CN101504409A - Fast detection ELISA reagent kit for methomyl residue - Google Patents
Fast detection ELISA reagent kit for methomyl residue Download PDFInfo
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- CN101504409A CN101504409A CNA2008102469598A CN200810246959A CN101504409A CN 101504409 A CN101504409 A CN 101504409A CN A2008102469598 A CNA2008102469598 A CN A2008102469598A CN 200810246959 A CN200810246959 A CN 200810246959A CN 101504409 A CN101504409 A CN 101504409A
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Abstract
The invention discloses an ELISA kit for quickly detecting methomyl residue, and belongs to a novel quick detection method established in the technical field of enzyme immunoassay. The method overcomes the defects of complex operation, high detection cost, low speed and the like of a physical and chemical analytical method for methomyl, and can quickly and accurately detect the methomyl residue in a sample such as water, soil and the like. In the detection process, the coating antigen and the methomyl to be detected adsorbed on the pore wall of an enzyme label plate compete with each other to react with the antibody in solution, and a competitive result is shown by color development of the enzyme catalytic reaction. By detecting the methomyl with known concentration and drawing a standard curve, the concentration of the methomyl to be detected can be calculated. The retention period of the kit is over 6 months.
Description
One, technical field
The present invention relates to a kind of fast detecting ELISA kit of methomyl residue, mainly be applicable to the fast measuring of methomyl residue in the samples such as water and soil earth in enormous quantities.Belong to a kind of new rapid assay methods of being founded in the EIA enzyme immunoassay technical field.
Two, background technology
Methomyl (methomyl) is an absorption broad spectrum pesticide in the carbamates, has strong tagging and stomach poison function.Be mainly used in Homoptera, Lepidoptera, coleoptera and other insects such as cotton, tobacco, fruit tree, vegetables anti-ly eliminate aphis, moth, cutworm.To female, great and mighty or powerful mouse acute oral LD
50Be respectively: 23.5mg/kg and 17.0mg/kg, rabbit is acute through skin LD
505000mg/kg, high toxicity N monomethyl carbamate insecticides belonged to.China only allows to use on specific crop.Because this pesticide uses in recent years more widely, its caused food poisoning, wrongly take and the incident of poisoning happens occasionally.The maximum residue limit(MRL) of Methomyl is respectively 2mg/kg and 5mg/kg in mandatory national Specification wild cabbage of China and the oranges and tangerines.
The detection method of Methomyl has liquid chromatography, gas chromatography, reaches colourimetry etc., these method pre-treatment relative complex are consuming time, sample clean is required high, need set up different analytical approachs because of the sample matrix components is complicated, be difficult to satisfy the on-the-spot needs of sample fast detecting in batches, therefore, press for development and application high-level efficiency residues of pesticides express-analysis technology.
ELISA adsorption analysis method (ELISA) high specificity, highly sensitive and easyly fast can remedy the defective of chromatogram analysis method, the present invention has important practical significance to the methomyl residue on-site supervision technology that solves batch samples.
Three, summary of the invention
For solve present methomyl residue instrument analytical method cost height, complicated operation, poor specificity, sensitivity is low and shortcoming such as testing result instability, the invention provides that a kind of to have a high specific, high sensitivity, pin-point accuracy, high precision, method of operating easy, and can be used for the ELISA kit of the methomyl residue of batch samples fast detecting.
The ELISA kit of methomyl residue comprises box body, is located at 96 hole ELISA Plate, the sponge bracket in the box body and includes the Methomyl titer of concentrated cleaning solution (PBST), variable concentrations, anti-Methomyl polyclonal antibody, horseradish peroxidase-labeled goat anti-rabbit antibody, A liquid, B liquid and reaction terminating liquid.
The technical solution adopted for the present invention to solve the technical problems is: at first Methomyl is transformed into the haptens Methomyl monomester succinate (MOSE) that is applicable to carrier protein (BSA and OVA) coupling, prepare artificial immunogen after utilizing water-soluble carbodiimide (EDC) method with haptens (MOSE) and bovine serum albumin(BSA) coupling, prepare the Methomyl polyclonal antibody with this immunogen immune new zealand white rabbit.Again with mixed anhydride method with haptens (MOSE) and ovalbumin coupling, their compound is fixed on the ELISA Plate hole wall as envelope antigen.In advance with Methomyl sample to be measured and polyclonal antibody hybrid reaction 30 minutes, again reacted antigen-antibody is joined in the ELISA Plate hole, fixedly envelope antigen and Methomyl to be measured are vied each other and antibody response, because the solid phase antigen content in each hole and the antibody content of adding are all consistent, so when Methomyl concentration to be measured is high, antibody (once the anti-) amount that is bonded on the solid phase antigen is few, after adding horseradish peroxidase-labeled goat anti-rabbit antibody (two is anti-), two is anti-and few once anti-binding capacity, add substrate solution (A liquid) and colour developing liquid (B liquid) at last, chromogenic reaction shallow (the OD value is low) shows the inhibiting rate height; Otherwise when Methomyl concentration to be measured was low, the OD value of then being surveyed was high, and inhibiting rate is low.Detect and the drawing standard curve with known Methomyl concentration, can extrapolate the concentration of Methomyl to be measured.
Advantage of the present invention is accurately to detect methomyl residue in the water and soil earth delicately, and pre-treatment process in sample ground is easy, and is consuming time few, can detect sample in large quantities simultaneously, and the sample detection cost is far below traditional instrument detecting method.The storage life of kit was greater than 6 months.
Four, description of drawings
Accompanying drawing is that the standard of Methomyl suppresses curve, and the regression equation of curve is y=12.55x-3.2714 (R
2=0.9984, I
50=500.0ng/mL, I
20=100.0ng/mL).
Five, embodiment
Kit operation and result calculate: testing sample is after pre-treatment, and is standby with the PBST constant volume.Take vacuum packaging bag apart and take out ELISA Plate, put room temperature (20～25 ℃) more than the balance 30min.(0.0,10.0,50.0,100.0,250.0,500.0,1000.0,2000.0,5000.0, Methomyl titer 10000.0ng/mL) or Methomyl liquid to be measured mix, and hatch 30 minutes for 37 ℃ with variable concentrations respectively with the antibody of same concentrations; Then this reaction mixture of 100 μ L is moved in the ELISA Plate, hatched 30 minutes for 37 ℃; Take out back washing 3 times, pat dry; Every hole adds the goat anti-rabbit igg-HRP liquid of 100 μ L through 2000 times of PBS dilutions, puts into 37 ℃ behind the shrouding and hatches 1h, and washing pats dry; Get A liquid and B liquid equal-volume mixing, every hole adds 100 μ L, in the dark develops the color 10～15 minutes, and the sulfuric acid cessation reaction that every hole adds 50 μ L 2moL/L leaves standstill 2min, measures the OD in each hole in stop buffer 30min with microplate reader
492Value.
The OD value that will contain 0.0ng/mL standard items hole deducts the OD value that contains Cmax standard items hole and is decided to be B
0, the OD value after all the other apertures are proofreaied and correct with quadrat method is decided to be B: with standard solution concentration logarithm is horizontal ordinate, inhibiting rate I (I=(B
0-B)/B
0) be ordinate, draw out the Methomyl standard and suppress curve.The concentration of respective sample can be extrapolated according to the regression equation of curve, also I can be extrapolated
50(B/B
0=50.0%) and minimum detectable level I
20(B/B
0=80.0%).I wherein
50For suppressing the required Methomyl concentration of 50.0% antigen-antibody reaction, I
20For suppressing the required Methomyl concentration of 20.0% antigen-antibody reaction.
Embodiment 1
The Methomyl haptens is synthetic:
Being MHTA with the former medicine purification of Methomyl posthydrolysis at first, is Methomyl monomester succinate (MOSE) with succinic anhydride with its esterification then.
(1) the former medicine of Methomyl adds the methyl alcohol of 1.5～3 times of volumes, and 6h is extracted in vibration, and the filtration rear filtrate is concentrated into 0.5～1.0 times of former filtrate volume, adds the water of 0.7～1.7 times of volume of concentrated filtrate again, the white Methomyl crystal after obtaining purifying after leaving standstill;
(2) get the pure product 0.03mol of the Methomyl that makes and be dissolved in the NaOH solution of 70mL0.67mol/L, solvent is methyl alcohol: water=1:3 (v/v).25 ℃ of vibration 36h, mixed liquor extract with the 80mL isopropyl ether after regulating pH=7 with hydrochloric acid, with organic phase extract concentrating under reduced pressure, obtain MHTA;
(3) add succinic anhydride in MHTA, the mol ratio of MHTA and succinic anhydride is 1:1～1.2, adds in the anhydrous tetrahydrofuran of 4mL triethylamine and 20mL again.Back flow reaction 24h;
(4) boil off tetrahydrofuran and triethylamine, residue is dissolved in the 30mL ethyl acetate, regulates pH=3～4 with hydrochloric acid under 4 ℃ of stirrings, uses ethyl acetate extraction behind the separatory again, and organic phase is in anhydrous Na
2SO
4After the last dry also filtration, boil off ethyl acetate, add the ice-cold ether of 10mL in the crude product, remove by filter the precipitation of white, filtrate boils off ether, and the solid vacuum drying that obtains obtains Methomyl haptens-Methomyl monomester succinate (MOSE).
Embodiment 2
Methomyl and carrier protein couplet:
(1) water-soluble carbodiimide (EDC) legal system is equipped with immunogene (MOSE-BSA)
Take by weighing synthetic MOSE 100mg and be dissolved in the anhydrous dioxane of 10mL, add several of NaOH then, vibration makes it abundant dissolving, takes by weighing carrier protein BSA100mg and is dissolved in the borate buffer of 10mL 0.1moL/L pH9.0, and EDC 50mg is dissolved in the 10mL distilled water.With the mixed liquor of Methomyl and carrier protein at the room temperature 18h that vibrates, the centrifugal 10min of 4000rpm then, get in the EDC solution that supernatant dropwise adds vibration, add follow-up persistent oscillation 5h, the centrifugal 10min of 4000rpm, with the reactant liquor bag filter of packing into, using distill water dialysis 3 times earlier, is the PBS dialysis 3d of 0.01moL/L pH7.4 with dislysate then, changes liquid every day four times, identify synthetic result with polyacrylamide gel electrophoresis at last, and measure the calculations incorporated ratio with ultraviolet spectrophotometer.Packing is stored in 4 ℃ the refrigerator standby.
(2) the mixed anhydride method coupling prepares coating antigen (MOSE-OVA)
Take by weighing 450mgMOSE and be dissolved in the 3mL anhydrous dimethyl base acid amides (DMF), open and stir, add 64 μ L tri-n-butylamines then, ice bath slowly drips the isobutyl chlorocarbonate 35 μ L by the dissolving of 1mL anhydrous dimethyl base acid amides down, finish, 4 ℃ of hybrid reaction 1h get mixed acid anhydride.
Take by weighing 450mg OVA and be dissolved in the carbonate buffer solution of 2mL 0.05moL/L pH9.5, dropwise add the above-mentioned mixed anhydride reaction liquid of 100 μ L, the limit edged stirs, and the stirring at room reaction is spent the night.
Next day, with the reactant liquor bag filter of packing into, 4 ℃ down earlier with distill water dialysis 3 times, is the PBS of the 0.01moL/LpH7.4 3d that dialyses with dislysate then, changes liquid every day 4 times, till can not surveying haptens and getting uv absorption.After the accurate Calculation dialysis conjugate solution get volume, measure the calculations incorporated ratio with ultraviolet spectrophotometer, packing is stored in 4 ℃ the refrigerator standby.
Embodiment 3
The preparation of Methomyl polyclonal antibody:
With the MOSE-BSA conjugate as immunogene, carry out fundamental immunity earlier, immunizing dose 0.5mg/kg (in carrier protein) rabbit body weight, mix with the equivalent complete Freund's adjuvant, the injection of fully emulsified back, adopt auricular vein injection and neck, the subcutaneous multi-point injection in back combines, every 2 all booster immunizations once, using incomplete Freund's adjuvant instead mixes with immunogene, from booster immunization for the third time, back 7 days of each immunity is got blood 0.2～0.5mL from rabbit ear edge vein, measure the antiserum quality with indirect elisa method, adopt whole blood from rabbit heart behind the booster immunization 5 times, slightly carry rabbit anti-serum with 35% saturated ammonium sulfate salting out method earlier, be further purified with the DE-52 anion exchange chromatography again, obtain purer Methomyl polyclonal antibody.
Embodiment 4
The storage life experiment:
Kit is put into 37 ℃ of constant incubators do storage preservation experiment, 4 ℃ of refrigerator cold-storages compare.With best antigen-antibody working concentration serves as to measure concentration, and every 24h detects 3 samples of high, medium and low content respectively with the kit of preserving in the 37 ℃ of kits stored and the refrigerator, and each sample is 3 repetitions, follow-on test 7d.At 37 ℃, 7d measures under preserving by kit, and it is still good to detect effect after preserving 7d, according to convention, preserves 7d for 37 ℃, is equivalent to 4 ℃ and preserves more than 6 months.To note the stable of maintenance condition in the practical application, prolong storage life.
Embodiment 5
The experiment of kit specificity
The analogue of selecting Methomyl is as determinand, records concentration (I in the inhibition of each material
50), use the cross reactivity of following formula calculating antibody to these materials again: cross reacting rate is more little, and then antibody is strong more to the specificity of Methomyl, on the contrary the poor specificity of antibody then.
Cross reaction (CR%)=I
50(Methomyl)/I
50(for the examination thing) * 100%.
Test determination the results are shown in Table 1, adopts indirect elisa method, and the agricultural chemicals of being surveyed all is lower than 5% to the cross reacting rate of Methomyl antibody, can illustrate that the antibody that test obtains has specificity preferably.
Table 1 antibody cross reaction measurement result
Embodiment 6
Add and reclaim experiment
Get an amount of Methomyl standard specimen and add in the sample, 0.5,5,50 a μ g/mL3 interpolation level is set, each level repeats 3 times, measures calculate recovery rate.
The pre-treatment of ELISA test sample:
Wild cabbage and rape: sample thief 5.0g, pulverize and make homogenate, add 20mL acetone behind the Methomyl of interpolation variable concentrations, vibration is filtered, and is concentrated into dried.Add the saturated NaCL aqueous solution of 20mL, the 5mL0.5mol/L sulfuric acid solution is with normal hexane 30mL extraction 3 times, discard the hexane phase, water continues the extraction with methylene chloride 50mL, aqueous phase discarded, dichloromethane layer changes over to concentrate in the 250mL round-bottomed flask and dries up, and is standby to 2mL with the PBS constant volume.
Water sample: filter and remove mechanical impurity, centrifugal behind the Methomyl of interpolation variable concentrations, be directly used in ELISA and detect.
Soil sample: take by weighing the 50g soil sample, add the aqueous solution of variable concentrations Methomyl standard specimen, air-dry, add 50mL distilled water and 100mL acetone, suction filtration behind the mechanical oscillation.With washing with acetone residue and filtration, pressure reducing and steaming acetone, use the PBS constant volume to 1mL then, elisa assay is carried out in sampling.
The testing result of kit sees Table 2,
The recovery of wild cabbage is 77%～85.36%, the coefficient of variation 5.23%～7.36%; The recovery of rape is 79.6%～85.4%, the coefficient of variation 4.28%～6.12%; The phreatic recovery is 86.32%～89%, the coefficient of variation 3.61%～4.82%; The recovery of soil is 65.6%～70.8%, the coefficient of variation 6.92%～8.34%.The recovery of kit meets the requirement of pesticide residue analysis in allowed band.
Table 2 kit adds the recovery measurement result
Claims (9)
1. the fast detecting ELISA kit of a methomyl residue is characterized in that being made up of the reagent bottle that includes the Methomyl titer of concentrated cleaning solution (PBST), variable concentrations, anti-Methomyl polyclonal antibody, horseradish peroxidase-labeled goat anti-rabbit antibody, A liquid, B liquid and reaction terminating liquid in box body, detachable 96 hole ELISA Plate, sponge bracket and the support.
2. a kind of fast detection ELISA reagent kit for methomyl residue according to claim 1 is characterized in that the hole slot that reagent bottle is housed is arranged on the described sponge bracket.
3. the fast detecting ELISA kit of a kind of methomyl residue according to claim 1, it is characterized in that described detachable 96 hole ELISA Plate aluminium foil bag vacuum packagings, the lath of ELISA Plate detachably gets off, and all is adsorbed with the envelope antigen of same amount in each hole.
4. envelope antigen according to claim 3 is characterized in that the coupled complex for Methomyl and ovalbumin (OVA).
5. the fast detecting ELISA kit of a kind of methomyl residue according to claim 1, it is characterized in that described anti-Methomyl preparation method of polyclonal antibody is as follows: Methomyl is joined in the NaOH solution, solvent is a methyl alcohol: water=1: 3 (v/v), 25 ℃ of reactions were transferred pH=7 after 36 hours.With the isopropyl ether extraction, obtain MHTA afterwards.Product and succinic anhydride and triethylamine are dissolved in the anhydrous tetrahydrofuran, back flow reaction 24h, residue is dissolved in the ethyl acetate, dry and concentrating under reduced pressure on anhydrous sodium sulfate, crude product is iced ether dissolution, is filtered the distillation filter liquor with 10mL, obtains haptens Methomyl succinic acid oxime ester (MOSE).As immunogene, immune new zealand white rabbit obtains the polyclonal antibody of Methomyl with the compound of haptens (MOSE) and bovine serum albumin(BSA) (BSA) coupling.
6. the fast detecting ELISA kit of a kind of methomyl residue according to claim 1 is characterized in that the Methomyl titer of described variable concentrations is prepared with methyl alcohol and distilled water, and the content of methyl alcohol is less than 1.0% in the solution, concentration is respectively 0.0,10.0,50.0,100.0,250.0,500.0,1000.0,2000.0,5000.0,10000.0ng/mL.
7. the fast detecting ELISA kit of a kind of methomyl residue according to claim 1 is characterized in that described concentrated cleaning solution (PBST) contains sodium chloride 7～9g, potassium dihydrogen phosphate 0.1～0.3g, sodium hydrogen phosphate 2～4g, potassium chloride 3～6g, tween-201mL, distilled water 50mL.
8. the fast detecting ELISA kit of a kind of methomyl residue according to claim 1 is characterized in that described A liquid: 30% hydrogen peroxide, 5 μ l, 96mg citric acid, 358mg Na
2HPO
4, tween-201 μ l, distilled water 10mL, pH5.0; B liquid: o-phenylenediamine (0PD) 4mg, distilled water 10mL.
9. the fast detecting ELISA kit of a kind of methomyl residue according to claim 1 is characterized in that described reaction terminating liquid is the sulfuric acid liquid of 2mol/L.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008102469598A CN101504409A (en) | 2008-12-31 | 2008-12-31 | Fast detection ELISA reagent kit for methomyl residue |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008102469598A CN101504409A (en) | 2008-12-31 | 2008-12-31 | Fast detection ELISA reagent kit for methomyl residue |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113447646A (en) * | 2021-06-22 | 2021-09-28 | 浙江理工大学 | By using C-SiO2Method for accelerating ELISA detection process by using adsorbing material |
| CN115073337A (en) * | 2022-05-16 | 2022-09-20 | 江南大学 | Methomyl hapten, holoantigen, monoclonal antibody, preparation method and application thereof |
-
2008
- 2008-12-31 CN CNA2008102469598A patent/CN101504409A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113447646A (en) * | 2021-06-22 | 2021-09-28 | 浙江理工大学 | By using C-SiO2Method for accelerating ELISA detection process by using adsorbing material |
| CN113447646B (en) * | 2021-06-22 | 2024-03-19 | 浙江理工大学 | By using C-SiO 2 Method for accelerating ELISA detection process by adsorption material |
| CN115073337A (en) * | 2022-05-16 | 2022-09-20 | 江南大学 | Methomyl hapten, holoantigen, monoclonal antibody, preparation method and application thereof |
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Application publication date: 20090812 |