CN101412673A - Deltamethrin antigen and antibody, as well as use thereof - Google Patents
Deltamethrin antigen and antibody, as well as use thereof Download PDFInfo
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Abstract
The invention discloses a decamethrin antigen, an antibody and application thereof, which belong to the technical field of immune chemical analysis, and provides a method special for preparing decamethrin specific antibody and detecting enzyme coupled immunity. The method comprises the following steps: synthesizing hapten 1-carboxyl-(3'-phenoxyl phenyl) methyl-3-(2', 2'-dibromo vinyl)-2, 2-dimethyl cyclopropyl carboxylic ester (Med for short) and N-2-(carboxyl propyl) carbamoyl-(3'-phenoxyl phenyl) methyl-3-(2', 2'-dibromo vinyl)-2, 2-dimethyl cyclopropyl carboxylic ester (Di for short); preparing artificial immune antigen by coupling the Di and protein; obtaining specific polyclonal antibody of decamethrin by an immune rabbit; preparing envelope antigen by coupling the Med and the protein; and finally establishing an enzyme coupled immunity analytic method by using the synthesized envelope antigen and the specific antibody, wherein the detection limit of the decamethrin reaches 0.004 milligram per liter, the linearity is between 0.005 and 100 milligrams per kilogram, and the decamethrin antigen has no remarkable cross reaction for most pyrethroid and metabolite thereof.
Description
One, technical field
The present invention relates to a kind of deltamethrin antigen, antibody and application thereof, belong to fields of immunochemistry analysis, be exclusively used in the preparation and the enzyme-linked immune detection method of Deltamethrin specific antibody.
Two, background technology
Deltamethrin (deltamethrin) is the synthetic pyrethroid insecticides of people such as ELLIOT exploitation in 1974, the trade(brand)name Decamethrin, chemical name and structure See Figure, pure product are white rhombus needle crystal, mp:101～102 ℃, be slightly soluble in water, can be dissolved in most of organic solvents, stable under acidic conditions.Have stomach toxicity and action of contace poison, knock down soon, and refuse to keep away activity.Effective especially to lepidopterous larvae, be one of pyrethroid insecticides the most efficiently in the present age.
Retention analysis to Deltamethrin mainly is a vapor-phase chromatography at present, and this method testing cost height is not suitable for carrying out the rapid detection of sample in batches.And being immunoassay, the pesticide residue immuno analytical method is applied to the new technology in pesticide residue analysis field, simple, quick, cheap with it, advantages such as particularly suitable batch samples detection have obtained developing rapidly comprehensively, and American Chemical Society (AOAC) props up column technologies greatly with immunoassay and gas-chromatography, same three of the Detecting Pesticide of classifying as of liquid chromatography.But up to the present, the immune analysis method of Deltamethrin, domestic not appearing in the newspapers has report abroad, but different with method with synthetic haptens structure of the present invention.
The structure of Deltamethrin
Three, summary of the invention
Technical problem
The preparation and the enzyme-linked immune analytic method that the purpose of this invention is to provide a kind of Deltamethrin specific antibody.
Technical scheme
The molecular structural formula of Deltamethrin artificial semiantigen is:
The 1-carboxyl-(3 '-Phenoxyphenyl) methyl-3-(2 ', 2 '-dibromo vinyl)-2,2-dimethyl cyclopropyl carboxylic acid esters Med
Or
N-2-(carboxyl propyl group) carbamyl-(3 '-Phenoxyphenyl) methyl-3-(2 ', 2 '-dibromo vinyl)-2,2-dimethyl cyclopropyl carboxylic acid esters Di
Artificial semiantigen 1-carboxyl-(3 '-Phenoxyphenyl) methyl-3-(2 ', 2 '-dibromo vinyl)-2, the preparation process of 2-dimethyl cyclopropyl carboxylic acid esters Med is as follows:
1) gets 0.5～5g cyanalcohol in the 100mL there-necked flask, add 5～15mL concentrated hydrochloric acid, 50～70 ℃ of magnetic agitation reaction 2h;
2) reacted mixed solution extracted with diethyl ether;
3) ether layer washes with water again, and anhydrous Na is crossed on the upper strata
2SO
4, concentrate near doing, get the crude product of cyanic acid;
4) column chromatography purification, sherwood oil wet method dress post, collecting elutriant is the wash-out phase of acetone, concentrate near do clear crystal;
5) take by weighing the 0.5-5g dibromo chrysanthemic acid in the 100mL there-necked flask, add the 5-15mL sulfur oxychloride, stirring and refluxing 2h concentrates, yellow liquid 3-(2 ', 2 '-dibromo vinyl)-2,2-dimethyl cyclopropyl acyl chlorides is with 15mL THF dissolving;
6) in the 100mL there-necked flask, add 0.5-5g cyanic acid and stir,, above-mentioned acyl chlorides is added stirring reaction 4h under the ice bath with 2mol/L NaOH dissolving;
7) use the ethyl acetate extraction product, regulate pH value to 2.0, washing, anhydrous MgSO
4Drying, concentrating under reduced pressure obtain the Med crude product;
8) column chromatography purification, use ethyl acetate: (10:90 → 100:0) drip washing concentrates and obtains xanchromatic Med product sherwood oil.Artificial semiantigen N-2-(carboxyl propyl group) carbamyl-(3 '-Phenoxyphenyl) methyl-3-(2 ', 2 '-dibromo vinyl)-2, the preparation process of 2-dimethyl cyclopropyl carboxylic acid esters Di is as follows:
1) takes by weighing 0.5-5g Med in the 100mL there-necked flask, add the 10mL sulfur oxychloride, stirring and refluxing 2h;
2) concentrating under reduced pressure gets yellow oily liquid, with the 15mLTHF dissolving and 4 ℃ of coolings down;
3) take by weighing the 0.5-5g4-aminobutyric acid in the 100mL there-necked flask, add the 4mol/L NaOH solution of 5mL, ice bath stirs down.
4) divide the THF solution that adds acyl chlorides for 5 times, each 10min at interval finishes and continues reaction 3h adding 2mL 4mol/L NaOH liquid feeding for back 4 times in reinforced.
5) with the pH value to 2.0 of dense HCl conditioned reaction liquid, use ethyl acetate extraction, washing, anhydrous MgSO
4Drying, concentrating under reduced pressure obtains xanchromatic oily matter.
6) (developping agent is the thin-layer chromatography purifying: ethyl acetate: sherwood oil: acetate (30:79.5:0.5)), concentrate and obtain yellow Di product.
The molecular structural formula of porcess for artificial antigen of cyanobromide chrysanthemum ester is:
Immunogen Di-BSA envelope antigen Med-OVA
Synthesizing of artificial antigen
Immunogenic synthetic employing carbodlimide method.The haptens Di of 0.2 mmole is dissolved in N, in the dinethylformamide, add 1.5 normal dicyclohexylcarbodiimides and 3 normal N-hydroxy-succinamides, reaction is spent the night under the room temperature, and is centrifugal, gets in the bovine serum albumin carbonate buffer solution that supernatant liquor joins 10～20mg/mL, stirring reaction 1～6 hour, the dialysis tubing of packing into, respectively with PBS buffered soln dialysis 3～5d of distilled water and 0.01mol/L, packing is stored in-20 ℃ the refrigerator.
The synthetic mixed anhydride method of utilizing of envelope antigen.Respectively 0.25 mmole haptens Med is dissolved in N, in the dinethylformamide, add positive Tributylamine of 60uL and 30uL butyl chlorocarbonate, reacted under the room temperature 1～2 hour, reaction solution joins in ovalbumin (OVA) carbonate buffer solution of 10～20mg/mL, stirring reaction 1～4 hour, the dialysis tubing of packing into, respectively with PBS buffered soln dialysis 3～5d of distilled water and 0.01mol/L, packing is stored in-20 ℃ the refrigerator.
Prepare the Deltamethrin specific antibody with above-mentioned deltamethrin antigen, the Deltamethrin specific antibody be can with the immunoglobulin (Ig) of Deltamethrin generation specific immune response.It is used for detecting the residual quantity of food, agricultural-food and environmental sample (as soil and water sample) Deltamethrin.
Beneficial effect the present invention produces specific antibody by design synthetic bromide Cyano chrysanthemate artificial semiantigen and artificial antigen through immune animal, and purified to make tiring behind the lyophilized powder be 2.5 * 10
5Based on the antigen and antibody specific immunological response, and introduce marker and amplify this reaction of demonstration, then can be used for sample and measure.Its selectivity depends on the specificity of immunological response, and the affinity of antibody and the property examined of marker are depended in its sensitivity.
Antibody is to the cross reacting rate of some similar agricultural chemicals: bromine cyanogen chrysanthemumic acid is 9.46%, and Cypermethrin is 4.88%, and lambda-cyhalothrin, fenvalerate, Fenvalerate, bifenthrin are all＜0.1%.Thereby as can be known, prepared antibodies specific is stronger, rapidly and accurately the residual quantity of analyzing and testing Deltamethrin in sample.Highly sensitive, the high specificity of this method, sample pre-treatments is simple, is convenient to carry out on-site supervision, can complement one another with ordinary method.
This method can be used for the residue detection of Deltamethrin in environment and the plant sample, and pre-treating process is simple than instrument analytical method, is fit to screening in enormous quantities.The rate of recovery of this method Deltamethrin is 75.6～106.8%, and average coefficient of variation is 1.79～7.21%, meets the retention analysis standard.
Four, embodiment
Synthesizing of 1 Deltamethrin artificial semiantigen
For the Stereo structure Characteristics that makes Deltamethrin fully expose and contain can with protein link coupled carboxyl, with α-Qing Ji-3-Ben Yangjibianchun, bromine cyanogen chrysanthemumic acid and chlorination sulfone is raw material, through three-step reaction synthesized haptens 1-carboxyl-(3 '-Phenoxyphenyl) methyl-3-(2 ', 2 '-dibromo vinyl)-2,2-dimethyl cyclopropyl carboxylic acid esters (being called for short Med).Cross the allos reaction for enable pass and improve detection sensitivity; again the reaction of haptens Med and 4-aminobutyric acid has been synthesized N-2-(carboxyl propyl group) carbamyl-(3 '-Phenoxyphenyl) methyl-3-(2 '; 2 '-dibromo vinyl)-2,2-dimethyl cyclopropyl carboxylic acid esters (being called for short Di).Behind product Med and the Di purifying respectively through mass spectrum (ESI) and proton nmr spectra (
1H-NMR) identify, and relatively prove conclusively with similar compound known data.
Haptenic structure is as follows:
The 1-carboxyl-(3 '-Phenoxyphenyl) methyl-3-(2 ', 2 '-dibromo vinyl)-2,2-dimethyl cyclopropyl carboxylic acid esters Med
Or
N-2-(carboxyl propyl group) carbamyl-(3 '-Phenoxyphenyl) methyl-3-(2 ', 2 '-dibromo vinyl)-2,2-dimethyl cyclopropyl carboxylic acid esters Di
Haptens Med's is synthetic
1) gets 0.5～5g cyanalcohol in the 100mL there-necked flask, add 5～15mL concentrated hydrochloric acid, 50～70 ℃ of magnetic agitation reaction 2h;
2) reacted mixed solution extracted with diethyl ether;
3) ether layer washes with water again, and anhydrous Na is crossed on the upper strata
2SO
4, concentrate near doing, get the crude product of cyanic acid;
4) column chromatography purification, sherwood oil wet method dress post, collecting elutriant is the wash-out phase of acetone, concentrate near do clear crystal;
5) take by weighing the 0.5-5g dibromo chrysanthemic acid in the 100mL there-necked flask, add the 5-15mL sulfur oxychloride, stirring and refluxing 2h concentrates, yellow liquid 3-(2 ', 2 '-dibromo vinyl)-2,2-dimethyl cyclopropyl acyl chlorides is with 15mL THF dissolving;
6) in the 100mL there-necked flask, add 0.5-5g cyanic acid and stir,, above-mentioned acyl chlorides is added stirring reaction 4h under the ice bath with 2mol/L NaOH dissolving;
7) use the ethyl acetate extraction product, regulate pH value to 2.0, washing, anhydrous MgSO
4Drying, concentrating under reduced pressure obtain the Med crude product;
8) column chromatography purification, use ethyl acetate: (10:90 → 100:0) drip washing concentrates and obtains xanchromatic Med product sherwood oil.
With the haptens Di behind the purifying through ESI-MS,
1H-NMR (500MHz, CDCl
3) (Finnigan LC-MS) measures, to identify its molecular structure.The H of haptens Med
1-NMR (CDCl
3): δ 7.0-7.4 (m ,-ArH), 6.82 (d ,=CH), 5.90 (2 * s, CHCOO), 2.15 (m, CHC), 2.05 (d, CHCO), 1.27 (m, 2 * CH
3).
From above as can be known analysis integrated, institute's synthetic product is a target compound.
Haptens LBy's is synthetic:
1) takes by weighing 0.5-5g Med in the 100mL there-necked flask, add the 10mL sulfur oxychloride, stirring and refluxing 2h;
2) concentrating under reduced pressure gets yellow oily liquid, with the 15mLTHF dissolving and 4 ℃ of coolings down;
3) take by weighing the 0.5-5g4-aminobutyric acid in the 100mL there-necked flask, add the 4mol/L NaOH solution of 5mL, ice bath stirs down.
4) divide the THF solution that adds acyl chlorides for 5 times, each 10min at interval finishes and continues reaction 3h adding 2mL 4mol/L NaOH liquid feeding for back 4 times in reinforced.
5) with the pH value to 2.0 of dense HCl conditioned reaction liquid, use ethyl acetate extraction, washing, anhydrous MgSO
4Drying, concentrating under reduced pressure obtains xanchromatic oily matter.
6) (developping agent is the thin-layer chromatography purifying: ethyl acetate: sherwood oil: acetate (30:79.5:0.5)), concentrate and obtain yellow Di product.
With the haptens LBy behind the purifying respectively through ESI-MS,
1H-NMR (500MHz, CDCl
3) measure, to identify its molecular structure.The ESI-MS:[Found:m/z 632 of haptens Di, [M+Na
+], 1243[2M+Na
+], M=609], and have 634 and 630 liang to locate isotopic peak by this peak, its peak height is the last 1/3 at 632 peaks, hence one can see that, and this material has 2 bromine atoms.
1H-NMR(500MHz，CDCl
3)，δ?7.0-7.4(m，8H，-ArH)，6.53(d，1H，＝CH)，5.98，5.95(2s，CHCOO)，4.72(dd，CH
2N)，3.17(m，CH
2CO)，2.22(s，6H，CH
2CH
2CH
2)，1.25，1.24(2s，2×CH
3)；
From above as can be known analysis integrated, institute's synthetic product is a target compound.
2. artificial antigen is synthetic
The structural formula of artificial antigen is as follows:
Immunogen Di-BSA envelope antigen Med-OVA
2.1 immunogenic synthetic and purifying
The immunogenic synthetic carbodlimide method that utilizes.With 0.2 mmole haptens Di, be dissolved in the N of 1mL, in the dinethylformamide, add 1.5 normal dicyclohexylcarbodiimides and 3 normal N-hydroxy-succinamides, reaction is spent the night under the room temperature, centrifugal, get in bovine serum albumin (BSA) carbonate buffer solution that supernatant liquor 100～800 μ L slowly join 4mL10mg/mL, under magnetic agitation, reacted 1～6 hour, the dialysis tubing of packing into, earlier with distill water dialysis 2～4 times, then with PBS buffered soln dialysis 3～5d of 0.01mol/L, packing is stored in-20 ℃ the refrigerator.
2.2 the synthetic and purifying of envelope antigen
The synthetic mixed anhydride method of utilizing of envelope antigen.0.25 mmole haptens Med is dissolved in the N of 1mL respectively, in the dinethylformamide, add positive Tributylamine of 60uL and 30uL butyl chlorocarbonate, reacted under the room temperature 1～2 hour, reaction solution 100 μ L～800 μ L join in ovalbumin (OVA) carbonate buffer solution of 5mL10mg/mL, and reaction is 1 hour under magnetic agitation, the dialysis tubing of packing into then, earlier with distill water dialysis 3 times, then with PBS buffered soln dialysis 3～5d of 0.01mol/L, packing is stored in-20 ℃ the refrigerator.
2.3 the evaluation of artificial antigen
The ratio of reactant and product during according to synthetic bromide Cyano chrysanthemate immunogen and envelope antigen is got reactant and product respectively and is carried out ultraviolet (200nm～400nm) scanning.Conjugate is compared with the absorption peak of haptens Di, Med, BSA and OVA, and obvious variation has taken place, and shows that the synthetic of artificial antigen Di-BSA and Med-OVA is successful.
Haptens and combination of proteins are such as following as calculated:
LBc-BSA 10～40:1 LBy-OVA 10～30:1
3. the preparation of antibody
3.1 immune animal prepares antiserum(antisera)
Experiment was selected for use about half cycle year, and body weight is 2-3 kilograms, healthy male new zealand white rabbit.Immunity three rabbits (being responsible for the raising work of rabbit by the academy of agricultural sciences, Jiangsu Province) are numbered rabbit 1-3 respectively.
Experiment immunization dosage fundamental immunity is 0.25～4.0mg/kg, booster immunization dosage is 0.5～4.0mg/kg, dilutes corresponding dosage immunogen mixture (Di-BSA) respectively with physiological saline, adds the equal-volume Freund's complete adjuvant, fully emulsified, emulsion droplet does not disperse in splashing into water.The method that adopts the subcutaneous multi-point injection in back to combine with the leg muscle injection.Carry out booster immunization after 3～4 weeks,, adopt Freund's incomplete adjuvant during booster immunization later on every 2 weeks booster immunization once more.From immunity for the third time, each immunity back the 8th～10 day from rabbit hearts or ear edge vein exploitating blood, is measured and is tired and specificity.Treat immune serum tire qualified after, just take a blood sample.
The heart extracting blood method is adopted in this experiment.Every rabbit can get about blood 80mL.After the blood sampling, after waiting to be collected in the blood coagulation in the Erlenmeyer flask earlier, clot and glass are broken away from along the Erlenmeyer flask edge with inoculating needle then, be positioned over half an hour in 37 ℃ of incubators, be put into again in 4 ℃ of refrigerators 3～4 hours, treat blood clot retraction after, with suction pipe serum is sucked in the test tube, with 3000rpm centrifugal 15 minutes, isolate serum.
3.2 purifying antibody and evaluation
Sad-the ammonium sulfate salting-out process of general employing also can adopt the albumin A column chromatography.Sad-ammonium sulfate salting-out process is a classic methods.Sad during protein except that IgG all precipitates in can be with serum under the condition of slant acidity, have only IgG in the supernatant liquor.Sad adding is different because of the source of antibody, and human serum is 70ul/ml, and rabbit anteserum is 75ul/ml, and mice serum is 40ul/ml, and mouse ascites is 33ul/ml.The rate of recovery of this method IgG reaches more than 90%.
3.3 antibody titer is measured
Immunogen mixture (Di-BSA) according to a conventional method the immunity three rabbits.From booster immunization for the second time, serum was through suitably tiring with indirect ELISA mensuration after the dilution in the rabbit ear edge vein exploitating blood in the 8th day in each immunity back.Treat the 4th when immunity, rabbit has obtained high antibody of tiring, and purified to make tiring behind the lyophilized powder be 2.5 * 10
5
4 Deltamethrin enzyme-linked immunosorbent assay for measuring are set up and are identified
4.1 the principle of Deltamethrin ELISA measuring method
Adopt the indirect competitive enzyme-linked immunosorbent analytical procedure.It is that the mixture that pesticide molecule and macromolecular carrier (as protein) coupling make is adsorbed on the solid phase carrier (96 hole enzyme plate) as envelope antigen, be prepared into solid phase antigen, add agricultural chemicals to be measured and corresponding antibodies then, agricultural chemicals in the solid phase antigen, agricultural chemicals to be measured, with the antibody association reaction that is at war with, pesticide concentration to be measured is many, the antibody that is bonded on the solid phase antigen is just few, otherwise the antibody that is combined in solid phase antigen is many, and the reaction back adds ELIAS secondary antibody (can only combine with the antibody on being combined in solid phase antigen), develops the color with substrate at last and is measured, when one timing of antibody amount, the pesticide volume to be measured that adds is many more, and just few more with solid phase antigen bonded antibody, color reaction just weakens, inhibiting rate increases, otherwise then color reaction strengthens, and inhibiting rate lowers, thereby can extrapolate the concentration of agricultural chemicals to be measured according to the standard lines of known quantity agricultural chemicals and the inhibiting rate of sample to be checked.
4.2 determining of optimum antibody working concentration and envelope antigen complex concentration
Select envelope antigen Med-OVA and antibody square formation volumetry, dilute antibody and solid phase antigen coating buffer simultaneously.
Under same coating buffer concentration, along with the dilution of antibody, the OD value of gained is on a declining curve, and under same antibody dilution concentration, along with the decline of coating buffer concentration, gained OD value is also on a declining curve equally.Through test, envelope antigen concentration 1.5 μ g.mL
-1, antibody concentration is with 2 μ g.mL
-1As the suitableeest working concentration.
4.3 typical curve and detection sensitivity
4.3.1 the preparation of typical curve, its basic operation steps is as follows:
4.3.1.1 bag quilt
1) preparation of envelope antigen solution
From cryogenic refrigerator, take out the Med-OVA coupled complex, after making it to thaw fully, be diluted to relevant work concentration.
2) the bag quilt of micro-reaction plate
After 96 hole polystyrene micro-reaction plates wash with PBST, the antigen coated liquid 100 μ L that every hole is joined above adding, incubation 2h in 37 ℃ of incubators discards liquid in the hole, uses 300ul PBST solution washing 5 times, pats dry.
4.3.1.2 sealing
Take out bag by good micro-reaction plate, get rid of coating buffer, after the PBST washing, every hole adds 1.0% OVA200 μ L, and incubation 0.5h in 37 ℃ of incubators discards liquid in the hole, uses 300ul PBST solution washing 5 times, pats dry.
4.3.1.3 some plate
1) standardized solution of preparation Deltamethrin
Get the standardized solution of Deltamethrin standard specimen preparation 100ppm, be diluted to some (5～8) individual concentration, make in each concentration solution and contain 30% methyl alcohol with PBS.
2) preparation of Deltamethrin antibody diluent
From refrigerator, take out antibody, be diluted to working concentration with PBS.
3) some plate
Taking-up is through the plate of sealing, and after 200ul PBST washing 5 times, every hole adds the Deltamethrin reference liquid 50 μ L of series concentration, adds antibody diluent 50 μ L again, and control wells adds PBS50 μ L and the antibody diluent 50 μ L that contain 30% methyl alcohol.Put into 37 ℃ of incubator incubation 1h, discard liquid in the hole, use 300ul PBST solution washing 5 times, pat dry.
4.3.1.4 add ELIAS secondary antibody
Every hole adds the goat-anti rabbit horseradish peroxidase PBS solution 100 μ L through the 1:2000 dilution, puts into 37 ℃ of incubator 1h, discards liquid in the hole, uses 200ul PBST solution washing 5 times, pats dry.
4.3.1.5 colour developing
Every hole adds substrate OPD-superoxol 100 μ L, and incubation 15min in 37 ℃ of incubators is with 50 μ L 2MH
2SO
4Termination reaction.On enzyme connection instrument, measure the light absorption value under the 490nm wavelength.Mapping promptly obtains typical curve according to the relation of the semilog between inhibiting rate and the pesticide concentration.
The typical curve of ELISA method represents that with the semilog plot of inhibiting rate and pesticide concentration inhibiting rate calculates with following formula:
In the formula: OD
MaxLight absorption value during for not dosing, OD
xLight absorption value during for agricultural chemicals x, OD
MinLight absorption value for the blank hole.
Calculating the inhibiting rate of each concentration of Deltamethrin by above-mentioned formula, is X-coordinate with Deltamethrin concentration, and inhibiting rate is the ordinate zou mapping.Deltamethrin is in 0.005ppm～100ppm scope, and inhibiting rate and Deltamethrin concentration are linear, and relation conefficient is r=0.9955.Concentration is 0.55mg/L in the inhibition of Deltamethrin, and lowest detection is limited to 0.004mg/L,
4.4 the specificity of antibody
Sero-fast specificity be meant its homospecificity antigen bonded ability with the comparison of this antigen-analogues ability.Cross-reactivity commonly used is as the major criterion of estimating.Cross reaction is more little, and sero-fast specificity is then good more.
Deltamethrin and analogue thereof are done serial dilution, react respectively with a kind of antibody competition, by system 4.3 method production standard curve, and the consumption when on curve, finding out the dosage of inhibiting rate 50% and analogue inhibiting rate 50%, calculate the cross reacting rate of each analogue then.
Antibody is to the cross reacting rate of some similar agricultural chemicals: bromine cyanogen chrysanthemumic acid is 9.46%, and Cypermethrin is 4.88%, and lambda-cyhalothrin, fenvalerate, Fenvalerate, bifenthrin are all＜0.1%.Thereby as can be known, prepared antibodies specific is stronger.
5 environmental sample rapid determination
5.1 extracting method
5.1.1 water sample
Water sample (or after filtration) can directly detect
5.1.2 soil or plant sample
(1) takes by weighing soil or plant sample 5g, in the triangular flask of packing into.
(2) the Deltamethrin methanol solution of three different levelss of preparation.Liquor strength is 10ppm, 0.2ppm, therefrom gets 1mL respectively and joins in every class sample, repeats 3 times, and remaining compares.
(3) behind the certain hour, in sample, add the vibration of 20mL methyl alcohol and extract 0.5h.
(4) centrifugal, get supernatant liquor, with 3 times of the PBS dilutions that contains 30% methyl alcohol, be used for detecting.
5.2 the ELISA method of sample is measured
Method is with the operation of typical curve.The every hole of the plate that has sealed adds the sample liquid 50 μ L of serial known interpolation concentration, adds antibody diluent 50 μ L again, and control wells adds PBS50 μ L and the antibody diluent 50 μ L that contain 30% methyl alcohol.37 ℃ of incubations 1 hour, the same 4.3.1 of surplus back step.By analysis as can be known, the Deltamethrin rate of recovery of this method is 75.6～106.8%, and average coefficient of variation is 1.79～7.21%, meets the retention analysis standard.This method can be used for the residue detection of Deltamethrin in the environmental sample, and pre-treating process is simple than instrument analytical method, is fit to screening in enormous quantities.
Claims (5)
1. Deltamethrin artificial semiantigen is characterized in that its molecular structural formula is:
1-carboxyl-(3 '-Phenoxyphenyl) methyl-3-(2 ', 2 '-dibromo vinyl)-2,2-dimethyl cyclopropyl carboxylic acid esters Med or
N-2-(carboxyl propyl group) carbamyl-(3 '-Phenoxyphenyl) methyl-3-(2 ', 2 '-dibromo vinyl)-2,2-dimethyl cyclopropyl carboxylic acid esters Di
2. the preparation method of the described Deltamethrin artificial semiantigen of claim 1 is characterized in that,
Artificial semiantigen 1-carboxyl-(3 '-Phenoxyphenyl) methyl-3-(2 ', 2 '-dibromo vinyl)-2, the preparation process of 2-dimethyl cyclopropyl carboxylic acid esters Med is as follows:
1) gets 0.5～5g cyanalcohol in the 100mL there-necked flask, add 5～15mL concentrated hydrochloric acid, 50～70 ℃ of magnetic agitation reaction 2h;
2) reacted mixed solution extracted with diethyl ether;
3) ether layer washes with water again, and anhydrous Na is crossed on the upper strata
2SO
4, concentrate near doing, get the crude product of cyanic acid;
4) column chromatography purification, sherwood oil wet method dress post, collecting elutriant is the wash-out phase of acetone, concentrate near do clear crystal;
5) take by weighing the 0.5-5g dibromo chrysanthemic acid in the 100mL there-necked flask, add the 5-15mL sulfur oxychloride, stirring and refluxing 2h concentrates, yellow liquid 3-(2 ', 2 '-dibromo vinyl)-2,2-dimethyl cyclopropyl acyl chlorides is with 15mL THF dissolving;
6) in the 100mL there-necked flask, add 0.5-5g cyanic acid and stir,, above-mentioned acyl chlorides is added stirring reaction 4h under the ice bath with 2mol/L NaOH dissolving;
7) use the ethyl acetate extraction product, regulate pH value to 2.0, washing, anhydrous MgSO
4Drying, concentrating under reduced pressure obtain the Med crude product;
8) column chromatography purification, use ethyl acetate: (10:90 → 100:0) drip washing concentrates and obtains xanchromatic Med product sherwood oil.Artificial semiantigen N-2-(carboxyl propyl group) carbamyl-(3 '-Phenoxyphenyl) methyl-3-(2 ', 2 '-dibromo vinyl)-2, the preparation process of 2-dimethyl cyclopropyl carboxylic acid esters Di is as follows:
1) takes by weighing 0.5-5g Med in the 100mL there-necked flask, add the 10mL sulfur oxychloride, stirring and refluxing 2h;
2) concentrating under reduced pressure gets yellow oily liquid, with the 15mLTHF dissolving and 4 ℃ of coolings down;
3) take by weighing the 0.5-5g4-aminobutyric acid in the 100mL there-necked flask, add the 4mol/L NaOH solution of 5mL, ice bath stirs down.
4) divide the THF solution that adds acyl chlorides for 5 times, each 10min at interval finishes and continues reaction 3h adding 2mL4mol/L NaOH liquid feeding for back 4 times in reinforced.
5) with the pH value to 2.0 of dense HCl conditioned reaction liquid, use ethyl acetate extraction, washing, anhydrous MgSO
4Drying, concentrating under reduced pressure obtains xanchromatic oily matter.
6) (developping agent is the thin-layer chromatography purifying: ethyl acetate: sherwood oil: acetate (30:79.5:0.5)), concentrate and obtain yellow Di product.
3. deltamethrin antigen; it is characterized in that; respectively by claim 1 or 2 described haptens N-2-(carboxyl propyl group) carbamyls-(3 '-Phenoxyphenyl) methyl-3-(2 '; 2 '-dibromo vinyl)-2; 2-dimethyl cyclopropyl carboxylic acid esters Di and protein coupling, 1-carboxyl-(3 '-Phenoxyphenyl) methyl-3-(2 '; 2 '-dibromo vinyl)-2,2-dimethyl cyclopropyl carboxylic acid esters Med and protein coupling form, and its molecular structural formula is:
Immunogen Di-BSA envelope antigen Med-OVA
4. use the Deltamethrin specific antibody of the described deltamethrin antigen preparation of claim 3.
5. the application in the residual quantity of the described Deltamethrin specific antibody of claim 4 Deltamethrin in detecting food, agricultural-food and environmental sample.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008102341857A CN101412673A (en) | 2008-11-25 | 2008-11-25 | Deltamethrin antigen and antibody, as well as use thereof |
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| CNA2008102341857A CN101412673A (en) | 2008-11-25 | 2008-11-25 | Deltamethrin antigen and antibody, as well as use thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105319350A (en) * | 2014-07-24 | 2016-02-10 | 江苏维赛科技生物发展有限公司 | Deltamethrin colloidal gold test card |
| CN113774030A (en) * | 2021-09-23 | 2021-12-10 | 江南大学 | Hybridoma cell strain secreting anti-picloram monoclonal antibody and application thereof |
| CN115669676A (en) * | 2021-07-30 | 2023-02-03 | 江苏扬农化工股份有限公司 | Insecticidal composition and application thereof |
| CN117384061A (en) * | 2023-12-11 | 2024-01-12 | 深圳市通量检测科技有限公司 | Dioxamine hapten, antigen, antibody, detection device and preparation and application thereof |
-
2008
- 2008-11-25 CN CNA2008102341857A patent/CN101412673A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105319350A (en) * | 2014-07-24 | 2016-02-10 | 江苏维赛科技生物发展有限公司 | Deltamethrin colloidal gold test card |
| CN115669676A (en) * | 2021-07-30 | 2023-02-03 | 江苏扬农化工股份有限公司 | Insecticidal composition and application thereof |
| CN113774030A (en) * | 2021-09-23 | 2021-12-10 | 江南大学 | Hybridoma cell strain secreting anti-picloram monoclonal antibody and application thereof |
| CN113774030B (en) * | 2021-09-23 | 2022-05-24 | 江南大学 | Hybridoma cell strain secreting anti-picloram monoclonal antibody and application thereof |
| CN117384061A (en) * | 2023-12-11 | 2024-01-12 | 深圳市通量检测科技有限公司 | Dioxamine hapten, antigen, antibody, detection device and preparation and application thereof |
| CN117384061B (en) * | 2023-12-11 | 2024-03-15 | 深圳市通量检测科技有限公司 | Dioxamine hapten, antigen, antibody, detection device and preparation and application thereof |
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Open date: 20090422 |