CN105388286A - Test strip for detecting iprodione and preparation method and application of test strip - Google Patents
Test strip for detecting iprodione and preparation method and application of test strip Download PDFInfo
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Abstract
The invention discloses a test strip for detecting iprodione and a preparation method and application of the test strip. The test strip comprises a sample absorption cushion (1), a compound release cushion (2), a reaction film (3), a water absorption cushion (4) and a bottom plate (5). The test strip is characterized in that a detection area (6) wrapped by iprodione hapten-carrier protein conjugate and a quality control area (7) wrapped by goat anti-mouse antibodies are arranged on the reaction film, and the compound release cushion (2) is coated with iprodione monoclonal antibody-colloidal gold markers. The invention further provides a method for detecting iprodione residues in tobacco leaves through the prepared iprodione test strip. The test strip has the advantages of being easy to operate, free of sample pretreatment, high in sensitivity, high in detection speed, low in cost and the like, and is suitable for screening and on-site monitoring of a large number of samples.
Description
Technical field
The present invention relates to the detection of iprodione, specifically a kind of detect iprodione test strips and preparation method and its application in tobacco leaf detects.
Background technology
Iprodione, has another name called iprodione, and molecular formula is C
13h
13cl
2n
3o
3, belong to dicarboximide class, be a kind of broad spectrum activity contact killing type protective fungicide, be widely used in tobacco, fruit tree, the disease control of vegetables and the preservation and freshness of fruit.Iprodione can play systemic action by root absorption, can effectively prevent and treat fungi benzimidazole systemic fungicide being had to resistance.Its main controlling object is the disease caused by botrytis, Alternariaspp, sclerotinite etc., as gray mold, early blight, black spot, sclerotiniose etc.Iprodione agricultural chemicals is the test item that Japan, the U.S., Korea S, Australia, Canada and state of Codex Committee on Food finite quantity require, its maximum residue limit(MRL) in varieties of food items is 0.05 ~ 15mg/kg.At present, the detection method of iprodione is mostly liquid phase chromatography and vapor-phase chromatography, and the matrix species that can detect is less, to the Patents of the detection method of iprodione in tobacco and bibliographical information also less.Analytical instrument method testing cost is high, and need professional and technical personnel to operate, detection time is long, has limitation for carrying out of Site Detection work.
Summary of the invention
Object of the present invention is to provide a kind of test strips detecting iprodione and its preparation method and application based on above-mentioned prior art situation just, the present invention's application colloidal gold immunity chromatography measures the residual quantity of iprodione in tobacco, tool accuracy is high, there have to be simple to operate, without the need to professional's operation, without the need to other checkout equipments supporting, be convenient for carrying short, the feature such as testing cost is low detection time, is applicable to the examination of on-the-spot a large amount of sample.
The object of the invention is to be achieved through the following technical solutions:
A kind of test strips detecting iprodione, comprise absorption of sample pad, bond release pad, reaction film, adsorptive pads and base plate, described reaction film there are detection zone and quality control region, detection zone is coated with iprodione hapten-carrier protein conjugate, quality control region is coated with antiantibody, and described bond release pad is coated with iprodione specific antibody-colloid gold label thing.Iprodione specific antibody in iprodione specific antibody-colloid gold label thing prepares using iprodione hapten-carrier protein conjugate as immunogene, and described iprodione hapten-carrier protein conjugate prepares with iprodione haptens and carrier protein couplet.
Described test strips discharges pad by absorption of sample pad, bond, reaction film, adsorptive pads are pasted successively and formed on base plate, and bond discharges under pad 1/3 ~ 1/2 is capped on absorption of sample pad.
Described detection zone is near absorption of sample pad one end, and quality control region is near adsorptive pads one end.
Described iprodione hapten-carrier protein conjugate is obtained by iprodione haptens and carrier protein couplet, and described carrier protein can be bovine serum albumin(BSA), ovalbumin, hemocyanin, thyroprotein, human serum albumins.
Described antiantibody is sheep anti mouse antiantibody.
Described iprodione specific antibody can be iprodione monoclonal antibody or iprodione polyclonal antibody.
Described iprodione haptens is by 2, the chloro-4-nitrophenol of 6-bis-is initiation material, through obtaining hydroxyl 2 with the hydrocarbyl reaction of the 4-bromo-butyric acid tert-butyl ester, the chloro-4-nitrobenzene of 6-bis-, after reduction nitro, obtain alkyl 2, 6-dichloroaniline, alkyl 2, 6-dichloroaniline is under the effect of phosgene, with aminoacetic acid condensation, obtain alkyl 2, 6-dichloro-benzenes glycinate, again through annulation, obtain iprodione agent structure compound, this compound is again through phosgene effect, with isopropylamine condensation, obtain iprodione mother nucleus structure compound, hydrolysis reaction obtains in acid condition, molecular structure is such as formula I:
(formula I).
Described base plate can be the material that PVC base plate or other hard do not absorb water; Described absorption of sample pad can be nonwoven fabrics or filter paper for oil; Described bond release pad can be glass fibre or polyester material; Described adsorptive pads is thieving paper; Described reaction film can be nitrocellulose filter or cellulose acetate membrane.
Prepare a method for above-mentioned test strips, its step comprises:
(1) preparation is coated with the bond release pad of iprodione monoclonal antibody-colloid gold label thing;
(2) preparation has the reaction film of the detection zone being coated with iprodione hapten-carrier protein conjugate and the quality control region being coated with sheep anti mouse antiantibody;
(3) reaction film (2) prepared and bond discharge pad, absorption of sample pad, adsorptive pads, base plate be assembled into test strips;
Specifically, step comprises:
(1) by iprodione and bromo valeral generation nucleophilic substitution, iprodione haptens is obtained;
(2) by iprodione haptens and carrier protein couplet, iprodione hapten-carrier protein conjugate is obtained;
(3) with iprodione hapten-carrier albumen coupling immunity thing immune mouse, mouse boosting cell and myeloma cell are obtained the strain of iprodione monoclonal hybridoma by merging, screening;
(4) extract mouse IgG immune health goat, obtain sheep anti mouse antiantibody;
(5) react with trisodium citrate and gold chloride and prepare collaurum;
(6) iprodione monoclonal antibody prepared by step (3) is added in collaurum prepared by step (5), obtain iprodione monoclonal antibody-colloid gold label thing;
(7) iprodione monoclonal antibody-colloid gold label thing is sprayed on bond release pad, takes out after 37 DEG C of baking 1h, be placed in dry environment and save backup;
(8) iprodione hapten-carrier protein conjugate bag is formed detection zone by reaction film again, sheep anti mouse antiantibody is coated on reaction film and forms quality control region;
(9) by absorption of sample pad be 7.2 containing 0.5% bovine serum albumin(BSA) (volume fraction), pH, 0.1mol/L phosphate buffer soaks 2h, dries 2h at 37 DEG C;
(10) on base plate, paste absorption of sample pad, bond release pad, reaction film, adsorptive pads in order, absorption of sample pad covers bond release pad, is finally cut into the little bar that 3mm is wide, adds plastic casing, vacuum packaging, can preserve 12 months under 4 ~ 30 DEG C of conditions.
Apply the method that in above-mentioned ELISA test strip tobacco leaf, iprodione is residual, comprise step:
(1) detect by test strips;
(2) testing result is analyzed.
Iprodione residue detection test strips of the present invention adopts antibody antigen reaction and the Competitive assays immunochromatographiassays assays technology of high degree of specificity, iprodione monoclonal antibody-colloid gold label thing is fixed on bond release pad, iprodione in sample is in flow process, discharge the monoclonal antibody-colloid gold label thing of the iprodione on padding with bond to be combined, form drug-antibody-colloid gold label thing.Iprodione hapten-carrier albumen coupling competition binding iprodione monoclonal antibody-colloid gold label thing on medicine in sample and reaction film detection zone, according to detection zone red stripes with or without or shade judge whether remain containing iprodione in analyte sample fluid.
During detection, sample instills absorption of sample pad after treatment, when iprodione concentration in the sample to which lower than ELISA test strip limit or be zero time, monoclonal antibody-colloid gold label thing in chromatography process can be fixed on the iprodione hapten-carrier protein conjugate on reaction film and be combined, each appearance red stripes in detection zone (T line) and quality control region (C line), and the colour developing of T line is darker than the colour developing of C line or the colour developing depth is consistent; If iprodione concentration is in the sample to which equal to or higher than ELISA test strip limit, monoclonal antibody-colloid gold label thing can be combined with iprodione, thus at T line place because competitive reaction can not or partly be combined with iprodione hapten-carrier protein conjugate and not occur that the color of the colour developing of red stripes or T line is more shallow than C line.As shown in Figure 2.
Negative: when quality control region (C line) demonstrates red stripes, detection zone (T line) also demonstrates red stripes simultaneously, and the colour developing of T line is darker than the colour developing of C line or colour developing is consistent, is all judged to feminine gender, shows that in sample, the concentration of iprodione is limit lower than ELISA test strip.
Positive: when quality control region (C line) demonstrates red stripes, and detection zone (T line) colour developing is than C line, and shallow or T line does not develop the color, and is judged to the positive, shows that in sample, the concentration of iprodione is equal to or higher than ELISA test strip limit.
Invalid: when quality control region (C line) does not demonstrate red stripes, then no matter whether detection zone (T line) demonstrates red stripes, and it is invalid that this test strips is all judged to.
Test strips beneficial effect of the present invention: test strips of the present invention is passed through 2, the chloro-4-nitrophenol of 6-bis-carries out structure of modification, synthesize the carboxyl haptens with four carbon chain lengths, highlight the molecular structure of iprodione itself, enhance immunogenicity, make the iprodione monoclonal antibody for preparing highly sensitive, high specificity, test strips cost of the present invention is low, simple to operate, sample pre-treatments is simple, detection time is short, only need 10min, be convenient for carrying, be applicable to the examination of on-the-spot a large amount of sample, store simple, long shelf-life, can extensively promote the use of in food safety Regulation work.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of test strips cross-section structure of the present invention.
In Fig. 1: 1 is absorption of sample pad, 2 is bond release pad, and 3 is reaction film, and 4 is adsorptive pads, and 5 is base plate, and 6 is detection zone, and 7 is quality control region.
Fig. 2 is the schematic diagram that ELISA test strip result of the present invention judges.
Fig. 3 is the schematic diagram of iprodione hapten synthesis route of the present invention.
Embodiment
Below by embodiment, and 1-3 by reference to the accompanying drawings, technical scheme of the present invention is further described in detail.The explanation of following reference accompanying drawing to embodiment of the present invention is intended to make an explanation to general plotting of the present invention, and not should be understood to one restriction of the present invention.
The experimental technique used in following embodiment if no special instructions, is conventional method.
the formation of the test strips of embodiment 1, detection iprodione
As shown in Figure 1, pasted according to the test strips of the detection iprodione of the preferred embodiment of the present invention successively formed on base plate 5 by absorption of sample pad 1, bond release pad 2, reaction film 3, adsorptive pads 4.The end of absorption of sample pad is connected with reaction film, and the end of reaction film is connected with adsorptive pads, and the top of absorption of sample pad aligns with the top of base plate, and the end of adsorptive pads aligns with the end of base plate; Described reaction film has detection zone 6 and quality control region 7, detection zone and quality control region are the ribbon detection zone vertical with the appearance of described test strips and are positioned at the side being bordering on absorption of sample pad, and quality control region is positioned at the side being bordering on adsorptive pads; Described detection zone is coated with iprodione hapten-carrier protein conjugate (conjugate of iprodione haptens-bovine serum albumin(BSA)), and quality control region is coated with sheep anti mouse antiantibody; Described base plate is PVC base plate, and absorption of sample pad is nonwoven fabrics, and reaction film is nitrocellulose filter, the glass fibre of bond release pad for being coated with iprodione monoclonal antibody-colloid gold label thing; Described iprodione residue detection test strips stores 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 haptenic preparation of iprodione
The synthesis of compd A: get 5.0g2, the chloro-4-nitrophenol of 6-bis-and the 3.2g4-bromo-butyric acid tert-butyl ester stir in acetone, then add 2.6g Anhydrous potassium carbonate wherein as catalyzer at 60 DEG C of reaction 8h, after reaction stops, solvent evaporated, add water again and extraction into ethyl acetate, and with anhydrous sodium sulfate drying, concentrated, cross 200-300 order silicagel column, chromatography purifying, obtains compound A-45 .23g, yield 87%.1HNMR（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 compd B: add 3.1g zinc powder and 1mL glacial acetic acid in 20mL water, at 90 DEG C of reaction 30min, then adds the ethanolic solution 80mL of 5.2g compd A, at 60 DEG C of reaction 4h, question response stops, suction filtration, evaporate to dryness, add water wherein and dichloromethane extraction, leaving standstill, with anhydrous sodium sulfate drying, is sherwood oil by volume ratio: ethyl acetate=20:1 recrystallization, obtain compd B 4.8g, yield 91%.1HNMR（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: get 4.7g compd B 100mL acetonitrile and dissolve, stir, add 2.16g aminoacetic acid wherein, under nitrogen protection; pass into phosgene, at 50 DEG C, react 7h, after stopping reaction; add 50mL sodium hydrate aqueous solution again, concussion, revolves steaming; removing acetonitrile, adds acetic acid ethyl dissolution, adds water washing; concentrated, evaporate to dryness, crosses 200-300 order silicagel columns; chromatography, obtains Compound C 3.57g, yield 73%.1HNMR（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: get 3.4g Compound C, dissolves with 1,2-ethylene dichloride, adds 0.5mLDMF, 3mL oxalyl chloride, stirring at room temperature 3h, revolve steaming evaporate to dryness, and removing organic solvent, adds 80mL pyridine, at 80 DEG C of reaction 7h.Stop reaction, cool to room temperature, revolve steaming, removing pyridine, adding volume ratio is ethanol: the solvothermal of sherwood oil=10:3 dissolves, spend the night in 4 DEG C of placements, suction filtration, obtain Compound D 2.97g, yield 82%.1HNMR(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 compd E: get 2.8g Compound D and add in 200mL ethylene dichloride and dissolve, then add 2.7mL isopropylamine, pass into nitrogen, discharge air, pass into phosgene, stirring at room temperature reaction 6h, stop reaction, add the concussion of 50mL potassium hydroxide aqueous solution, leave standstill, isolate organic phase, washing, uses anhydrous sodium sulfate drying evaporate to dryness, cross 200-300 order silicagel columns, be sherwood oil by volume ratio: the solvent elution of ethyl acetate=1:1 is separated, and obtains compd E 1.53g, yield 64%.1HNMR（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),1.1693H,d,J=6.794),1.169(3H,d,J=6.794)。
The synthesis of compound F 17-hydroxy-corticosterone: get 1.53g compound, adds 100mL70% aqueous formic acid and dissolves, in 70 DEG C of stirring reaction 6h, stop reaction, revolve steaming, except desolventizing, hydro-oxidation sodium water solution, adds extraction into ethyl acetate, divides and goes organic phase, aqueous phase regulates pH value to 4, adds the extraction of 1,2-ethylene dichloride, washing, dry, evaporate to dryness, be ethanol by volume ratio: the solvent recrystallization of normal hexane=6:4, obtains iprodione haptens 0.81g, yield 77%.1HNMR（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)。
Get above-mentioned product through nuclear magnetic resonance hydrogen spectruming determining, chemical shift δ=11.0 be carboxyl hydrogen, δ=4.0,1.9,2.4 be the resonance absorbing peak of the hydrogen of methylene on spacerarm, these peaks prove hapten synthesis success there is the existence coordinating other iprodione hydrogen inherent absorption peaks.
2. immunogenic preparation
Iprodione haptens and bovine serum albumin(BSA) coupling obtain immunogene
Get 9.0mg iprodione haptens, be dissolved in the dimethyl formamide (DMF) of 1.0mL, obtain solution I; Add solution I, stirred at ambient temperature 24h after getting 30mg carbodiimides (EDC) and the water-soluble solution of 30mgN-N-Hydroxysuccinimide (NHS) 0.2mL, obtain reactant liquor II; Get 50mL bovine serum albumin(BSA) (BSA), the phosphate buffer PBS(pH being fully dissolved in 3.8mL is 7.2) in, reactant liquor II is dropwise slowly added drop-wise in bovine serum albumin solution, and in stirred at ambient temperature 24h, obtains reactant liquor III; Dialyse 3 days at 4 DEG C with the PBS of 0.01mol/L, to remove unreacted small-molecule substance, obtain immunogene; Save backup in-20 DEG C.
Iprodione haptens and hemocyanin coupling obtain immunogene
Take hemocyanin 50mg, make it the phosphate buffer PBS(PH7.2 being fully dissolved in 3.8mL0.1M) in, obtain solution A; Get 30mg carbodiimides (EDC) and N-hydroxy-succinamide (NHS) 0.2mL water fully dissolve after in adding in solution A, stirred at ambient temperature 30min.Get 3mg haptens, be dissolved in 1mLN, in dinethylformamide (DMF), then slowly join in protein dissolution, stirring at room temperature reaction 24h.3 dislysates are changed every day with 0.01mol/LPBS4 DEG C of dialysis 3d.Packing, saves backup in-20 DEG C.
Iprodione haptens and thyroprotein coupling obtain immunogene
Take thyroprotein 50mg, make it the phosphate buffer PBS(PH7.2 being fully dissolved in 3.8mL0.1M) in, obtain solution A; Get 30mg carbodiimides (EDC) and N-hydroxy-succinamide (NHS) 0.2mL water fully dissolve after in adding in solution A, stirred at ambient temperature 30min.Get 4mg haptens, be dissolved in 1mLN, in dinethylformamide (DMF), then slowly join in protein dissolution, stirring at room temperature reaction 24h.3 dislysates are changed every day with 0.01mol/LPBS4 DEG C of dialysis 3d.Packing, saves backup in-20 DEG C.
3. the preparation of coating antigen
Iprodione haptens and oralbumin coupling obtain coating antigen
Get 7.0mg iprodione haptens, be dissolved in the dimethyl formamide (DMF) of 1.0mL, obtain reactant liquor I; Be added in reactant liquor I after N-hydroxy-succinamide (NHS) the 0.2mL water getting 30mg carbodiimides (EDC) and 30mg fully dissolves, stirred at ambient temperature 24h, obtains reactant liquor II; Get 50mL oralbumin (OVA), the phosphate buffer PBS(pH being fully dissolved in 3.8mL is 7.2) in, reactant liquor II is dropwise slowly added drop-wise in oralbumin solution, and in stirred at ambient temperature 24h, obtains reactant liquor III; With the PBS of 0.01mol/L by reactant liquor III 4 DEG C dialysis 3 days, to remove unreacted small-molecule substance, obtain coating antigen; Save backup in-20 DEG C.
Iprodione haptens and human serum albumins coupling obtain coating antigen
Take human serum albumins 50mg, make it fully to be dissolved in 3.8mL0.1MPBS(PH7.2) in, obtain solution B; Get 30mgEDC and NHS 0.2mL water fully dissolve after in adding in solution B, stirred at ambient temperature 30min.Get 9mg haptens, be dissolved in 1mLDMF, then slowly join in protein dissolution, stirring at room temperature reaction 24h.3 dislysates are changed every day with 0.01mol/LPBS4 DEG C of dialysis 3d.Packing, saves backup in-20 DEG C.
Iprodione haptens and albumin rabbit serum coupling obtain coating antigen
Take albumin rabbit serum 50mg, make it fully to be dissolved in 3.8mL0.1MPBS(PH7.2) in, obtain solution B; Get 30mgEDC and NHS 0.2mL water fully dissolve after in adding in solution B, stirred at ambient temperature 30min.Get 9mg haptens, be dissolved in 1mLDMF, then slowly join in protein dissolution, stirring at room temperature reaction 24h.3 dislysates are changed every day with 0.01mol/LPBS4 DEG C of dialysis 3d.Packing, saves backup in-20 DEG C.
4. the qualification of iprodione hapten-carrier protein conjugate
The PBS of iprodione haptens, carrier protein, iprodione hapten-carrier protein conjugate pH7.6 is made into the solution of 0.5mg/mL, return to zero with 0.01mol/LpH7.4PBS, with ultraviolet spectrophotometer in the interscan of wavelength 200 ~ 800nm scope, obtain the absorption curve of iprodione haptens, carrier protein, iprodione hapten-carrier protein conjugate.There is different absorption curves in three, shows iprodione haptens and carrier protein couplet success.
5. the preparation of iprodione monoclonal antibody
(1) animal immune
Immunogene step 2 obtained is injected in Balb/c Mice Body, and immunizing dose is 150 μ g/, makes it produce antiserum.
(2) Fusion of Cells and cloning
Get immune Balb/c mouse boosting cell, in 8:1(quantitative proportion) ratio and SP2/0 myeloma cell fusion, adopt indirect competitive ELISA method to measure cell conditioned medium liquid, screen positive hole.Limiting dilution assay is utilized to carry out cloning to positive hole, until obtain the hybridoma cell strain of stably excreting monoclonal antibody.
(3) cell cryopreservation and recovery
Hybridoma cryopreserving liquid is made 1 × 10
6the cell suspension of individual/mL, preserves for a long time in liquid nitrogen.Take out cryopreservation tube during recovery, put into 37 DEG C of water-bath middling speeds immediately and melt, after centrifugal segregation cryopreserving liquid, move into and cultivate culture in glassware.
(4) preparation and purification of monoclonal antibody
Increment cultivation: hybridoma is placed in cell culture medium, cultivates under 37 DEG C of conditions, by sad-saturated ammonium sulfate method, the nutrient solution obtained is carried out purifying, obtains monoclonal antibody ,-20 DEG C of preservations.
Described cell culture medium, for add calf serum and sodium bicarbonate in RPMI1640 nutrient culture media, makes the final concentration of calf serum in cell culture medium be 20%(massfraction), the final concentration of sodium bicarbonate in cell culture medium is 0.2%(massfraction); The pH of described cell culture medium is 7.4.
6, the preparation of sheep anti mouse antiantibody
Using sheep as immune animal, for immunogene, immunity is carried out to pathogen-free domestic sheep with mouse source antibody, obtain sheep anti mouse antiantibody.
7. the preparation of iprodione monoclonal antibody-colloid gold label thing
(1) preparation of collaurum
With two ionized water that boils off, 1% gold chloride (is purchased from sigma company, catalog number T09041) be diluted to 0.01%(mass percentage), get 100mL and be placed in conical flask, boiling is heated to thermostatic electromagnetic stirrer, under continuous high temperature, Keep agitation, add 2.5mL1% trisodium citrate (be purchased from Guangzhou Chemical Reagent Factory, catalog number BG11-AG-01KG), continue at the uniform velocity to be heated with stirring to solution be bright red time stopping, original volume is returned to deionized water, 4 DEG C of preservations after being cooled to room temperature.The collaurum outward appearance prepared is pure, bright, nothing precipitates and floating thing.
(2) preparation of iprodione monoclonal antibody-colloid gold label thing
Under magnetic stirring, the pH value to 7.0 of collaurum is adjusted with 0.2mol/L solution of potassium carbonate, in colloidal gold solution, iprodione monoclonal antibody is added by the standard adding 30 ~ 60 μ g in every milliliter of colloidal gold solution, continue to stir and evenly mix 30min, add 10%BSA, its final concentration in colloidal gold solution is made to be 1%(volume fraction), leave standstill 10min.12000r/min, 4 DEG C of centrifugal 30min, abandon supernatant, precipitation with redissolve buffer solution twice, with volume be initial colloid gold volume 1/20 redissolution damping fluid will precipitate resuspended, put 4 DEG C for subsequent use.
Redissolve damping fluid: casein containing protein 0.02% ~ 0.1%(massfraction), Tween-80 0.02% ~ 0.2%(massfraction), the 0.02mol/L phosphate buffer of pH7.2.
8. the preparation of bond release pad
Bond is discharged pad to be soaked in and to be 7.2 containing bovine serum albumin(BSA) (concentration of bovine serum albumin(BSA) in damping fluid is 1.0%), pH, in the phosphate buffer of 0.2mol/L, evenly to soak 1h, 37 DEG C to dry 3h for subsequent use.Spray film instrument with Isoflow the iprodione monoclonal antibody-colloid gold label thing even application prepared is padded in bond release, after every 1cm bond release pad spraying 0.01 ~ 0.04mL iprodione monoclonal antibody-colloid gold label thing, take out after being placed in 37 DEG C of environment (humidity < 20%) 60min, be placed in dry environment (humidity < 20%) and save backup.
9. the preparation of reaction film
Iprodione haptens-ovalbumin conjugate bag is formed detection zone by reaction film, sheep anti mouse antiantibody is coated on reaction film and forms quality control region.
Bag is by process: with phosphate buffer, iprodione haptens-ovalbumin conjugate is diluted to 10mg/mL, be coated in the detection zone (T line) on nitrocellulose filter with Isoflow point film instrument, package amount is 0.5 ~ 0.7 μ L/cm; With the phosphate buffer of 0.01mol/L, pH7.4, sheep anti mouse antiantibody is diluted to 200 μ g/mL, be coated in the quality control region (C line) on nitrocellulose filter with Isoflow point film instrument, package amount is 0.6 ~ 1.0 μ L/cm.Bag is placed in dry 2h under 37 DEG C of conditions by good reaction film, for subsequent use.
10. absorption of sample pad
Be placed in by absorption of sample pad and soak 2h containing 0.5% bovine serum albumin(BSA) (volume fraction), pH7.2,0.2mol/L phosphate buffer, 37 DEG C of baking 2h are for subsequent use.
The assembling of 11. test strips
Absorption of sample pad, bond release pad, reaction film, adsorptive pads are pasted onto on PVC base plate successively in order; Bond release pad has 1/3 region to be covered by absorption of sample pad from initiating terminal, the end of bond release pad is connected with the top of reaction film, the end of reaction film is connected with the top of adsorptive pads, the top of absorption of sample pad aligns with the top of PVC base plate, and the end of adsorptive pads aligns with the end of PVC base plate; Described reaction film has detection zone and quality control region, detection zone (T line) is the strip tape vertical with the appearance of described test strips with quality control region (C line); Detection zone is positioned at the side of the end near bond release pad; Quality control region is positioned at the side of the end away from bond release pad; Test strips machine is cut into the little bar that 3mm is wide, is contained in special plastics fabrication, under 4 ~ 30 DEG C of conditions, can 12 months be preserved.
the detection that in embodiment 3, sample, iprodione is residual
1. the pre-treatment of sample
Before detecting, tobacco sample need be risen again room temperature 20-25 DEG C; Take the sample of 1.0 ± 0.05g pulverizing in polystyrene centrifuge tube; Add 10mL sample extraction liquid (50% methyl alcohol), with refiner, it is fully smashed; 0.1mL supernatant is pipetted and 0.4mL Sample dilution is mixed to get sample liquid to be checked after leaving standstill.
2. detect by test strips
Get measuring samples solution 100 μ L(dropper 2-3 with pipettor to drip) vertically drip in well, start timing during liquid flow, reaction 10min, result of determination, more than 10min, result can be used as reference.
3. analyze testing result
Negative (-): the colour developing of T line is darker than the colour developing of C line or colour developing is consistent, all represents that in sample, iprodione concentration is lower than detectability, as Fig. 2 (a) and 2(b).
Positive (+): T line colour developing or T line more shallow than the colour developing of C line does not develop the color, and represents that in sample, iprodione concentration is equal to or higher than detectability, as Fig. 2 (c) and 2(d).
Invalid: not occur C line, show the deterioration failure of incorrect operating process or test strips, as Fig. 2 (e) and 2(f).In the case, again should read over instructions, and retest by new test strips.
embodiment 4, sample detection example
1. detectability test
Get blank tobacco sample, adding iprodione final concentration respectively is wherein 2 μ g/g, 4 μ g/g and 8 μ g/g, is undertaken detecting each sample replication three times by the test strips described in embodiment 1.The negative test strips quality control region colour developing of testing result, detection zone colour developing is darker than quality control region colour developing or colour developing is consistent, and testing result is positive, and test strips quality control region develops the color, and do not develop the color in detection zone, testing result is in table 1.
The mensuration of table 1 ELISA test strip limit
During with ELISA test strip tobacco sample, when wherein iprodione interpolation concentration is 2.0 μ g/g, the C line colour developing of test strips, the colour developing of T line is darker than C line, and testing result is negative; When wherein iprodione interpolation concentration is 4.0 μ g/g; Test strips C line develops the color, and the colour developing of T line is more shallow than C line, and testing result is positive; When wherein iprodione interpolation concentration is 8.0 μ g/g, test strips C line develops the color, and T line does not develop the color, and testing result is positive, shows that this test strips is limited to 4 μ g/g to the detection of iprodione in tobacco leaf.
2. false positive rate and false negative rate test
Get known iprodione content and be greater than each 20 parts of the tobacco sample that the tobacco sample of 4.0 μ g/g each 20 parts and iprodione content is less than 4.0 μ g/g, detect by 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.
Table 2 false positive rate measurement result
Result shows: when being less than 4.0 μ g/g tobacco sample by the concentration of three batches of ELISA test strip iprodiones, and result is negative entirely, and known test strips negative match-rate is 100% false positive rate is 0.
Table 3 false negative rate measurement result
Result shows: during by the concentration of three batches of ELISA test strip iprodiones higher than 4.0 μ g/g tobacco sample, and result is positive entirely, and known positive coincidence rate is 100%, and false negative rate is 0.
3. specific test
Be that the phosphate buffer of the 0.2mol/L of 7.2 dilutes with pH by iprodione in tobacco leaf and other drug (probenazole, thiophanate methyl, thiophanate, 2-aminobenzimidazole, thiabendazolum).Testing result display probenazole, thiophanate methyl, thiophanate, 2-aminobenzimidazole, thiabendazolum is carried out when 500 μ g/L concentration by the test strips described in embodiment 1, test strips C line develops the color, the colour developing of T line is darker than C line, can show that this test strips cross reaction does not occur to these medicines.
Claims (9)
1. one kind is detected the test strips of iprodione, comprise absorption of sample pad, bond release pad, reaction film, adsorptive pads and base plate, it is characterized in that: described reaction film is provided with detection zone and quality control region, detection zone is coated with iprodione hapten-carrier protein conjugate, quality control region is coated with antiantibody, described bond release pad is coated with iprodione specific antibody-colloid gold label thing, iprodione specific antibody in described iprodione specific antibody-colloid gold label thing prepares using iprodione hapten-carrier protein conjugate as immunogene, described iprodione hapten-carrier protein conjugate prepares with iprodione haptens and carrier protein couplet.
2. test strips according to claim 1, is characterized in that: described test strips discharges pad by absorption of sample pad, bond, reaction film, adsorptive pads are pasted successively and formed on base plate, and bond discharges under pad 1/3 ~ 1/2 is capped on absorption of sample pad.
3. test strips according to claim 1, is characterized in that: described detection zone is near absorption of sample pad one end, and quality control region is near adsorptive pads one end.
4. test strips according to claim 1, is characterized in that: described carrier protein can be bovine serum albumin(BSA), ovalbumin, hemocyanin, thyroprotein, human serum albumins, albumin rabbit serum.
5. test strips according to claim 1, is characterized in that: described antiantibody is sheep anti mouse antiantibody.
6. test strips according to claim 1, is characterized in that: described iprodione specific antibody can be iprodione monoclonal antibody.
7. test strips according to claim 1, it is characterized in that: described iprodione haptens is by 2, the chloro-4-nitrophenol of 6-bis-is initiation material, through obtaining hydroxyl 2 with the hydrocarbyl reaction of the 4-bromo-butyric acid tert-butyl ester, the chloro-4-nitrobenzene of 6-bis-, after reduction nitro, obtain alkyl 2, 6-dichloroaniline, alkyl 2, 6-dichloroaniline is under the effect of phosgene, with aminoacetic acid condensation, obtain alkyl 2, 6-dichloro-benzenes glycinate, again through annulation, obtain iprodione agent structure compound, this compound is again through phosgene effect, with isopropylamine condensation, obtain iprodione mother nucleus structure compound, hydrolysis reaction obtains in acid condition, molecular structure as shown in the formula:
。
8. prepare a method for the test strips described in any one of claim 1-7, it is characterized in that: comprise the following steps:
(1) preparation is coated with the bond release pad of iprodione specific antibody-colloid gold label thing;
(2) preparation has the detection zone of iprodione hapten-carrier protein conjugate and bag by the reaction film of the quality control region of antiantibody;
(3) reaction film (2) prepared and bond discharge pad, absorption of sample pad, adsorptive pads, base plate be assembled into test strips.
9. application rights requires the detection method that in the ELISA test strip tobacco sample described in 1-7, iprodione is residual, and it comprises step:
(1) pre-treatment is carried out to sample;
(2) detect by test strips;
(3) testing result is analyzed.
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| CN109061156A (en) * | 2018-09-21 | 2018-12-21 | 中国烟草总公司郑州烟草研究院 | A kind of time-resolved fluoroimmunoassay chromatograph test strip and its preparation method and application detecting pendimethalin |
| CN109265401A (en) * | 2018-09-21 | 2019-01-25 | 中国烟草总公司郑州烟草研究院 | A kind of preparation method and application of iprodione haptens and antigen |
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