CN109061149A - A kind of time-resolved fluoroimmunoassay chromatograph test strip and its preparation method and application detecting butralin - Google Patents

A kind of time-resolved fluoroimmunoassay chromatograph test strip and its preparation method and application detecting butralin Download PDF

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CN109061149A
CN109061149A CN201811104551.7A CN201811104551A CN109061149A CN 109061149 A CN109061149 A CN 109061149A CN 201811104551 A CN201811104551 A CN 201811104551A CN 109061149 A CN109061149 A CN 109061149A
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butralin
detection
test strip
time
preparation
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CN109061149B (en
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陈黎
范子彦
刘惠民
唐纲岭
崔华鹏
樊美娟
聂聪
赵乐
郭军伟
王洪波
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Zhengzhou Tobacco Research Institute of CNTC
National Tobacco Quality Supervision and Inspection Center
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Zhengzhou Tobacco Research Institute of CNTC
National Tobacco Quality Supervision and Inspection Center
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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 butralin.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 butralin 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 butralin hapten-carrier protein conjugate, the quality control region is coated with sheep anti mouse antiantibody, the butralin haptens is to be reacted by chloro- 3, the 5- dinitrobenzene acetic acid of 4- with butylamine.The present invention also provides the methods of butralin 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 butralin in batch samples.

Description

It is a kind of detect butralin time-resolved fluoroimmunoassay chromatograph test strip and its preparation side Method and application
Technical field
The invention belongs to Detecting Pesticide fields, and in particular to it is a kind of detection tobacco and tobacco product in butralin when Between resolved fluorometric immuno-chromatographic test paper strip and its preparation method and application.
Background technique
Butralin (Butralin) also known as Amex820, butralin, nitre Chem hoe, than reaching, peaceful, only bud element, chemical name are N- sec-butyl -4- tertiary butyl -2,6- dinitroaniline, molecular formula C14H21N3O4.Butralin is dinitroaniline herbicide, Sterling is orange-yellow crystalline solid, is soluble in organic solvent, and is insoluble in water, has volatility, easily decomposes after light, dynamic to people and animals etc. Species toxicity is lower.The medicine is that selectivity sprouts pro-herbicide, is acted on similar to trefanocide.After medicament enters plant, mainly Inhibit the cell division of separate living tissue, to inhibit young shoot and the young root growth of weeds, causes weeds dead.Butralin can be applied Inhibit weed growth in field crop, it can also be used to which the miscellaneous algae in water body is removed in aquaculture, and butralin is answered in tobacco leaf production With more, it is mainly used to inhibit the growth of tobacco leaf bud.It is secondary in international tobacco scientific research Cooperation Centre (CORESTA) regulation tobacco The guiding residue limits of fourth spirit are 5 mg/kg.Currently, common detection method has gas chromatography, high performance liquid chromatography, gas Phase chromatography-tandem mass spectrometry, Liquid Chromatography-Tandem Mass Spectrometry etc..Since above method is both needed to advanced detecting instrument, testing cost Valuableness, complex steps, time-consuming, and it is professional more demanding to operator, it is not suitable for the high throughput of enterprises and institutions of base Rapid screening detection.Therefore, it develops a kind of not examined equipment limit and can be realized and batch samples are quickly examined The product and method of survey become problem in the urgent need to address.
Fluorescent micro-ball immune chromatography technology is grown up in fluorochrome label technology, is examined as a kind of immunology Survey method, it is the combination of affine in immunity technology, immunolabelling technique, immunochromatography technique, is had quick, easy to operate etc. excellent Point.Compared to conventional tag object, the luminous intensity of fluorescent microsphere can enhance with the enhanced strength of exciting light, so fluorescent microsphere Label is expected to improve the detection limit of immunochromatography technique；And under the action of microballoon shell structure, fluorescent microsphere has relatively stable Morphosis, homogeneous grain diameter, monodispersity are good, stability is good, luminous efficiency is high, reproducible, there is preferable bio-compatible Property；Dyestuff fluorescent quenching greatly reduces after forming microballoon, and transmitting is strong and stablizes, and substantially not by the shadow of external environment media variations It rings.Therefore above-mentioned detection method is compared, fluorescent micro-ball immune chromatography technology has detection sensitivity high, easy to operate, steady simultaneously Qualitative 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 butralin；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 butralin 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 butralin 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 butralin 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 butralin hapten-carrier protein conjugate, the quality control region is coated with sheep anti mouse antiantibody.
The butralin monoclonal antibody is to prepare to obtain using butralin hapten-carrier protein conjugate as immunogene ?；The butralin hapten-carrier protein conjugate is to be obtained by butralin haptens with carrier protein couplet, the carrier Albumen is bovine serum albumin(BSA), ovalbumin, keyhole limpet hemocyanin, thyroprotein or human serum albumins, the butralin Haptens is to react to obtain with butylamine by chloro- 3, the 5- dinitrobenzene acetic acid of 4-, molecular structural formula are as follows:
。
The preparation of the butralin haptens specifically includes the following steps:
Chloro- 3,5- dinitrobenzene acetic acid, 1.00 g of 4- is taken, adds 50 mL ethyl alcohol to dissolve, adds 0.45 g of sodium carbonate, stirs, adds butylamine 0.31 g, 80 DEG C of oil bath heatings stir 3 h, detect, and raw material fundamental reaction is complete；Stop reaction, be cooled to room temperature, rotates, remove Ethyl alcohol is removed, 80 mL water are added, with 1 mol/L salt acid for adjusting pH value to 4, there are a large amount of muddinesses, adds 80 mL ethyl acetate to extract, water It washes, upper silicagel column, elutes separation with the methylene chloride/methanol that volume ratio is 5:1, obtain 1.05 g of butralin haptens product.
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.
Another technical solution that the present invention takes is to provide a kind of time-resolved fluorescence for preparing above-mentioned detection butralin The method of immuno-chromatographic test paper strip, it includes the following steps:
1) preparation of conjugate release pad: butralin 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: butralin 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 butralin 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) by 4- chloro- 3,5- dinitrobenzene acetic acid is reacted with butylamine, prepares butralin haptens；
2) by butralin haptens and carrier protein couplet, butralin hapten-carrier protein conjugate is prepared；
3) mouse is immunized with butralin hapten-carrier protein conjugate, mouse boosting cell and murine myeloma cell is passed through Fusion, screening, obtain the hybridoma cell strain of secretion butralin monoclonal antibody；
4) mouse IgG immune health goat is extracted, sheep anti mouse antiantibody is obtained；
5) butralin 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 butralin 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 butralin 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 butralin Application of the chromatograph test strip in detection butralin, it includes the following steps:
1) sample pre-treatments；
2) it is detected with the time-resolved fluoroimmunoassay chromatograph test strip of the detection butralin；
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 butralin 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 butralin 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 butralin, it is easy, quickly, it is intuitive, quasi- Really, applied widely, 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 butralin 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 butralin
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 butralin hapten-carrier egg White conjugate (butralin 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 butralin
The preparation method for detecting the time-resolved fluoroimmunoassay chromatograph test strip of butralin mainly comprises the steps that
1) preparation of conjugate release pad: butralin 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: butralin 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 butralin 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 butralin hapten-carrier protein conjugate
Butralin 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 butralin haptens
Chloro- 3,5- dinitrobenzene acetic acid, 1.00 g of 4- is taken, adds 50 mL ethyl alcohol to dissolve, adds 0.45 g of sodium carbonate, stirs, adds butylamine 0.31 g, 80 DEG C of oil bath heatings stir 3 h, detect, and raw material fundamental reaction is complete；Stop reaction, be cooled to room temperature, rotates, remove Ethyl alcohol is removed, 80 mL water are added, with 1 mol/L salt acid for adjusting pH value to 4, there are a large amount of muddinesses, adds 80 mL ethyl acetate to extract, water It washes, upper silicagel column, elutes separation with the methylene chloride/methanol that volume ratio is 5:1, obtain 1.05 g of butralin haptens product, Yield 92.11%.
Nuclear-magnetism identification1H NMR(CDCl3, 300MHz) and δ: 11.01(1H, d, J=0.000), 8.281(1H, d, J= 0.000), 4.011(1H, tq, J=6.914, J=6.757), 8.281(1H, d, J=0.000), 3.822(t, 2H), 2.791(1H, ddd, J=7.114, J=6.914), 1.591(2H, d, J=6.757), 1.251(3H, t, J=7.114), 0.901(3H, t, J=7.114).
In map, chemical shift δ=1.591,1.251,0.901,2.791 are methyl and methylene hydrogen on raw material butylamine Resonance absorbing peak, δ=4.011 are the resonance absorbing peak of formation imines hydrogen after the reaction of raw material butylamine, between the presence at these peaks proves It is coupled successfully every arm, butralin haptens structure is correct.
(2) preparation of immunogene
Butralin haptens and bovine serum albumin(BSA) (BSA) coupling obtain immunogene.
11 mg of butralin haptens is taken, adds 0.2 mL of dimethyl sulfoxide (DMSO) to dissolve, adds 20 μ L of triethylamine, is stirred, It mixes, 6 mg of chlorination iso-butyl formate, stirs 2 h, obtain haptens activating solution A liquid；50 mg BSA are taken to add 0.1 mol/L pH The PB buffer solution that value is 7.2, obtains B liquid, A liquid is slowly dropped in B liquid, continues to stir 5 h, stops reacting, 0.02 Mol/L PBS 3 d of dialysis, change liquid 3 times daily, obtain butralin-BSA conjugate, as immunogene, dispense, -20 DEG C of preservations, It is spare.
(3) preparation of coating antigen
Butralin haptens and ovalbumin (OVA) coupling obtain coating antigen.
6 mg of butralin haptens is taken, adds 0.2 mL of n,N-Dimethylformamide (DMF) to dissolve, adds carbodiimide (EDC) 4.5 mg are stirred, and clarification adds 2.24 mg of N- succinimide (NHS), and 2 h of activation are stirred at room temperature, obtain A liquid；Take OVA 50 Mg, adding 6 mL, 0.05 mol/L pH value is 7.2 PB buffer solution, obtains B liquid, A liquid is slowly dropped in B liquid, room Temperature is stirred to react 5 h.Stop reaction, 0.02 mol/L PBS buffer solution, 3 d of dialysis change liquid 3 times daily, obtain butralin-OVA Conjugate, as coating antigen, packing, -20 DEG C of preservations are spare.
(4) it identifies
In the ratio of synthesis butralin 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.It is even Join object butralin hapten-carrier albumen maximum absorption band with butralin haptens, carrier protein maximum absorption band compared with Apparent variation has occurred, shows that the synthesis of butralin hapten-carrier albumen is successful.It is computed, haptens and BSA's In conjunction with than for 12:1, and the combination ratio of OVA is 9:1.
2, the preparation of butralin monoclonal antibody
(1) acquisition of hybridoma
First immunisation: butralin haptens-BSA conjugate (immunogene) and the Freund's complete adjuvant of equivalent is fully emulsified, skin The Balb/c mouse of 6 week old of lower injection, every 0.2 mL；
Booster immunization is twice: since first immunisation, booster immunization is primary every two weeks, replaces Freund complete with not formula Freund's incomplete adjuvant Full adjuvant, method and the same first immunisation of dosage；
Potency and inhibition are surveyed in eyeground vein blood sampling to last time booster immunization after a week, have inhibition and potency reach 1:10000 with Following final immunization was carried out when upper: 0.1 mL of immunogen solution of any adjuvant is not added in intraperitoneal injection, puts to death mouse after three days, takes Its spleen is merged with myeloma cell；
Cell supernatant is measured using indirect competitive enzyme-linked immunosorbent analysis method, screens positive hole.Using limiting dilution assay to sun Property hole carry out cloning, obtain and establish the hybridoma cell strain of stably excreting butralin monoclonal antibody, take raw in logarithm Cell suspension is made with frozen stock solution in long-term hybridoma, is sub-packed in cryopreservation tube, saves for a long time in liquid nitrogen.
(2) preparation of monoclonal antibody
Cell recovery: taking out butralin monoclonal antibody hybridoma cell strain cryopreservation tube, be immediately placed in 37 DEG C of water-bath middling speeds and melt, from After the heart removes frozen stock solution, culture culture in glassware is moved into；
Preparation ascites and antibody purification: 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 butralin 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:(150000 ~ 400000).
Indirect competitive ELISA method: using butralin haptens-OVA conjugate coated elisa plate, and butralin standard items are added The sheep anti mouse antiantibody solution of solution, butralin 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 other dinitroaniline herbicides (flumetralim, isopropalin, two common in butralin and tobacco Penta spirit of first, benfluralin, trefanocide) it is serially diluted, indirect competitive ELISA is carried out with monoclonal antibody respectively, makes standard Curve, analysis obtain IC50, cross reacting rate is then calculated as follows:
The cross reacting rate of other dinitroaniline herbicides common in butralin and tobacco as the result is shown are as follows: butralin 100%, flumetralim < 1%, isopropalin < 1%, pendimethalin < 1%, benfluralin < 1%, trefanocide < 1%.Antibody of the present invention Other dinitroaniline herbicides common to flumetralim, isopropalin, pendimethalin, benfluralin, trefanocide etc. are without friendship Fork reaction, has specific binding just for butralin.
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 butralin 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 butralin 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 butralin 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
Butralin 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.Butralin 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 butralin
1, tobacco sample pre-treatment
The tobacco sample of 2.0 ± 0.05 g crushing is weighed into polystyrene centrifuge tube；10 mL, 50% methanol aqueous solution is added, It is sufficiently smashed with refiner, 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
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 butralin 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 Zhong Ding in sample Spirit 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, butralin 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, detection limit test
Butralin standard items are added respectively into blank tobacco sample to final concentration of 2.5,5,10 mg/kg, it is glimmering with time resolution Light immuno-chromatographic test paper strip is detected, as a result are as follows: when butralin concentration is 2.5 mg/kg, fluorescence detector is detected as feminine gender； When butralin concentration is 5 and 10 mg/kg, fluorescence detector test positive shows inspection of this test strips to butralin in tobacco Survey is limited to 5 mg/kg.
2, false positive rate, false negative rate test
Take known butralin content greater than 20 parts of positive tobacco sample of 5 mg/kg, it is known that the negative tobacco sample without butralin It this 20 parts, 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 butralin of the invention can carry out butralin in tobacco Quickly detection.
3, specific test
By other dinitroanilines common in the tobaccos such as flumetralim, isopropalin, pendimethalin, benfluralin, trefanocide Herbicide is diluted to 500 mg/L with the phosphate buffer that pH value is 7.2,0.2 mol/L, is examined with butralin test strips It surveys.The results show that when detecting 500 mg/L flumetralims, isopropalin, pendimethalin, benfluralin, trefanocide with the test strips, Test strips T line colour developing develops the color deep or develops the color unanimously with C line than C line, is negative.Illustrate this test strips to flumetralim, isopropalin, Other common dinitroaniline herbicide no cross reactions in the tobaccos such as pendimethalin, benfluralin, trefanocide.

Claims (7)

1. a kind of time-resolved fluoroimmunoassay chromatograph test strip for detecting butralin, 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 butralin 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 butralin hapten-carrier protein conjugate, and the quality control region is coated with sheep anti mouse antiantibody；It is described Butralin monoclonal antibody is prepared using butralin hapten-carrier protein conjugate as immunogene；The butralin Hapten-carrier protein conjugate is to be obtained by butralin haptens with carrier protein couplet, the butralin haptens be by Chloro- 3, the 5- dinitrobenzene acetic acid of 4- reacts to obtain with butylamine, molecular structural formula are as follows:
。
2. the time-resolved fluoroimmunoassay chromatograph test strip of detection butralin as described in claim 1, it is characterised in that: described The preparation reaction process of butralin haptens is as follows:
。
3. the time-resolved fluoroimmunoassay chromatograph test strip of detection butralin 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 ~ 300 nm, and surface is connected with-COOH Group.
4. the time-resolved fluoroimmunoassay chromatograph test strip of detection butralin 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 butralin 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 butralin of claim 1-5 Method, it is characterised in that the following steps are included:
1) preparation of conjugate release pad: butralin 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: butralin 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 butralin 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 time-resolved fluoroimmunoassay chromatograph test strip using any detection butralin of claim 1-5 is answered With, it is characterised in that the following steps are included:
1) sample pre-treatments；
2) it is detected with the test strips；
3) fluorescence detector analysis detection result is used.
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US20210215678A1 (en) * 2020-01-10 2021-07-15 Tobacco Research Institute Of Chinese Academy Of Agricultural Sciences Time-resolved fluorescence immunochromatography test paper card for detecting butralin
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