CN109061158A - A kind of time-resolved fluoroimmunoassay chromatograph test strip and its preparation method and application detecting Acetamiprid - Google Patents
A kind of time-resolved fluoroimmunoassay chromatograph test strip and its preparation method and application detecting Acetamiprid Download PDFInfo
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Abstract
The invention discloses a kind of time-resolved fluoroimmunoassay chromatograph test strips and its preparation method and application for detecting Acetamiprid.The test strips include bottom plate and successively overlap the sample absorption pad of stickup, conjugate release pad, nitrocellulose filter and water absorption pad on bottom plate, the Acetamiprid monoclonal antibody of fluorescent microsphere label is embedded in the conjugate release pad, detection zone and quality control region are fixed on the nitrocellulose filter, the detection zone is coated with Acetamiprid hapten-carrier protein conjugate, the quality control region is coated with sheep anti mouse antiantibody, and the Acetamiprid haptens is that first Acetamiprid is gone to react with aminobutyric acid by N-.The present invention also provides the methods of Acetamiprid in the above-mentioned test strips test sample of the preparation method and application of the test strips.Test strips and detection method provided by the invention have the advantages that easy to operate, high sensitivity, detection speed are fast, at low cost, can be realized quick detection and on-site supervision to Acetamiprid in batch samples.
Description
Technical field
The invention belongs to Detecting Pesticide fields, and in particular to it is a kind of detection tobacco and tobacco product in Acetamiprid when
Between resolved fluorometric immuno-chromatographic test paper strip and its preparation method and application.
Background technique
Acetamiprid (acetamiprid) belongs to anabasine insecticide, and anabasine insecticide is after organic phosphorus, amino first
The important insecticide of one kind after acid esters and pyrethroid insecticides mainly passes through selectivity control insect nervous centralis
System nicotinic acetylcholine esterase receptor (nAChRs) causes to do harm to block the normal conduction of insect CNS
There is general paralysis and then death in worm.Acetamiprid is the representative medicaments of anabasine insecticide, extensive in agricultural production
For the prevention and treatment of various pests in seed, blade face and soil, to the pest controling effects such as aphid, leafhopper, Bemisia tabaci, leaf miner compared with
For ideal, it is mainly used for the prevention and treatment of cigarette aphid on tobacco.Pesticide residue control is the important content of Product quality and safety control, is
The emphasis that government organs, manufacturing enterprise and consumer pay close attention to jointly.Acetamiprid uses extensively in recent years, and recall rate is high, has repeatly super
Limit happens.To maintain strict control over gate of the quality monitoring, GB 2763-2016 " national food safety standard Pesticide maximum residue limit "
Middle regulation Acetamiprid is in maximum residue limit of the maximum residue limit (MRL) between 0.2 ~ 1 mg/kg, in fruit in vegetables
For amount between 0.5 ~ 2 mg/kg, the maximum residue limit in brown rice, wheat, cottonseed, tealeaves is respectively 0.5,0.5,0.1 and
10 mg/kg, China not yet formulate the maximum residue limit of Acetamiprid in tobacco, international tobacco scientific research Cooperation Centre
(CORESTA) the guiding residue limits for providing Acetamiprid in tobacco are 3 mg/kg.Therefore, to avoid acetamiprid residue to people
Body causes damages and breaks through foreign trade barrier, establishes the detection side of simple, quick, accurate, reliable acetamiprid residue amount
Method is necessary.
Currently, common detection method has high performance liquid chromatography, Liquid Chromatography-Tandem Mass Spectrometry, gas chromatography, gas phase
Chromatograph-mass spectrometer coupling method etc..Since above method is both needed to advanced detecting instrument, testing cost valuableness, complex steps, time-consuming, and
It is professional to operator more demanding, it is not suitable for the high-throughput rapid screening detection of enterprises and institutions of base.
Fluorescence immune chromatography technology is grown up in fluorochrome label technology, as a kind of immunology detection side
Method, it is the combination of affine in immunity technology, immunolabelling technique, immunochromatography technique, is had many advantages, such as quick, easy to operate.
Compared to conventional tag object, the luminous intensity of fluorescent microsphere can enhance with the enhanced strength of exciting light, so fluorescent microsphere mark
Note is expected to improve the detection limit of immunochromatography technique；And under the action of microballoon shell structure, fluorescent microsphere has metastable
Morphosis, homogeneous grain diameter, monodispersity are good, stability is good, luminous efficiency is high, reproducible, there is preferable biocompatibility；
Dyestuff fluorescent quenching greatly reduces after forming microballoon, and transmitting is strong and stablizes, and is not influenced substantially by external environment media variations.
Therefore above-mentioned detection method is compared, fluorescent micro-ball immune chromatography technology has that detection sensitivity is high, easy to operate, stability simultaneously
Good advantage.
Summary of the invention
It is an object of the invention in view of the above-mentioned defects in the prior art, provide a kind of high sensitivity, easy to operate, inspection
Survey the time-resolved fluoroimmunoassay chromatograph test strip of quick, cheap detection Acetamiprid；Another object of the present invention is
The preparation method of above-mentioned test strips is provided；It is also another object of the present invention to provide above-mentioned test strips answering in detection Acetamiprid
With.
To achieve the goals above, the technical solution that the present invention takes is:
A kind of time-resolved fluoroimmunoassay chromatograph test strip for detecting Acetamiprid is provided, it includes bottom plate and successively takes on bottom plate
Sample absorption pad, conjugate release pad, nitrocellulose filter and the water absorption pad of stickup are connect, is embedded in the conjugate release pad
The Acetamiprid monoclonal antibody of fluorescent microsphere label, is fixed with detection zone and quality control region, the inspection on the nitrocellulose filter
It surveys area and is coated with Acetamiprid hapten-carrier protein conjugate, the quality control region is coated with sheep anti mouse antiantibody.
The Acetamiprid monoclonal antibody is to prepare to obtain using Acetamiprid hapten-carrier protein conjugate as immunogene
?；The Acetamiprid hapten-carrier protein conjugate is to be obtained by Acetamiprid haptens with carrier protein couplet, the carrier
Albumen is bovine serum albumin(BSA), ovalbumin, keyhole limpet hemocyanin, thyroprotein or human serum albumins, the Acetamiprid
Haptens is that first Acetamiprid is gone to react to obtain with aminobutyric acid by N-, molecular structural formula are as follows:
。
The preparation of the Acetamiprid haptens specifically includes the following steps:
It takes N- to remove 0.50 g of first Acetamiprid, adds 50 mL of methanol to dissolve, add 0.30 g of aminobutyric acid, stir, add 37% formaldehyde water-soluble
0.37 mL of liquid is stirred, and is mixed, 80 DEG C of 4 h of reaction；Stop reaction, revolving removes methanol, adds 50 mL of water, add 100 mL acetic acid
Ethyl ester extraction, extraction three times, merge organic phase, and revolving is evaporated, upper silicagel column, methylene chloride/methanol (V/V, 10/1) and elution point
From obtaining 0.69 g of haptens product carboxyl Acetamiprid.
The fluorescent microsphere is the microballoon that fluorescent material is wrapped up with polystyrene that diameter is 100 ~ 300nm, and surface connects
It is connected to-COOH group, the fluorescent material is group of the lanthanides.
The sheep anti mouse antiantibody is to carry out immune prepare to goat using source of mouse antibody as immunogene.
Another technical solution that the present invention takes is to provide a kind of time-resolved fluorescence for preparing above-mentioned detection Acetamiprid
The method of immuno-chromatographic test paper strip, it includes the following steps:
1) preparation of conjugate release pad: Acetamiprid monoclonal antibody is marked with commercially available fluorescent microsphere, and by it with specific slow
After rushing system dilution, conjugate release pad is soaked in dilution buffer, is prepared after vacuum freeze drying；
2) preparation of nitrocellulose filter: Acetamiprid hapten-carrier protein conjugate is sprayed on nitrocellulose filter
Detection zone range, is made detection zone；Sheep anti mouse antiantibody is sprayed into the quality control region range on nitrocellulose filter, Quality Control is made
Area；
3) it assembles and shears: pasting sample absorption pad with successively overlapping on bottom plate, be embedded with fluorescent microsphere label Acetamiprid list
The conjugate release pad of clonal antibody, the nitrocellulose filter and water absorption pad for being fixed with detection zone and quality control region, and cut into institute
The width needed is time-resolved fluoroimmunoassay chromatograph test strip.
Specifically, step includes:
1) it goes first Acetamiprid to react with aminobutyric acid by N-, prepares Acetamiprid haptens；
2) by Acetamiprid haptens and carrier protein couplet, Acetamiprid hapten-carrier protein conjugate is prepared；
3) mouse is immunized with Acetamiprid hapten-carrier protein conjugate, mouse boosting cell and murine myeloma cell is passed through
Fusion, screening, obtain the hybridoma cell strain of secretion Acetamiprid monoclonal antibody；
4) mouse IgG immune health goat is extracted, sheep anti mouse antiantibody is obtained；
5) Acetamiprid hapten-carrier protein conjugate and sheep anti mouse antiantibody are sprayed to the detection of nitrocellulose filter respectively
Area's range (T) and quality control region range (C)；
6) sample absorption pad is used and contains 0.5% bovine serum albumin(BSA) (volume fraction), pH 7.2,0.1 mol/L phosphate-buffered
Liquid impregnates 2 h, dries 2 h at 37 DEG C；
7) with commercially available fluorescent microsphere mark Acetamiprid monoclonal antibody, and by its with specific buffer system dilution after, will combine
Object release pad is soaked in dilution buffer, spare after vacuum freeze drying；
8) combination that successively overlap joint pastes sample absorption pad, is embedded with fluorescent microsphere label Acetamiprid monoclonal antibody on bottom plate
Object release pad, the nitrocellulose filter and water absorption pad for being fixed with detection zone and quality control region, and required width is cut into when being
Between resolved fluorometric immuno-chromatographic test paper strip.
Another technical solution that the present invention takes is to provide a kind of time-resolved fluoroimmunoassay of above-mentioned detection Acetamiprid
Application of the chromatograph test strip in detection Acetamiprid, it includes the following steps:
1) sample pre-treatments；
2) it is detected with the time-resolved fluoroimmunoassay chromatograph test strip of the detection Acetamiprid；
3) fluorescence detector analysis detection result is used.
Compared with prior art, the invention has the following advantages:
(1) high specificity, high sensitivity: this test strips, which is used, is embedded in knot for the Acetamiprid monoclonal antibody that fluorescent microsphere marks
Close object release pad on, have hydrophily it is good, can large capacity absorption antibody coupling matter, rapidly again moisten, antibody conjugates release fill
Point, performance is good, release is fast, form is good etc., and advantages reduce cost to reduce error, increase the reaction sensitivity of whole system.
(2) time-resolved fluorescence is displaced with larger stock, reduces the special stray light as caused by exciting light to detection
Interference, improve fluorescence detection stability；Its service life is long, eliminates interference of the fluorescent material to determinand in environment；It is excited
Wavelength is wider, and emission spectrum range is relatively narrow, reduces background fluorescence, improves resolution ratio.
(3) polystyrene has been wrapped up on fluorescent microsphere surface, realizes the protection to fluorescent material group of the lanthanides, reduces extraneous ring
The interference in border increases the stability and fluorescence lifetime of fluorescent microsphere.
(4) fluorescent microsphere surface modified active group-COOH is formed anti-using the method for chemical coupling come labelled antibody
The stable bond of body and microballoon.
It there is no the time-resolved fluoroimmunoassay chromatograph test strip for detecting Acetamiprid in tobacco and tobacco product at present, this
The blank has been filled up in invention.Test strips of the invention are at low cost, easy to operate, detection time is short, various units is suitble to make
With the advantages of, storage is simple, long shelf-life, with the method for test strips of the present invention detection Acetamiprid, it is easy, quickly, it is intuitive, quasi-
Really, without large-scale instrument, at low cost, easy popularization and use.
Detailed description of the invention
Fig. 1 is time-resolved fluoroimmunoassay chromatograph test strip the schematic diagram of the section structure；
Fig. 2 is Acetamiprid hapten synthesis route map (figure is as Figure of abstract).
Specific embodiment
Further detailed description is done to the present invention below with reference to examples and drawings, but embodiments of the present invention are unlimited
In this.
Embodiment 1 detects the composition of the time-resolved fluoroimmunoassay chromatograph test strip of Acetamiprid
One, test strips
Referring to Fig. 1: the test strips are by bottom plate, sample absorption pad, conjugate release pad, nitrocellulose filter and water absorption pad group
At；
Successively overlap joint is pasted onto order for the sample absorption pad 1, conjugate release pad 2, nitrocellulose filter 3 and water absorption pad 4
On bottom plate 7, conjugate release pad has 1/3 region to be absorbed by the sample pad covering, the end of conjugate release pad and nitre from starting point
The beginning of acid cellulose film connects, and the end of nitrocellulose filter is connected with the beginning of water absorption pad, the beginning of sample absorption pad and
The beginning of PVC bottom plate is aligned, and the end of water absorption pad is aligned with the end of PVC bottom plate；
Detection zone 5 and quality control region 6 are fixed on the nitrocellulose filter, detection zone is coated with Acetamiprid hapten-carrier egg
White conjugate (Acetamiprid haptens-ovalbumin conjugate), quality control region is coated with sheep anti mouse antiantibody；
The bottom plate is PVC bottom plate；The conjugate release pad is mineral wool；The water absorption pad is blotting paper.
Embodiment 2 detects the preparation of the time-resolved fluoroimmunoassay chromatograph test strip of Acetamiprid
The preparation method for detecting the time-resolved fluoroimmunoassay chromatograph test strip of Acetamiprid mainly comprises the steps that
1) preparation of conjugate release pad: Acetamiprid monoclonal antibody is marked with commercially available fluorescent microsphere, and by it with specific slow
After rushing system dilution, conjugate release pad is soaked in dilution buffer, is prepared after vacuum freeze drying；
2) preparation of nitrocellulose filter: Acetamiprid hapten-carrier protein conjugate is sprayed on nitrocellulose filter
Detection zone range, is made detection zone；Sheep anti mouse antiantibody is sprayed into the quality control region range on nitrocellulose filter, Quality Control is made
Area；
3) it assembles and shears: pasting sample absorption pad with successively overlapping on bottom plate, be embedded with fluorescent microsphere label Acetamiprid list
The conjugate release pad of clonal antibody, the nitrocellulose filter and water absorption pad for being fixed with detection zone and quality control region, and cut into institute
The width needed is time-resolved fluoroimmunoassay chromatograph test strip.
Substep narration in detail below:
(1) preparation of each component
1, the synthesis and identification of Acetamiprid hapten-carrier protein conjugate
Acetamiprid is small-molecule substance, only immunoreactivity, without immunogenicity, cannot induce body and generate immune response,
Must with after macromolecular carrier albumen coupling just have immunogenicity.
(1) preparation of Acetamiprid haptens
It takes N- to remove 0.50 g of first Acetamiprid, adds 50 mL of methanol to dissolve, add 0.30 g of aminobutyric acid, stir, add 37% formaldehyde water-soluble
0.37 mL of liquid is stirred, and is mixed, 80 DEG C of 4 h of reaction；Stop reaction, revolving removes methanol, adds 50 mL of water, add 100 mL acetic acid
Ethyl ester extraction, extraction three times, merge organic phase, and revolving is evaporated, upper silicagel column, methylene chloride/methanol (V/V, 10/1) and elution point
From obtaining 0.69 g of haptens product carboxyl Acetamiprid, yield 92.81%.
Above-mentioned haptens is taken to identify through nuclear magnetic resonance spectroscopy, 1H NMR(CDCl3, 300MHZ) δ: 11.0 (1H ,-
COOH), 8.53 (1H, s, ArH), 7.86 (1H, d, ArH), 7.21 (1H, d, ArH), 4.16 (1H, dd,
=CH-), 3.91 (1H, s, -CH2-), 3.21 (2H, d, -CH2-), 2.82 (2H, t, -CH2-), 2.49
(2H, t, -CH2-), 2.26 (3H, s, -CH3), 2.0 (1H, s, -NH-).Chemical shift δ=11 are on spacerarm
The resonance absorbing peak of carboxyl hydrogen, δ=2.82,2.49 be the resonance absorbing peak of methylene hydrogen on spacerarm, the presence at these peaks, card
Bright spacerarm is coupled successfully.
(2) preparation of immunogene
Acetamiprid haptens and bovine serum albumin(BSA) (BSA) coupling obtain immunogene.
11 mg of haptens carboxyl Acetamiprid is taken, 1 mL of dimethyl sulfoxide is added to dissolve, chlorination iso-butyl formate 0.18 mL, three
Ethamine 0.1 mL, 0 ~ 4 DEG C of reaction 1h obtain haptens activating solution A liquid；50 mg of BSA is taken, adds 0.8% salt water, 3 mL to dissolve, obtains
To B liquid；A drop is added in B liquid, continues to be stirred to react 5h, 0.02 mol/L PB dialysis purification 3 days is changed liquid 3 times daily, obtained
To immunogene, packing, -20 DEG C of preservations.
(3) preparation of coating antigen
Acetamiprid haptens and ovalbumin (OVA) coupling obtain coating antigen.
5 mg of haptens carboxyl Acetamiprid is taken, adds 1 mL of dimethyl sulfoxide to dissolve, adds dicyclohexylcarbodiimide (DCC) 9
Mg, 6 mg of n-hydroxysuccinimide (NHS) react at room temperature 1h, obtain haptens activating solution A liquid；50 mg of OVA is taken, is added
0.8% salt water, 3 mL dissolution, obtains B liquid；A drop is added in B liquid, continues to be stirred to react 5h, 0.02 mol/L PB dialysis is pure
Change 3 days, change daily liquid 3 times, obtain coating antigen, dispenses, -20 DEG C of preservations.
(4) it identifies
In the ratio of synthesis Acetamiprid coupled antigen reaction haptens used, carrier protein and coupled product, ultraviolet (200 are carried out
The nm of nm ~ 400) sweep measuring, by comparing three respectively the light absorption value of 260 nm and 280 nm calculate its combine than.Coupling
The maximum absorption band of object Acetamiprid hapten-carrier albumen with Acetamiprid haptens, carrier protein maximum absorption band compared with send out
Apparent variation has been given birth to, has shown that the synthesis of Acetamiprid hapten-carrier albumen is successful.It is computed, the knot of haptens and BSA
Composition and division in a proportion is 14:1, and the combination ratio of OVA is 9:1.
2, the preparation of Acetamiprid monoclonal antibody
(1) acquisition of hybridoma
1) first immunisation: Acetamiprid haptens-BSA conjugate (immunogene) and the Freund's complete adjuvant of equivalent is fully emulsified,
The Balb/c mouse of 6 week old, every 0.2 mL is subcutaneously injected；
2) booster immunization is twice: since first immunisation, booster immunization is primary every two weeks, replaces Freund with not formula Freund's incomplete adjuvant
Freund's complete adjuvant, method and the same first immunisation of dosage；
3) potency and inhibition are surveyed in eyeground vein blood sampling to last time booster immunization after a week, have inhibition and potency reaches 1:10000
Following final immunization is carried out when above: 0.1 mL of immunogen solution of any adjuvant is not added in intraperitoneal injection, puts to death mouse after three days,
Its spleen is taken to merge with myeloma cell；
4) cell supernatant is measured using indirect competitive enzyme-linked immunosorbent analysis method, screens positive hole.Utilize limiting dilution assay pair
Positive hole carries out cloning, obtains and establishes the hybridoma cell strain of stably excreting Acetamiprid monoclonal antibody, take in logarithm
Cell suspension is made with frozen stock solution in the hybridoma in growth period, is sub-packed in cryopreservation tube, saves for a long time in liquid nitrogen.
(2) preparation of monoclonal antibody
1) cell recovery: taking out Acetamiprid monoclonal antibody hybridoma cell strain cryopreservation tube, be immediately placed in 37 DEG C of water-bath middling speeds and melt,
After centrifugation removal frozen stock solution, culture culture in glassware is moved into；
2) ascites and antibody purification are prepared: using method is induced in vivo, by Balb/c mouse (8 week old) Intraperitoneal injection sterilizing paraffin oil
Only, hybridoma 5 × 10 is injected intraperitoneally in 0.5 mL/ after 7 days5A/only, ascites is acquired after 7 days.With octanoic acid-saturated ammonium sulfate
Method is purified, and Acetamiprid monoclonal antibody solution (- 20 DEG C of preservations) is obtained.
(3) measurement of antibody titer
Potency with indirect competitive ELISA method measurement antibody is 1:(100000 ~ 300000).
Indirect competitive ELISA method: using Acetamiprid haptens-OVA conjugate coated elisa plate, and Acetamiprid standard items are added
The sheep anti mouse antiantibody solution of solution, Acetamiprid monoclonal antibody solution and horseradish peroxidase-labeled, 25 DEG C of reactions 30
Min pours out liquid in hole, is washed 3 ~ 5 times with cleaning solution, is patted dry with blotting paper；Substrate developing solution, 25 DEG C of 15 min of reaction are added
Afterwards, terminate liquid is added and terminates reaction；Setting microplate reader measures every hole absorbance value at 450 nm of wavelength.
(4) measurement of monoclonal antibody specificity
Antibody specificity refer to the ability of its homospecificity antigen binding compared with such antigen-analogues ability, often
Use cross reacting rate as evaluation criterion.Cross reaction is smaller, and the specificity of antibody is then higher.
This experiment is by nicotinic insecticide (Acetamiprid, imidacloprid, Nitenpyram, thiacloprid, Diacloden, clothianidin, furan worm
Amine) it is serially diluted, indirect competitive ELISA is carried out with monoclonal antibody respectively, makes standard curve, analysis obtains IC50, then
Cross reacting rate is calculated as follows:
The cross reacting rate of each analog as the result is shown are as follows: Acetamiprid 100%, imidacloprid < 1%, Nitenpyram < 1%, thiacloprid
< 1%, Diacloden < 1%, clothianidin < 1%, dinotefuran < 1%.Antibody of the present invention is to imidacloprid, Nitenpyram, thiacloprid, thiophene worm
Other nicotinic insecticide no cross reactions such as piperazine, clothianidin, dinotefuran, have specific binding just for Acetamiprid.
3, the preparation of sheep anti mouse antiantibody
Using sheep as immune animal, pathogen-free domestic sheep is immunized using source of mouse antibody as immunogene, obtains sheep anti mouse antiantibody.
4, the preparation of fluorescent microsphere label Acetamiprid monoclonal antibody
(1) it activates: commercially available internal embedding fluorescent dye, surface modification being taken to have the 100 μ L of microsphere suspensions of carboxyl functional group to be suspended
In 900 μ L activation buffers, supernatant is abandoned after 4 DEG C of 10000 r/min is centrifuged 10min, it is slow in 1 mL activation that microballoon is resuspended
It in fliud flushing, washs microballoon 2 times in this way, appropriate activator is added, shaken at room temperature activates 10 min after mixing；
(2) it is coupled: (1) described suspension being abandoned into supernatant after 4 DEG C of 10000 r/min is centrifuged 10min, is resuspended in coupling buffering
It in liquid, washs microballoon 2 times in this way, 10 ~ 20 μ L Acetamiprid monoclonal antibody solutions (1 mg/mL of protein concentration) is added, mix
120 min of room temperature concussion coupling afterwards；
(3) it closes: (2) described suspension being abandoned into supernatant after 4 DEG C of 10000 r/min is centrifuged 10 min, is resuspended in closing buffering
It in liquid, washs microballoon 1 time in this way, 30 min of room temperature concussion closing after mixing；
(4) it stores: (3) described suspension being abandoned into supernatant after 4 DEG C of 10000 r/min is centrifuged 10 min, is resuspended in storage buffering
In liquid, washs microballoon 1 time, be kept in dark place after mixing in 4 DEG C in this way.
The activation buffer is 2- (N- morpholine) ethanesulfonic acid (MES) buffering that pH value is 5.5 ~ 6.5,0.05 mol/L
Liquid.
The activator is water-soluble carbodiimide, wherein molal weight ratio EDC: NHS: COOH=(1.5 ~ 3): (8 ~
20): 1, required concentration is diluted to activation buffer before use.
The coupling buffer is that the borate buffer solution that pH value is 7.5 ~ 8.5 0.05 mol/L (is avoided using in the presence of trip
Solvent from amine).
The Block buffer is containing 0.1 ~ 0.4 mol/L primary amine (hydroxylamine hydrochloride, ethanol amine or ethylaminoethanol), 1% ~ 10%
The PB buffer that the pH value of BSA is 7.4.
The storage buffer is containing 0.01% NaN3, 0.1% BSA pH value be 7.4 PB buffer.
5, the preparation of conjugate release pad
After the Acetamiprid monoclonal antibody that the fluorescent microsphere of storage marks is diluted with storage buffer, conjugate release pad is soaked
It steeps in dilution buffer, it is spare after vacuum freeze drying.
6, the preparation of nitrocellulose (NC) film
Acetamiprid haptens-ovalbumin conjugate is diluted to 100 μ with 0.05 mol/L, the PBS buffer solution that pH value is 7.2
G/mL is sprayed at the detection zone (T) on NC film with Isoflow point film instrument, and spray film amount is 1.0 μ L/cm；With 0.01 mol/
L, sheep anti mouse antiantibody is diluted to 200 μ g/mL by the PBS buffer solution that pH value is 7.4, is sprayed at Isoflow point film instrument
Quality control region (C) on NC film, spray film amount are 1.0 μ L/cm.Dry 2 h, spare under the conditions of the NC film prepared is placed in 37 DEG C.
7, the preparation of sample absorption pad
Sample absorption pad is used and contains 0.5% bovine serum albumin(BSA) (volume fraction), pH value 7.2,0.1 mol/L phosphate-buffered
Liquid impregnates 2 h, dries 2 h at 37 DEG C.
(2) assembling of test strips
By sample absorption pad, conjugate release pad, nitrocellulose filter, water absorption pad, successively overlap joint is pasted and fixed on bottom from left to right
On plate, conjugate release pad has 1/3 region to be absorbed by the sample pad covering from starting point, and the end of conjugate release pad and nitric acid are fine
The beginning for tieing up plain film is connected, and the end of nitrocellulose filter is connected with the beginning of water absorption pad, the beginning and bottom plate of sample absorption pad
Beginning alignment, the end of water absorption pad is aligned with the end of bottom plate, the small item of 3.96 mm wide is then cut into machine, mounted in spy
In the plastics fabrication of system, test card is formed.Acetamiprid fluorescent micro-ball immune chromatography test paper is stuck in 2 ~ 8 DEG C of cool places and is protected from light dry guarantor
It deposits, validity period is 12 months.
Embodiment 3 detects the application of the time-resolved fluoroimmunoassay chromatograph test strip of Acetamiprid
1, tobacco sample pre-treatment
The tobacco leaf sample of 1.0 ± 0.05 g crushing is weighed into polystyrene centrifuge tube；5 mL, 50% methanol aqueous solution is added, uses
Refiner sufficiently smashes it, obtains sample liquid；It pipettes to be checked after 75 μ L sample liquid are mixed with 425 μ L deionized waters.
2, it is detected with test strips
It draws 100 μ L sample to be examined solution to be vertically added dropwise in test card well, liquid starts timing, reaction 10 when flowing
min；By in the carrier of test card insertion KFT-100A type fluorescence detector, project to be checked is selected by touch display screen, is pressed
Under " start to detect " key, fluorescence detector will be scanned test to test card automatically, by reading on the display screen of instrument
Take or print testing result.
3, Analysis of test results
(1) quantitative detection
After the completion of test, instrument obtains detection zone time-resolved fluorescence intensity and quality control region time-resolved fluorescence on fluorescent test paper strip
The ratio of intensity, detection zone time-resolved fluorescence intensity and quality control region time resolution are glimmering on the fluorescent test paper strip based on preset configuration
The ratio of luminous intensity and the relation curve of Acetamiprid concentration, obtain the content of Acetamiprid in sample to be tested extracting solution, most afterwards through changing
It calculates up to the content of Acetamiprid in sample to be tested.
(2) half-quantitative detection
After the completion of test, detection zone time-resolved fluorescence intensity and quality control region time-resolved fluorescence that instrument will be obtained according to detection
The ratio of intensity calculates the concentration value of Acetamiprid in extracting solution automatically, and provides yin and yang attribute judgement according to preset threshold value.
Negative (-): it if being as the result is shown feminine gender on the display screen of fluorescence detector, indicates not containing pyridine worm in sample
Amidine or its concentration are lower than detection limit.
Positive (+): if being as the result is shown the positive on the display screen of fluorescence detector, Acetamiprid concentration etc. in sample is indicated
In or higher than detection limit.
It is invalid: if fluorescence signal intensity is not detected in quality control region, to show that incorrect operating process or test card have been failed.
4 sample detection example of embodiment
1, sensitivity test
Acetamiprid standard items are diluted to 1,2,4 μ g/L respectively, used diluent is the phosphoric acid that pH value is 7.2,0.2 mol/L
Salt buffer.
It is detected with time-resolved fluorescence immuno-chromatographic test paper strip, as a result are as follows: Acetamiprid standard concentration is 1 μ g/L
When, fluorescence detector is detected as feminine gender；When Acetamiprid standard concentration is 2,4 μ g/L, fluorescence detector test positive, table
The sensitivity of bright test strips detection Acetamiprid is 2 μ g/L.
2, detection limit test
Acetamiprid standard items are added respectively into blank tobacco sample to final concentration of 0.05,0.1,0.2 mg/kg, with the time point
Distinguish that fluorescence immune chromatography test paper bar is detected, as a result are as follows: when Acetamiprid concentration is 0.05 mg/kg, fluorescence detector is detected as
It is negative；When Acetamiprid concentration is 0.1,0.2 mg/kg, fluorescence detector test positive shows this test strips to pyridine in tobacco
The detection of worm amidine is limited to 0.1 mg/kg.
3, false positive rate, false negative rate test
Take known Acetamiprid content greater than 20 parts of positive tobacco leaf sample of 0.1 mg/kg, it is known that the negative tobacco leaf without Acetamiprid
It 20 parts of sample, is detected respectively with the time-resolved fluoroimmunoassay chromatograph test strip that 3 batches produce, calculates its yin and yang attribute
Rate.It the results are shown in Table 2.
Table 2 detects positive, negative sample result
The result shows that: when detecting positive sample with the test strips of 3 batch productions, as a result it is all positive, it is known that positive coincidence rate
It is 100%, false negative rate 0；When detecting negative sample, as a result it is all negative, it is known that negative match-rate 100%, false positive rate
It is 0.Illustrate that the time-resolved fluoroimmunoassay chromatograph test strip of detection Acetamiprid of the invention can carry out Acetamiprid in tobacco leaf
Quickly detection.
4, specific test
Imidacloprid, Nitenpyram, thiacloprid, Diacloden, the clothianidin, dinotefuran of 10 mg/L are detected with Acetamiprid test strips.Knot
Fruit shows that fluorescence detector is detected as feminine gender.Illustrate this test strips to imidacloprid, Nitenpyram, thiacloprid, Diacloden, thiophene worm
Amine, dinotefuran no cross reaction.
Claims (7)
1. a kind of time-resolved fluoroimmunoassay chromatograph test strip for detecting Acetamiprid, including bottom plate and successively overlap joint is viscous on bottom plate
Sample absorption pad, conjugate release pad, nitrocellulose filter and the water absorption pad of patch, it is characterised in that in the conjugate release pad
It is embedded with the Acetamiprid monoclonal antibody of fluorescent microsphere label, is fixed with detection zone and quality control region on the nitrocellulose filter,
The detection zone is coated with Acetamiprid hapten-carrier protein conjugate, and the quality control region is coated with sheep anti mouse antiantibody；It is described
Acetamiprid monoclonal antibody is prepared using Acetamiprid hapten-carrier protein conjugate as immunogene；The Acetamiprid
Hapten-carrier protein conjugate is to be obtained by Acetamiprid haptens with carrier protein couplet, the Acetamiprid haptens be by
N- goes first Acetamiprid to react to obtain with aminobutyric acid, molecular structural formula are as follows:
。
2. the time-resolved fluoroimmunoassay chromatograph test strip of detection Acetamiprid as described in claim 1, it is characterised in that: described
The preparation reaction process of Acetamiprid haptens is as follows:
。
3. the time-resolved fluoroimmunoassay chromatograph test strip of detection Acetamiprid according to claim 1, it is characterised in that: institute
Stating fluorescent microsphere is the microballoon that fluorescent material is wrapped up with polystyrene that diameter is 100 ~ 300nm, and surface is connected with-COOH
Group.
4. the time-resolved fluoroimmunoassay chromatograph test strip of detection Acetamiprid according to claim 3, it is characterised in that: institute
Stating fluorescent material is group of the lanthanides.
5. the time-resolved fluoroimmunoassay chromatograph test strip of detection Acetamiprid according to claim 1, it is characterised in that: institute
Stating carrier protein is bovine serum albumin(BSA), ovalbumin, keyhole limpet hemocyanin, thyroprotein or human serum albumins.
6. a kind of preparation side of the time-resolved fluoroimmunoassay chromatograph test strip of any detection Acetamiprid of claim 1-5
Method, it is characterised in that the following steps are included:
1) preparation of conjugate release pad: Acetamiprid monoclonal antibody is marked with fluorescent microsphere, and by it with specific buffer system
After dilution, conjugate release pad is soaked in dilution buffer, is prepared after vacuum freeze drying；
2) preparation of nitrocellulose filter: Acetamiprid hapten-carrier protein conjugate is sprayed on nitrocellulose filter
Detection zone range, is made detection zone；Sheep anti mouse antiantibody is sprayed into the quality control region range on nitrocellulose filter, Quality Control is made
Area；
3) it assembles and shears: pasting sample absorption pad with successively overlapping on bottom plate, be embedded with fluorescent microsphere label Acetamiprid list
The conjugate release pad of clonal antibody, the nitrocellulose filter and water absorption pad for being fixed with detection zone and quality control region, and cut into institute
The width needed is time-resolved fluoroimmunoassay chromatograph test strip.
7. a kind of application of the time-resolved fluoroimmunoassay chromatograph test strip of any detection Acetamiprid of claim 1-5,
Characterized by the following steps:
1) sample pre-treatments；
2) it is detected with the test strips；
3) fluorescence detector analysis detection result is used.
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