CN109061157A - A kind of time-resolved fluoroimmunoassay chromatograph test strip and its preparation method and application detecting flumetralim - Google Patents
A kind of time-resolved fluoroimmunoassay chromatograph test strip and its preparation method and application detecting flumetralim Download PDFInfo
- Publication number
- CN109061157A CN109061157A CN201811104814.4A CN201811104814A CN109061157A CN 109061157 A CN109061157 A CN 109061157A CN 201811104814 A CN201811104814 A CN 201811104814A CN 109061157 A CN109061157 A CN 109061157A
- Authority
- CN
- China
- Prior art keywords
- flumetralim
- detection
- test strip
- time
- resolved fluoroimmunoassay
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
- 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
- G01N33/558—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
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
-
- 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
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- 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
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- 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
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
Abstract
A kind of time-resolved fluoroimmunoassay chromatograph test strip and its preparation method and application detecting flumetralim.The test strips include bottom plate, sample absorption pad, conjugate release pad, nitrocellulose filter and water absorption pad, the flumetralim monoclonal antibody of fluorescent microsphere label is embedded in conjugate release pad, detection zone and quality control region are fixed on nitrocellulose filter, detection zone is coated with flumetralim hapten-carrier protein conjugate, quality control region is coated with sheep anti mouse antiantibody, flumetralim haptens is by 2- chloro- 1, 3- dinitro -5- trifluoromethylbenzene is reacted with 3- amino -3- (the chloro- 6- fluorophenyl of 2-)-propionic acid generates 3- (the chloro- 6- fluorophenyl of 2-) -3- (2, 6- dinitro -4- trifluoromethyl-phenylamino)-propionic acid, it reacts to obtain with iodoethane again.Test strips and detection method of the invention have the advantages that easy to operate, high sensitivity, detection speed are fast, at low cost, are able to achieve the quick detection to flumetralim 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 flumetralim when
Between resolved fluorometric immuno-chromatographic test paper strip and its preparation method and application.
Background technique
Flumetralim (flumetralin) is that a kind of contact and local internal-suction type inhibit the dinitroaniline of tobacco lateral bud to plant
Object growth regulator, it is excellent tobacco germination inhibitor.It is developed by vapour Ba-Jia Ji (Ciba-Geigy) company of Switzerland within 1977
Success.Nineteen ninety, registration number was PDll6-90 using maleic Min (Prime) as trade name in China's formal registration.It is a kind of
More popular new and effective Suckering agents in the world are suitable for flue-cured tobacco, fire-cured tobacco, Maryland, suncured tabacco, cigar.State
Interior Zhejiang Provincial Chemical Engineering Research Inst is researched and developed earliest, and is succeeded in developing in 1998, is obtained pesticide within 1999 and is temporarily stepped on
Note.It is administered in tobacco is manually pinched 24 hours once, bud picking is not had in the entire season of growth.Per acre with 25% flumetralim missible oil
60-70 mL, effect rapidly, absorb it is fast, as long as after application 2 hours without rain, can prove effective, it is convenient to be administered in rainy season.Medicament connects
The blade of touching full extension will not generate phytotoxicity, be free of the Harmful Residue.It is artificial that a large amount of bud pickings can be saved using flumetralim, and
Keep natural maturity degree consistent, increase yield, improves the interior quality of ratio and tobacco leaf intermediate on tobacco leaf.In addition, using fluorine section
Amine also can reduce the contagion of field mosaic disease.Compared with other Suckering agents, flumetralim drug effect is very high.International tobacco science is ground
The guiding residue limits for studying carefully flumetralim in Cooperation Centre (CORESTA) regulation tobacco are 5 mg/kg, and China not yet formulates food
The maximum residue limit of middle flumetralim.Currently, the method for common detection flumetralim have gas chromatography, gas chromatography-mass spectrography,
Gas-chromatography tandem mass spectrometry, etc..Since above method is both needed to advanced detecting instrument, testing cost valuableness, complex steps, consumption
When, and it is professional more demanding to operator, it is not suitable for the high-throughput rapid screening detection of enterprises and institutions of base.Cause
This, develop a kind of not examined equipment limit and can be realized the product that batch samples are used for quickly detecting and method at
For 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.So far, there has been no the appearance of flumetralim time-resolved fluoroimmunoassay chromatograph test strip in the market.
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 flumetralim；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 flumetralim
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 flumetralim 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 flumetralim 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 flumetralim hapten-carrier protein conjugate, the quality control region is coated with sheep anti mouse antiantibody.
The flumetralim monoclonal antibody is to prepare to obtain using flumetralim hapten-carrier protein conjugate as immunogene
?；The flumetralim hapten-carrier protein conjugate is to be obtained by flumetralim haptens with carrier protein couplet, the carrier
Albumen is bovine serum albumin(BSA), ovalbumin, keyhole limpet hemocyanin, thyroprotein or human serum albumins, the flumetralim
Haptens is to react generation with 3- amino -3- (the chloro- 6- fluorophenyl of 2-)-propionic acid by the chloro- 1,3- dinitro -5- trifluoromethylbenzene of 2-
3- (the chloro- 6- fluorophenyl of 2-) -3- (2,6- dinitro -4- trifluoromethyl-phenylamino)-propionic acid, then react to obtain with iodoethane,
Molecular structural formula are as follows:
。
The preparation of the flumetralim haptens specifically includes the following steps:
1) chloro- 1,3- dinitro -5- trifluoromethylbenzene, 1.00 g of 2- is taken, adds 20 mL dehydrated alcohols to dissolve, obtains A liquid；Separately take 3-
0.88 g of amino -3- (the chloro- 6- fluorophenyl of 2-)-propionic acid adds 10 mL dehydrated alcohols to dissolve, 1 mL is added to contain 0.37 g bicarbonate
The aqueous solution of sodium obtains B liquid, and A drop is added in B liquid, reacts at room temperature 3 h；TLC detection, raw material fundamental reaction are complete；Stop
Reaction, revolving remove ethyl alcohol, and 80 mL water is added to dissolve, and with 1 mol/L salt acid for adjusting pH value to 6,80 mL ethyl acetate are added to vibrate
Layering, organic phase washing, revolving, upper silicagel column, n-hexane in a volume ratio of 10:1 and ethyl acetate elutes and separates, and obtains
1.53 g of mesosome 3- (the chloro- 6- fluorophenyl of 2-) -3- (2,6- dinitro -4- trifluoromethyl-phenylamino)-propionic acid；
2) 1.50 g of intermediate 3- (the chloro- 6- fluorophenyl of 2-) -3- (2,6- dinitro -4- trifluoromethyl-phenylamino)-propionic acid is taken to add
The dissolution of 50 mL acetonitriles, adds 0.20 g of potassium hydroxide, adds 0.57 g of iodoethane, 50 DEG C of 4 h of reaction, detects, raw material fully reacting,
Stop reaction, revolving removes acetonitrile, adds 100 mL water, dissolves, with 1 mol/L salt acid for adjusting pH value to 6, adds 80 mL acetic acid second
Ester extraction, organic phase washing and drying are evaporated, obtain yellow oil, the n-hexane and dichloromethane solution for being 1:1 with volume ratio
Recrystallization, obtains 1.51 g of flumetralim haptens product.
The fluorescent microsphere is the microballoon that fluorescent material is wrapped up with polystyrene that diameter is 100 ~ 300 nm, 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 flumetralim
The method of immuno-chromatographic test paper strip, it includes the following steps:
1) preparation of conjugate release pad: flumetralim 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: flumetralim 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 flumetralim 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) the chloro- 1,3- dinitro -5- trifluoromethylbenzene of 2- is reacted with 3- amino -3- (the chloro- 6- fluorophenyl of 2-)-propionic acid and generates 3-
(the chloro- 6- fluorophenyl of 2-) -3- (2,6- dinitro -4- trifluoromethyl-phenylamino)-propionic acid, then reacted with iodoethane, prepare fluorine section
Amine haptens；
2) by flumetralim haptens and carrier protein couplet, flumetralim hapten-carrier protein conjugate is prepared；
3) mouse is immunized with flumetralim hapten-carrier protein conjugate, mouse boosting cell and murine myeloma cell is passed through
Fusion, screening, obtain the hybridoma cell strain of secretion flumetralim monoclonal antibody；
4) mouse IgG immune health goat is extracted, sheep anti mouse antiantibody is obtained；
5) flumetralim 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 flumetralim 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 flumetralim 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 flumetralim
Application of the chromatograph test strip in detection flumetralim, it includes the following steps:
1) sample pre-treatments；
2) it is detected with the time-resolved fluoroimmunoassay chromatograph test strip of the detection flumetralim；
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 flumetralim 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 flumetralim 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 flumetralim, 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 flumetralim 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 flumetralim
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 flumetralim hapten-carrier egg
White conjugate (flumetralim 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 flumetralim
The preparation method for detecting the time-resolved fluoroimmunoassay chromatograph test strip of flumetralim mainly comprises the steps that
1) preparation of conjugate release pad: flumetralim 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: flumetralim 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 flumetralim 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 flumetralim hapten-carrier protein conjugate
Flumetralim 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 flumetralim haptens
Chloro- 1,3- dinitro -5- trifluoromethylbenzene, 1.00 g of 2- is taken, adds 20 mL dehydrated alcohols to dissolve, obtains A liquid；Separately take 3- ammonia
0.88 g of base -3- (the chloro- 6- fluorophenyl of 2-)-propionic acid adds 10 mL dehydrated alcohols to dissolve, 1 mL is added to contain 0.37 g sodium bicarbonate
Aqueous solution, obtain B liquid, A drop be added in B liquid, react at room temperature 3 h；TLC detection, raw material fundamental reaction are complete；Stop anti-
It answers, rotates, remove ethyl alcohol, 80 mL water is added to dissolve, with 1 mol/L salt acid for adjusting pH value to 6, add the oscillation point of 80 mL ethyl acetate
Layer, organic phase washing, revolving, upper silicagel column, n-hexane in a volume ratio of 10:1 are separated with ethyl acetate elution, obtain centre
1.53 g of body 3- (the chloro- 6- fluorophenyl of 2-) -3- (2,6- dinitro -4- trifluoromethyl-phenylamino)-propionic acid, yield 92.17%；
1.50 g of intermediate 3- (the chloro- 6- fluorophenyl of 2-) -3- (2,6- dinitro -4- trifluoromethyl-phenylamino)-propionic acid is taken to add 50
The dissolution of mL acetonitrile, adds 0.20 g of potassium hydroxide, adds 0.57 g of iodoethane, 50 DEG C of 4 h of reaction, detects, raw material fully reacting, stops
Reaction, revolving remove acetonitrile, add 100 mL water, dissolve, and with 1 mol/L salt acid for adjusting pH value to 6,80 mL ethyl acetate are added to extract
It takes, organic phase washing and drying is evaporated, obtains yellow oil, and the n-hexane for being 1:1 with volume ratio is tied again with dichloromethane solution
Crystalline substance obtains 1.51 g of flumetralim haptens product, yield 94.97%.
Nuclear-magnetism identifies 1H NMR(CDCl3, 300MHZ) and δ: 11.00 (1H, s), 7.141 (1H, dd, J=8.271, J=
1.347), 7.373 (2H, dd, J=8.373, J=8.271), 4.23 (2H, dd, J=8.373, J=1.347), 2.880
(2H, d, J=6.843), 3.631 (2H, q, J=7.108), 1.238 (3H, t, J=7.108), 8.75 (2H, d, J
=0.000), chemical shift δ=11.0 are carboxyl hydrogen resonance absorbing peak on spacerarm, and δ=2.88 are methylene hydrogen on spacerarm
Resonance absorbing peak, the presence at these peaks, it was demonstrated that spacerarm is coupled successfully.
(2) preparation of immunogene
Flumetralim haptens and bovine serum albumin(BSA) (BSA) coupling obtain immunogene.
18 mg of flumetralim haptens is taken, 0.3 mL dimethylformamide (DMF) is added to dissolve, clarification adds carbodiimide
(EDC) 8.6 mg is stirred, and clarification adds 5.2 mg of n-hydroxysuccinimide (NHS), and 3 h of activation are stirred at room temperature, obtain A liquid；
50 mg of BSA is taken, adds the phosphate buffer (PB) that 8 mL, 0.05 mol/L pH value is 7.2 to dissolve, obtains B liquid, A liquid is delayed
Slowly it is added drop-wise in B liquid, 5 h of reaction is stirred at room temperature.Stop reaction, 0.02 M phosphate buffer (PBS) is dialysed 3 days, changed daily
Liquid three times, obtains flumetralim-BSA immunogene, packing, -20 DEG C of preservations, spare.
(3) preparation of coating antigen
Flumetralim haptens and ovalbumin (OVA) coupling obtain coating antigen.
8 mg of flumetralim haptens is taken, 0.2 mL DMF is added to dissolve, clarification adds dicyclohexylcarbodiimide (DCC) 4.13
Mg adds 2.3 mg of NHS, and 2 h are stirred at room temperature, and filtering removes sediment, obtains haptens activating solution A liquid；50 mg of OVA is taken,
Adding 0.05 mol/L pH value of 8mL is 7.2 PB buffer solution, obtains B liquid, A liquid is slowly dropped in B liquid, room temperature is stirred
Mix 5 h of reaction.Stopping reaction, 0.02 M PBS buffer solution dialyses 3 days, changes liquid daily three times, obtain flumetralim-OVA coating antigen,
Packing, -20 DEG C of preservations are spare.
2, the preparation of flumetralim monoclonal antibody
(1) animal immune
In the immunogene injection Balb/c Mice Body that step 2 is obtained, immunizing dose is 150 μ g/, its is made to generate antiserum.
(2) cell fusion and cloning
Immune Balb/c mouse boosting cell being taken, in 8:1(quantitative proportion) ratio merges with SP2/0 myeloma cell, using indirect
Inhibition ELISA measures cell supernatant, screens positive hole.Cloning is carried out to positive hole using limiting dilution assay, until
To the hybridoma cell strain of stably excreting monoclonal antibody.
(3) cell cryopreservation and recovery
Hybridoma is made 1 × 10 with frozen stock solution6The cell suspension of a/mL, saves for a long time in liquid nitrogen.It is taken out when recovery
Cryopreservation tube is immediately placed in 37 DEG C of water-bath middling speeds and melts, and after centrifugation removal frozen stock solution, moves into culture culture in glassware.
(4) preparation and purification of monoclonal antibody
Increment cultivation: hybridoma is placed in cell culture medium, is cultivated under the conditions of 37 DEG C, with octanoic acid-saturation
Ammonium sulfate method purifies obtained culture solution, obtains monoclonal antibody, -20 DEG C of preservations.
The cell culture medium is that calf serum and sodium bicarbonate are added into RPMI1640 culture medium, and calf serum is made to exist
Final concentration of 20%(mass fraction in cell culture medium), the final concentration of 0.2%(mass of sodium bicarbonate in cell culture medium
Score)；The pH value of the cell culture medium is 7.4.
(5) measurement of antibody titer
Potency with indirect competitive ELISA method measurement antibody is 1:(100000 ~ 400000).
Indirect competitive ELISA method: using flumetralim haptens-OVA conjugate coated elisa plate, and flumetralim standard items are added
The sheep anti mouse antiantibody solution of solution, flumetralim 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.
(6) 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.
Dinitroaniline herbicide (flumetralim, butralin, pendimethalin, trefanocide) is serially diluted by this experiment,
Indirect competitive ELISA is carried out with monoclonal antibody respectively, makes standard curve, analysis obtains IC50, intersection is then calculated as follows
Reactivity:
The cross reacting rate of each analog as the result is shown are as follows: flumetralim 100%, butralin < 1%, pendimethalin < 1%, trefanocide
< 1%.Antibody of the present invention is to other dinitroaniline herbicide no cross reactions such as butralin, pendimethalin, trefanocide, only
There is specific binding for flumetralim.
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 flumetralim 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 flumetralim 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 flumetralim 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
Flumetralim 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.Flumetralim 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 flumetralim
1, tobacco sample pre-treatment
The tobacco sample of 1.0 ± 0.05 g crushing is weighed into polystyrene centrifuge tube；10 mL, 50% methanol aqueous solution is added,
With 3 min of vortex instrument whirling motion, 6000 rpm room temperatures are centrifuged 5 min；It pipettes 100 μ L supernatants and 400 μ L deionized waters mixes
It is to be checked afterwards.
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) 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 flumetralim 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 fluorine section in sample
Amine 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, flumetralim 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.
(2) 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 flumetralim concentration, obtain the content of flumetralim in sample to be tested extracting solution, most afterwards through changing
It calculates up to the content of flumetralim in sample to be tested.
4 sample detection example of embodiment
1, detection limit test
Flumetralim 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 flumetralim concentration is 2.5 mg/kg, fluorescence detector is detected as feminine gender；
When flumetralim concentration is 5 and 10 mg/kg, fluorescence detector test positive shows inspection of this test strips to flumetralim in tobacco
Survey is limited to 5 mg/kg.
2, false positive rate, false negative rate test
Take known flumetralim content greater than 20 parts of positive tobacco sample of 5 mg/kg, it is known that the negative tobacco sample without flumetralim
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 flumetralim of the invention can carry out flumetralim in tobacco
Quickly detection.
3, specific test
The dinitroaniline herbicides such as pendimethalin, butralin, the trefanocide of 500 μ g/L are detected with flumetralim test strips.Knot
Fruit shows that test strips nature controlling line and detection line develop the color, and is negative.Illustrate this test strips to the pendimethalin of 500 μ g/L, secondary
The dinitroaniline herbicides no cross reaction such as Ding Ling, trefanocide, specificity are good.
Claims (7)
1. a kind of time-resolved fluoroimmunoassay chromatograph test strip for detecting flumetralim, 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: the conjugate release pad
On be embedded with the flumetralim monoclonal antibody of fluorescent microsphere label, be fixed with detection zone and Quality Control on the nitrocellulose filter
Area, the detection zone are coated with flumetralim hapten-carrier protein conjugate, and the quality control region is coated with sheep anti mouse antiantibody；
The flumetralim monoclonal antibody is prepared using flumetralim hapten-carrier protein conjugate as immunogene；The fluorine
Saving amine hapten-carrier protein conjugate is to be obtained by flumetralim haptens with carrier protein couplet, the flumetralim haptens
It is to be reacted to generate 3- (2- with 3- amino -3- (the chloro- 6- fluorophenyl of 2-)-propionic acid by the chloro- 1,3- dinitro -5- trifluoromethylbenzene of 2-
Chloro- 6- fluorophenyl) -3- (2,6- dinitro -4- trifluoromethyl-phenylamino)-propionic acid, then reacted with iodoethane, point
Subformula are as follows:
。
2. the time-resolved fluoroimmunoassay chromatograph test strip of detection flumetralim as described in claim 1, it is characterised in that: described
The preparation reaction process of flumetralim haptens is as follows:
。
3. the time-resolved fluoroimmunoassay chromatograph test strip of detection flumetralim 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 flumetralim 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 flumetralim 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 flumetralim of claim 1-5
Method, it is characterised in that the following steps are included:
1) preparation of conjugate release pad: flumetralim 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: flumetralim 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 flumetralim 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 flumetralim of claim 1-5,
It is characterized by comprising following steps:
1) sample pre-treatments；
2) it is detected with the test strips；
3) fluorescence detector analysis detection result is used.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811104814.4A CN109061157B (en) | 2018-09-21 | 2018-09-21 | Time-resolved fluorescence immunochromatographic test strip for detecting flumetralin and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811104814.4A CN109061157B (en) | 2018-09-21 | 2018-09-21 | Time-resolved fluorescence immunochromatographic test strip for detecting flumetralin and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109061157A true CN109061157A (en) | 2018-12-21 |
| CN109061157B CN109061157B (en) | 2021-04-30 |
Family
ID=64763304
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811104814.4A Active CN109061157B (en) | 2018-09-21 | 2018-09-21 | Time-resolved fluorescence immunochromatographic test strip for detecting flumetralin and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109061157B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3674707A1 (en) * | 2018-12-26 | 2020-07-01 | Beijing Diagreat Biotechnologies Co., Ltd. | Time-resolved fluorescent immunochromatographic test strip for detecting paclitaxel drugs as well as preparation method and application thereof |
| US11614444B2 (en) | 2020-01-10 | 2023-03-28 | Tobacco Research Institute Of Chinese Academy Of Agricultural Sciences | Time-resolved fluorescence immunochromatography test paper card for detecting butralin |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104849467A (en) * | 2014-02-15 | 2015-08-19 | 北京勤邦生物技术有限公司 | Fluorescent microsphere immunochromatography test paper strip for detecting clenbuterol hydrochloride residue, preparation method and applications thereof |
| CN104897863A (en) * | 2014-03-06 | 2015-09-09 | 北京勤邦生物技术有限公司 | Fluorescent microsphere immunochromatographic test strip for detecting aflatoxin M1, and method thereof |
| CN104977410A (en) * | 2014-04-14 | 2015-10-14 | 北京勤邦生物技术有限公司 | Melamine fluorescent microsphere immunochromatography test paper strip, preparation method and application thereof |
| US20160330976A1 (en) * | 2013-12-24 | 2016-11-17 | Indigo Ag, Inc. | Method for propagating microorganisms within plant bioreactors and stably storing microorganisms within agricultural seeds |
| CN106771238A (en) * | 2016-12-14 | 2017-05-31 | 北京勤邦生物技术有限公司 | Detect fluorescent micro-ball immune chromatography test paper strip of melamine residual and its preparation method and application |
-
2018
- 2018-09-21 CN CN201811104814.4A patent/CN109061157B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160330976A1 (en) * | 2013-12-24 | 2016-11-17 | Indigo Ag, Inc. | Method for propagating microorganisms within plant bioreactors and stably storing microorganisms within agricultural seeds |
| CN104849467A (en) * | 2014-02-15 | 2015-08-19 | 北京勤邦生物技术有限公司 | Fluorescent microsphere immunochromatography test paper strip for detecting clenbuterol hydrochloride residue, preparation method and applications thereof |
| CN104897863A (en) * | 2014-03-06 | 2015-09-09 | 北京勤邦生物技术有限公司 | Fluorescent microsphere immunochromatographic test strip for detecting aflatoxin M1, and method thereof |
| CN104977410A (en) * | 2014-04-14 | 2015-10-14 | 北京勤邦生物技术有限公司 | Melamine fluorescent microsphere immunochromatography test paper strip, preparation method and application thereof |
| CN106771238A (en) * | 2016-12-14 | 2017-05-31 | 北京勤邦生物技术有限公司 | Detect fluorescent micro-ball immune chromatography test paper strip of melamine residual and its preparation method and application |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3674707A1 (en) * | 2018-12-26 | 2020-07-01 | Beijing Diagreat Biotechnologies Co., Ltd. | Time-resolved fluorescent immunochromatographic test strip for detecting paclitaxel drugs as well as preparation method and application thereof |
| US11614444B2 (en) | 2020-01-10 | 2023-03-28 | Tobacco Research Institute Of Chinese Academy Of Agricultural Sciences | Time-resolved fluorescence immunochromatography test paper card for detecting butralin |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109061157B (en) | 2021-04-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109324182B (en) | Fluorescent microsphere immunochromatography test strip for detecting pendimethalin and preparation method and application thereof | |
| CN109061152A (en) | A kind of time-resolved fluoroimmunoassay chromatograph test strip and its preparation method and application detecting carbendazim | |
| CN108508215A (en) | A kind of time-resolved fluoroimmunoassay chromatograph test strip and its preparation method and application of detection tetracycline medication | |
| JPH04230855A (en) | Reagent and method for detecting polychlorinated biphenyl | |
| CN109111394B (en) | Preparation method and application of acetamiprid hapten and antigen | |
| CN109206332B (en) | Preparation method and application of flumetralin hapten and antigen | |
| CN109061169B (en) | Enzyme linked immunosorbent assay kit for detecting acetamiprid and application thereof | |
| CN109061146B (en) | Test strip for detecting acetamiprid and preparation method and application thereof | |
| CN109061147B (en) | Test strip for detecting pendimethalin and preparation method and application thereof | |
| CN109239336B (en) | Test strip for detecting isoprocarb and application thereof | |
| CN109265401B (en) | Preparation method and application of iprodione hapten and antigen | |
| CN108912090B (en) | Test strip for rapidly detecting total amount of alternariol and alternariol monomethyl ether | |
| CN109265404A (en) | A kind of preparation method and application of carbendazim haptens and antigen | |
| CN109061157A (en) | A kind of time-resolved fluoroimmunoassay chromatograph test strip and its preparation method and application detecting flumetralim | |
| CN109061149A (en) | A kind of time-resolved fluoroimmunoassay chromatograph test strip and its preparation method and application detecting butralin | |
| CN109061156B (en) | Time-resolved fluorescence immunochromatographic test strip for detecting pendimethalin and preparation method and application thereof | |
| CN109061158B (en) | Time-resolved fluorescence immunochromatographic test strip for detecting acetamiprid and preparation method and application thereof | |
| CN110551220A (en) | Preparation and application of DDT monoclonal antibody | |
| CN109061154B (en) | Fluorescent microsphere immunochromatography test strip for detecting metalaxyl and preparation method and application thereof | |
| CN109061150B (en) | Time-resolved fluorescence immunochromatographic test strip for detecting metalaxyl and preparation method and application thereof | |
| CN109061171A (en) | It is a kind of detect flumetralim enzyme linked immunological kit and its application | |
| CN109061153B (en) | Time-resolved fluorescence immunochromatographic test strip for detecting iprodione and preparation method and application thereof | |
| CN109061145B (en) | Test strip for detecting flumetralin, preparation method and application thereof | |
| CN111735951B (en) | Test strip for detecting fenpropathrin and application thereof | |
| CN109061148B (en) | Test strip for detecting butralin and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |