CN105388286B - A kind of test strips for detecting iprodione and its preparation method and application - Google Patents
A kind of test strips for detecting iprodione and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of test strips for detecting iprodione and its preparation method and application, test strips include sample absorption pad（1）, conjugate release pad（2）, reaction film（3）, adsorptive pads（4）And base plate（5）, it is characterised in that：There is the detection zone for being coated with iprodione hapten-carrier protein conjugate on the reaction film（6）With the quality control region for being coated with sheep anti mouse antiantibody（7）, the conjugate release pad（2）It is coated with iprodione monoclonal antibody colloid gold label thing.Present invention also offers a kind of method for preparing iprodione residual in above-mentioned iprodione ELISA test strip tobacco leaf.Test strips provided by the present invention have it is simple to operate, without sample pre-treatments, sensitivity is high, detection speed is fast, low cost the features such as, be adapted to a large amount of samples examination and on-site supervision.
Description
Technical field
The present invention relates to the detection of iprodione, specifically a kind of test strips and preparation method for detecting iprodione are with it in cigarette
Application in leaf detection.
Background technology
Iprodione, also known as iprodione, molecular formula are C13H13Cl2N3O3, belong to dicarboximide class, it is that a kind of broad spectrum activity is touched
Type protective fungicide is killed, is widely used in tobacco, fruit tree, the disease control of vegetables and the preservation and freshness of fruit.Iprodione can
Systemic action is played with by root absorption, the fungi resistant to benzimidazole systemic fungicide can be effectively prevented and treated.Its is main
Controlling object is the disease caused by botrytis, Alternariaspp, sclerotinite etc., such as gray mold, early blight, black spot, sclerotium
Disease etc..Iprodione agricultural chemicals is Japan, the U.S., South Korea, Australia, the state-owned limitation requirement of Canada and Codex Committee on Food
Detection project, its MRL in varieties of food items is 0.05 ~ 15 mg/kg.At present, the detection method of iprodione is most
It is liquid chromatography and gas chromatography, the matrix species that can be detected are less, to the phase of the detection method of iprodione in tobacco
Close patent and document report is also less.Analytical instrument method testing cost is high, it is necessary to professional and technical personnel's operation, detection time
It is long, there is limitation for the development that Site Detection works.
The content of the invention
The purpose of the present invention be based on above-mentioned prior art situation be to provide it is a kind of detect iprodione test strips and its
Preparation method and application, the present invention application colloidal gold immunity chromatography determine tobacco in iprodione residual quantity, tool the degree of accuracy it is high,
Have it is simple to operate, without professional's operation, without supporting other testing equipments, be convenient for carrying that detection time is short, testing cost
Low feature, it is adaptable to the examination of a large amount of samples in scene.
The purpose of the present invention is achieved through the following technical solutions：
A kind of test strips for detecting iprodione, including sample absorption pad, conjugate release pad, reaction film, adsorptive pads and bottom
Plate, there is detection zone and quality control region on the reaction film, detection zone is coated with iprodione hapten-carrier protein conjugate, Quality Control
Area is coated with antiantibody, and the conjugate release pad is coated with iprodione specific antibody-colloid gold label thing.Iprodione is special
Iprodione specific antibody in property antibody-colloidal gold label is using iprodione hapten-carrier protein conjugate as exempting from
Epidemic focus is prepared, and the iprodione hapten-carrier protein conjugate is with iprodione haptens and carrier protein couplet system
It is standby to obtain.
The test strips paste the group on base plate successively by sample absorption pad, conjugate release pad, reaction film, adsorptive pads
Into, and conjugate release pad 1/3 ~ 1/2 is capped under sample absorption pad.
, near sample absorption pad one end, quality control region is near adsorptive pads one end for described detection zone.
The iprodione hapten-carrier protein conjugate is obtained by iprodione haptens and carrier protein couplet, described
Carrier protein can be bovine serum albumin(BSA), ovalbumin, hemocyanin, thyroprotein, human serum albumins.
The antiantibody is sheep anti mouse antiantibody.
The iprodione specific antibody can be iprodione monoclonal antibody or iprodione polyclonal antibody.
The iprodione haptens be by the chloro- 4- nitrophenols of 2,6- bis- be initiation material, by with the tertiary fourth of 4- bromo-butyric acids
The hydrocarbyl reaction of ester obtains hydroxyl 2, and the chloro- 4- nitrobenzene of 6- bis- after reduction nitro, obtains alkyl 2,6-DCA, alkyl
2,6-DCA, with amion acetic acid condensation, obtains alkyl 2,6- dichloro-benzenes glycinates, then pass through in the presence of phosgene
Annulation, obtains iprodione agent structure compound, and the compound is acted on through phosgene again, with isopropylamine condensation, obtains different
Bacterium urea mother nucleus structure compound, in acid condition hydrolysis obtain, molecular structure such as formula I：
（Formula I）.
The base plate can be the material that PVC base plates or other hard do not absorb water；The sample absorption pad can for non-woven fabrics or
Filter paper for oil；The conjugate release pad can be glass fibre or polyester material；The adsorptive pads are blotting paper；The reaction film can
It is nitrocellulose filter or CAM.
A kind of method for preparing above-mentioned test strips, its step includes：
（1）Preparation is coated with the conjugate release pad of iprodione monoclonal antibody-colloid gold label thing；
（2）Preparing, there is the detection zone for being coated with iprodione hapten-carrier protein conjugate to resist with sheep anti mouse is coated with
The reaction film of the quality control region of antibody；
（3）Will（2）The reaction film for preparing is assembled into test paper with conjugate release pad, sample absorption pad, adsorptive pads, base plate
Bar；
Specifically, step includes：
（1）Be there is into nucleophilic substitution in iprodione and bromo valeral, obtain iprodione haptens；
（2）By iprodione haptens and carrier protein couplet, iprodione hapten-carrier protein conjugate is obtained；
（3）With the immune thing immune mouse of iprodione hapten-carrier albumen coupling, by mouse boosting cell and myeloma cell
The strain of iprodione monoclonal hybridoma is obtained by fusion, screening；
（4）Mouse IgG immune health goat is extracted, sheep anti mouse antiantibody is obtained；
（5）Collaurum is prepared with trisodium citrate and gold chloride reaction；
（6）By step（3）The iprodione monoclonal antibody of preparation adds step（5）In the collaurum of preparation, different bacterium is obtained
Urea monoclonal antibody-colloid gold label thing；
（7）Iprodione monoclonal antibody-colloid gold label thing is sprayed in conjugate release pad, is taken after 37 DEG C of baking 1h
Go out, be placed in dry environment and save backup；
（8）Detection zone will be constituted in iprodione hapten-carrier protein conjugate coating again reaction film, sheep anti mouse is anti-
Body is coated on and quality control region is constituted on reaction film；
（9）By sample absorption pad with containing 0.5% bovine serum albumin(BSA)（Volume fraction）, pH be 7.2,0.1 mol/L phosphate
Buffer solution soaks 2 h, and 2 h are dried at 37 DEG C；
（10）Paste sample absorption pad, conjugate release pad, reaction film, adsorptive pads in order on base plate, sample is inhaled
Receive pad and cover conjugate release pad, be finally cut into 3 mm small bars wide, plus plastic casing, be vacuum-packed, can be protected under the conditions of 4 ~ 30 DEG C
Deposit 12 months.
Using the method that iprodione in above-mentioned ELISA test strip tobacco leaf is remained, including step：
（1）Detected with test strips；
（2）Analysis testing result.
Iprodione residue detection test strips of the invention are reacted using the antibody antigen of high degree of specificity and Competitive assays are exempted from
Epidemic disease Chromatographic techniques, iprodione monoclonal antibody-colloid gold label thing is fixed in conjugate release pad, different in sample
Bacterium urea is combined in flow process with the iprodione monoclonal antibody-colloid gold label thing in conjugate release pad, forms medicine
Thing-antibody-colloidal gold label.The iprodione hapten-carrier albumen coupling on medicine and reaction film detection zone in sample
Competition binding iprodione monoclonal antibody-colloid gold label thing, whether there is according to detection zone red stripes or shade judges
Whether contain iprodione residual in analyte sample fluid.
During detection, sample instills sample absorption pad after treatment, when iprodione concentration in the sample is examined less than test strips
When surveying limit or being zero, monoclonal antibody-colloid gold label thing can be with the iprodione being fixed on reaction film half in chromatography process
Antigen-carrier protein conjugate is combined, in detection zone（T lines）And quality control region（C lines）One red stripes of interior each appearance, and T lines
Colour developing is developed the color deeply than C line or the colour developing depth is consistent；If iprodione concentration in the sample is limited equal to or higher than ELISA test strip,
Monoclonal antibody-colloid gold label thing can be combined with iprodione, so as at T lines because competitive reaction will not or part with it is different
The color that bacterium urea hapten-carrier protein conjugate is combined and occurs without the colour developing of red stripes or T lines is more shallow than C line.Such as Fig. 2 institutes
Show.
It is negative：Work as quality control region（C lines）Show red stripes, detection zone（T lines）Also show that red stripes, and T simultaneously
Line colour developing develops the color deep or colour developing unanimously than C line, is judged to feminine gender, shows that the concentration of iprodione in sample is less than ELISA test strip
Limit.
It is positive：Work as quality control region（C lines）Show red stripes, and detection zone（T lines）Or the T line more shallow than C line that develop the color does not develop the color,
The positive is judged to, shows that the concentration of iprodione in sample is limited equal to or higher than ELISA test strip.
It is invalid：Work as quality control region（C lines）Do not show red stripes, then no matter detection zone（T lines）Show red stripes with
No, it is invalid that the test strips are judged to.
Test strips beneficial effect of the invention：Test strips of the invention are tied by the chloro- 4- nitrophenols of 2,6- bis-
Structure is transformed, and has been synthesized with four carboxyl haptens of carbon chain lengths, highlights iprodione molecular structure in itself, is enhanced and is exempted from
Epidemic focus so that the iprodione monoclonal antibody sensitivity for preparing is high, high specificity, test strips low cost of the present invention, behaviour
Make simple, sample pre-treatments are simple, and detection time is short, only need 10 min, be convenient for carrying, it is adaptable to the sieve of a large amount of samples in scene
Look into, storage is simple, long shelf-life, can be widely popularized in food safety Regulation work and used.
Brief description of the drawings
Fig. 1 is the schematic diagram of test strips cross-section structure of the invention.
In Fig. 1：1 is sample absorption pad, and 2 is conjugate release pad, and 3 is reaction film, and 4 is adsorptive pads, and 5 is base plate, and 6 is inspection
Area is surveyed, 7 is quality control region.
Fig. 2 is the schematic diagram of ELISA test strip result judgement of the invention.
Fig. 3 is the schematic diagram of iprodione hapten synthesis route of the invention.
Specific embodiment
Below by embodiment, and with reference to accompanying drawing 1-3, technical scheme is further described in detail.It is following
Referring to the drawings the explanation of embodiment of the present invention is intended to explain the present general inventive concept, and be not construed as it is right
A kind of limitation of the invention.
Experimental technique used in following embodiments is conventional method unless otherwise specified.
The composition of embodiment 1, the test strips of detection iprodione
As shown in figure 1, the test strips of the detection iprodione according to the preferred embodiment of the present invention are by sample absorption pad
1st, conjugate release pad 2, reaction film 3, adsorptive pads 4 are pasted constituted on base plate 5 successively.The end of sample absorption pad and reaction film
Be connected, the end of reaction film is connected with adsorptive pads, and the top of sample absorption pad aligns with the top of base plate, the end of adsorptive pads and
The end alignment of base plate；There are detection zone 6 and quality control region 7 on the reaction film, detection zone and quality control region are and the test strips
The perpendicular ribbon detection zone of length be located at the side for being bordering on sample absorption pad, quality control region is located at the side for being bordering on adsorptive pads；
The detection zone is coated with iprodione hapten-carrier protein conjugate（The coupling of iprodione haptens-bovine serum albumin(BSA)
Thing）, quality control region is coated with sheep anti mouse antiantibody；The base plate is PVC base plates, and sample absorption pad is non-woven fabrics, and reaction film is nitre
Acid cellulose film, conjugate release pad is the glass fibre for being coated with iprodione monoclonal antibody-colloid gold label thing；It is described different
Bacterium urea residue detection test strips are stored in 4 DEG C ~ 30 DEG C environment, and the term of validity is 12 months.
The preparation method of test strips described in embodiment 2, embodiment 1
1. the preparation of iprodione haptens
The synthesis of compound A：The chloro- 4- nitrophenols of 5.0 g 2,6- bis- and the 3.2 g 4- bromo-butyric acid tert-butyl esters are taken in acetone
Middle stirring, then 2.6 g Anhydrous potassium carbonates are added thereto to as catalyst in 60 DEG C of 8 h of reaction, after reaction stops, it being evaporated molten
Agent, adds water and is extracted with ethyl acetate, and uses anhydrous sodium sulfate drying, concentrates, and crosses 200-300 mesh silicagel columns, and chromatography is pure
Change, obtain the g of compound A 5.23, yield 87%.1H NMR（CDCl3,300MHZ）δ: 7.945 ( 1H, d, J=0.000),
4.144 ( 2H, t, J=7.500), 7.945 ( 1H, d, J=0.000), 1.973 ( 2H, tt, J=7.500, J=
7.367), 2.359 (2H, t, J=7.367), 1.414 ( 3H), 1.414 ( 3H), 1.414 ( 3H,s) 。
The synthesis of compound B：To 3.1 g zinc powders and 1 mL glacial acetic acids is added in 20 mL water, 30 min are reacted at 90 DEG C,
The mL of ethanol solution 80 of 5.2 g compounds A is added, 4 h are reacted at 60 DEG C, question response stops, and suction filtration is evaporated, and adds thereto
Water is extracted with dichloromethane, is stood, and is petroleum ether with volume ratio with anhydrous sodium sulfate drying：Ethyl acetate=20:1 recrystallization,
Obtain the g of compound B 4.8, yield 91%.1H NMR（CDCl3,300MHZ）δ:6.898 (1H, d, J=0.000), 4.039
(2H, t, J=7.500), 6.898 (1H, d, J=0.000), 1.965 ( 2H, tt, J=7.500, J=7.367),
2.361 ( 2H, t, J=7.367), 1.414 ( 3H), 1.414 ( 3H,s), 1.414 ( 3H,s)。
The synthesis of compound C：Take 4.7 g compounds B to be dissolved with 100 mL acetonitriles, stir, be added thereto to 2.16 g ammonia
Guanidine-acetic acid, under nitrogen protection, is passed through phosgene, and 7 h are reacted at 50 DEG C, after stopping reaction, adds 50 mL NaOH
The aqueous solution, concussion, revolving removes acetonitrile, plus ethyl acetate dissolving, and add water washing, concentration, is evaporated, and crosses 200-300 mesh silica gel
Post, chromatography obtains compound C3.57 g, yield 73%.1H NMR（CDCl3,300MHZ）δ:7.536 ( 1H, d, J=
0.000), 4.037 ( 2H, t, J=7.500), 7.535 ( 1H, d, J=0.000), 1.968 ( 2H, tt, J=
7.500, J=7.367), 2.362 ( 2H, t, J=7.367), 3.751 ( 2H,s), 1.414 (3H,s), 1.414
( 3H,s), 1.414 ( 3H,s)。
The synthesis of compound D：3.4 g compound C are taken, is dissolved with 1,2- dichloroethanes, add 0.5 mL DMF, 3 mL grass
Acyl chlorides, is stirred at room temperature 3 h, and revolving is evaporated, and removes organic solvent, adds 80 mL pyridines, and 7 h are reacted at 80 DEG C.Stop reaction,
Room temperature is cooled to, is rotated, remove pyridine, addition volume ratio is ethanol：Petroleum ether=10:3 solvent heating for dissolving, in 4 DEG C of placements
Overnight, suction filtration, obtains the g of compound D 2.97, the 1H NMR of yield 82%.（CDCl3,300MHZ）δ:7.439 ( 1H, d, J=
0.000), 4.044 (2H, t, J=7.500), 7.439 (1H, d, J=0.000), 1.969 (2H, tt, J=
7.500, J=7.367), 2.362 (2H, t, J=7.367), 4.191 (1H, d, J=17.742), 4.196 (1H,
d, J=17.742), 1.414 (3H,s), 1.414 ( 3H,s), 1.414 ( 3H,s)。
The synthesis of compound E：Take during 2.8 g compounds D add 200 mL dichloroethanes and dissolve, add 2.7 mL isopropyls
Amine, is passed through nitrogen, discharges air, is passed through phosgene, and 6 h of reaction are stirred at room temperature, and stops reaction, adds 50 mL potassium hydroxide waters
Solution shakes, and stands, and isolates organic phase, washes, and is evaporated with anhydrous sodium sulfate drying, crosses 200-300 mesh silicagel columns, uses body
Product is than being petroleum ether：Ethyl acetate=1:1 solvent wash-out is separated, and obtains the g of compound E 1.53, yield 64%.1H NMR
（CDCl3,300MHZ）δ:7.441 ( 1H, d, J=0.000), 4.044 ( 2H, t, J=7.500), 7.441 ( 1H,
d, J=0.000), 1.970 ( 2H, tt, J=7.500, J=7.367), 2.362 ( 2H, t, J=7.367),
4.527 ( 1H, d, J=0.000), 4.528 ( 1H, d, J=0.000), 4.214 (1H, hept, J=6.794),
1.414 (3H, s), 1.414 (3H, s), 1.414 (3H, s), 1.169 3H, d, J=6.794), 1.169 (
3H, d, J=6.794)。
The synthesis of compound F：1.53 g compounds are taken, the dissolving of 100 mL70% aqueous formic acids is added, it is anti-in 70 DEG C of stirrings
6 h are answered, stops reacting, revolving removes solvent, be hydrogenated with aqueous solution of sodium oxide, plus ethyl acetate extraction, divided and go organic phase, water phase
Regulation pH value plus 1 to 4, and 2- dichloroethanes extraction, washing is dried, and is evaporated, and is ethanol with volume ratio：N-hexane=6:4 solvent
Recrystallization, obtains the g of iprodione haptens 0.81, yield 77%.1H NMR（CDCl3,300MHZ）δ: 11.0 (1H, s)7.4
(1H, d, J=0.000), 4.0 (2H, t, J=7.500), 7.4(1H, d, J=0.000), 1.9 (2H, tt, J=
7.500, J=7.367), 2.4 (2H, t, J=7.367), 4.5(1H, d, J=0.000), 4.5(1H, d, J=
0.000), 4.2(1H, hept, J=6.794), 1.1 (3H, d, J=6.794), 1.1(3H, d, J=6.794)。
Above-mentioned product is taken through nuclear magnetic resonance hydrogen spectruming determining, chemical shift δ=11.0 are carboxyl hydrogen, δ=4.0,1.9,2.4
It is the resonance absorbing peak of the hydrogen of methylene on spacerarm, these peaks coordinate the presence at other iprodione hydrogen inherent absorption peaks existing
Prove hapten synthesis success.
2. the preparation of immunogene
Iprodione haptens obtains immunogene with bovine serum albumin(BSA) coupling
9.0 mg iprodione haptens are taken, the dimethylformamide of 1.0 mL is dissolved in（DMF）In, obtain solution I；Take 30
Mg carbodiimides（EDC）With 30 mg N-hydroxy-succinamides（NHS）With addition solution I, room temperature after 0.2 mL water dissolves
24 h of lower stirring, obtain reaction solution II；Take 50 mL bovine serum albumin(BSA)s（BSA）, it is substantially dissolved in the phosphate-buffered of 3.8 mL
Liquid PBS（PH is 7.2）In, reaction solution II is dropwise slowly dropped in bovine serum albumin solution, and stir 24 at room temperature
H, obtains reaction solution III；Dialysed 3 days at 4 DEG C with the PBS of 0.01 mol/L, to remove unreacted small-molecule substance, exempted from
Epidemic focus；Saved backup in -20 DEG C.
Iprodione haptens obtains immunogene with hemocyanin coupling
The mg of hemocyanin 50 is weighed, is allowed to be substantially dissolved in the phosphate buffer PBS of 3.8 mL 0.1M（PH 7.2）
In, obtain solution A；Take 30 mg carbodiimides（EDC）And N-hydroxy-succinamide（NHS）Fully dissolved with 0.2 mL water
In solution A is added, 30 min are stirred at room temperature.3mg haptens is taken, 1 mL DMFs are dissolved in（DMF）
In, it is then slowly added in protein dissolution, 24 h of reaction are stirred at room temperature.It is daily with 0.01 mol/L PBS, 4 DEG C of 3 d of dialysis
Change 3 dialyzates.Packing, saves backup in -20 DEG C.
Iprodione haptens obtains immunogene with thyroprotein coupling
The mg of thyroprotein 50 is weighed, is allowed to be substantially dissolved in the phosphate buffer PBS of 3.8 mL 0.1M（PH
7.2）In, obtain solution A；Take 30 mg carbodiimides（EDC）And N-hydroxy-succinamide（NHS）It is abundant with 0.2 mL water
Dissolving stirs 30 min at room temperature in solution A is added.4mg haptens is taken, 1 mL DMFs are dissolved in
（DMF）In, it is then slowly added in protein dissolution, 24 h of reaction are stirred at room temperature.With 0.01 mol/L PBS, 4 DEG C of 3 d of dialysis
Change 3 dialyzates daily.Packing, saves backup in -20 DEG C.
3. the preparation of coating antigen
Iprodione haptens obtains coating antigen with oralbumin coupling
7.0 mg iprodione haptens are taken, the dimethylformamide of 1.0 mL is dissolved in（DMF）In, obtain reaction solution I；Take
30 mg carbodiimides（EDC）With the N-hydroxy-succinamide of 30 mg（NHS）Added after fully being dissolved with 0.2 mL water to
In reaction solution I, 24 h are stirred at room temperature, obtain reaction solution II；Take 50 mL oralbumins（OVA）, it is substantially dissolved in 3.8 mL
Phosphate buffer PBS（PH is 7.2）In, reaction solution II is dropwise slowly dropped in oralbumin solution, and in room
Temperature 24 h of lower stirring, obtain reaction solution III；Dialysed 3 days at III 4 DEG C by reaction solution with the PBS of 0.01 mol/L, it is not anti-to remove
The small-molecule substance answered, obtains coating antigen；Saved backup in -20 DEG C.
Iprodione haptens obtains coating antigen with human serum albumins coupling
The mg of human serum albumins 50 is weighed, is allowed to be substantially dissolved in 3.8 mL 0.1M PBS（PH 7.2）In, obtain molten
Liquid B；Take 0.2 mL water of 30mg EDC and NHS fully to dissolve in solution B is added, 30 min are stirred at room temperature.Take 9mg half
Antigen, is dissolved in 1mL DMF, is then slowly added in protein dissolution, and 24 h of reaction are stirred at room temperature.Use 0.01 mol/L
4 DEG C of 3 d of dialysis of PBS change 3 dialyzates daily.Packing, saves backup in -20 DEG C.
Iprodione haptens obtains coating antigen with albumin rabbit serum coupling
The mg of albumin rabbit serum 50 is weighed, is allowed to be substantially dissolved in 3.8 mL 0.1M PBS（PH 7.2）In, obtain molten
Liquid B；Take 0.2 mL water of 30mg EDC and NHS fully to dissolve in solution B is added, 30 min are stirred at room temperature.Take 9mg half
Antigen, is dissolved in 1mL DMF, is then slowly added in protein dissolution, and 24 h of reaction are stirred at room temperature.Use 0.01 mol/L
4 DEG C of 3 d of dialysis of PBS change 3 dialyzates daily.Packing, saves backup in -20 DEG C.
4. the identification of iprodione hapten-carrier protein conjugate
Iprodione haptens, carrier protein, iprodione hapten-carrier protein conjugate are made into the PBS of pH7.6
The solution of 0.5 mg/mL, is returned to zero, with ultraviolet specrophotometer in the nm models of wavelength 200 ~ 800 with 0.01 mol/L pH7.4 PBS
Interior scanning is enclosed, iprodione haptens, carrier protein, the absorption curve of iprodione hapten-carrier protein conjugate is obtained.Three
There are different absorption curves, show iprodione haptens with carrier protein couplet success.
5. the preparation of iprodione monoclonal antibody
（1）Animal immune
In the immunogene injection Balb/c Mice Bodies that step 2 is obtained, immunizing dose is 150 μ g/, produces it anti-
Serum.
（2）Cell fusion and cloning
Immune Balb/c mouse boosting cells are taken, by 8:1（Quantitative proportion）Ratio is merged with SP2/0 myeloma cell, is used
Indirect competitive ELISA method determines cell supernatant, the positive hole of screening.Cloning is carried out to positive hole using limiting dilution assay, directly
To the hybridoma cell strain for obtaining stably excreting monoclonal antibody.
（3）Cell cryopreservation and recovery
Hybridoma is made 1 × 10 with frozen stock solution6The cell suspension of individual/mL, preserves for a long time in liquid nitrogen.During recovery
Cryopreservation tube is taken out, 37 DEG C of water-bath middling speeds is immediately placed in and is melted, after centrifugation removal frozen stock solution, move into culture culture in glassware.
（4）The 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-
The nutrient solution that saturated ammonium sulfate method will be obtained is purified, and obtains monoclonal antibody, -20 DEG C of preservations.
The cell culture medium is, to addition calf serum and sodium acid carbonate in RPMI1640 culture mediums, calf serum is existed
In cell culture medium final concentration of 20%（Mass fraction）, sodium acid carbonate in cell culture medium final concentration of 0.2%（Quality
Fraction）；The pH of the cell culture medium is 7.4.
6th, the preparation of sheep anti mouse antiantibody
Using sheep as immune animal, pathogen-free domestic sheep is immunized by immunogene of mouse source antibody, obtains sheep anti mouse and resist
Antibody.
7. the preparation of iprodione monoclonal antibody-colloid gold label thing
（1）The preparation of collaurum
With double distilled deionized water by 1% gold chloride（It is purchased from sigma companies, catalog number T09041）It is diluted to 0.01%
（Weight/mass percentage composition）, take 100 mL and be placed in conical flask, boiling is heated to thermostatic electromagnetic agitator, in continuous high temperature, hold
Continuous stirring is lower to add the trisodium citrates of 2.5 mL 1%（It is purchased from Guangzhou Chemical Reagent Factory, catalog number BG11-AG-01KG）, after
It is continuous be at the uniform velocity heated with stirring to solution in it is bright red when stop, returning to original volume, 4 DEG C with deionized water after being cooled to room temperature
Preserve.The collaurum outward appearance for preparing is pure, bright, without precipitation and floating object.
（2）The preparation of iprodione monoclonal antibody-colloid gold label thing
Under magnetic stirring, the pH value of collaurum is adjusted to 7.0 with 0.2 mol/L solution of potassium carbonate, by every milliliter of collaurum
Add the standard of 30 ~ 60 μ g to iprodione monoclonal antibody is added in colloidal gold solution in solution, continue to stir and evenly mix 30
Min, adds 10%BSA, makes its final concentration of 1% in colloidal gold solution（Volume fraction）, stand 10 min.12000 r/
Min, 4 DEG C of 30 min of centrifugation, abandon supernatant, and precipitation redissolution buffer solution is washed twice, and are initial colloid gold volume 1/ with volume
20 redissolution buffer solution will precipitate resuspended, put 4 DEG C it is standby.
Redissolve buffer solution：Casein containing protein 0.02% ~ 0.1%（Mass fraction）, Tween-80 0.02% ~ 0.2%（Mass fraction）、
The 0.02 mol/L phosphate buffers of pH7.2.
8. the preparation of conjugate release pad
Conjugate release pad is soaked in and contains bovine serum albumin(BSA)（Concentration of the bovine serum albumin(BSA) in buffer solution is
1.0%）, in the phosphate buffer that pH is 7.2,0.2 mol/L, uniformly soak 1 h, 37 DEG C to dry 3 h standby.Sprayed with Isoflow
Iprodione monoclonal antibody-colloid gold label thing even application that film instrument will be prepared is combined in conjugate release pad per 1cm
After thing release pad spraying 0.01 ~ 0.04 mL iprodiones monoclonal antibody-colloid gold label thing, it is placed in 37 DEG C of environment（Humidity ＜
20%）Taken out after 60 min, be placed in dry environment（Humidity ＜ 20%）In save backup.
9. the preparation of reaction film
Detection zone will be constituted in iprodione haptens-ovalbumin conjugate coating to reaction film, by sheep anti mouse antiantibody
It is coated on and quality control region is constituted on reaction film.
Coating process：Iprodione haptens-ovalbumin conjugate is diluted to 10 mg/mL with phosphate buffer, is used
Isoflow point film instruments are coated in the detection zone on nitrocellulose filter（T lines）, package amount is 0.5 ~ 0.7 μ L/cm；With
Sheep anti mouse antiantibody is diluted to 200 μ g/mL by the phosphate buffer of 0.01 mol/L, pH7.4, will with Isoflow point film instruments
It is coated in the quality control region on nitrocellulose filter（C lines）, package amount is 0.6 ~ 1.0 μ L/cm.The reaction film that will be coated with is put
2 h are dried under the conditions of 37 DEG C, it is standby.
10. sample absorption pad
Sample absorption pad is placed in containing 0.5% bovine serum albumin(BSA)（Volume fraction）, pH7.2,0.2 mol/L phosphate delay
2 h are soaked in fliud flushing, 37 DEG C of 2 h of baking are standby.
The assembling of 11. test strips
Sample absorption pad, conjugate release pad, reaction film, adsorptive pads are pasted onto on PVC base plates in order successively；With reference to
Thing release pad has 1/3 region to be absorbed by the sample pad covering from initiating terminal, and the end of conjugate release pad connects with the top of reaction film
Connect, the end of reaction film is connected with the top of adsorptive pads, the top of sample absorption pad aligns with the top of PVC base plates, adsorptive pads
End alignd with the end of PVC base plates；There are detection zone and quality control region, detection zone on the reaction film（T lines）And quality control region（C
Line）It is the strip tape perpendicular with the length of the test strips；Detection zone is located at the side near the end of conjugate release pad；
Quality control region is located remotely from the side of the end of conjugate release pad；Test strips machine is cut into 3 mm small bars wide, mounted in spy
In the plastics fabrication of system, can be preserved 12 months under the conditions of 4 ~ 30 DEG C.
The detection of iprodione residual in embodiment 3, sample
1. the pre-treatment of sample
Tobacco sample need to rise again to 20-25 DEG C of room temperature before detection；The sample of 1.0 ± 0.05 g crushing is weighed to polystyrene
In centrifuge tube；10 mL sample extractions liquid (50% methyl alcohol) are added, it is fully smashed with refiner；Pipetted after standing on 0.1 mL
Clear liquid and 0.4 mL Sample dilutions mix to obtain sample liquid to be checked.
2. detected with test strips
The μ L of measuring samples solution 100 are taken with pipettor（Dropper 2-3 drops）Vertically it is added dropwise in well, when liquid flows
Beginning timing, 10 min of reaction, result of determination, more than 10min, as a result can be used as reference.
3. testing result is analyzed
It is negative（-）：The colour developing of T lines develops the color than C line, and deep or colour developing is consistent, and iprodione concentration is less than detection in representing sample
Limit, such as Fig. 2（a）With 2（b）.
It is positive（+）：The colour developing of T lines does not develop the color than the C line shallow or T lines of colour developing, and iprodione concentration is equal to or higher than in representing sample
Test limit, such as Fig. 2（c）With 2（d）.
It is invalid：There are not C lines, show the deterioration failure of incorrect operating process or test strips, such as Fig. 2（e）With 2
（f）.In the case, specification should be again read over, and is retested with new test strips.
Embodiment 4, sample detection example
1. test limit experiment
Blank tobacco sample is taken, the final concentration of 2 μ g/g of iprodione, 4 μ g/g and 8 μ g/g is added respectively wherein, with reality
Applying the test strips described in example 1 carries out detecting that each sample is repeated three times.Testing result feminine gender test strips quality control region colour developing,
Detection zone colour developing develops the color than quality control region, and deep or colour developing is consistent, and testing result is positive, and test strips quality control region colour developing, detection zone does not show
Color, testing result is shown in Table 1.
The measure of the ELISA test strip of table 1 limit
During with ELISA test strip tobacco sample, when wherein iprodione addition concentration is 2.0 μ g/g, the C lines of test strips show
Color, than C line depths, testing result is feminine gender to the colour developing of T lines；When wherein iprodione addition concentration is 4.0 μ g/g；Test strips C lines show
Color, T lines colour developing is more shallow than C line, and testing result is the positive；When wherein iprodione addition concentration is 8.0 μ g/g, test strips C lines show
Color, T lines do not develop the color, and testing result is the positive, shows that detection of this test strips to iprodione in tobacco leaf is limited to 4 μ g/g.
2. false positive rate and false negative rate are tested
Take each 20 parts of tobacco sample of the known iprodione content more than 4.0 μ g/g and iprodione content is less than 4.0 μ g/g
Each 20 parts of tobacco sample, detected with the test strips described in three batches of embodiments 1, calculate its false positive rate and false negative
Rate.The results are shown in Table 2 and table 3.
The false positive rate measurement result of table 2
Result shows：When being less than 4.0 μ g/g tobacco samples with three batches of concentration of ELISA test strip iprodione, as a result it is all
It is negative, it is known that test strips negative match-rate is that 100% false positive rate is 0.
The false negative rate measurement result of table 3
Result shows：During with three batches of concentration of ELISA test strip iprodione higher than 4.0 μ g/g tobacco samples, as a result it is all
It is positive, it is known that positive coincidence rate is 100%, and false negative rate is 0.
3. specific test
By iprodione in tobacco leaf and other drugs（Probenazole, thiophanate methyl, thiophanate, 2- aminobenzimidazoles, thiophene benzene
Up to azoles）It is diluted with the phosphate buffer of 0.2 mol/L that pH is 7.2.Examined with the test strips described in embodiment 1
Result display probenazole, thiophanate methyl, thiophanate, 2- aminobenzimidazoles, thiabendazolum are surveyed in 500 μ g/L concentration, is tried
Paper slip C lines develop the color, and T lines develop the color than C line depths, it can be deduced that this test strips does not occur cross reaction to these medicines.
Claims (7)
1. it is a kind of detect iprodione test strips preparation method, it is characterised in that：The test strips include sample absorption pad, combine
Thing release pad, reaction film, adsorptive pads and base plate, the reaction film are provided with detection zone and quality control region, and detection zone is coated with different bacterium
Urea hapten-carrier protein conjugate, quality control region is coated with antiantibody, comprises the following steps that：
（1）Preparation is coated with the conjugate release pad of iprodione specific antibody-colloid gold label thing；
（2）Prepare the quality control region of the detection zone and coating antiantibody with coating iprodione hapten-carrier protein conjugate
Reaction film；
（3）Will（2）The reaction film for preparing is assembled into test strips with conjugate release pad, sample absorption pad, adsorptive pads, base plate；
Wherein, the iprodione specific antibody in iprodione specific antibody-colloid gold label thing is with iprodione haptens-load
Body protein conjugate is prepared as immunogene, iprodione hapten-carrier protein conjugate be with iprodione haptens with
Carrier protein couplet is prepared, and the preparation method of the iprodione haptens is as follows：
The synthesis of compound A：The chloro- 4- nitrophenols of 5.0 g 2,6- bis- are taken to be stirred in acetone with the 3.2 g 4- bromo-butyric acid tert-butyl esters
Mix, then be added thereto to 2.6 g Anhydrous potassium carbonates and 8 h are reacted at 60 DEG C as catalyst, after reaction stops, solvent evaporated, then
Add water and ethyl acetate extraction, and use anhydrous sodium sulfate drying, concentrate, cross 200-300 mesh silicagel columns, chromatography purifying is obtained
To the g of compound A 5.23；
The synthesis of compound B：To 3.1 g zinc powders and 1 mL glacial acetic acids is added in 20 mL water, 30 min are reacted at 90 DEG C, then add
Enter the mL of ethanol solution 80 of 5.2 g compounds A, 4 h reacted at 60 DEG C, question response stops, and suction filtration is evaporated, add water thereto with
Dichloromethane is extracted, and is stood, and is petroleum ether with volume ratio with anhydrous sodium sulfate drying:Ethyl acetate=20:1 recrystallization, obtains
The g of compound B 4.8；
The synthesis of compound C：Take 4.7 g compounds B to be dissolved with 100 mL acetonitriles, stir, be added thereto to 2.16 g amino second
Acid, under nitrogen protection, is passed through phosgene, and 7 h are reacted at 50 DEG C, after stopping reaction, adds 50 mL NaOH water-soluble
Liquid, concussion, revolving removes acetonitrile, plus ethyl acetate dissolving, and add water washing, concentration, is evaporated, and crosses 200-300 mesh silicagel columns, layer
Analysis is separated, and obtains the g of compound C 3.57；
The synthesis of compound D：3.4 g compound C are taken, is dissolved with 1,2- dichloroethanes, add 0.5 mL DMF, 3 mL oxalyl
Chlorine, is stirred at room temperature 3 h, and revolving is evaporated, and removes organic solvent, adds 80 mL pyridines, and 7 h are reacted at 80 DEG C, stops reaction, cold
But room temperature is arrived, is rotated, remove pyridine, addition volume ratio is ethanol:Petroleum ether=10:3 solvent heating for dissolving, placed in 4 DEG C
Night, suction filtration obtains the g of compound D 2.97；
The synthesis of compound E：Take during 2.8 g compounds D add 200 mL dichloroethanes and dissolve, add 2.7 mL isopropylamines,
Nitrogen is passed through, air is discharged, phosgene is passed through, 6 h of reaction are stirred at room temperature, stop reaction, add 50 mL potassium hydroxide aqueous solutions
Concussion, stands, and isolates organic phase, washes, and is evaporated with anhydrous sodium sulfate drying, crosses 200-300 mesh silicagel columns, is with volume ratio
Petroleum ether:Ethyl acetate=1:1 solvent wash-out is separated, and obtains the g of compound E 1.53；
The synthesis of compound F：1.53 g compound E are taken, the dissolving of the aqueous formic acids of 100 mL 70% is added, in 70 DEG C of stirring reactions
6 h, stop reacting, and revolving removes solvent, are hydrogenated with aqueous solution of sodium oxide, plus ethyl acetate extraction, divide and go organic phase, and water is mutually adjusted
Section pH value plus 1 to 4, and 2- dichloroethanes extraction, washing is dried, and is evaporated, and is ethanol with volume ratio:N-hexane=6:4 solvent weight
Crystallization, obtains the g of iprodione haptens 0.81, and its molecular structural formula is：
。
2. preparation method according to claim 1, it is characterised in that：The test strips are released by sample absorption pad, conjugate
Put pad, reaction film, adsorptive pads and paste the composition on base plate successively, and conjugate release pad 1/3 ~ 1/2 is capped on sample absorption
Under pad.
3. preparation method according to claim 1, it is characterised in that：The detection zone is near sample absorption pad one end, institute
Quality control region is stated near adsorptive pads one end.
4. preparation method according to claim 1, it is characterised in that：The carrier protein is bovine serum albumin(BSA), egg white
Albumen, hemocyanin, thyroprotein, human serum albumins or albumin rabbit serum.
5. preparation method according to claim 1, it is characterised in that：The antiantibody is sheep anti mouse antiantibody.
6. preparation method according to claim 1, it is characterised in that：The iprodione specific antibody is iprodione Dan Ke
Grand antibody.
7. iprodione in the ELISA test strip tobacco sample that prepared by the preparation method described in a kind of application claim any one of 1-6
The detection method of residual, it includes step：
（1）Pre-treatment is carried out to sample；
（2）Detected with test strips；
（3）Analysis testing result.
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| CN107119022B (en) * | 2017-04-26 | 2021-02-09 | 江南大学 | Isobacteriumurea monoclonal antibody hybridoma cell strain ZXL-2 and application thereof |
| CN109061156B (en) * | 2018-09-21 | 2021-07-13 | 中国烟草总公司郑州烟草研究院 | Time-resolved fluorescence immunochromatographic test strip for detecting pendimethalin and preparation method and application thereof |
| CN109265401B (en) * | 2018-09-21 | 2021-09-03 | 中国烟草总公司郑州烟草研究院 | Preparation method and application of iprodione hapten and antigen |
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