CN108535474A - Time-resolved fluorescence test strip for detecting permethrin and application thereof - Google Patents

Time-resolved fluorescence test strip for detecting permethrin and application thereof Download PDF

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CN108535474A
CN108535474A CN201810217657.1A CN201810217657A CN108535474A CN 108535474 A CN108535474 A CN 108535474A CN 201810217657 A CN201810217657 A CN 201810217657A CN 108535474 A CN108535474 A CN 108535474A
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permethrin
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room temperature
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张静
王晓磊
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North China University of Technology
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    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms

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Abstract

The invention discloses a time-resolved fluorescence test strip for detecting permethrin and application thereof. The test strip comprises a sample absorption pad, a reaction membrane and a water absorption pad, wherein the reaction membrane is positioned in the middle, and the sample absorption pad and the water absorption pad are respectively lapped at the left end and the right end of the reaction membrane; a sample adding area and a microsphere area are arranged on the sample absorption pad, and permethrin time-resolved fluorescent microspheres are loaded on the microsphere area; the reaction membrane is provided with a detection line (T line) and a quality control line (C line), the T line is connected with a coated permethrin antigen (coating antigen), and the C line is coated with an anti-rabbit antibody. The invention also provides a method for detecting permethrin residues in samples such as apples, corns, tea leaves, tomatoes, spinach, Chinese cabbages and the like by applying the permethrin test strip. The test strip provided by the invention has the characteristics of simple operation, high sensitivity, quantitative detection, rapid detection, low cost and the like, and is suitable for screening and on-site monitoring of a large number of samples.

Description

It is a kind of detection Permethrin time-resolved fluorescence test strips and its application
Technical field
The present invention relates to a kind of time-resolved fluorescence test strips of detection Permethrin and its applications, belong to time-resolved fluorescence Immunoassay (TRFIA) technical field contains for Permethrin in the samples such as apple, corn, tealeaves, tomato, spinach, Chinese cabbage Amount detection.
Background technology
Permethrin also known as permethrin, deinsectization essence, hundred are gone out spirit, are that a kind of pyrethroid of not cyano-containing structure kills Worm agent.Be suitable for prevent the unstability insecticides of agricultural pests as first in chrysanthemum ester insecticide, Permethrin with compared with It is strong to tag and stomach poison function, it can be used for preventing cotton, vegetables, tealeaves, various pests on fruit tree, be particularly suitable for hygienic evil The prevention of worm.Pyrethrin pesticide is compared with organo-chlorine pesticide, and to the less pollution of environment, but it can enter machine by food chain Body to cause the toxic side effects such as reproduction, immune and angiocarpy to mammal, while will also result in environmental pollution.Therefore, I State's correlation quality testing department has carried out limitation regulation to the substance.GB 2763-2016《National food safety standard Pesticide Maximum residue limit》Define the maximum residue limit of Permethrin：Cereal (part) and soybean are 2mg/kg, great Bai in vegetables Dish, cabbage are 5mg/kg, and citrus, nuts and kernels, kernel approaches, melon and fruit class etc. are 2mg/kg in fruit, and tealeaves is 20mg/kg。
Currently, the method for detection Permethrin has gas chromatography, gas chromatography-mass spectrography, Liquid Chromatography-Tandem Mass Spectrometry Deng.The accuracy of instrument detection method is higher, but expensive equipment, needs technical professional, detecting step complicated, it is difficult to Batch detection sample is not suitable for laboratories batch, quickly detects sample.And immunological detection method is easy to operate, fast It is fast, sensitive, most samples can be detected simultaneously, be ideal quick screening means.
Invention content
The object of the present invention is to provide a kind of high sensitivity, the Permethrin times that easy to operate, at low cost, detection time is short Resolved fluorometric test strips.
A kind of Permethrin time-resolved fluorescence test strips provided by the present invention, which includes sample absorption pad, anti- Answer film and water absorption pad three parts.Reaction film is located at centre, and sample absorption pad is overlapped in reaction film or so two respectively with water absorption pad End.Wherein, sample absorption pad is equipped with sample application zone and microballoon area, and microballoon area is loaded with time-resolved fluorescence microballoon；On reaction film Equipped with detection line (T lines) and nature controlling line (C lines), T lines connect coating Permethrin antigen, and C lines are coated with anti-rabbit antibody.
The Permethrin time-resolved fluorescence microballoon, including detection microballoon and Quality Control microballoon, detection microballoon are pan coating It is the fluorescent microsphere that pan coating has rabbit-anti labelled protein to have the fluorescent microsphere of Permethrin monoclonal antibody, Quality Control microballoon.
Bright-coloured series elements compound is filled in the fluorescent microsphere；Preferably, which is europium chelating object； Optimal, which can be Eu (TTA) 3/TOPO or Eu (TTA) 3/Phen.
The albumen can be cow's serum Y- globulin (BGG) or balf serum albumin (BSA).
The time-resolved fluorescence microspherulite diameter ranging from 100-1000nm.
On the nitrocellulose filter, coated antibody is Permethrin antigen on T lines, coated anti-rabbit antibody on C lines.
The Permethrin time-resolved fluoroimmunoassay quantitative testing test paper bottom is equipped with plastic bottom board.
The T lines, which connect coating Permethrin antigen, to be obtained with carrier protein couplet by Permethrin haptens, the carrier Albumen can be bovine serum albumin(BSA), ovalbumin, hemocyanin, thyroprotein, human serum albumins.
The Permethrin monoclonal antibody is to prepare to obtain using Permethrin hapten-carrier protein conjugate as immunogene , it is to be secreted to obtain by Permethrin monoclonal antibody hybridoma cell strain；The anti-rabbit antibody is to obtain rabbit source antibody mediated immunity sheep It arrives.
The sample absorption pad is Fusion5 films or the identical film of other functions；The water absorption pad is water absorption pad；It is described anti- It can be nitrocellulose filter or cellulose acetate film to answer film.
It is a further object to provide a kind of methods preparing above-mentioned test strips comprising step：
(1) prepared by Quality Control microballoon：
1. using biotinylated protein；
2. being coated with aldehyde group modified fluorescent microsphere using above-mentioned labelled protein；
(2) prepared by haptens：Permethrin haptens product is obtained by series of chemical；
(3) by Permethrin haptens and carrier protein couplet, Permethrin hapten-carrier protein conjugate is obtained；
(4) use Permethrin hapten-carrier protein conjugate that new zealand white rabbit is immunized, by new zealand white rabbit splenocyte With myeloma cell by merging, screening, Permethrin monoclonal hybridoma strain is obtained；
(5) new zealand white rabbit IgG immune health goats are extracted, anti-rabbit antibody is obtained；
(6) prepared by detection microballoon：Aldehyde group modified fluorescent microsphere is coated with using the monoclonal antibody of Permethrin；
(7) blank kilocalorie is pasted：Nitrocellulose filter is pasted onto among plastic bottom board, Fusion5 films and water absorption pad point It is not overlapped in nitrocellulose filter left and right ends；
(8) film is sprayed：By the fluorescent microsphere of rabbit-anti labelled protein and the fluorescent microsphere of Permethrin monoclonal antibody using release Buffer solution mixed diluting is sprayed onto the microballoon area of Fusion5 films to a certain concentration；After Permethrin antigen and the dilution of anti-rabbit antibody, point It is not sprayed onto the T lines and C line positions of nitrocellulose filter；
(9) dry and slitting：By the above-mentioned kilocalorie drying for having sprayed reagent, slitting.
The release buffer solution used in spray film step contains 10%-20% sucrose, 3%-10% trehaloses, 0.5%-1% The bis- trimethylsilyl acetamides (BSA) of N, 0-, 0.2%-0.5% gentamicins.
The final concentration of 0.5mg/mL-2mg/mL of microballoon, Quality Control microballoon final concentration 0.05mg/ are detected after spraying the dilution of film step mL-0.2mg/mL；T line antigen final concentration 0.5mg/mL-2mg/mL, C line antigen final concentrations 0.5mg/mL-2mg/mL；C, T line spray Film liquid amount is 0.5 μ L/cm-1.5 μ L/cm, and it is 2 μ L/cm-8 μ L/cm that microballoon, which sprays film amount,.
Drying described in dry and slitting step can be in constant temperature oven or drying chamber, and 37 DEG C dry 8-12 hours.
It is a further object to provide a kind of above-mentioned ELISA test strip apple of application, corn, tealeaves, tomato, The remaining method of Permethrin in the samples such as spinach, Chinese cabbage, it includes step：
(1) sample pre-treatments；
(2) it is detected with test strips；
(3) testing result is analyzed.
The Permethrin time-resolved fluorescence test strips of the present invention are using the antibody antigen reaction of high degree of specificity and immune layer Analytical technology is analysed, Permethrin monoclonal antibody-time-resolved fluorescence marker is fixed on Fusion5 films, the chlorine in sample Pyrethroids is combined in flow process with Permethrin monoclonal antibody-time-resolved fluorescence marker on Fusion5 films, is formed Drug-antibody-time-resolved fluorescence marker.Drug in sample and the Permethrin hapten-carrier in reaction film detection line Protein conjugate competitive binding Permethrin monoclonal antibody-time-resolved fluorescence marker, final application immunochromatography detector Calculate the Permethrin residual quantity contained in analyte sample fluid.
The test strips of the present invention have high specificity, high sensitivity, quantitative detection, at low cost, easy to operate, detection time The advantages of short, suitable various units use, storage is simple, long shelf-life.With the remaining side of ELISA test strip Permethrin of the present invention Method is easy, it is quick, intuitive, accurate, applied widely, at low cost, easily promote the use of.
Description of the drawings
Fig. 1 is Permethrin hapten synthesis route map.
Fig. 2 is Permethrin haptens hydrogen nuclear magnetic resonance spectrogram.
Specific implementation mode
With reference to specific embodiment, the present invention is further explained.It should be understood that these embodiments are merely to illustrate this Invention, and be not used to limit the scope of the invention.
The preparation of 1 Permethrin test strip of embodiment
The preparation method of the test strips mainly includes the following steps that：
1) the sample absorption pad for being coated with Permethrin monoclonal antibody-time-resolved fluorescence marker is prepared；
2) preparing has the detection line for being coated with Permethrin hapten-carrier protein conjugate and is coated with anti-rabbit antibody The reaction film of nature controlling line；
3) will test strips 1) be assembled into sample absorption pad, reaction film and the water absorption pad 2) prepared.
Substep narration in detail below：
1, the preparation of Permethrin haptens
(1) 24.3g 3- (4-nitrophenoxy) benzaldehyde is dissolved in 100ml absolute methanols, dissolving is stirred at room temperature, point It criticizes and sodium borohydride 0.4g is added, react at room temperature 5h, solvent evaporated, column chromatography purifying obtains 23.3g aromatic alcohol objects, yield 95%；
(2) the aromatic alcohol 24.5g obtained in step 1 is added in 100ml chloroforms, dissolving is stirred at room temperature, be added The 30ml chloroform solns containing 22.6g dichloro chrysanthemum acyl chlorides are added dropwise in Py8.5g at room temperature；It is added dropwise, room temperature continues anti- 5h, solvent evaporated, column chromatography purifying is answered to obtain pyrethroids object 39.2g, yield 90%；
(3) it by the pyrethroids compound 43.5g obtained by step 2, is added in 120ml absolute methanols, room-temperature dissolution pours into Add in hydrogen kettle, Pd/C 3g are added, add 1 atmospheric pressure, react at room temperature 6h, be filtered to remove Pd/C, decompression removes solvent, obtains amino Object 36.5g, yield 90%；
(4) amino-compound 40.5g obtained by step 3 is added in 200ml chloroforms, dissolving is stirred at room temperature；It is added Succinic anhydride 12g is added in 1g p-methyl benzenesulfonic acids, and reaction is stirred at room temperature overnight；Solvent evaporated, column chromatography, which purifies to obtain, contains carboxyl Haptens object 43g, yield 85%.By Permethrin haptens hydrogen nuclear magnetic resonance spectrogram it is found that 12.2 carboxyl peak, 7-8 Aromatic ring peak and 0.97 methyl peak, illustrate hapten synthesis success.
There is no the structures for changing Permethrin for haptens prepared by this method, but from the stronger two aryl oxides structure of rigidity Draw linking arm so that whole active structures of Permethrin can be exposed to except antigen after coupling carrier albumen, be drawn Stronger immune response is played, the antibody of high-affinity can be obtained.
2, the preparation of immunogene
133mg BSA are dissolved in 5ml 0.01M PBS, stirring and dissolving.Permethrin haptens and BSA ratios can 1: 1~ It is adjusted in the range of 60: 1, the application selects that 50mg Permethrins haptens (being 50: 1 with BSA molar ratios) is being added, and EDC is added Hydrochloride 10mg, is stirred at room temperature 2h.The 48h that dialyses is moved into bag filter.Permethrin immunogene is obtained under the ratio can be in antigen Surface exposes more Permethrin haptens, to preferably cause the immune response for Permethrin.
3, the preparation of coating antigen
86mg OVA are dissolved in 5ml 0.01M PBS, stirring and dissolving.Permethrin haptens and OVA ratios can be 1: 1~40 : it is adjusted in the range of 1, the application selects that 40mg Permethrins haptens (being 40: 1 with OVA molar ratios) is being added, and EDC salt is added Hydrochlorate 10mg, is stirred at room temperature 2h.The 48h that dialyses is moved into bag filter.Obtained under the ratio Permethrin coating antigen with sample moderate resistance When original competition antibody, can fully and antibody response will not be excessive because of coating antigen exposed sites to avoid false positive, Steric hindrance is formed, to avoid false negative.
4, the preparation of Permethrin monoclonal antibody
Immunogene is injected in Balb/c new zealand white rabbit bodies, antiserum is generated.Take immune Balb/c New Zealand great Bai Rabbit splenocyte is merged with SP2/0 myeloma cell, is screened positive hole, is obtained the hybridoma of stably excreting monoclonal antibody Strain.Cell suspension is made with frozen stock solution in hybridoma, it is spare.Hybridoma is placed in cell culture medium, at 37 DEG C Under the conditions of cultivated, obtained culture solution is purified with octanoic acid-saturated ammonium sulfate method, obtains monoclonal antibody, -20 DEG C It preserves.
The cell culture medium is that calf serum and sodium bicarbonate are added into RPMI1640 culture mediums, and calf serum is made to exist Final concentration of 20% (mass fraction) in cell culture medium, the final concentration of 0.2% (matter of sodium bicarbonate in cell culture medium Measure score)；The pH of the cell culture medium is 7.4.It is immunogene to pathogen-free domestic using rabbit source antibody using sheep as immune animal Sheep is immunized, and anti-rabbit antibody is obtained.
5, the preparation of microspheres solution, detection line (T lines) solution and nature controlling line (C lines) solution
(1) configuration of microspheres solution
With release buffer solution (containing 20% sucrose, 5% trehalose, 0.5%BSA, 0.2% gentamicin) by Quality Control microballoon It is configured to time-resolved fluorescence microballoon mixed liquor, the final concentration of 0.1mg/mL-0.3mg/mL of Quality Control microballoon, detection with detection microballoon The final concentration of 0.8mg/mL-1.2mg/mL of microballoon.
(2) preparation of detection line (T lines) solution
Permethrin antigenic compound is diluted to 0.4mg/mL-0.6mg/mL with 0.01M PB solution.
(3) preparation of nature controlling line (C lines) solution
Streptavidin is diluted to 0.4mg/mL-0.6mg/mL with 0.01M PB solution.
6, the preparation of test strips
Reaction film is located at centre, and sample absorption pad is overlapped in reaction film left and right ends with blotting paper, is pasted onto and carries respectively On the plastic bottom board of gum.It is 0.6 μ L/cm-1.2 μ L/cm that C, T line, which spray film liquid amount, and it is 2 μ L/cm-6 μ L/ that microspheres solution, which sprays film amount, cm.The Permethrin for having sprayed reagent is stuck in greatly in constant temperature oven and is dried 12 hours for 37 DEG C.The Permethrin kilocalorie of drying is cut into The paper slip of 3mm-5mm width is to get to Permethrin test strips.
The remaining detection of Permethrin in 2 sample of embodiment
1, sample treatment
(1) apple pre-treating method.
With homogenizer homogeneous samples；The apple sample after (2.00 ± 0.05) g homogeneous is weighed to centrifuge to 10mL polystyrene Guan Zhong is separately added into 4mL 0.01M PBS, 2mL methanol, with vortex instrument whirling motion 5min；4000r/min centrifuges 5min at room temperature； 200 μ L of supernatant are taken, is added in 800 μ L 0.01M PBS, mixes well, take its 50 μ L for analyzing.
(2) corn pre-treating method.
With homogenizer homogeneous samples；The corn sample after (2.00 ± 0.05) g homogeneous is weighed to centrifuge to 10mL polystyrene Guan Zhong, is separately added into 10% sodium chloride of 4mL, and 2mL methanol vibrates 5min with vortex instrument；4000r/min centrifuges 5min at room temperature； 100 μ L of supernatant are taken, is added in 900 μ L 0.01M PBS, mixes well, take its 50 μ L for analyzing.
(3) vegetables/tealeaves pre-treating method.
With homogenizer homogeneous samples；The vegetable sample after (1.00 ± 0.05) g homogeneous is weighed to centrifuge to 10mL polystyrene Guan Zhong is added 2mL 0.1mol/L sulfuric acid, 5mL methanol is added, with vortex instrument whirling motion 5min；, 1000r/min centrifugations at room temperature 5min；200 μ L of supernatant are taken, is added in 800 μ L 0.01MPBS, mixes well, take 50 μ L for analyzing.
2, it is detected with test strips
(1) test strips and extracting solution are restored to room temperature.
(2) 60 μ L measuring samples are accurately drawn with pipettor, (sucking bubble is careful not to when sampling) is vertically slowly added dropwise In well (S), start timing after sample-adding, reads result within 8 minutes after sample-adding.
(3) reagent card is inserted into the carrier of immunochromatography detector, presses detection key, instrument will automatically stick into test Row scanning.
(4) testing result is read from the display screen of immunochromatography detector.
3, testing result is analyzed
Detection limit range：1ng/ml-30ng/ml.
3 sample detection example of embodiment
1, detection limit experiment
The samples such as blank apple/corn/tealeaves/tomato/spinach/Chinese cabbage are taken, add Permethrin respectively to final concentration of 0.5ng/ml, 1ng/ml, 5ng/ml, 10ng/ml, 15ng/ml, 25ng/ml, 30ng/ml, 35ng/ml take test strips to be examined It surveys, each sample is repeated three times.
When with samples such as ELISA test strip apple/corn/tealeaves/tomato/spinach/Chinese cabbages, is shown and tied according to test strips Fruit determines detection limit range, shows that this ELISA test strip limits range：1ng/ml-30ng/ml.
2, sample preci-sion and accuracy is tested
Using the rate of recovery as accuracy estimating index, the testing result relative standard deviation of a certain concentration samples of replication (RSD%) it is used as precision evaluation index.Calculation formula is：The rate of recovery (%)=actual measured value/theoretical value × 100%, Middle theoretical value is the addition concentration of sample；Relative standard deviation RSD%=SD/X × 100%, wherein SD are standard deviation, and X is The average value of determination data.
By five concentration Permethrins of 1ng/ml, 5ng/ml, 10ng/ml, 20ng/ml, 30ng/ml respectively to apple/corn/ The samples such as tealeaves/tomato/spinach/Chinese cabbage be added recycling measure, each sample do 4 it is parallel, with three batches of different reagents It is measured, the average recovery rate and precision result for calculating sample see the table below.
1 sample precision of table and accuracy test
With the Permethrin of five concentration of 1ng/ml, 5ng/ml, 10ng/ml, 20ng/ml, 30ng/ml respectively to apple/jade The samples such as rice/tealeaves/tomato/spinach/Chinese cabbage are added, and average recovery rate is between 90.3%~106.9%；In batch, Relative standard deviation is respectively less than 15% between batch.
3, cross reacting rate is tested
Selection with Permethrin there are similar structures and the drug of similar functions to carry out time-resolved fluorescence test strips measurement, lead to The standard curve for crossing various drugs respectively obtains its 50% inhibition concentration, and it is anti-to the intersection of other medicines to calculate kit with following formula It should rate.
2 cross reacting rate of table is tested
Medicine name Cross reacting rate (%)
Permethrin 100
Cypermethrin ＜ 1
Decis ＜ 1
Fenpropathrin ＜ 1
Biphenthrin ＜ 1
Cyhalothrin ＜ 1
Lambda-cyhalothrin ＜ 1
Fenvalerate ＜ 1

Claims (7)

1. a kind of time-resolved fluorescence test strips of detection Permethrin, including sample absorption pad, reaction film and water absorption pad, special Sign is that the reaction film is located at centre, and sample absorption pad is overlapped in reaction film left and right ends respectively with water absorption pad；The sample Absorption pad is equipped with sample application zone and microballoon area, and microballoon area is loaded with Permethrin time time-resolved fluorescence microballoon；The reaction film It is equipped with detection line (T lines) and nature controlling line (C lines), T lines connect coating Permethrin antigen (coating antigen), and C lines are coated with anti-rabbit antibody, The Permethrin antigen is Permethrin hapten-carrier protein conjugate, and the preparation method of the Permethrin haptens is as follows：
(1) 24.3g 3- (4-nitrophenoxy) benzaldehyde is dissolved in 100ml absolute methanols, dissolving is stirred at room temperature, added in batches Enter sodium borohydride 0.4g, react at room temperature 5h, solvent evaporated, column chromatography purifying obtains 23.3g aromatic alcohol objects, yield 95%；
(2) the aromatic alcohol 24.5g obtained in step 1 is added in 100ml chloroforms, dissolving is stirred at room temperature, be added The 30ml chloroform solns containing 22.6g dichloro chrysanthemum acyl chlorides are added dropwise in Py8.5g at room temperature；It is added dropwise, room temperature continues anti- 5h, solvent evaporated, column chromatography purifying is answered to obtain pyrethroids object 39.2g, yield 90%；
(3) it by the pyrethroids compound 43.5g obtained by step 2, is added in 120ml absolute methanols, room-temperature dissolution, pours into and add hydrogen In kettle, Pd/C 3g are added, add 1 atmospheric pressure, react at room temperature 6h, are filtered to remove Pd/C, decompression removes solvent, obtains amino target Object 36.5g, yield 90%；
(4) amino-compound 40.5g obtained by step 3 is added in 200ml chloroforms, dissolving is stirred at room temperature；It is added 1g pairs Succinic anhydride 12g is added in toluenesulfonic acid, and reaction is stirred at room temperature overnight；Solvent evaporated, column chromatography purify to obtain carboxylic half Antigen targets 43g, yield 85%.
2. the time-resolved fluorescence test strips of detection Permethrin as described in claim 1, it is characterised in that the Permethrin half The molecular structural formula of antigen is：
3. the time-resolved fluorescence test strips of detection Permethrin as described in claim 1, it is characterised in that when the Permethrin Between resolved fluorometric microballoon, including detection microballoon and Quality Control microballoon, detection microballoon, which is pan coating, Permethrin monoclonal antibody Fluorescent microsphere, Quality Control microballoon are the fluorescent microsphere that pan coating has rabbit-anti labelled protein.
4. the time-resolved fluorescence test strips of detection Permethrin as described in claim 1, Permethrin monoclonal antibody is with chlorine Pyrethroids hapten-carrier protein conjugate is prepared as immunogene, and the preparation method of the immunogene is as follows：
133mg BSA are dissolved in 5ml 0.01M PBS, stirring and dissolving；Permethrin haptens and BSA ratios can be 1: 1~60: 1 In the range of adjust, the application select be added 50mg Permethrins haptens (be 50: 1 with BSA molar ratios), addition EDC hydrochloric acid Salt 10mg, is stirred at room temperature 2h；The 48h that dialyses is moved into bag filter.
5. the time-resolved fluorescence test strips of detection Permethrin as described in claim 1, the preparation method of the coating antigen is such as Under：
86mg OVA are dissolved in 5ml 0.01M PBS, stirring and dissolving；Permethrin haptens and OVA ratios can be 1: 1~40: 1 Adjustment in range, the application select that 40mg Permethrins haptens (being 40: 1 with OVA molar ratios) is being added, and EDC hydrochlorides are added 2h is stirred at room temperature in 10mg；The 48h that dialyses is moved into bag filter.
6. a kind of method preparing any one of claim 1-5 test strips comprising step：
1) the sample absorption pad for being coated with Permethrin monoclonal antibody-time-resolved fluorescence marker is prepared；
2) it prepares to have and is coated with the detection line of Permethrin hapten-carrier protein conjugate and is coated with the Quality Control of anti-rabbit antibody The reaction film of line；
3) will test strips 1) be assembled into sample absorption pad, reaction film and the water absorption pad 2) prepared.
7. the remaining method of Permethrin in the samples such as a kind of detection apple, corn, tealeaves, tomato, spinach, Chinese cabbage comprising Step：
1) Sample pretreatment；
2) it is detected with claim 1-6 any one of them test strips；
3) testing result is analyzed.
CN201810217657.1A 2018-03-16 2018-03-16 Time-resolved fluorescence test strip for detecting permethrin and application thereof Pending CN108535474A (en)

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CN110684110A (en) * 2019-09-20 2020-01-14 北京勤邦生物技术有限公司 Preparation and application of pirimiphos-methyl monoclonal antibody
CN112858537A (en) * 2021-04-14 2021-05-28 上海市质量监督检验技术研究院 Rapid detection method for permethrin in anti-mosquito fabric
CN114107368A (en) * 2021-11-29 2022-03-01 重庆大学 Combined expression vector for expressing trans-chrysanthemic acid and application of combined expression vector in regulation and control of synthesis of trans-chrysanthemic acid by tomato type VI glandular hairs
CN115093347A (en) * 2022-06-20 2022-09-23 云南省烟草质量监督检测站 Hapten for detecting cypermethrin content and application thereof

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CN114107368B (en) * 2021-11-29 2023-05-26 重庆大学 Combined expression vector for expressing trans-chrysanthemic acid and application thereof in regulation and control of synthesis of trans-chrysanthemic acid by tomato VI glandular wool
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CN115093347B (en) * 2022-06-20 2023-09-12 云南省烟草质量监督检测站 Hapten for detecting cypermethrin content and application thereof

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