CN1687782A - Analytical kit of enzyme linked immunosorbent assay for residual carbaryl - Google Patents
Analytical kit of enzyme linked immunosorbent assay for residual carbaryl Download PDFInfo
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- CN1687782A CN1687782A CN 200510041981 CN200510041981A CN1687782A CN 1687782 A CN1687782 A CN 1687782A CN 200510041981 CN200510041981 CN 200510041981 CN 200510041981 A CN200510041981 A CN 200510041981A CN 1687782 A CN1687782 A CN 1687782A
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- carbaryl
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- linked immunosorbent
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Abstract
This inventon shows a reagent box , which is used for enzyme linked immunosorbent assay of remained carbaryl. It belongs to enzyme linked immunosorbent assay technology. The common methods of carbaryl rationalize analyze are too complex, slow and its cost is high. This invention overcomes these defaults. It can quickly detect the remained carbaryl in water, soil and food samples. In the process, the antigen on the enzyme target rivals carbaryl . It reacts with the antigens in the solution. And the result is showed by enzyme catalysis color reaction. You can estimate the consistency of carbaryl by detecting the solution, whose consistency of carbaryl is known and drawing calibration vurve. The retention period of the box is more than 6 months.
Description
One, technical field
The present invention relates to a kind of Enzyme Linked Immunoadsorbent Assay (ELISA) kit of carbaryl residue, mainly be applicable to the fast measuring of carbaryl residue in the samples such as vegetables in enormous quantities, water, soil.Belong to EIA enzyme immunoassay
Technical field.
Two, background technology
Carbamate insecticides is the main pesticide type of China's production application, and carbaryl is wherein a kind of, and its trade name is a sevin.Turnout at China's carbaryl is big, use wider, be used to prevent and treat multiple kinds of crops insects such as rice hoppers, rice leafhopper, cotton pink bollworm, eating-core bean worm, also be used to prevent and treat livestock and poultry parasite and domestic sanitary pesticide, its main poisonous metabolic product is the 1-naphthols.Carbaryl is considered to a kind of safe pesticide, but a lot of scholars studies show that carbaryl is to people and animals' toxic side effect: as long-term contact carbaryl, can cause the variation of mouse liver microsomal enzyme, under isolated culture condition, cause the variation of large-scale granular lymphocyte immunologic function; Animal had inferior chronic neurotoxicity.The widespread use of carbaryl also causes the concern of people to soil, pollution of waterhead, and its residual frequency of occurrences in food, fruit and manufactured goods thereof constantly increases.It is as follows that the youngster of the mandatory national Specification of China plants in the agricultural product the high residue amount of carbaryl.
The high residue amount of carbaryl in several agricultural product of table 1
| The agricultural product title | High residue amount (MRL, mg/kg) | Standard No. |
| Grain edible oil vegetable and fruit tobacco | ????5 ????0.5 ????2 ????2.5 ????1 | ?GB14971-94 ?GB14971-94 ?GB14971-94 ?GB14971-94 ?GB14971-94 |
The analytical approach that carbamate pesticide residue is commonly used mainly is that application gas chromatography (GC) and high performance liquid chromatography instruments such as (HPLC) carry out in the laboratory.Using these physico-chemical analysis technology analyzes trace carbaryl residue in the samples such as environment, biology, food, not only the instrumentation degree is had relatively high expectations, and need through complicated separation, extraction, the pre-treatment process such as purify, derive, analysis speed is slow, cost is high, the pre-treatment process need uses a large amount of organic solvents, has caused new environmental pollution again.Many physico-chemical analysis technology itself just have limitation, as the poor heat stability of carbaryl, are difficult to use gc analysis, and liquid chromatography still lacks the good high sensitivity detector of selectivity etc.Along with sample to be checked, particularly require increasing sharply of field quick detection sample size, traditional pesticide residue analysis means are difficult to adapt to requirement, therefore, press for development and application high-level efficiency residues of pesticides express-analysis technology.
Enzyme inhibition method is present the most widely used rapid detection for pesticide residue method, it is a kind of Detecting Pesticide method of utilizing organophosphorus and carbamate chemicals for agriculture that the degree of inhibition of AchE principle is set up, its sensitivity is in 0.1～10mg/L scope, the shortcoming that enzyme suppresses rapid method is that the enzyme great majority derive from animal or plant, material expensive, the sensitivity and the degree of stability of the enzyme that obtains from different materials are different, and preserve difficulty, this method can only be used for the qualitative detection of a class agricultural chemicals.
The present invention adopts ELISA adsorption analysis method based on the carbaryl monoclonal antibody.This method high specificity, highly sensitive and easyly fast can remedy the defective of chromatogram analysis method and the enzyme method of inhibitioning, thus the present invention detection has great importance for the carbaryl residue in actual environment and the food samples.
Three, summary of the invention
In order to solve that present carbaryl residues of pesticides chromatogram analysis method cost height, complicated operation and enzyme suppress poor specificity in the method for quick, sensitivity is low and shortcoming such as testing result instability, the invention provides and a kind ofly have high specific, high sensitivity, pin-point accuracy, cost is low, method of operating simple and can be used for the analytical kit of enzyme linked immunosorbent assay of the carbaryl residue of batch samples fast detecting.
The analytical kit of enzyme linked immunosorbent assay of carbaryl residue comprises box body, is located at test solutions such as the concentrated cleaning solution that reagent bottle is equipped with in detachable 96 hole ELISA Plate, sponge carriage and the carriage in the box body, the carbaryl titer of variable concentrations, anti-carbaryl monoclonal antibody, horseradish peroxidase-labeled haptens, A liquid, B liquid, reaction terminating liquid.
The technical solution adopted for the present invention to solve the technical problems is: at first haptens N-(1-the naphthalene oxygen carbonyl)-6-aminocaprolc acid of carbaryl is synthesized in design, to prepare artificial immunogen after haptens and bovine serum albumin(BSA) (BSA) coupling again, immunity Balb/c small white mouse, get its splenocyte and Sp2/0 myeloma cell and merge, prepare the carbaryl monoclonal antibody.Monoclonal antibody is adsorbed in the ELISA Plate hole, and the content of every hole antibody is identical.Haptens and horseradish peroxidase are prepared enzyme mark haptens.The carbaryl titer that adds concentration known in the ELISA Plate hole earlier adds enzyme mark haptens then; Carbaryl and enzyme mark haptens is vied each other and the solid phase monoclonal antibody reactive in the hole, because the uniform content of the insolubilized antibody in each hole causes, so when the carbaryl concentration in the hole is high, then just few by the enzyme of antibodies mark haptens, add substrate solution (A liquid) and colour developing liquid (B liquid), chromogenic reaction shallow (the OD value is low) shows the inhibiting rate height; Otherwise when carbaryl concentration in the hole was low, the OD value of then being surveyed was high, and inhibiting rate is low.When the adding testing sample detects in the hole,, can extrapolate the concentration of carbaryl to be measured according to detecting the typical curve of being done with known carbaryl concentration.
Advantage of the present invention is accurately to detect carbaryl residue in the food such as water, soil and vegetables delicately, and the pre-treatment process of sample is simple, and is consuming time few, can detect a large amount of samples simultaneously, and the sample detection cost is far below traditional instrument detecting method.Kit under 4 ℃ condition storage life above 6 months.
Four, description of drawings
Accompanying drawing is that the standard of carbaryl suppresses curve, and the regression equation of curve is Y=-0.3515X+1.0635 (R
2=0.9812), suppresses 50% antigen-antibody reaction desired concn IC
50=39.8ng/mL suppresses 20% antigen-antibody reaction desired concn IC
20=5.6ng/mL.
Five, embodiment
Kit operation: take out kit from 4 ℃ of refrigerators, at room temperature balance 5～10 minutes is standby; Testing sample is after pre-treatment, and is standby with the PBST constant volume after diluting 20 times.Take vacuum packaging bag apart and take out ELISA Plate, add the carbaryl titer of 50 μ L and the sample handled well in each hole, standard specimen and sample are done 2～4 repetitions; Enzyme mark haptens dilutes certain multiple by kit operation requirements with PBST, add again 50 μ L to above-mentioned standard specimen with treat in the test sample hole, hatched 30 minutes for 37 ℃; Pour out the liquid in the hole, the PBST good with dilution washes 2～6 times, ELISA Plate is upside down in pat dry on the thieving paper; Get A liquid and B liquid equal-volume mixing, every hole adds 100 μ L mixed liquors, under 37 ℃ of conditions, and dark place colour developing 10～15 minutes.Last every hole adds the stop buffer cessation reaction of 50 μ l, and measuring each hole on the microplate reader is the OD value at 450nm place at wavelength.
The result calculates: the OD value that will contain 0ng/mL titer hole deducts the OD value that contains Cmax titer 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 B/B
0Value is ordinate, and the logarithm value (logC) of respective standard liquid concentration is a horizontal ordinate, draws the carbaryl standard and suppresses curve.Can extrapolate the concentration of counter sample according to the regression equation of curve, also can extrapolate carbaryl and suppress 50% antigen-antibody reaction desired concn IC
50(B/B
0=50%) and minimum detectable level IC
20(B/B
0=80%).
Embodiment 1
Synthetic haptens
5.6g NaOH (0.139mol) is dissolved in the 56mL distilled water; In solution, add 20g1-naphthols (0.139mol).Potpourri is cooled to room temperature at 85 ℃ of following stirring reaction 1h.In fuming cupboard, slowly add excessive triphosgene (19.3g triphosgene+100mL toluene; The triphosgene severe toxicity, careful), stirring reaction 1h under the room temperature.The organic phase anhydrous Na
2SO
4Drying, vacuum decompression is taken off organic solvent, gets brown oil matter.It is dissolved in methylene chloride, and 100 ℃ cut is collected in vacuum distillation then (1mmHg), gets the faint yellow oily intermediate product of 13.7g (48%) (chloro-carbonic acid naphthyl ester).
Claim 7.37g 6-aminocaprolc acid (56.1mmol) to be dissolved among the NaOH of 7.5mL 4mol/L, be cooled to 4 ℃.(30.3mmol) is dissolved in 11mL1 with the 6.25g intermediate product, in the 4-dioxane, be cooled to 4 ℃, mix with the cold 4mol/L NaOH of 7.5mL then, divide 5 inferior batches to add in the 6-aminocaprolc acid alkali lye, each 5min at least at interval that adds is behind the mixed liquor stirring reaction 3h, with concentrated hydrochloric acid the pH value is transferred to 4, this two-step reaction all carries out in ice bath.Gained oily product with ethyl acetate extraction (3 * 35mL), organic phase with the salt acid elution of 1mol/L (2 * 15mL), use the NaHCO of 1mol/L then
3(3 * 50mL), extract is used the concentrated hydrochloric acid acidifying of pH3 at last in ice bath, get yellow solid in extraction, with distilled water washing 2 times, get the 7.5g crude product after the drying, use the absolute ethyl alcohol recrystallization, get 2g white product, productive rate 65%, 128～130 ℃ of fusing points after the drying.White product promptly is a haptens.
Embodiment 2
Preparation immunogene and enzyme mark haptens
(1) immunogene
Get the about 12.2 μ mol of haptens and be dissolved in the anhydrous N of 200 μ L, among the N '-dimethyl formamide (DMF), be sequentially added into 2.9 μ L (about 12.2 μ mol) tri-n-butylamine, 1.6 μ L (about 12.3 μ mol) isobutyl chlorocarbonate then, stirring reaction 1h under the room temperature.Reactant liquor dilutes with the 1.8mL dry DMF.
Claim 30mg BSA be dissolved in the 2mL carbonate buffer solution (0.05mol/L, pH9.6) in, in BSA solution, dropwise add 100 μ L first step reactant liquors respectively under the stirring, about 20min adds, stirring reaction 3h under the room temperature.Dialysis, centrifugal, freeze drying then, 4 ℃ of preservations.
(2) enzyme mark haptens
The method that adopts is synthetic identical with immunogene, and horseradish peroxidase (HRP) is substituted BSA.In conjugate, add isopyknic glycerine ,-20 ℃ of preservations at last.
Embodiment 3
The carbaryl Monoclonal Antibody
(1) immune Balb/c small white mouse, immunogene dosage are 100 μ g/ at every turn, before the immunity immunogene are diluted to certain concentration.Use Freund's complete adjuvant and immunogene mixing and emulsifying during initial immunity, the booster immunization incomplete Freund, per 2 all immunity once are total to immunity 5 times, and the injection site is that mouse back is subcutaneous.Last immunity need not any adjuvant, the mouse peritoneal injection.
(2) preparation feeder cells, the splenocyte and the Sp2/0 myeloma of getting immune mouse merge, and splenocyte and myeloma cell's ratio are 10: 1～3, and fusion agent is PEG2000;
(3) merged the back the 8th day, detect fused cell culture supernatant antibody horizontal with indirect non-competing ELISA method earlier, the OD value is decided to be the positive greater than 1.0 hole.Detect the positive cell hole with the indirect competitive ELISA method again, the carbaryl concentration of adding is 1 μ g/mL, selects inhibiting rate to clone greater than 70% positive cell;
(4) the limiting dilution assay subclone is 3 times, and behind the 3rd time cloning, non-competing ELISA detects the still all positive person of each cell hole, is the monoclonal strain that filters out, then enlarged culture and frozen;
(5) induce method in the animal body and prepare monoclonal antibody.The inoculation hybridoma: the positive colony hybridoma of cultivating in the cell bottle is blown down, and collecting cell transfers to 10 with cell number
6Individual/mL, injection 1mL is to the mouse peritoneal of handling with paraffin oil in advance.
(6) 7～9d begins to collect ascites behind the inoculation hybridoma, and ascites is collected monoclonal antibody with saturated ammonium sulfate (SAS) salting out method precipitation.
Embodiment 4
Coated antibody
(1) phosphate buffer (PBS) of preparation 0.02mol/L pH7.2, dilute monoclonal antibody with damping fluid, the concentration of antibody is 2 μ g/mL, with the micro sample-adding rifle above-mentioned dilution is added in the ELISA Plate (100 μ L/ hole), 4 ℃ are spent the night, abandon damping fluid, wash plate 3 times, on thieving paper, pat dry with PBST.
(2) preparation confining liquid: 1g BSA is dissolved among the 0.02mol/L pH7.2 PBS of 100mL.In above-mentioned ELISA Plate hole, add confining liquid (150 μ L/ hole), at room temperature seal 2h, abandon confining liquid, with PBST washing 3 times.Vacuumize packing after patting dry the liquid in the hole.
Embodiment 5
The experiment of kit storage life
Kit is positioned over 4 ℃ of preservations, get respectively 0,10,20,30,60,90,120,150 and the kit of 180d experimentize, drawing standard suppresses curve, and according to regression equation calculation IC
50The storage life measurement result sees Table 2:
Table 2 kit storage life experimental result
| Time (d) | ??0 | ???10 | ???20 | ???30 | ???60 | ??90 | ???120 | ???150 | ???180 |
| B 0(450nm) | ??1.42 | ???1.38 | ???1.40 | ???1.40 | ???1.40 | ??1.38 | ???1.37 | ???1.42 | ???1.39 |
| IC 50(ng/mL) | ??41.0 | ???40.4 | ???39.2 | ???39.8 | ???41.9 | ??42.5 | ???42.2 | ???42.6 | ???42.0 |
As can be seen from the above results, the hole OD value variation that does not add the carbaryl standard items is little, IC
50Change also not quite, kit can be preserved 6 months under 4 ℃ at least.
Embodiment 6
The experiment of kit specificity
Select the metabolin alpha-Naphthol of carbaryl and carbamate chemicals for agriculture carbofuran commonly used, isoprocarb, Methomyl, Aldicarb as determinand, the standard of drawing these determinands respectively suppresses curve, obtains concentration (IC in the inhibition of various materials
50), calculate the cross reactivity of monoclonal antibody with following formula again to these materials; Cross reacting rate is littler, and then antibody is stronger to the specificity of carbaryl, on the contrary the poor specificity of antibody then.
Cross reaction (CR%)=IC
50(carbaryl)/IC
50(for the examination thing) * 100%.
This measuring the results are shown in Table 3, can know from table 3, adopt direct ELISA method, monoclonal antibody to the cross reaction of carbamate chemicals for agriculture commonly used and alpha-Naphthol all less than 1%, the specificity that this kit is described is good, can guarantee the reliability to carbaryl residue measurement result in the sample.
Table 3 cross reaction
| The compound title | IC 50(μg/mL) | Cross reaction (%) |
| Carbaryl alpha-Naphthol carbofuran Methomyl Aldicarb isoprocarb | ????0.042 ????68.7 ????7.76 ????＞50 ????＞60 ????21.3 | ????100 ????0.06 ????0.54 ????＜0.1 ????＜0.1 ????0.197 |
Embodiment 7
Add and reclaim experiment
Get an amount of carbaryl standard specimen and add in the sample, 50 μ g/kg are set, 100 μ g/kg and three concentration of 200 μ g/kg, each concentration is established 6 repetitions, measures.Kit measurement result and high performance liquid chromatography (HPLC) measurement result relatively.
The pre-treatment of ELISA test sample:
Water sample: add the ethylenediamine tetraacetic acid (EDTA) of 10mmol/L, can take a sample after the filtration and carry out elisa assay.
Soil sample: get 10g soil with 20～40mL methanol extraction three times, merge extract, concentrate, be settled to 10mL with the PBST dilution then, carry out elisa assay.
Vegetable sample: get and take by weighing 10g after vegetable sample rubs with comminutor, 20～40mL methanol extraction three times merges extract, concentrates, and is settled to 10mL with PBST, and elisa assay is carried out in sampling.
The testing result of kit sees Table 4, and the water sample recovery is 87.45～93.46%, the coefficient of variation 5.64～6.89%, the pedotheque recovery is 85.3～89.7%, the coefficient of variation 5.30～5.88%, the vegetable sample recovery are 88.9～92.45%, the coefficient of variation 4.5～5.20%.The recovery of kit is in allowed band, and is and consistent with the HPLC measurement result, meets the requirement of pesticide residue analysis to degree of accuracy.
The comparison of table 4 kit measurement result and HPLC measurement result
| Sample | Add concentration (μ g/kg) | The recovery (%) | The coefficient of variation (%) | ||
| The ELISA kit | ?HPLC | The ELISA kit | ??HPLC | ||
| Water | ????50 ????100 ????200 | ????87.45 ????88.6 ????93.46 | ?92.6 ?96.3 ?106.5 | ???5.64 ???6.89 ???6.77 | ??6.35 ??5.60 ??5.00 |
| Soil | ????50 ????100 ????200 | ????86.8 ????89.7 ????85.3 | ?107.0 ?102.3 ?91.2 | ???5.88 ???5.28 ???5.30 | ??8.62 ??9.34 ??7.54 |
| Vegetables | ????50 ????100 ????200 | ????92.45 ????90.4 ????88.9 | ?105.0 ?89.3 ?87.6 | ???4.50 ???4.85 ???5.20 | ??8.86 ??8.75 ??9.56 |
Claims (9)
1. the Enzyme Linked Immunoadsorbent Assay of a carbaryl residue (ELISA) kit is characterized in that being made up of box body, detachable 96 hole ELISA Plate, sponge carriage and reagent bottle that carbaryl titer, horseradish peroxidase-labeled haptens, A liquid, B liquid and the reaction terminating liquid of concentrated cleaning solution, variable concentrations be housed.All reagent bottles are placed in the hole slot of sponge carriage, and sponge carriage and ELISA Plate all are placed in the box body.
2. the analytical kit of enzyme linked immunosorbent assay of a kind of carbaryl residue according to claim 1 is characterized in that having on the described sponge carriage hole slot of installed reagents bottle.
3. the analytical kit of enzyme linked immunosorbent assay of a kind of carbaryl residue according to claim 1 is characterized in that described horseradish peroxidase-labeled haptens is the compound of horseradish peroxidase and haptens N-(1-naphthalene oxygen carbonyl)-6-aminocaprolc acid coupling.Haptens chemistry structural formula:
Application mix acid anhydrides method coupling horseradish peroxidase and haptens.
4. the analytical kit of enzyme linked immunosorbent assay of a kind of carbaryl 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 can disassemble from grillage, all wraps in each hole by the anti-carbaryl monoclonal antibody of same amount.
5. anti-carbaryl monoclonal antibody according to claim 4, its preparation method is as follows: with the compound of haptens N-(1-naphthalene oxygen carbonyl)-6-aminocaprolc acid and bovine serum albumin(BSA) coupling as immunogene, behind the immunity Balb/c small white mouse, get its splenocyte and Sp2/0 myeloma cell and hybridize fusion, filter out can the anti-carbaryl monoclonal antibody of stably excreting hybridoma cell strain, hybridoma is injected mouse peritoneal, collect ascites then, can obtain a large amount of monoclonal antibodies.
6. the analytical kit of enzyme linked immunosorbent assay of a kind of carbaryl residue according to claim 1 is characterized in that the carbaryl titer of described variable concentrations is prepared with methyl alcohol and distilled water, and the content of methyl alcohol is less than 0.5% in the solution.Concentration is respectively 0ng/mL, 3ng/mL, 10ng/mL, 30ng/mL, 90ng/mL, 270ng/mL, 810ng/mL.
7. the analytical kit of enzyme linked immunosorbent assay of a kind of carbaryl 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-20 1mL, distilled water 50mL.
8. the analytical kit of enzyme linked immunosorbent assay of a kind of carbaryl residue according to claim 1 is characterized in that described A liquid: peroxidating urine 10mg, 103mg citric acid, 358mg Na
2HPO
412 H
2O, Tween-20 1 μ L, distilled water 10mL, pH5; B liquid: tetramethyl benzidine (TMB) 4mg (with the dissolving of 500 μ L dimethyl sulfoxides), 103mg citric acid, distilled water 9.5mL, pH2.4.
9. the analytical kit of enzyme linked immunosorbent assay of a kind of carbaryl 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 |
|---|---|---|---|
| CN 200510041981 CN1687782A (en) | 2005-04-21 | 2005-04-21 | Analytical kit of enzyme linked immunosorbent assay for residual carbaryl |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510041981 CN1687782A (en) | 2005-04-21 | 2005-04-21 | Analytical kit of enzyme linked immunosorbent assay for residual carbaryl |
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| CN106680484A (en) * | 2016-11-15 | 2017-05-17 | 中国农业大学 | ELISA (enzyme-linked immuno sorbent assay) detection kit for analyzing residual carbaryl and application thereof |
| CN106872706A (en) * | 2017-01-25 | 2017-06-20 | 中国农业科学院油料作物研究所 | A kind of FPIA method for detecting carbaryl |
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| CN106680484A (en) * | 2016-11-15 | 2017-05-17 | 中国农业大学 | ELISA (enzyme-linked immuno sorbent assay) detection kit for analyzing residual carbaryl and application thereof |
| WO2018121677A1 (en) * | 2016-12-31 | 2018-07-05 | 中国农业科学院油料作物研究所 | Time-resolved fluorescence immunochromatography kit for simultaneous detection of mixed pollutant of aflatoxin and carbaryl, preparation method therefor, and application thereof |
| CN106872706A (en) * | 2017-01-25 | 2017-06-20 | 中国农业科学院油料作物研究所 | A kind of FPIA method for detecting carbaryl |
| CN106872706B (en) * | 2017-01-25 | 2019-01-04 | 中国农业科学院油料作物研究所 | It is a kind of for detecting the fluorescence polarization immunoassay method of carbaryl |
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| CN109752527A (en) * | 2017-11-05 | 2019-05-14 | 镇江华维检测技术有限公司 | The preparation of the kit of antistaling agent biphenyl in a kind of detection fruits and vegetables |
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| CN113049812A (en) * | 2021-03-26 | 2021-06-29 | 信达安检测技术(天津)有限公司 | ELISA method for detecting carbofuran and kit thereof |
| CN113049812B (en) * | 2021-03-26 | 2024-03-19 | 信达安检测技术(天津)有限公司 | ELISA method for detecting carbofuran and kit thereof |
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