organic solvent).Get optimum working buffer liquid for sample liquid (containing the organic solvent) dilution afterwards of part constant volume, for ic-ELISA, detect.
Adopt butocarboxim ELISA detection method to detect above sample, operate as follows:
Coated: with coated damping fluid (0.05mol/L, pH9.6), envelope antigen (H5-OVA) is diluted to 1000 times and add enzyme plate, every hole 50 μ l, hatch 2h in 37 ℃ of incubators;
(2) wash plate: with washings PBST (0.05% polysorbas20,0.01mol/L, pH7.4), wash 5 times, thieving paper pats dry;
(3) sealing: every hole adds 120 μ L1% gelatin, hatches 1.5h for 37 ℃;
(4) wash plate: with (2);
(5) add agricultural chemicals and mixtures of antibodies: the apple sample solution that has added butocarboxim with 8 times of dilutions of PBS phosphoric acid buffer of optimizing, every hole adds 25 μ L, add again the antibody-solutions 25 μ L that dilute certain multiple, hatch 1h for 37 ℃, PBST washing 5 times, and be arranged in parallel positive control and negative control;
(6) wash plate: with (2);
(7) add ELIAS secondary antibody body: every hole adds 50 μ L through the horseradish peroxidase-goat anti-mouse igg (Wuhan doctor's moral company) of 1: 20000 times of PBST dilution, hatches 1h for 37 ℃;
(8) wash plate: with (2);
(9) colour developing: every hole adds the freshly prepared nitrite ion of 50 μ L, 37 ℃ of incubation 10min;
(10) stop: every hole adds the H of 25 μ L2mol/L
2sO
4solution;
In microplate reader, 450nm reads light absorption value in place, positive value B0=0.998, testing sample light absorption value B=0.477.
11) data processing
Calculate combination rate B/B
0=0.4784, substitution formula
B/B
0＝-0.4222LogC+1.687
Wherein C is the butocarboxim concentration recording in enzyme plate, and trying to achieve butocarboxim residual quantity in testing sample is C=0.7288 μ g/mL, and theoretical interpolation value is 0.75 μ g/mL, and obtaining the rate of recovery is 97.17%.
Claims (1)
1. the preparation method of butocarboxim monoclonal antibody, comprising:
1.1H1's is synthetic
1g propionaldoxime and 1.88g Succinic anhydried add triethylamine 2.15ml in 20ml THF (anhydrous) N2 environment, reflux 24h, after acidifying, ethyl acetate extracts to obtain 2.6g product, by ethyl acetate and 1: 1 heating for dissolving of sherwood oil, take advantage of then recrystallization of heat filtering, obtain 1.74g product.1H?NMR(300MH，CDCl3)δ：2.97(s，3H)，3.11(s，3H)，4.00(s，4H)。
1.2H2's is synthetic
2.95g triphosgene is dissolved in 20m1 methylene dichloride together with 8m1 pyridine, with 20m1 methylene dichloride, dissolve 3.3mmol acetoxime, under-15 ℃ of conditions, slowly this solution is dripped in the mixed solution of triphosgene and pyridine, after dropping, slowly rise to room temperature, reaction 5h, filters, with cold wash three times, dry with anhydrous Na 2S04.
Take product acyl chlorides 3.275g (about 12.1mmo1), dissolve in 4ml1,4-dioxane, puts 5 ℃ of refrigerators cooling, obtains A liquid; Take β-Beta Alanine 1.989g (about 22.3mmo1), dissolve into 4ml4mol/L NaOH solution, put 5 ℃ of refrigerators cooling, obtain B liquid; Separately get 3ml 4mol/L NaOH solution, put 5 ℃ of refrigerators cooling, obtain C liquid.A liquid and C liquid are divided into 5 equal portions, and at interval of respectively adding 1 part in 5 minutes clockwise B liquid, whole reaction is put in frozen water, under magnetic agitation, carries out simultaneously.Application of sample all completes and starts to clock, and finishes reaction after 2 hours.With concentrated hydrochloric acid, regulating the pH value of the whole liquid of reaction is 4, ethyl acetate for target product (50mL * 3) extraction; Ethyl acetate with rare HCl washing for several times, extracts with 1mol/LNaHCO3 solution (50mL * 2) mutually; Water is used concentrated hydrochloric acid acidifying again, and ethyl acetate extracts again; Anhydrous Na 2SO4 is dry, and gains underpressure distillation is concentrated into 13.8ml, in concentrated solution, adds gradually 31ml normal hexane, and white crystals thing is separated out, and filters.Crystallisate obtains 3.898g through cryodrying; Through at 5 ℃ of toluene and deionized waters, wash, weigh and obtain Hapten1 compared with sterling 1.871g after cryodrying, productive rate is 48%.1H?NMR(300MHz，DMSO)δ：2.22(2H，t，J＝7.2Hz)，1.67(m，2H)，3.07(m，2H)，1.98(s，3H)，1.97(s，3H)。
1.3H3's is synthetic
1.3.13-first sulfydryl-2-butanone oxime 1.
3-first sulfydryl-2-butanone (0.2g, 1.69mmo1) is dissolved in the pyridine of 2ml, adds oxammonium hydrochloride (0.36g, 5.13mmo1), stirs 24h.Stopped reaction, with 3mol/L salt acid for adjusting pH value, is extracted with ethyl acetate (10ml * 3) under ice bath, and anhydrous sodium sulfate drying, filters, and is spin-dried for.Obtain colorless oil 1. (0.2,88.9%).1H?NMR(300MHz，CDCl3)δ：3.43(m，2H)，1.97(s，3H)，1.93(s，3H)，1.36(d，3H，J＝7.2Hz)。
1.3.20-[N-(2-oxyethyl group-2-oxoethyl) carboxamide]-3-first sulfydryl Diacetylmonoxime is (2.
Product 1. (0.2g, 1.50mrno1) is dissolved in the methylene dichloride of I0ml with 0.1ml triethylamine, drips wherein the mixing solutions of the methylene dichloride of isocyanide ethyl acetoacetic acid ethyl ester (0.2g, 1.55mmo1) and 4ml.Under normal temperature, stir 1h, be then warming up to 40-45 ℃ and react again 1h.Cooling, wash (20ml x3) with water.Organic layer anhydrous sodium sulfate drying, filters, and concentrated white oily matter is (0.20g, 50.9%) 2..1H?NMR(300MHz，CDCl3)δ：4.14(m，2H)，3.97(m，2H)，3.41(m，1H)，1.98(s，3H)，1.91(s，3H)，1.32(d，3H，J＝6.9Hz)，1.20(t，3H，J＝6.9Hz)。
1.3.30-(N-acetic acid carboxamide)-3-first sulfydryl Diacetylmonoxime H3
Product 2. (0.20g, 0.76mmol) joins in the aqueous solution of sodium carbonate (0.58g sodium carbonate, 18ml water), adds methyl alcohol to material dissolution.At 55 ℃, stir half hour.Be cooled to normal temperature, organic phase is spin-dried for.Water layer uses 3mol/L salt acid for adjusting pH to 3-4.Ethyl acetate extraction (20ml * 3), anhydrous sodium sulfate drying, filters, and concentrates to obtain crude product.Through column chromatography separated [eluent: ethyl acetate: sherwood oil (1: 2, v/v)], obtain faint yellow oily matter H3 (0.16g, 89.9%).1H?NMR(300MHz，CDCl3)δ：4.11(d，2H，J＝5.1Hz)，3.48(m，1H)，2.06(s，3H)，1.97(s，3H)，1.38(d，3H，J＝6.9Hz)。
1.4H4's is synthetic
1.4.13-first sulfydryl-2-butanone oxime 3.
3-first sulfydryl-2-butanone (0.2g, 1.69mmo1) is dissolved in the pyridine of 2ml, adds oxammonium hydrochloride (0.36g, 5.13mmo1), stirs 24h.Stopped reaction, with 3mol/L salt acid for adjusting pH value, is extracted with ethyl acetate (10ml * 3) under ice bath, and anhydrous sodium sulfate drying, filters, and is spin-dried for.Obtain colorless oil 3. (0.2g, 85.9%).1H?NMR(300MHz，CDCl3)δ：3.43(m，1H)，1.97(s，3H)，1.93(s，3H)，1.36(d，3H，J＝7.2Hz)。
1.4.20-[N-(4-ketobutyric acid)]-3-first sulfydryl Diacetylmonoxime H4
Product is (0.2g, 1.50mmo1) 3., and Succinic anhydried (0.15g, 1.50mmo1) is dissolved in the tetrahydrofuran (THF) of 5ml, and adding 1m1 triethylamine is catalyzer.Stirring at normal temperature 24h.After reaction finishes, be spin-dried for solvent, add ethyl acetate dilution, use 3mol/L salt acid for adjusting pH value to 3-4.Be extracted with ethyl acetate (10ml * 3), anhydrous sodium sulfate drying, filters, and is spin-dried for.Through the separated [eluent: ethyl acetate: sherwood oil (1: 2, v/v)), obtains faint yellow oily matter H4 (0.2g, 57.3%) of column chromatography.1H?NMR(300MHz，CDCl3)δ：3.60(m，1H)，2.75(s，4H)，2.02(s，3H)，2.00(s，3H)，1.38(d，?3H，J＝7.2Hz)。
1.5H5's is synthetic
1.5.13-[(1-methyl-2-oxopropyl)], mercapto-propionate 4.
The chloro-2-butanone of 3-(1.06g, 10mmo1), 3-mercapto-propionate (1.22g, 10mmo1), is dissolved in 11ml methylene dichloride, adds 1ml triethylamine.Ice bath stirs 30min, removes ice bath, under normal temperature, stirs and spends the night, and has insolubles in process always.After reaction finishes, add 2.5ml water, insolubles dissolves.Separatory, gets organic layer.Water 10ml extraction once again.Get organic layer, anhydrous sodium sulfate drying, filters, and concentrates to obtain crude product.Through chromatographic column separated [eluent: ethyl acetate: sherwood oil (1: 4, v/v)], obtain colorless oil 4. (0.68g, 35.8%).1H?NMR(300MHz，CDCl3)δ：3.70(s，3H)，3.55(m，1H)，2.72(m，2H)，2.59(m，2H)，2.29(s，3H)，1.40(d，3H，J＝3.9Hz)。
1.5.23-1 (1-methyl-2-isonitroso propyl group)] mercapto-propionate is 5.
Product is (0.68g, 3.58mmo1) 4., is dissolved in the pyridine of 3.5ml, adds oxammonium hydrochloride (0.74g, 10.7mmo1) to stir 24h.Stopped reaction, with 3mol/L salt acid for adjusting pH value, is extracted with ethyl acetate (10ml * 3) under ice bath, and anhydrous sodium sulfate drying, filters, and is spin-dried for.Obtain colorless oil 5. (0.57g, 78%).1H?NMR(300MHz，CDCl3)δ：3.69(s，3H)，3.60(m，1H)，2.71(m，2H)，2.60(m，2H)，1.93(s，3H)，1.35(d，3H，J＝7.2Hz)。
1.5.30-sulfydryl Diacetylmonoxime 6. for (N-ethyl carboxamide)-3-(3-methoxyl group-3-oxopropyl)
Product is (0.57g, 2.78mmol) 5., and ethyl isocyanate (0.21g, 3.02mmol) is dissolved in 5ml ether, adds 3ml triethylamine, stirs 12h.After reaction finishes, water: ether (20ml: 10ml) extractive reaction liquid, water layer is used ethyl acetate (20mlx2) washing again, and organic layer anhydrous sodium sulfate drying filters, and concentrates to obtain crude product.Through column chromatography separated [eluent: ethyl acetate: sherwood oil (1: 5, v/v)], obtain colorless oil 6. (0.51g, 67.1%).1H?NMR(300MHz，CDCl3)δ：3.69(s，3H)，3.60(m，1H)，3.34(m，2H)，2.71(m，2H)，2.68(m，2H)，2.05(s，3H)，1.38(d，3H，J＝7.2Hz)，1.21(t3H，J＝7.2Hz)。
1.5.40-(N-ethyl carboxamide)-3-(2-propyloic) sulfydryl Diacetylmonoxime H5
Product 6. (0.51g, 1.84mmol) joins in the aqueous solution of sodium carbonate (1.43g sodium carbonate, 36ml water), adds methyl alcohol to material dissolution.At 75 ℃, stir one hour.Be cooled to normal temperature, organic phase is spin-dried for.Water layer uses 3mol/L salt acid for adjusting pH to 3-4.Ethyl acetate extraction (20mlx3), anhydrous sodium sulfate drying, filters, and concentrates to obtain thick product.Through chromatographic column separated [eluent: ethyl acetate: sherwood oil (1: 2, v/v)], obtain faint yellow oily matter H5 (0.28g, 58.1%).1H?NMR(300MHz，CDCl3)δ：3.63(m，1H)，(m，2H)，2.66(m，4H)，2.06(s，3H)，1.38(d，3H，J＝7.2Hz)，1.20(t，3H，J＝7.2Hz)。
The preparation of immunizing antigen: take haptens 0.25mmol and 0.25mmol NHS, dissolve with 1.5ml DMF solution.Take 0.25mmol DCC and be dissolved in 1ml DMF, this solution is slowly added in the solution of above-mentioned haptens and NHS.Under magnetic agitation, room temperature sealed reaction is 7 hours.Reacting whole liquid, to put 4 ℃ of refrigerators cooling more than 2 hours, through 12000rpm centrifugal 5 minutes, gets the active ester liquid of supernatant and join very slowly in the BSA albumen of 3ml15mg/ml, reaction stirring at room 4 hours under magnetic agitation.After question response completes, reaction solution is placed in dialysis tubing, in the PB of 0.2mol/LpH6.8 damping fluid, 4 ℃ are stirred dialysis, within every four hours, change dialyzate one time, dialyse altogether 60 hours.After dialysis finishes, the white emulsion (HA-BSA) in dialysis tubing being taken out to packing is stored in-20 ℃ of refrigerators.
Synthesizing of envelope antigen: method, with the coupling of immunizing antigen, obtains H-OVA.
Using the H3-BSA preparing as immunogen immune 6-8 age in week female Balb/C mouse, the immunogen consumption 200 μ g of each every mouse, immunity is five times altogether, immunity for the first time adopts by isopyknic immunogen and Freund's complete adjuvant emulsification, abdominal injection.Immune interval is after three weeks for the first time, and 2 weeks, five each immune intervals of for the second time to the, adopt isopyknic immunogen and Freund's incomplete adjuvant emulsification;
From for the third time, after each immunity one week, the specificity of indirect non-competing ELISA method detection serum antibody for caudal vein blood sampling: return to zero with blank; Using without immune serum as negative control, if treat, gaging hole 0D450 value is more than or equal to 2.1 times of negative hole values and is decided to be the positive.
By positive value screening Balb/C mouse, the high Balb/C mouse of positive value is directly used dH1-BSA immunity, after three days, get spleen, the Sp2/0 myeloma cell of splenocyte and logarithmic phase prepares hybridoma by hybridoma technology, 37 ℃, 5% CO2gas incubator was cultivated after 10-12 days, by detection, merged hole supernatant screening hybridoma;
Coating antigen H3-OVA coated elisa plate with 1000 times of dilutions, 1% gelatin sealing, get the culture supernatant of hybridoma, adopt non-competing indirect elisa method tentatively to screen that (as mentioned above, wherein primary antibodie is hybridoma supernatant to method, with the positive contrast of serum of immune mouse, with the negative contrast of myeloma cell's supernatant), choose the cell hole being positive, cell conditioned medium is retained for next step and is detected, if the hole of being negative is still negative after repeating once, can give up.
The cell hole being positive for previous step adopts indirect competitive ELISA method further to confirm, the hole that agricultural chemicals butocarboxim is occurred to obvious competitive inhibition reaction is the positive hole that produces anti-carboxylamine oxime lipid agricultural chemicals butocarboxim antibody, subclone is carried out by limiting dilution assay in positive hole, and the supernatant of positive hole secretion is prepared butocarboxim monoclonal antibody.
Hybridoma cell strain 1C9D8 described in generation claim is preserved in Chinese Typical Representative culture collection center (China, Wuhan, Wuhan University, postcode 430072) on December 25th, 2012, and deposit number is CCTCC NO:C2012190.
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| CN108931640A (en) * | 2018-09-27 | 2018-12-04 | 北京勤邦生物技术有限公司 | A kind of test strips and its application detecting organophosphorus pesticide |
| CN111505297A (en) * | 2020-05-13 | 2020-08-07 | 北京勤邦生物技术有限公司 | Application of endosulfan artificial antigen in enzyme linked immunosorbent assay kit |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108383754A (en) * | 2018-04-18 | 2018-08-10 | 马鞍山南大高新技术研究院有限公司 | The preparation method and application of a kind of aryl oxime compound |
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| CN108931640A (en) * | 2018-09-27 | 2018-12-04 | 北京勤邦生物技术有限公司 | A kind of test strips and its application detecting organophosphorus pesticide |
| CN108931640B (en) * | 2018-09-27 | 2022-10-18 | 北京勤邦生物技术有限公司 | Test strip for detecting organophosphorus pesticide and application thereof |
| CN111505297A (en) * | 2020-05-13 | 2020-08-07 | 北京勤邦生物技术有限公司 | Application of endosulfan artificial antigen in enzyme linked immunosorbent assay kit |
| CN111505297B (en) * | 2020-05-13 | 2022-11-18 | 北京勤邦生物技术有限公司 | Application of endosulfan artificial antigen in enzyme linked immunosorbent assay kit |
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