CN105388281A - Enzyme linked immunosorbent assay kit for detecting iprodione and application thereof - Google Patents
Enzyme linked immunosorbent assay kit for detecting iprodione and application thereof Download PDFInfo
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Abstract
The invention provides an enzyme linked immunosorbent assay kit for detecting iprodione and application thereof. The enzyme linked immunosorbent assay kit comprises an elisa plate coated with a coating antigen, an iprodione standard substance solution, an enzyme-labeled secondary antibody, an iprodione specific antibody, a substrate color developing solution, a stop solution, a scrubbing solution and a redissolved solution. The coating antigen is an iprodione coupling antigen, and the enzyme-labeled secondary antibody is an enzyme-labeled goat-anti-mouse antibody. The invention further discloses a method using the enzyme linked immunosorbent assay kit for detecting iprodione. The enzyme linked immunosorbent assay kit can be used for detecting the content of iprodione in tobacco leaf samples, and is easy to operate, low in cost, high in sensitivity, capable of achieving on-site monitoring and suitable for screening a great number of samples.
Description
Technical field
The present invention relates to enzyme linked immunosorbent detection technology, being specifically related to a kind of enzyme linked immunological kit for detecting iprodione and application thereof, can the residual quantity of iprodione in qualitative and quantitative analysis tobacco leaf.
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.
Summary of the invention
Object of the present invention provides a kind of enzyme linked immunological kit that can detect iprodione drug residue in tobacco based on above-mentioned prior art situation just, and provide a kind of efficient, accurate, easy, be suitable for batch samples screening qualitative and quantitative analysis method, apply the residual quantity that euzymelinked immunosorbent assay (ELISA) of the present invention measures iprodione in tobacco, there is the advantages such as specificity is good, simple to operate, detection is quick, testing cost is low.
The object of the invention is to be achieved through the following technical solutions: kit of the present invention comprises: the ELISA Plate being coated with coating antigen, iprodione standard solution, ELIAS secondary antibody, iprodione specific antibody, substrate nitrite ion, stop buffer, cleansing solution, redissolution liquid, described coating antigen is iprodione coupled antigen, described coating antigen is prepared by iprodione haptens and carrier protein couplet, described ELIAS secondary antibody is the sheep anti mouse antiantibody of enzyme labeling, 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.
Described carrier protein can be thyroprotein, bovine serum albumin(BSA), rabbit serum proteins, human albumin, ovalbumin, hemocyanin.
The haptenic molecular structure of described iprodione is such as formula I:
(formula I).
Described iprodione specific antibody prepares using iprodione coupled antigen as immunogene, and described iprodione specific antibody is iprodione monoclonal antibody.
The marker enzyme of described enzyme labeling is that horseradish peroxidase or bacterium extract alkaline phosphatase, when marker enzyme is horseradish peroxidase, substrate nitrite ion A liquid is hydrogen peroxide or urea peroxide, substrate nitrite ion B liquid is o-phenylenediamine or tetramethyl benzidine, and stop buffer is sulfuric acid or the hydrochloride buffer of 2mol/L; When marker enzyme is bacterium extraction alkaline phosphatase, substrate nitrite ion is to nitro phosphate buffer, and stop buffer is 2mol/L sulfuric acid.
Described cleansing solution pH value is 7.4, the phosphate buffer containing 0.5% ~ 1.0% Tween-20,0.01 ‰ ~ 0.03 ‰ sodium azide preservatives, 0.1 ~ 0.3mol/L; Number percent is wherein percent weight in volume, unit g/mL.
To be pH value be described redissolution liquid 7.0, the phosphate buffer of 0.02mol/L.
The concentration of described iprodione standard solution is respectively 0 μ g/L, 0.5 μ g/L, 1.5 μ g/L, 4.5 μ g/L, 13.5 μ g/L, 40.5 μ g/L.
It is pH value is 9.6 that wherein used in ELISA Plate preparation process bag is buffered liquid, the carbonate buffer solution of 0.05mol/L, confining liquid is pH value is 7.1 ~ 7.5, phosphate buffer containing 1% ~ 3% (g/mL) casein, 0.1 ~ 0.3mol/L, described number percent is percent weight in volume.
In the present invention, the preparation process of ELISA Plate is: be buffered liquid with bag and coating antigen is diluted to 20 μ g/mL, every hole adds 100 μ L, 25 DEG C of lucifuges are hatched 2h or 4 DEG C and are spent the night, and liquid in hole of inclining, washs 2 times with cleansing solution, each 30s, pat dry, in every hole, then add 150 ~ 200 μ L confining liquids, 25 DEG C of lucifuges hatch 1 ~ 2h, the liquid in hole that inclines pats dry, and preserves after dry with the vacuum seal of aluminium film.
Present invention also offers a kind of method applying above-mentioned enzyme linked immunological kit detection iprodione, it comprises step:
(1) sample pre-treatments;
(2) detect with kit;
(3) testing result is analyzed.
The enzyme linked immunological kit Cleaning Principle that the present invention detects iprodione is: adopt indirect competitive ELISA method, pre-coated coupled antigen on ELISA Plate capillary strip, coupled antigen pre-coated on iprodione residual in sample and capillary strip competes the antibody of anti-iprodione, after adding ELIAS secondary antibody, develop the color with tmb substrate, sample light absorption value becomes negative correlation with the content of iprodione contained by it, its corresponding extension rate is multiplied by more again with typical curve, can be qualitative or quantitatively detect the residual quantity of iprodione in sample.Kit of the present invention passes through 2, the chloro-4-nitrophenol of 6-bis-carries out structure of modification, synthesize the carboxyl haptens with four carbon chain lengths, highlighted the molecular structure of iprodione itself, enhance immunogenicity, make the iprodione monoclonal antibody for preparing highly sensitive, high specificity, kit is simple to the pre-treating method of sample, without the need to using organic solvent, sample handling processes environmental protection, can detect batch samples fast; Main agents provides with the form of working fluid, the method of inspection is convenient and easy, be easy to carry, kit has qualitative, the quantitative examination that the features such as specificity is high, highly sensitive, degree of accuracy is high, accuracy is high, simple to operate, detection time is short, testing cost is low are suitable for tobacco sample in enormous quantities.
Accompanying drawing explanation
Fig. 1 is iprodione hapten synthesis route map;
Fig. 2 is kit standard curve map.
Embodiment
The present invention is set forth further below in conjunction with specific embodiment.Should be understood that these embodiments are only for illustration of the present invention, and be not used for limiting the scope of the invention.
the preparation of embodiment 1 reagent constituents
1, the haptenic preparation of iprodione
The synthesis of compound 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 5.23g, 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 compound 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 compound 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 compound b4.8g, 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 compound 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 c3.57g, 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 d2.97g, 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 Verbindung: 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 Verbindung 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: 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, the preparation of iprodione coupled antigen
Prepared by immunogene---and iprodione haptens and bovine serum albumin(BSA) (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.
Prepared by coating antigen---and iprodione haptens and ovalbumin (OVA) 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.
3, the preparation of iprodione monoclonal antibody
Animal immune: immunogene above-mentioned steps obtained is injected in Balb/c Mice Body, immunizing dose is 150 μ g/, makes it produce antiserum.
Fusion of Cells and cloning: after mice serum measurement result is higher, get its splenocyte, in 8:1(quantitative proportion) ratio and SP2/0 myeloma cell fusion, adopt indirect competitive ELISA 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 secreting iprodione monoclonal antibody.
Cell cryopreservation and recovery: monoclonal hybridoma strain 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.
The production of monoclonal antibody and purifying: only Balb/c mouse peritoneal is injected sterilizing paraffin oil 0.5mL/, within 7 days, pneumoretroperitoneum injects stable monoclonal hybridoma strain 5 × 10
5individual/only, gather ascites after 7 days.Carry out ascites by sad-saturated ammonium sulfate method to purify ,-20 DEG C of preservations.
4, the preparation of enzyme mark disome
The Over-voltage protection after improveing is adopted to carry out coupling sheep anti mouse antiantibody and horseradish peroxidase (HRP).
5, the preparation of ELISA Plate
Be buffered liquid with bag and coating antigen is diluted to 20 μ g/mL, every hole adds 100 μ L, 25 DEG C of lucifuges hatch 2h, and liquid in hole of inclining washs 2 times with cleansing solution, each 30s, pat dry, in every hole, then add 200 μ L confining liquids, 25 DEG C of lucifuges hatch 2h, the liquid in hole that inclines pats dry, and preserves after dry with the vacuum seal of aluminium film.
embodiment 2 detects the establishment of the enzyme linked immunological kit of iprodione
Set up the enzyme linked immunological kit detecting iprodione, make it comprise following component:
(1) bag is by the ELISA Plate of iprodione coupled antigen;
(2) iprodione standard solution 6 bottles, concentration is respectively 0 μ g/L, 4 μ g/L, 12 μ g/L, 36 μ g/L, 108 μ g/L, 324 μ g/L;
(3) with the sheep anti mouse antiantibody of horseradish peroxidase-labeled;
(4) iprodione specific antibody;
(5) substrate nitrite ion is made up of A liquid and B liquid, and A liquid is urea peroxide, and B liquid is tetramethyl benzidine;
(6) stop buffer is 2mol/L sulfuric acid;
(7) cleansing solution is pH value is 7.4, the phosphate buffer containing 0.5% ~ 1.0% Tween-20,0.01 ‰ ~ 0.03 ‰ sodium azide preservatives, 0.1 ~ 0.3mol/L, and wherein number percent is percent weight in volume;
(8) redissolve liquid to be pH value be 7.0, the phosphate buffer of 0.02mol/L.
the detection of iprodione in embodiment 3 tobacco leaf
1, sample pre-treatments
Take 1.0 ± 0.05g tobacco leaf sample in 50mL polystyrene centrifuge tube, add 10mL tobacco leaf extract (measure 100ml methyl alcohol, add 100mL deionized water, fully mix); With refiner, it is fully smashed; The sample filter membrane smashed is filtered; Pipette filtrate 200mL and add 800mL redissolution liquid, fully mix; Get 50mL for analyzing.
2, detect with kit
Add standard items/sample 50 μ L in the micropore of correspondence, then add antibody working fluid 50 μ L/ hole, mixing of vibrating gently, react 30min with in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate.Carefully open cover plate film, dried by liquid in hole, with wash operating solution 250 μ L/ hole, fully washing 4-5 time, every minor tick 10s, pats dry with thieving paper.Add ELIAS secondary antibody 100 μ L/ hole, mixing of vibrating gently, react 30min with in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate.Carefully open cover plate film, dried by liquid in hole, with wash operating solution 250 μ L/ hole, fully washing 4-5 time, every minor tick 10s, pats dry with thieving paper.Add substrate solution A liquid 50 μ L/ hole, then add substrate solution B liquid 50 μ L/ hole, mixing of vibrating gently, react 15min with in cover plate membrane cover plate rearmounted 25 DEG C (77 ℉) light protected environment.Add stop buffer 50 μ L/ hole, mixing of vibrating gently, setting microplate reader is in 450nm place, and reference wavelength 620nm, measures every hole OD value.
3, Analysis of test results
The percentage absorptance of standard items or sample equals the mean value of mean value (diplopore) divided by the absorbance of first standard items (0 standard) of the absorbance of standard items or sample, then is multiplied by 100%, obtains the percentage absorbance of standard items or sample.With standard items percentage absorptance for ordinate, with the logarithm of iprodione standard concentration (μ g/L) for horizontal ordinate, drawing standard curve map.The percentage absorptance of sample substituted in typical curve, read the concentration corresponding to sample from typical curve, the extension rate being multiplied by its correspondence is the actual concentrations of iprodione in sample.
the determination test of embodiment 4 iprodione technical parameter
1, kit sensitivity and detectability
Conventionally measure kit sensitivity, the scope of typical curve is 4 ~ 324 μ g/L, IC
50(50% inhibition concentration) domain of walker is 10 ~ 30 μ g/L; Detect 20 increment product, find the concentration corresponding to each percentage absorbance from typical curve, add that 3 times of standard deviations represent detectability with the mean value of 20 parts of concentration of specimens, result shows, and the detectability of the method to tobacco leaf is 200 μ g/kg.
2, sample preci-sion and accuracy test
Using the recovery as accuracy estimating index, the testing result relative standard deviation (RSD%) of a certain concentration samples of replication is as precision evaluation index.Computing formula is: the recovery (%)=practical measurement value/theoretical value × 100%, and wherein theoretical value is the interpolation concentration of sample; Relative standard deviation RSD%=SD/X × 100%, wherein SD is standard deviation, and X is the mean value of determination data.
By 200 μ g/kg, 400 μ g/kg, two concentration iprodione respectively to tobacco sample carry out interpolation reclaim measure, each sample do 4 parallel, measure with three batches of different reagent, average recovery rate and the precision of calculation sample the results are shown in following table.
Table 1 tobacco leaf sample precision and accuracy test
Add tobacco leaf respectively with the iprodione of 200 μ g/kg, 400 μ g/kg, two concentration, average recovery rate is between 87.8% ~ 97.6%; Batch in, batch between relative standard deviation be all less than 10%.
3, stabilization of kit test
Kit preservation condition is 2 ~ 8 DEG C, and through the mensuration of 12 months, the maximum absorbance value (zero standard) of kit, 50% inhibition concentration, iprodione added practical measurement value all within normal range.Consider in transport and use procedure, have improper preservation condition and occur, placed 7 days under 37 DEG C of preservation conditions by kit, carry out accelerated aging tests, result shows that this kit indices meets the requirements completely.Consider that the freezing situation of kit occurs, kit is put into-20 DEG C of refrigerator freezings 7 days, measurement result also shows that kit indices is completely normal.Can show that kit at least can be preserved more than 12 months at 2 ~ 8 DEG C from above result.
Claims (8)
1. one kind is detected the enzyme linked immunological kit of iprodione, it is characterized in that: comprise the ELISA Plate being coated with coating antigen, iprodione standard solution, ELIAS secondary antibody, iprodione specific antibody, substrate nitrite ion, stop buffer, cleansing solution, redissolution liquid, described coating antigen is iprodione coupled antigen, described coating antigen is prepared by iprodione haptens and carrier protein couplet, described ELIAS secondary antibody is the sheep anti mouse antiantibody of enzyme labeling, 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.
2. kit as claimed in claim 1, it is characterized in that: described iprodione coupled antigen is obtained by iprodione haptens and carrier protein couplet, and described carrier protein is thyroprotein, bovine serum albumin(BSA), rabbit serum proteins, human albumin, ovalbumin, hemocyanin.
3. kit as claimed in claim 1 or 2, it is characterized in that, the haptenic molecular structure of described iprodione is such as formula I:
(formula I).
4. kit as claimed in claim 1, is characterized in that: described iprodione specific antibody prepares using iprodione coupled antigen as immunogene, and described iprodione specific antibody is iprodione monoclonal antibody.
5. kit as claimed in claim 1, it is characterized in that: the marker enzyme of described ELIAS secondary antibody is that horseradish peroxidase or bacterium extract alkaline phosphatase, when marker enzyme is horseradish peroxidase, substrate nitrite ion A liquid is hydrogen peroxide or urea peroxide, substrate nitrite ion B liquid is o-phenylenediamine or tetramethyl benzidine, and stop buffer is sulfuric acid or the hydrochloride buffer of 2mol/L; When marker enzyme is bacterium extraction alkaline phosphatase, substrate nitrite ion is to nitro phosphate buffer, and stop buffer is 2mol/L sulfuric acid.
6. kit as claimed in claim 1, is characterized in that: described cleansing solution is pH value is 7.4, the phosphate buffer containing 0.5% ~ 1.0% Tween-20,0.01 ‰ ~ 0.03 ‰ sodium azide preservatives, 0.1 ~ 0.3mol/L; To be pH value be described redissolution liquid 7.0, the phosphate buffer of 0.02mol/L, and the number percent in cleansing solution is percent weight in volume, unit g/mL.
7. kit as claimed in claim 1, is characterized in that: the concentration of described iprodione standard solution is respectively 0 μ g/L, 0.5 μ g/L, 1.5 μ g/L, 4.5 μ g/L, 13.5 μ g/L, 40.5 μ g/L.
8. utilize kit described in claim 1 to detect a method for iprodione content in tobacco sample, it is characterized in that: comprise step:
(1) sample pre-treatments;
(2) detect with kit;
(3) testing result is analyzed.
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| CN201510973027.3A CN105388281B (en) | 2015-12-23 | 2015-12-23 | Detect enzyme linked immunological kit and its application of iprodione |
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| CN107022024A (en) * | 2017-04-25 | 2017-08-08 | 江南大学 | A kind of synthetic method of iprodione artificial antigen |
| CN107119022A (en) * | 2017-04-26 | 2017-09-01 | 江南大学 | One plant of iprodione monoclonal antibody hybridoma cell strain ZXL 2 and its application |
| CN109061153A (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 iprodione |
| 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 |
| CN109813922A (en) * | 2019-01-16 | 2019-05-28 | 北京勤邦生物技术有限公司 | Detect enzyme linked immunological kit and its application of Clorprenaline |
| CN110172092A (en) * | 2019-05-22 | 2019-08-27 | 江南大学 | A kind of synthetic method of eugenol artificial antigen |
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| CN107022024A (en) * | 2017-04-25 | 2017-08-08 | 江南大学 | A kind of synthetic method of iprodione artificial antigen |
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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 |
| CN109061153A (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 iprodione |
| 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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| CN109813922A (en) * | 2019-01-16 | 2019-05-28 | 北京勤邦生物技术有限公司 | Detect enzyme linked immunological kit and its application of Clorprenaline |
| CN109813922B (en) * | 2019-01-16 | 2023-01-20 | 北京勤邦生物技术有限公司 | Enzyme linked immunosorbent assay kit for detecting chlorpromazine and application thereof |
| CN110172092A (en) * | 2019-05-22 | 2019-08-27 | 江南大学 | A kind of synthetic method of eugenol artificial antigen |
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