CN101334408A - Enzyme linked immunological adsorption detection method for analyzing residuals of cyano pyrethroid pesticides - Google Patents
Enzyme linked immunological adsorption detection method for analyzing residuals of cyano pyrethroid pesticides Download PDFInfo
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- CN101334408A CN101334408A CNA2008100212226A CN200810021222A CN101334408A CN 101334408 A CN101334408 A CN 101334408A CN A2008100212226 A CNA2008100212226 A CN A2008100212226A CN 200810021222 A CN200810021222 A CN 200810021222A CN 101334408 A CN101334408 A CN 101334408A
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Abstract
The invention discloses an enzyme-linked immunosorbent assay method for multi-residue analysis of a cyano-containing pyrethroid pesticide, belonging to the technical field of immunoassay. The enzyme-linked immunosorbent assay method uses a coupled complex of hapten m-phenoxy benzal cyanohydrin succinate ester and ovalbumin as a coating antigen, samples of the cyano-containing pyrethroid and a polyclonal antibody of the m-phenoxy benzal cyanohydrin succinate ester are added, then the coating antigen and the cyano-containing pyrethroid to be detected are competed and reacted with the antibody, an enzyme-labeled secondary antibody is added to be combined with the fixed antibody, washing liquid is used for washing, developer is added, an enzyme-labeled instrument is used for detecting OD value, the OD value is compared with a standard curve of a standard product of the cyano-containing pyrethroid, and the concentration of the cyano-containing pyrethroid of the samples to be detected is calculated. The enzyme-linked immunosorbent assay method can accurately and sensitively detect the residues of the cyano-containing pyrethroid pesticide in the samples to be detected, the pre-treatment process of the samples is simple with less time-consuming, a large number of samples can be simultaneously detected, and the detection cost of the samples is far lower than the detection method of the traditional instrument; furthermore, the method of the invention has good stability and no radioactive pollution.
Description
Technical field
A kind of enzyme-linked immunosorbent assay method of analyzing residuals of cyano pyrethroid pesticides, specifically, relate to a kind of enzyme-linked immunosorbent assay method that is applicable to that cyano-containing pyrethroid (a-Cyano-Pyrethroid) pesticide multi-residues is analyzed, belong to the immuno analytical method field.
Background technology
Pyrethroid (Pyrethroid) is that a class develops on natural pyrethrin chemical constitution research basis and next bionical medicine.The application of pyrethrum has long history, because it is strong that it has the power of knocking down, insecticidal action is fast, broad spectrum activity, easily degraded is to higher mammal and birds hypotoxicity, safe in utilization, characteristics such as environmental pollution is little are widely used in the controls of insects such as tea place, orchard, farmland, and especially cyano-containing pyrethroid (a-Cyano-Pyrethroid) is used more extensive.Raising along with international standard, the internationalization of China's trade, green barrier is stern, China's agricultural byproducts particularly residues of pesticides problem in textile, tealeaves, the fruit become the major obstacle of the export trade, require the quick residue detection technology of Application and Development to strengthen the residual monitoring of Multiple Pesticides in the agricultural byproducts of the place of production.And traditional pesticide residue analysis mainly relies on gas chromatography, liquid chromatography, gas chromatography mass spectrometry, LC-MS etc., these methods to need expensive instrument, and needs through loaded down with trivial details pre-treatment, and requirement is detected at the scene that is difficult to reach fast and convenient.Therefore, be necessary the more effective detection method efficiently of research, promptly be applicable to the enzyme-linked immunosorbent assay method of analyzing residuals of cyano pyrethroid pesticides.
Summary of the invention
(1) technical matters that will solve
The objective of the invention is to set up a kind of have high sensitivity, high specific, pin-point accuracy, pinpoint accuracy, the simple enzyme-linked immunosorbent assay method of method of operating, be used for the how residual batch of cyano-containing pyrethroid pesticide, fast detecting.
(2) technical scheme
For achieving the above object, the present invention has set up a kind of enzyme-linked immunosorbent assay method of analyzing residuals of cyano pyrethroid pesticides, and this method comprises the optimization to detection method.
(1) the bag quilt of antigen
With the coupled complex of haptens m-phenoxybenzoic cyanohydrin ester succinate and ovalbumin OVA as envelope antigen, sodium carbonate buffer with 0.05M, pH 9.6 diluted envelope antigen 1: 100～1: 200, concentration is that 4～8 μ g/mL are as coating buffer, in every hole of ELISA Plate, add 100 μ L coating buffers, 4 ℃ of overnight incubation are sealed as confining liquid with the above-mentioned sodium carbonate buffer that contains 2%OVA;
(2) competitive reaction
M-phenoxybenzoic cyanohydrin ester succinate polyclonal antibody antibody diluent with immunity preparation: contain 1% gelatin, contain the phosphate buffered solution PBST of 0.5% Tween-20, pH=7.4, by after 1: 100～1: 200 dilution proportion, add in the ELISA Plate, every hole adds 50 μ L; Every hole adds the 0ng/mL with the standard items diluted respectively simultaneously, 0.1ng/mL, 1.0ng/mL, 10ng/mL, 100ng/mL, 1000ng/mL, 10000ng/mL, the cyano-containing pyrethroid pesticide standard items of one of a series of concentration of 100000ng/mL, every hole adds 50 μ L, hatches behind the 1h with PBST cleansing solution washing 3～5 times for 37 ℃;
(3) add ELIAS secondary antibody
Adding the goat-anti rabbit GAR-HRP of horseradish peroxidase-labeled, is 1: 5000 with the antibody diluent dilution, and every hole adds 100 μ L, washs 3～5 times with the PBST cleansing solution behind 37 ℃ of incubation 1h;
(4) colour developing
Every hole adds 100 μ L colour developing liquid, and the 37 ℃ of reactions in dark place 15min takes out the sulfuric acid stop buffer that every hole, back adds 100 μ L, 2mol/L, measures light absorption value A with microplate reader
450, calculate the concentration of cyano-containing pyrethroid to be measured with the typical curve contrast of being done.
The prescription of standard items dilution is 5% sodium chloride for add mass ratio in the PBS of the pH=6.5 of volume content 30% methyl alcohol.
The prescription of PBST cleansing solution is to add sodium chloride 4～6g, potassium dihydrogen phosphate 0.1～0.3g, sodium hydrogen phosphate 2～4g, potassium chloride 3～6g, Tween-20 0.5～3mL in the 1000mL distilled water.
Colour developing liquid comprises A liquid and B liquid, and the A formula of liquid is to add 0.933g citric acid, 3.68g Na in every 100mL water
2HPO
412H
2O, 18 μ L30%H
2O
2The B formula of liquid is dissolved in 100mL ethylene glycol for the 60mg tetramethyl benzidine; During use in A: B=1: 1 ratio is used.
Cyano-containing pyrethroid polyclonal antibody is the coupled complex immunity new zealand white rabbit preparation with m-phenoxybenzoic cyanohydrin ester succinate and bovine serum albumin(BSA) (BSA).
The check and analysis principle of the inventive method is:
Each Kong Jun on the ELISA Plate is coated with the antigen of same amount, after adding cyano-containing pyrethroid sample to be measured and m-phenoxybenzoic cyanohydrin ester succinate polyclonal antibody, solid-phase coating antigen and cyano-containing pyrethroid 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 the cyano-containing pyrethroid ester concentration of testing sample 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 antibodies amount that is fixed, add substrate solution (liquid A liquid promptly develops the color) and colour developing liquid (being B liquid) with cleansing solution washing back, chromogenic reaction is shallow, the OD value that detects with microplate reader is low, shows the inhibiting rate height; Otherwise when the cyano-containing pyrethroid ester concentration of testing sample hanged down, the OD value of then being surveyed was high, and inhibiting rate is low.According to the typical curve of being done, can extrapolate the concentration of cyano-containing pyrethroid to be measured.
(3) beneficial effect
The how residual inspection detection method of cyano-containing pyrethroid provided by the invention has adopted the m-phenoxybenzoic cyanohydrin ester succinate polyclonal antibody, can accurately detect pyrethroid pesticide residue containing cyan in the testing sample delicately, the pre-treatment process of sample is simple, consuming time few, can detect a large amount of samples simultaneously, the sample detection cost is far below traditional instrument detecting method, and the inventive method good stability, "dead" pollution.The present invention has important practical significance to the residual on-site supervision technology of cyano-containing pyrethroid that solves batch samples.
Description of drawings
The standard of Fig. 1 m-phenoxybenzoic cyanohydrin ester succinate suppresses curve.
Embodiment
Embodiment 1: the operation of detection method and result calculate:
(1) the bag quilt of antigen
With the coupled complex of haptens m-phenoxybenzoic cyanohydrin ester succinate and ovalbumin (OVA) as envelope antigen, sodium carbonate buffer with 0.05M, pH 9.6 diluted envelope antigen 1: 100～1: 200, concentration is that 4～8 μ g/ml are as coating buffer, in every hole of ELISA Plate, add 100 μ L coating buffers, 4 ℃ of overnight incubation are sealed as confining liquid with the above-mentioned sodium carbonate buffer that contains 2%OVA;
(2) competitive reaction
After the m-phenoxybenzoic cyanohydrin ester succinate polyclonal antibody of immunity preparation pressed 1: 100～1: 200 dilution proportion with antibody diluent (contain 1% gelatin, contain the phosphate buffered solution PBST of 0.5% Tween-20, pH=7.4), add in the ELISA Plate, every hole adds 50 μ L, add a series of concentration 0ng/mL simultaneously respectively, 0.1ng/mL, 1.0ng/mL, 10ng/mL, 100ng/mL, 1000ng/mL, 10000ng/mL, the cyano-containing pyrethroid pesticide standard items of 100000ng/mL, every hole 50 μ L are hatched behind the 1h with PBST cleansing solution washing 3～5 times for 37 ℃;
(3) add ELIAS secondary antibody
Adding the goat-anti rabbit (GAR-HRP) of horseradish peroxidase-labeled, is 1: 5000 with the antibody diluent dilution, and every hole adds 100 μ L, washs 3～5 times with the PBST cleansing solution behind 37 ℃ of incubation 1h;
(4) colour developing
Every hole adds 100 μ L colour developing liquid, and the 37 ℃ of reactions in dark place 15min takes out every hole, back and adds 100 μ L stop buffers (sulfuric acid of 2mol/L), measures light absorption value A with microplate reader
450
The OD value that will contain 0ng/mL standard items hole deducts the OD value that contains Cmax 100000ng/mL standard items hole and is decided to be B
0, the OD value (the OD value in 0ng/mL standard items hole deducts the OD value that each hole is measured) after all the other each apertures are proofreaied and correct with quadrat method is decided to be B; With B/B
0Value is ordinate, and respective standard product concentration is horizontal ordinate, draws the m-phenoxybenzoic cyanohydrin ester succinate standard and suppresses curve.The concentration of counter sample can be obtained according to the regression equation of curve, also m-phenoxybenzoic cyanohydrin ester succinate IC can be obtained
50(B/Bo=50%) and minimum detectable level IC
90(B/Bo=90%).IC
50Be 70.12ng/mL, LOD (concentration of 90% inhibiting rate correspondence) is 4.31ng/mL, and the concentration of the range of linearity (20%～80%) inhibiting rate correspondence is 5～1000ng/mL.
Embodiment 2: the specificity experiment of detection method:
Select the cyano-containing pyrethroid as determinand, record concentration (IC in the inhibition of various materials
50), use the cross reactivity of following formula calculating antibody again to these materials; Cross reacting rate is bigger, and then antibody is stronger to the compatibility of cyano-containing pyrethroid, otherwise then the compatibility of antibody is poor.
Cross reacting rate (CR%)=[IC
50(m-phenoxybenzoic cyanohydrin ester succinate)/IC
50(cyano-containing pyrethroid)] * 100%.
Measuring the results are shown in Table 1, adopt indirect elisa method, contain the m-phenoxybenzoic cyanohydrin ester succinate polyclonal antibody midbody derivant m-phenoxybenzoic cyanohydrin ester succinate and 8 kinds of cyano-containing pyrethroids of cyano-containing pyrethroid are all had cross reaction.But to cross reacting rates such as cyano-containing pyrethroid such as Permethrin not all below 0.1%.Generally speaking, the cyano-containing pyrethroid pesticide had certain group's selectivity.
Table 1 cross reaction
Embodiment 3: add and reclaim experiment:
(1) level of the interpolation of sample: 5.0ng/g, 10.0ng/g, 50.0ng/g, 100.0ng/g adds cyano-containing pyrethroid sample in the cotton that shreds, and at room temperature leaves standstill 15min at least.
(2) extraction and cleaning of sample: get the cotton sample that 1g has added the variable concentrations standard items respectively, in the 50mL centrifuge tube.Add 30mL again and contain the ethyl acetate of saturated acetonitrile, ultrasonic Extraction 30 minutes squeezes out extract with glass syringe, merges extract behind the triplicate, and nitrogen dries up, and adds the standard items dilution of 1mL, as sample for analyzing.Each concentration prepares five parts in sample respectively, measures its content, and measured concentration and interpolation concentration are compared.
The calculating of the recovery: add the sample OD value calculating corresponding inhibition ratio of concentration according to difference, find separately concentration according to corresponding inhibition ratio from typical curve again.Detectable concentration is the recovery of corresponding concentration with the ratio of actual concentration.
The results are shown in Table 2, as can be seen, the recovery of cyano-containing pyrethroid in cotton is between 61.32%～80.20%.This analytical approach repeatability is good, and relative standard deviation is lower than 3.30%.
Table 2 adds the mensuration of the recovery
Claims (4)
1. the enzyme-linked immunosorbent assay method of an analyzing residuals of cyano pyrethroid pesticides is characterized in that:
(1) the bag quilt of antigen
With the coupled complex of haptens m-phenoxybenzoic cyanohydrin ester succinate and ovalbumin OVA as envelope antigen, sodium carbonate buffer with 0.05M, pH 9.6 diluted envelope antigen 1: 100～1: 200, concentration is that 4～8 μ g/mL are as coating buffer, in every hole of ELISA Plate, add 100 μ L coating buffers, 4 ℃ of overnight incubation are sealed as confining liquid with the above-mentioned sodium carbonate buffer that contains 2%OVA;
(2) competitive reaction
M-phenoxybenzoic cyanohydrin ester succinate polyclonal antibody antibody diluent with immunity preparation: contain 1% gelatin, contain the phosphate buffered solution PBST of 0.5% Tween-20, pH=7.4, by after 1: 100～1: 200 dilution proportion, add in the ELISA Plate, every hole adds 50 μ L; Every hole adds the 0ng/mL with the standard items diluted respectively simultaneously, 0.1ng/mL, 1.0ng/mL, 10ng/mL, 100ng/mL, 1000ng/mL, 10000ng/mL, the cyano-containing pyrethroid pesticide standard items of one of a series of concentration of 100000ng/mL, every hole adds 50 μ L, hatches behind the 1h with PBST cleansing solution washing 3～5 times for 37 ℃;
(3) add ELIAS secondary antibody
Adding the goat-anti rabbit GAR-HRP of horseradish peroxidase-labeled, is 1: 5000 with the antibody diluent dilution, and every hole adds 100 μ L, washs 3～5 times with the PBST cleansing solution behind 37 ℃ of incubation 1h;
(4) colour developing
Every hole adds 100 μ L colour developing liquid, and the 37 ℃ of reactions in dark place 15min takes out the sulfuric acid stop buffer that every hole, back adds 100 μ L, 2mol/L, measures light absorption value A with microplate reader
450, contrast the cyano-containing pyrethroid ester concentration of calculating testing sample with the typical curve of being done.
2. enzyme-linked immunosorbent assay method according to claim 1, the prescription that it is characterized in that the standard items dilution is 5% sodium chloride for add mass ratio in the PBS of the pH=6.5 of volume content 30% methyl alcohol.
3. enzyme-linked immunosorbent assay method according to claim 1, the prescription that it is characterized in that the PBST cleansing solution are to add sodium chloride 4～6g, potassium dihydrogen phosphate 0.1～0.3g, sodium hydrogen phosphate 2～4g, potassium chloride 3～6g, Tween-20 0.5～3mL in the 1000mL distilled water.
4. enzyme-linked immunosorbent assay method according to claim 1, the liquid that it is characterized in that developing the color comprises A liquid and B liquid, the A formula of liquid is to add 0.933g citric acid, 3.68gNa in every 100mL water
2HPO
412H
2O, 18 μ L30%H
2O
2The B formula of liquid is dissolved in 100mL ethylene glycol for the 60mg tetramethyl benzidine; During use in A: B=1: 1 ratio is used.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102262089A (en) * | 2011-04-28 | 2011-11-30 | 华中农业大学 | Method for quickly and quantitatively detecting pesticide residue of cyanogens-containing synthetic pyrethroids in tea |
| CN102353767A (en) * | 2011-07-07 | 2012-02-15 | 贺福元 | Simultaneous calculation method for overall components group |
| CN102901811A (en) * | 2012-10-20 | 2013-01-30 | 江南大学 | Pyrethroid hapten design based on computer molecular simulation technique and application |
| CN103149351A (en) * | 2013-03-21 | 2013-06-12 | 江南大学 | Synthesis and application of envelope antigen universal for pyrethriods pesticide |
| CN104165986A (en) * | 2014-06-09 | 2014-11-26 | 浙江大学 | Preparation method and use method of visual detection chip for multiple pesticide residues based on membrane material |
| CN106198519A (en) * | 2016-07-05 | 2016-12-07 | 广州市食品检验所 | The ninhydrin colorimetry of cypermethrin content in a kind of quick detection vegetable or fruit |
| CN106525835A (en) * | 2016-11-09 | 2017-03-22 | 百奥森（江苏）食品安全科技有限公司 | Detection kit for gentamycin in food |
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2008
- 2008-07-22 CN CNA2008100212226A patent/CN101334408A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102262089A (en) * | 2011-04-28 | 2011-11-30 | 华中农业大学 | Method for quickly and quantitatively detecting pesticide residue of cyanogens-containing synthetic pyrethroids in tea |
| CN102262089B (en) * | 2011-04-28 | 2014-05-21 | 华中农业大学 | Method for quickly and quantitatively detecting pesticide residue of cyanogens-containing synthetic pyrethroids in tea |
| CN102353767A (en) * | 2011-07-07 | 2012-02-15 | 贺福元 | Simultaneous calculation method for overall components group |
| CN102353767B (en) * | 2011-07-07 | 2014-03-12 | 贺福元 | Simultaneous calculation method for overall components group |
| CN102901811A (en) * | 2012-10-20 | 2013-01-30 | 江南大学 | Pyrethroid hapten design based on computer molecular simulation technique and application |
| CN103149351A (en) * | 2013-03-21 | 2013-06-12 | 江南大学 | Synthesis and application of envelope antigen universal for pyrethriods pesticide |
| CN104165986A (en) * | 2014-06-09 | 2014-11-26 | 浙江大学 | Preparation method and use method of visual detection chip for multiple pesticide residues based on membrane material |
| CN106198519A (en) * | 2016-07-05 | 2016-12-07 | 广州市食品检验所 | The ninhydrin colorimetry of cypermethrin content in a kind of quick detection vegetable or fruit |
| CN106198519B (en) * | 2016-07-05 | 2019-01-25 | 广州市食品检验所 | The ninhydrin colorimetry of cypermethrin content in a kind of quick detection vegetables or fruit |
| CN106525835A (en) * | 2016-11-09 | 2017-03-22 | 百奥森（江苏）食品安全科技有限公司 | Detection kit for gentamycin in food |
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