Detect enzyme linked immunological kit and its application of carbendazimTechnical field
The present invention relates to enzyme linked immunosorbent detection technology, and in particular to a kind of enzyme linked immunological kit for detecting carbendazimAnd its application, can in qualitative and quantitative analysis tobacco leaf carbendazim medicine residual quantity.
Background technology
China is the big country for producing and using chemical pesticide, and long-term use agricultural chemicals is to ecological environment and the danger of healthEvil and influence have caused the highest attention of people.Carbendazim [carbendazim, N- (2- benzopyrazoles base) carbamic acid firstEster] it is benzopyrazoles class, it is a kind of good wide spectrum, systemic fungicide, in sac fungus, basidiomycetes and Fungi ImperfectiMost of pathogens are all effective, are widely used in the disease control work of crops and Chinese herbal medicine.Carbendazim chemical propertyStabilization, can be absorbed by the seed of plant, root and leaf, and the longevity of residure is more long, there is certain toxicity to people, animal, can cause tic, spiritDimly, the poisoning symptom such as n and V, uncomfortable in chest, dizzy.Therefore, the analysis about Determination of carbendazim residue is increasingly subject to weightDepending on.
Because carbendazim has a wide range of applications in terms of the preventing and treating of corps diseases, but it has certain poison to human body againEvil property, current world many countries have all formulated the highest limitation mark of carbendazim residual quantity in (class) agricultural byproducts not of the same raceIt is accurate.Determination of carbendazim residue per kilogram must not exceed 0.5 mg in the vegetables such as Canadian national Specification cucumber, cucurbita pepo；MalaysiaDetermination of carbendazim residue per kilogram must not exceed 1 mg in the regulation greengrocery of West Asia；Specify vegetable in China sanitary standard GB14870294Determination of carbendazim residue per kilogram must not exceed 0.5 mg in dish, fruit.But simple, efficiently detection method is there is no at present.
The content of the invention
The purpose of the present invention is just being directed to above-mentioned prior art situation and providing one kind can detect carbendazim medicine in tobacco leafThe enzyme linked immunological kit of thing residual quantity, and a kind of efficient, accurate, simplicity is provided, is suitable to the qualitative, fixed of batch samples screeningQuantity measuring method, the residual quantity of carbendazim medicine in tobacco leaf is determined using ELISA of the invention, with test limit is low, spyDifferent in nature strong, easy to operate, detection speed is fast, testing cost is low, is very easy to the advantages of promoting.
The purpose of the present invention is achieved through the following technical solutions：Kit of the invention includes：It is coated with coatingFormer ELISA Plate, carbendazim standard product solution, carbendazim antibody, ELIAS secondary antibody, substrate nitrite ion, terminate liquid, cleaning solution, redissolutionLiquid, the coating antigen is carbendazim coupled antigen, and the ELIAS secondary antibody is the carbendazim antiantibody of enzyme mark, the carbendazim idolAssociated antigen is obtained by carbendazim haptens and carrier protein couplet, and the carbendazim haptens is by carbendazim and trifluoroacetic acidAcid anhydride, ammonium nitrate reaction are obtained.
The carrier protein can be thyroprotein, bovine serum albumin(BSA), rabbit serum proteins, human albumin, egg white eggIn vain, hemocyanin.
The carbendazim hapten molecule structural formula is：
。
The carbendazim antibody is prepared as immunogene using carbendazim coupled antigen, and the carbendazim antibody is manyBacterium spirit monoclonal antibody or carbendazim polyclonal antibody, wherein it is preferred that carbendazim monoclonal antibody.
The marker enzyme of the ELIAS secondary antibody is that horseradish peroxidase or bacterium extract alkaline phosphatase, wherein it is preferred that pepperyRoot peroxidase；ELIAS secondary antibody is obtained by enzyme and the coupling of carbendazim antiantibody.
In order to be more convenient on-site supervision and great amount of samples examination, the kit also includes carbendazim standard product solution, bottomThing nitrite ion, terminate liquid, cleaning solution, redissolution liquid.
6 bottles of the carbendazim standard product solution, concentration is respectively 0 μ g/L, 0.1 μ g/L, 0.3 μ g/L, 0.9 μ g/L,2.7 μ g/L, 8.1 μ g/L.
When marker enzyme is horseradish peroxidase, the substrate nitrite ion is made up of substrate solution A liquid and substrate solution B liquid, AIt is hydrogen peroxide or urea peroxide, B liquid is o-phenylenediamine or tetramethyl benzidine, and the terminate liquid is the sulfuric acid of 1 ~ 2 mol/LSolution or hydrochloride buffer；When marker enzyme is that bacterium extracts alkaline phosphatase, the substrate nitrite ion is to nitro phosphoric acidSalt buffer, the terminate liquid is 1 ~ 2 mol/L sodium hydroxide solutions.
It is 7.4 that the cleaning solution is preferably pH value, contains 0.5% ~ 1.0% Tween-20,0.01 ‰ ~ 0.03 ‰ sodium azideThe phosphate buffer of preservative, 0.1 ~ 0.3 mol/L, percentage therein is percent weight in volume, unit g/mL.
The redissolution liquid is preferably the phosphate buffer that pH value is 7.0,0.02 mol/L.
Wherein used in ELISA Plate preparation process coating buffer solution is that pH value is the carbonic acid of 9.6,0.05 mol/LSalt buffer, confining liquid is that pH value is 7.1 ~ 7.5, and the phosphate containing 1% ~ 3% (g/mL) casein, 0.1 ~ 0.3 mol/L delaysFliud flushing.
The preparation process of ELISA Plate is in the present invention：Coating antigen is diluted to 20 μ g/mL with coating buffer solution, is added per holeEnter 100 μ L, 25 DEG C of lucifuges are incubated 2 h or 4 DEG C overnight, and liquid in hole of inclining is washed 2 times with cleaning solution, and 30 s, pats dry every time,Then 150 ~ 200 μ L confining liquids are added in every hole, 25 DEG C of lucifuges are incubated 1 ~ 2 h, and liquid is patted dry in hole of inclining, and is used after dryingAluminium film vacuum sealing is preserved.
Cleaning Principle of the invention is：
This kit uses direct competive ELISA method, and pre-coated coupled antigen, residual in sample on ELISA Plate capillary stripCoupled antigen pre-coated on the carbendazim and ELISA Plate capillary strip for staying competes the ELIAS secondary antibody of resisting carbendazim, aobvious with tmb substrateThe content of color, sample absorbance and its contained residue carbendazim compares into negative correlation with standard curve, multiplied by with its correspondenceExtension rate, you can draw the residual quantity of carbendazim in sample.
Present invention also offers a kind of method for applying above-mentioned enzyme linked immunological kit to detect carbendazim, it includes step：
（1）Sample pre-treatments；
（2）Detected with kit；
（3）Analysis testing result.
The enzyme linked immunological kit of present invention detection carbendazim is mainly qualitatively or quantitatively detected in sample using ELISA methodThe content of carbendazim；Pre-treatment requirement to sample is low, and sample pretreatment process is simple, can be while quick detection high-volume sampleProduct；Main agents are provided in the form of working solution, and the method for inspection is convenient and easy, with test limit is low, specific high, operation letterJust the features such as, sensitivity is high, accuracy is high, the degree of accuracy is high.Enzyme linked immunological kit of the invention, simple structure, it is easy to use,Cheap, carrying convenience, detection method are efficient, accurate, easy, be suitable to the qualitative, quantitative of batch samples screening.
Brief description of the drawings
Fig. 1：Carbendazim hapten synthesis route map,
Fig. 2：Kit standard curve map.
Specific embodiment
The present invention is expanded on further with reference to specific embodiment.It should be understood that these embodiments are merely to illustrate thisInvention, and it is not limited to the scope of the present invention.
The preparation of the reagent constituents of embodiment 1
1st, the preparation of carbendazim haptens
Carbendazim bulk drug introduces nitro through nitration reaction on phenyl ring, with aromatic amine half is obtained after carrying out reduction anti-Originate in thing.
The mL of trifluoroacetic acid 20 adds the mL of TFAA 2, ice-water bath to be down to 0 DEG C, plus the g of ammonium nitrate 0.5, stirs 1 h, plusEnter the trifluoroacetic acid solution containing the g of carbendazim 1.0, continue to stir, react 2 h.Stop reaction, diluted sodium hydroxide solution is neutralizedTo neutrality, dichloromethane extraction, washing is evaporated, ether wash crystallization, obtains the g of compound a 0.76, yield 67%.1H NMR（CDCl3,300MHZ）δ: 8.31 (1H, dd, J=1.616, J=1.239), 7.69 ( 1H, dd, J=8.716, J=1.616), 7.64 (1H, dd, J=8.716, J=1.239), 3.85 (3H, s).
The g of compound a 0.7 adds ethanol to dissolve, plus the mL of the 0.43 g stannic chlorides aqueous solution 10, is passed through nitrogen, adds backflow anti-Answer 3 h.Stop reaction, revolving removes ethanol, plus ethyl acetate extraction, silicagel column in concentration, petrol ether/ethyl acetate（1：1,v/v）Wash-out is separated, and obtains the g of hapten compound b products 0.54, yield, 83%.1H NMR（CDCl3,300MHZ）δ：3.79(3H, s), 6.27 (2H, s), 6.90 (1H, dd, J=2.225, J=1.850), 6.46 (1H, dd, J=8.422, J=2.225),7.34 ( 1H, dd, J=8.422, J=1.850), 5.00(1H,s), 9.15(1H,s)。
Chemical shift δ=6.27 is the resonance absorbing peak of aromatic amine on phenyl ring in collection of illustrative plates, and the presence of the absworption peak is proved,Hapten synthesis success.
2nd, the preparation of antigen
It is prepared by immunogene --- carbendazim haptens and bovine serum albumin(BSA)（BSA）Coupling obtains immunogene.
The mg of BSA 50 are weighed, is allowed to be substantially dissolved in the phosphate buffer PBS of 3.8 mL 0.1M（PH 7.2）In, obtainTo solution A；Take 30 mg carbodiimides（EDC）And N-hydroxy-succinamide（NHS）With 0.2 mL water fully dissolve after plusEnter in solution A, 30 min are stirred at room temperature.15 mg haptens are taken, 1 mL DMFs are dissolved in（DMF）In,It is then slowly added in protein dissolution, 24 h of reaction is stirred at room temperature.3 are changed with 0.01 mol/L PBS, 4 DEG C of 3 d of dialysis dailySecondary dialyzate.Packing, saves backup in -20 DEG C.
Carbendazim 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 waterIn solution A is added, 30 min are stirred at room temperature.5 mg haptens are 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 dialysisChange 3 dialyzates daily.Packing, saves backup in -20 DEG C.
Carbendazim 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（PH7.2）In, obtain solution A；Take 30 mg carbodiimides（EDC）And N-hydroxy-succinamide（NHS）It is abundant with 0.2 mL waterDissolving stirs 30 min at room temperature in solution A is added.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.With 0.01 mol/L PBS, 4 DEG C of 3 d of dialysisChange 3 dialyzates daily.Packing, saves backup in -20 DEG C.
It is prepared by coating antigen --- carbendazim haptens and ovalbumin（OVA）Coupling obtains coating antigen.
The mg of OVA 50 are weighed, is allowed to be substantially dissolved in 3.8 mL 0.1M PBS（PH 7.2）In, obtain solution B；Take0.2 mL water of 30mg EDC and NHS is fully dissolved in solution B is added, and 30 min are stirred at room temperature.Take 13 mg half anti-Original, is dissolved in 1mL DMF, is then slowly added in protein dissolution, and 24 h of reaction are stirred at room temperature.With 0.01 mol/L PBS4 DEG C of 3 d of dialysis change 3 dialyzates daily.Packing, saves backup in -20 DEG C.
Carbendazim 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 moltenLiquid 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 15mgHaptens, 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/L4 DEG C of 3 d of dialysis of PBS change 3 dialyzates daily.Packing, saves backup in -20 DEG C.
Carbendazim 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 moltenLiquid 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 15mgHaptens, 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/L4 DEG C of 3 d of dialysis of PBS change 3 dialyzates daily.Packing, saves backup in -20 DEG C.
3rd, the preparation of carbendazim monoclonal antibody
Animal immune：The immunogene that above-mentioned steps are obtained is injected into Balb/c Mice Bodies, immunizing dose is 150 μ g/Only, it is made to produce antiserum.
Cell fusion and cloning：After mice serum measurement result is higher, its splenocyte is taken, by 8:1（Quantitative proportion）ThanExample is merged with SP2/0 myeloma cell, and cell supernatant, the positive hole of screening are determined using indirect competitive ELISA.Using limited diluteInterpretation of the law carries out cloning to positive hole, the hybridoma cell strain until obtaining secreting carbendazim monoclonal antibody.
Cell cryopreservation and recovery：Monoclonal hybridoma strain is made 1 × 10 with frozen stock solution6The cell suspension of individual/mL,Preserved for a long time in liquid nitrogen.Cryopreservation tube is taken out during recovery, 37 DEG C of water-bath middling speeds is immediately placed in and is melted, after centrifugation removal frozen stock solution, movedEnter to cultivate culture in glassware.
The production of monoclonal antibody and purifying：By Balb/c mouse peritoneals injection sterilizing paraffin oil 0.5 mL/ only, after 7 daysThe monoclonal hybridoma strain 5 × 10 of intraperitoneal injection stabilization5Individual/only, gather ascites after 7 days.With octanoic acid-saturated ammonium sulfate methodCarry out ascites purifying, -20 DEG C of preservations.
4th, the preparation of ELIAS secondary antibody
Using goat as immune animal, pathogen-free domestic goat is immunized by immunogene of carbendazim monoclonal antibody,Obtain carbendazim antiantibody.By carbendazim antiantibody and horseradish peroxidase（HRP）Be coupled obtaining ELIAS secondary antibody.
5th, the preparation of ELISA Plate
Coating antigen is diluted to 20 μ g/mL with coating buffer solution, 100 μ L are added per hole, 25 DEG C of lucifuges are incubated 2 h, inclineLiquid in hole is removed, is washed with cleaning solution 2 times, 30 s, pats dry every time, 200 μ L confining liquids are then added in every hole, 25 DEG C are kept awayLight is incubated 2 h, and liquid is patted dry in hole of inclining, and is preserved with aluminium film vacuum sealing after drying.
The establishment of the enzyme linked immunological kit of the detection carbendazim of embodiment 2
The enzyme linked immunological kit of detection carbendazim is set up, it is included following components：
（1）It is coated with the ELISA Plate of carbendazim coupled antigen；
（2）6 bottles of carbendazim standard product solution, concentration is respectively 0 μ g/L, 0.1 μ g/L, 0.3 μ g/L, 0.9 μ g/L,2.7 μ g/L, 8.1 μ g/L；
（3）With the carbendazim antiantibody of horseradish peroxidase-labeled；
（4）Substrate nitrite ion is made up of A liquid and B liquid, and A liquid is urea peroxide, and B liquid is tetramethyl benzidine；
（5）Terminate liquid is 2 mol/L sulfuric acid；
（6）Cleaning solution is that pH value is 7.4, contains 0.5% ~ 1.0% Tween-20,0.01 ‰ ~ 0.03 ‰ sodium azide anti-corrosionsAgent, the phosphate buffer of 0.1 ~ 0.3 mol/L；
（7）Liquid is redissolved for phosphate buffer that pH value is 7.0,0.02 mol/L.
The detection of carbendazim in the tobacco leaf of embodiment 3
1st, sample pre-treatments
Weigh in 1.0 ± 0.05 g tobacco leaves samples to 50 ml polystyrene centrifuge tubes, add 10 mL tobacco leaf extract solutions, useRefiner fully smashes it；The sample that will be smashed is filtered with filter membrane；Pipette the mL of filtrate 50 and add 700 mL deionizationsWater, fully mixes；Pipetting the mL of above liquid 50 again adds 950 mL to redissolve working solution, fully mixes；50 mL are taken for analyzing.
2nd, detected with kit
Plus the mL of standard liquid/sample 50 adds the mL/ holes of antibody working solution 50 in corresponding micropore, gently vibratesMix, 30 min are reacted with the rearmounted 25 DEG C of light protected environments of cover plate membrane cover plate.Cover plate film carefully is opened, liquid in hole is dried,With the mL/ holes of wash operating solution 250, fully wash 4-5 times, per the s of minor tick 10, patted dry with blotting paper（It is not eliminated after patting dryBubble can gently be poked with original pipette tips）.The mL/ holes of ELIAS secondary antibody 100 are added, gently vibration is mixed, and uses cover plate film30 min are reacted in the rearmounted 25 DEG C of light protected environments of cover plate, is taken out and is repeated board-washing step.The μ L/ holes of substrate solution A liquid 50 are added, then is addedThe μ L/ holes of substrate solution B liquid 50, gently vibration is mixed, with 15 min that developed the color in the rearmounted 25 DEG C of light protected environments of cover plate membrane cover plate.AddThe μ L/ holes of terminate liquid 50, gently vibration is mixed, setting ELIASA Detection wavelength 450 nm, the nm of reference wavelength 620, please in 5 minInside run through data）, determine per hole OD values.
3rd, Analysis of test results
The percentage absorptance of standard items or sample is equal to the average value of the absorbance of standard items or sample（Diplopore）Divided byFirst standard items（0 standard）Absorbance average value, multiplied by with 100%, obtain the percentage absorbance of standard items or sampleValue.With standard items percentage absorptance as ordinate, with carbendazim standard product concentration（µg/L）Logarithm be abscissa, draw standardCurve map.By in the percentage absorptance substitution standard curve of sample, the concentration corresponding to sample is read from standard curve, be multiplied byIts corresponding extension rate is the actual concentrations of carbendazim in sample.
The determination experiment of the carbendazim technical parameter of embodiment 4
1st, kit sensitivity and test limit
Kit sensitivity is conventionally determined, the scope of standard curve is 0.1 ~ 8.1 μ g/L, IC50（50% suppressesConcentration）Domain of walker is 0.45 ~ 0.65 μ g/L；20 parts of samples are detected, is found from standard curve corresponding to each percentageThe concentration of absorbance, test limit is represented with 20 parts of average values of concentration of specimens plus 3 times of standard deviations, is as a result shown, the methodTest limit to carbendazim in tobacco leaf is 300 μ g/kg.
2nd, sample preci-sion and accuracy experiment
Using the rate of recovery as accuracy estimating index, the testing result relative standard deviation of a certain concentration samples of replication（RSD%）As precision evaluation index.Computing formula is：The rate of recovery（%）=actual measured value/theoretical value × 100%, wherein managingIt is the addition concentration of sample by value；Relative standard deviation RSD%=SD/X × 100%, wherein SD are standard deviation, and X is measure numberAccording to average value.
Recovery is added to tobacco sample by 300 μ g/kg, 600 μ g/kg, two carbendazim of concentration respectively to determine, oftenIndividual sample do 4 it is parallel, be measured with three batches of different reagents, calculate sample average recovery rate and precision result see belowTable.
The tobacco leaf sample precision of table 1 and accuracy test
Tobacco leaf is added respectively with 300 μ g/kg, 600 μ g/kg, two carbendazim of concentration, average recovery rate existsBetween 80.3% ~ 86.7%；Batch in, batch between relative standard deviation be respectively less than 10%.
3rd, stabilization of kit experiment
Kit preservation condition is 2 ~ 8 DEG C, by the measure of 12 months, the maximum absorbance value of kit（Zero standard）、50% inhibition concentration, carbendazim add actual measured value within normal range (NR).Consider during transport and use, haveImproper preservation condition occurs, and kit is placed 7 days under 37 DEG C of preservation conditions, carries out accelerated aging tests, as a result showsThe kit indices comply fully with requirement.Occur in view of kit freezing situation, kit is put into -20 DEG C of refrigerators coldFreeze 7 days, measurement result also indicates that kit indices are completely normal.Can show that kit can be at 2 ~ 8 DEG C from result aboveAt least preserve more than 12 months.