CN101526526B - Indirect competitive enzyme-linked immunosorbent assay kit for cyhalothrin residual - Google Patents
Indirect competitive enzyme-linked immunosorbent assay kit for cyhalothrin residual Download PDFInfo
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Abstract
The invention provides an indirect competitive enzyme-linked immunosorbent assay kit suitable for cyhalothrin residual, which comprises elisa plate coated with cyhalothrin antigen, concentrated cleaning liquid, cyhalothrin standard, cyhalothrin specific antibody, enzyme marker, substrate coloration liquid and reaction stopping solution. The invention has the advantages that the cyhalothrin residual in the water can be accurately and sensitively detected, the pretreatment process of the samples is simple and consumes less time, a great amount of the samples can be detected, and the detection cost of the samples is far lower than that of the detection method of the traditional instrument.
Description
Technical field
The present invention relates to a kind of indirect competitive enzyme-linked immunosorbent adsorption analysis (ELISA) kit of cyhalothrin residual, mainly be applicable to the Fast Measurement of cyhalothrin residual in the water sample in enormous quantities.
Background technology
Cyhalothrin is a kind of synthesis of pyrethrin ester pesticides, is widely used in Pest killing and the prevention of crops and animal house.In China, cyhalothrin is mainly used in cotton, tealeaves, fruit tree and the growing vegetables.Yet cyhalothrin is a kind of highly toxic substance, for the per os median lethal dose (LC of rat and mouse
50) be respectively 79mg/Kg and 56mg/Kg, and higher for the toxicity of fish (LC50=0.078-2.3 μ g/L) and aquatic invertabrate (LC50=0.0023-3.3 μ g/L).And the residence time of cyhalothrin in environment is also long, and it is at soil, and the half life period in plant and the adipose tissue was respectively 30,40 and 23 days.And have to studies show that cyhalothrin can accumulate in the body of fish, thereby might affect human health.
The analytical approach of cyhalothrin commonly used mainly contains high pressure lipuid chromatography (HPLC), gas chromatography connecting electronic acquisition detector method and Liquid Chromatography/Mass Spectrometry.Using these physico-chemical analysis technology analyzes trace cyhalothrin residual 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 pretreatment process such as purify, derive, analysis speed is slow, cost is high, pretreatment process need to use a large amount of organic solvents, has caused again new environmental pollution.Along with sample to be checked, particularly require increasing sharply of field quick detection sample size, traditional pesticide residue analysis means are difficult to meet the need, therefore, and in the urgent need to development and application high-level efficiency residues of pesticides express-analysis technology.
Summary of the invention
The technical matters that (one) will solve
For the shortcoming such as solve poor specificity in present residues of pesticides instrument analytical method cost high and complicated operation and the Fast Detection Technique, sensitivity is low and testing result is unstable, the invention provides that a kind of to have a high specific, high sensitivity, pin-point accuracy, pinpoint accuracy, method of operating simple, and can be used for the indirect competitive enzyme-linked immunosorbent assay kit of the cyhalothrin residual of batch samples fast detecting.
(2) technical scheme
Kit of the present invention comprises ELISA Plate, concentrated cleaning solution, cyhalothrin standard items, cyhalothrin specific antibody, enzyme labeling thing, substrate nitrite ion and the reaction terminating liquid of coated cyhalothrin antigen.
Wherein, described envelope antigen be 3-[(±) cyano group [(±)-(cis)-3-[(Z type)-3-chloro-4,4,4-three fluoro-1-ethyls-2, the 2-dimethyl]-cyclopropyl carbonyl oxygen base] phenoxy group] coupled complex of benzenpropanoic acid and carrier protein, described carrier protein is preferably ovalbumin.
Wherein said cyhalothrin specific antibody be by 3-[(±) cyano group [(±)-(cis)-3-[(Z type)-3-chloro-4,4,4-three fluoro-1-ethyls-2, the 2-dimethyl]-cyclopropyl carbonyl oxygen base] phenoxy group] benzenpropanoic acid and carrier protein couplet prepare as immunogene, can be the monoclonal antibody for preparing by hybridoma method, or the polyclonal antibody that obtains of direct immunization animal.Described, carrier protein can be the common carrier albumen such as mouse haemocyanin, thyroprotein (BCG), bovine serum albumin(BSA), rabbit anteserum albumen, human serum albumins, ovalbumin or hemocyanin, is preferably bovine serum albumin(BSA); Described polyclonal antibody can be mouse source, Ma Yuan, Yang Yuan, rabbit source or cavy source antibody, is preferably rabbit source polyclonal antibody.
Wherein, in the preparation process of the ELISA Plate of envelope antigen, used coating buffer can be 0.05M pH 9.6 sodium carbonate buffers, and used confining liquid is the above-mentioned coating buffer that contains 1% gelatin.
Wherein, the enzyme labeling thing is ELIAS secondary antibody, and marker enzyme can be horseradish peroxidase or alkaline phosphatase.
Wherein, the prescription of concentrated cleaning solution is to add sodium chloride 7～9g, potassium dihydrogen phosphate 0.1～0.3g, sodium hydrogen phosphate 2～4g, potassium chloride 3～6g, Tween-20 0.5～3mL in every 20mL distilled water.The concentration of concentrated cleaning solution is 50 times when normally using.
When marker enzyme was horseradish peroxidase, the substrate nitrite ion was comprised of A liquid and B liquid, and nitrite ion A liquid is hydrogen peroxide or urea peroxide, and nitrite ion B liquid is o-phenylenediamine or tetramethyl benzidine, and stop buffer is sulfuric acid or the hydrochloride buffer of 1～2mol/L.Specifically, the A formula of liquid can be and adds urea peroxide 1g, 10.3g citric acid, 35.8g Na in every 1000mL water
2HPO
412H
2O, Tween-20 100 μ L, pH5; The B formula of liquid can be and adds tetramethyl benzidine (TMB) 700mg (40mL DMSO dissolving), 10.3g citric acid, pH2.4-2.8 in every 1000mL distilled water.When marker enzyme is bacterium when extracting alkaline phosphatase, the substrate nitrite ion is for being the NaOH of 2mol/L to nitro phosphate buffer, stop buffer.
The analysis principle of kit of the present invention is:
When the pre-coated antigen of ELISA Plate, add cyhalothrin sample to be measured and polyclonal antibody, solid-phase coating antigen and cyhalothrin to be measured are vied each other and antibody response, because the solid phase antigen in each hole and the antibody content of adding are all consistent, so when cyhalothrin concentration to be measured is high, the antibody that then is bonded on the solid phase antigen is few, the ELIAS secondary antibody that adds is few with the antibody binding capacity that is fixed, add at last substrate solution and nitrite ion, chromogenic reaction is shallow, the OD value that detects with microplate reader is low, shows that inhibiting rate is high; Otherwise when cyhalothrin concentration to be measured was low, the OD value of then surveying was high, and inhibiting rate is low.According to detecting the typical curve of doing with known cyhalothrin concentration, can extrapolate the concentration of cyhalothrin to be measured.
(3) beneficial effect
Advantage of the present invention is accurately to detect delicately cyhalothrin residual in the water sample of separate sources, and the determination process is simple, and is consuming time few, can detect simultaneously a large amount of samples, and the sample detection cost is far below traditional instrument detection method.The present invention has important practical significance to the cyhalothrin residual on-site supervision technology that solves batch samples.
Description of drawings
The standard of Fig. 1 cyhalothrin suppresses curve.
Embodiment
Following embodiment is used for further specifying the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
The operation of embodiment 1 kit and result calculate
Testing sample is after pre-treatment, and is for subsequent use with the PBS constant volume that contains methyl alcohol 50%.Take vacuum packaging bag apart and take out ELISA Plate, at room temperature balance 5 minutes is for subsequent use.Preparation 0ng/mL, 5ng/mL, 10ng/mL, 100ng/mL, 1000ng/mL, the cyhalothrin titer of 10000ng/mL, add 50 μ L standard specimens or the sample handled well in each hole, standard specimen and sample are done 2～4 repetitions, add the antibody of 50 μ L dilution, hatch 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; Add by the good enzyme mark goat-anti rabbit two anti-100 μ L of dilution in 1: 1000, hatched 30 minutes for 37 ℃; Pour out the liquid in the hole, wash plate 2～6 times with PBST, pat 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 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 OD value that will contain 0 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 aperture same methods are proofreaied and correct is decided to be B; With B/B
0Value is ordinate, and the log value of respective standard product concentration is horizontal ordinate, draws the cyhalothrin standard and suppresses curve (accompanying drawing 1).Wherein, Regression Equations is Y=-0.448X+1.201, R
2=0.993,, concentration IC in the inhibition
50=37 μ g/L, lowest detectable limit IC
10=4.7 μ g/L.
Embodiment 2 haptens 3-[(±) cyano group [(±)-(cis)-3-[(Z)-3-chloro-4,4,4-three fluoro-1-ethyls-2,2-dimethyl] cyclopropyl carbonyl oxygen] phenoxy group] benzenpropanoic acid synthetic
Synthesizing of 4-hydroxy phenylpropionic acid benzene methyl
Add 14.4g para hydroxybenzene propionic acid in the 250mL there-necked flask, 27mL benzylalcohol, 15mL toluene splashes into 4 85% H
3PO
4, reflux, minute water, reaction needs 10h.Excess toluene is removed in distillation, and pressure reducing and steaming benzylalcohol, residue are dissolved in the ether, use respectively saturated NaHCO
3Solution, water washing three times.Anhydrous MgSO
4Drying boils off low-boiling-point substance, decompression distillation.Collect 234-236 ℃ of (4mm Hg) cut, get colourless mucus 4-hydroxy phenylpropionic acid benzene methyl 16.1g, yield 72.5%.
Synthesizing of 1-Australia-3-(dimethoxy-methyl) benzene
18.5g 3-bromobenzaldehyde, 12.2g trimethyl orthoformate, 15mL methyl alcohol splash into 2 dense H in the 250mL there-necked flask
2SO
4, stirring at normal temperature 3h.Potpourri dilutes with ether, uses respectively saturated Na
2CO
3Solution, water washing three times.Anhydrous MgSO
4Drying boils off low-boiling-point substance, decompression distillation.Collect 102-104 ℃ of (6mm Hg) cut, get colourless liquid 1-bromo-3-(dimethoxy-methyl) benzene 13.5g, yield 57.9%.
Synthesizing of 4-(3-formaldehyde phenoxy group) benzenpropanoic acid benzene methyl
In the 100mL there-necked flask, add 5.0g 4-hydroxy phenylpropionic acid benzene methyl, below the anhydrous DFM of 30mL, ice bath to 5 ℃, add NaH (50%) 1.0g in batches, add the recession deicing and bathe, add 2.7g 1-bromo-3-(dimethoxy-methyl) benzene, 1.8g Cu
2Cl
2, 0.9g copper powder, 4.8mL pyridine, back flow reaction 8h stops reaction, cooling, with the dilution of 150mL ethyl acetate, with 10%HCl washing three times, add 10%HCl 10mL vibration 2h, wash three times, tell organic layer, drying, precipitation, column chromatography gets 4-(3-formaldehyde phenoxy group) benzenpropanoic acid benzene methyl 1.27g, yield 30.2%.
3-[3-[cyano group (hydroxyl) methyl] phenoxy group] benzenpropanoic acid benzene methyl synthetic
In the 50mL there-necked flask, add 1.5g KCN, 4mL water, stirring and dissolving is below the ice bath to 5 ℃.Add 4-(3-formaldehyde phenoxy group) benzenpropanoic acid benzene methyl 1.27g, THF (tetrahydrofuran) 16mL slowly drips 40%H
2SO
44mL.After dripping off, react 30min below 5 ℃.Stop reaction, use extracted with diethyl ether three times, merge organic layer, drying, precipitation gets 3-[3-[cyano group (hydroxyl) methyl] phenoxy group] and benzenpropanoic acid benzene methyl 1.05g, yield 76.6%.
3-[(±) cyano group [(±)-(cis)-3-[(Z)-3-chloro-4,4,4-three fluoro-1-ethyls-2,2-dimethyl] cyclopropyl carbonyl oxygen] phenoxy group] benzenpropanoic acid benzene methyl synthetic
In the 50mL there-necked flask, add 2.38g 3-[3-[cyano group (hydroxyl) methyl] phenoxy group] the benzenpropanoic acid benzene methyl, 15mL CH
2Cl
2, 1.34g time chrysanthemumic acid ((±)-(cis)-3-[(Z)-3-chloro-4,4,4-three fluoro-1-ethyls-2,2-dimethyl] ethylene-acetic acid), drip the 15mLCH that is dissolved with 1.26g DCC under the ice bath
2Cl
2, stopping after dropwising sending out should.Precipitation, column chromatography get 3-[(±) cyano group [(±)-(cis)-3-[(Z)-3-chloro-4,4,4-three fluoro-1-ethyls-2,2-dimethyl] cyclopropyl carbonyl oxygen] phenoxy group] benzenpropanoic acid benzene methyl (Cwb-5) 2.54g, yield 75.1%.
3-[(±) cyano group [(±)-(cis)-3-[(Z)-3-chloro-4,4,4-three fluoro-1-ethyls-2,2-dimethyl] cyclopropyl carbonyl oxygen] phenoxy group] benzenpropanoic acid synthetic
In the 50mL there-necked flask, adding 2.38g 3-[(±) cyano group [(±)-(cis)-3-[(Z)-3-chloro-4,4,4-three fluoro-1-ethyls-2,2-dimethyl] cyclopropyl carbonyl oxygen] phenoxy group] the benzenpropanoic acid benzene methyl, CH
2Cl
210mL, Me
3SiZ 0.4mL, the N2 protection is lower, 35 ℃ of reaction 2h.Large plate layer chromatography separate 3-[(±) cyano group [(±)-(cis)-3-[(Z)-3-chloro-4,4,4-three fluoro-1-ethyls-2,2-dimethyl] cyclopropyl carbonyl oxygen] phenoxy group] benzenpropanoic acid 0.93g, yield 76.2%.
The preparation of embodiment 3 cyhalothrin polyclonal antibodies
With active ester method with haptens and bovine serum albumin(BSA) coupling, claim the 2mg conjugate to be dissolved in the 1mL physiological saline, mix with the 1mL complete Freund's adjuvant, fully emulsified rear injection New Zealand large ear rabbit thigh, later on every two all booster immunizations once, use incomplete Freund's adjuvant instead and mix with immunogene, immune position is that nape section is subcutaneous, from for the third time immunity, a week is detected serum titer from the rabbit ear vein blood sampling after each immunity.Immunity is 5 times altogether, a week adopts whole blood from rabbit arteria carotis after the last immunity, slightly carries rabbit anti-serum with 35% saturated ammonium sulfate salting out method, is further purified with the DE-52 anion exchange chromatography at last, obtains purer cyhalothrin polyclonal antibody.
DE-52 anion exchange antibody purification:
(1) DE-52 pre-service: need 5g weight in wet base cellulose to calculate with the thick IgG of 1mg, claim an amount of DE-52 to add 0.5mol/LNaOH and soak 30min, distilled water is washed till neutrality repeatedly; Then soak 30min with 0.5mol/LHCl, distilled water is washed till neutrality repeatedly; Soak 30min with 0.5mol/LNaOH again, distilled water is washed till neutrality repeatedly, uses at last the abundant equilibrate overnight of PBS of 0.01mol/L, pH7.4.
(2) dress post: take out bubble in the DE-52 solution with vacuum pump before the dress post, can not produce bubble in the dress post process, loading front pillar bed is with the PBS balance of 0.01mol/L, pH7.4.
(3) loading: the PBS with 4～6mL dilutes an amount of thick IgG, then slowly is added in post bed upper surface along the post arm, begins wash-out after sample enters the post bed.
(4) wash-out: the PBS with 0.01mol/L, pH7.4 makes eluent, and flow velocity 0.5mL/min collects the first and second absorption peaks.
(5) collect liquid freeze drying, 4 ℃ of preservations.
The preparation of embodiment 4 ELISA Plate
With coated damping fluid (0.05M pH 9.6 sodium carbonate buffers) the cyhalothrin antigen diluent is become 1 μ g/mL, every hole adds 100 μ L, 37 ℃ of incubation 2h and 4 ℃ spend the night, and the coating buffer that inclines is with cleansing solution washing 3 times, each 30 seconds, pat dry, then in every hole, add 150-200 μ L confining liquid, 37 ℃ of incubation 2h, the liquid in the hole that inclines is preserved with the vacuum seal of aluminium film after dry.
The establishment of embodiment 5 cyhalothrin residual enzyme analysis linked immuno sorbent assay kits
In this example, kit comprises following part:
(1) has been coated with the ELISA Plate of cyhalothrin antigen
(2) sponge bracket
(3) cyhalothrin standard items
(4) cyhalothrin polyclonal antibody
(5) horseradish peroxidase-labeled goat anti-rabbit antibody
(6) the thickening and washing formula of liquid is: sodium chloride 8g, potassium dihydrogen phosphate 0.2g, sodium hydrogen phosphate 3g, potassium chloride 5g, Tween-20 2mL, distilled water 20mL.
(7) nitrite ion A formula of liquid: urea peroxide 1g, 10.3g citric acid, 35.8g Na
2HPO
412H
2O, Tween-20 100 μ L, distilled water 1000mL, pH5.
(8) nitrite ion B formula of liquid: tetramethyl benzidine (TMB) 700mg (40mL DMSO dissolving), 10.3g citric acid, distilled water 1000mL, pH2.5.
The experiment of embodiment 6 kit specificitys
Selecting II type pyrethrin pesticide fenvalerate (fenvelerate) commonly used, Fenpropathrin (fenpropathrin), decis (deltmethrin), taufluvalinate (fluvalinate) and cypermethrin (cypermethrin) is determinand, records concentration (IC in the inhibition of various materials
50), use again the following formula calculating antibody to the cross reactivity of these materials; Cross reacting rate is less, and then antibody is stronger to the specificity of cyhalothrin, on the contrary the poor specificity of antibody then.
Cross reaction (CR%)=IC
50(cyhalothrin)/IC
50(for the examination thing) * 100%.
Measuring the results are shown in Table 1, adopts indirect elisa method, and polyclonal antibody all less than 3%, illustrates that the specificity of this kit is good to the cross reaction of II type pyrethrin pesticide commonly used, can guarantee the reliability to cyhalothrin residual measurement result in the sample.
Table 1 cross reaction
Ni: when used measured object concentration was 10mg/L, inhibiting rate was less than 10%
Embodiment 7 recovery tests
Tap water and well water water sample for examination pick up from school district, China Agricultural University west, and the river water sample picks up from Beijing's Xiaoqinghe River.The different water sample in source that adds cyhalothrin carries out carrying out the enzyme-linked immunoassay analysis after the different pre-treatments, the results are shown in Table 2.
The interpolation recovery of cyhalothrin in table 2 water sample
Table 2 Recovery of cyhalothrin from the spiked water samples by pretreatment
| Sample Analyte (n=4) | Addition Spiked (ppm) | Theoretical measured value Theoretical (ppb) | Practical measurement value Measured (ppb) (mean ± S.E.) | Recovery Recovery (the % of mean ± S.E.) |
| Tap water Tap water | 0.05 0.2 0.5 | 5 20 50 | 4.75±0.3 19.6±1.6 48.5±2.0 | 95±6 98±8 97±4 |
| Well water Well water | 0.05 0.2 0.5 | 5 20 50 | 5±0.4 19.6±2.2 48.5±1.5 | 100±8 98±11 97±3 |
| River River water | 0.25 1 2.5 | 5 20 50 | 5.7±0.25 21.6±1.0 55.5±3.5 | 114±5 108±5 111±7 |
Claims (6)
1. analytical kit of enzyme linked immunosorbent assay that detects cyhalothrin residual, the ELISA Plate, concentrated cleaning solution, cyhalothrin standard items, cyhalothrin specific antibody, enzyme labeling thing, substrate nitrite ion and the reaction terminating liquid that comprise coated cyhalothrin antigen, the immunogene that it is characterized in that preparing the cyhalothrin specific antibody be 3-[(±) cyano group [(±)-(cis)-3-[(Z type)-3-chloro-4,4,4-three fluoro-1-ethyls-2,2-dimethyl]-cyclopropyl carbonyl oxygen base] phenoxy group] coupled complex of benzenpropanoic acid and carrier protein;
Described cyhalothrin antigen be 3-[(±) cyano group [(±)-(cis)-3-[(Z type)-3-chloro-4,4,4-three fluoro-1-ethyls-2,2-dimethyl]-cyclopropyl carbonyl oxygen base] phenoxy group] coupled complex of benzenpropanoic acid and carrier protein.
2. kit according to claim 1 is characterized in that described enzyme labeling thing is that enzyme labeling two is anti-.
3. kit according to claim 1, the prescription that it is characterized in that described concentrated cleaning solution are to add in every 20mL distilled water sodium chloride 7～9g, potassium dihydrogen phosphate 0.1～0.3g, sodium hydrogen phosphate 2～4g, potassium chloride 3～6g, Tween-20 0.5～3mL are arranged.
4. kit according to claim 1, it is characterized in that, when the marker enzyme that uses in the enzyme labeling thing during as horseradish peroxidase, the substrate nitrite ion comprises A liquid and B liquid, and described A liquid is that hydrogen peroxide or urea peroxide, described B liquid are that o-phenylenediamine or tetramethyl benzidine, stop buffer are sulfuric acid or the hydrochloride buffer of 1～2mol/L; When the marker enzyme that uses in the enzyme labeling thing extracted alkaline phosphatase as bacterium, the substrate nitrite ion was for being the NaOH of 2mol/L to nitro phosphate buffer, stop buffer.
5. kit according to claim 4 is characterized in that, described A formula of liquid is to add urea peroxide 1g, 10.3g citric acid, 35.8g Na in every 1000mL water
2HPO
412H
2O, Tween-20 100 μ L, pH5; Described B formula of liquid is to add tetramethyl benzidine 700mg, 10.3g citric acid, pH2.4-2.6 in every 1000mL distilled water.
6. the application of each described kit of claim 1～5 in detecting cyhalothrin residual.
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| CN102417468A (en) * | 2011-09-14 | 2012-04-18 | 王利兵 | Synthetic method of lambdacyhalothrin hapten |
| CN103911348B (en) * | 2014-01-02 | 2016-04-20 | 河南科技大学 | The monoclonal antibody of No. 16, a kind of cell strain and generation thereof and application |
| CN105732788A (en) * | 2016-03-21 | 2016-07-06 | 贵州勤邦食品安全科学技术有限公司 | Preparation method and application of efficient cyhalothrin hapten |
| CN105807041A (en) * | 2016-03-21 | 2016-07-27 | 贵州勤邦食品安全科学技术有限公司 | Kit for detecting efficient cyhalothrin residue |
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| CN1624480A (en) * | 2004-10-20 | 2005-06-08 | 扬州大学 | Fenvalerate direct competition enzyme joint immune absorption analysis technology and its kit |
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Non-Patent Citations (2)
| Title |
|---|
| 李志茹.拟除虫菊酯类农药通用抗体制备及高效氯氟氰菊酯免疫检测技术研究.《中国优秀博硕士学位论文全文数据库（硕士） 工程科技I辑》.2005,(第2期),正文第32页第2.3节. * |
| 骆爱兰等.拟除虫菊酯类农药多残留酶免疫分析方法的建立.《中国农业科学》.2005,第38卷(第2期),全文. * |
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