Detect enzyme linked immunological kit and its application of chlopyrifosTechnical field
The present invention relates to enzyme linked immunosorbent detection technology, and in particular to a kind of ELISA reagent for being used to detect chlopyrifosBox, its detection particularly suitable for veterinary antibiotics and tealeaves Chlorpyrifos content.
Background technology
Chlopyrifos (Chlorpyrifos), chemical name O, O- diethyl-O- (3,5,6- trichloro-2-pyridyl) thio phosphorusAcid esters, trade name chlopyrifos, Le Siben, termite are clear etc., be the highly effective pesticides such as acephatemet and parathion-methyl it is new and effective,Less toxic substitute species, belong to broad spectrum type organophosphorus ester insecticides, available for grain, veterinary antibiotics and the evil of industrial cropsWorm prevents.Chlopyrifos is one of important goal thing of Monitoring Pesticide Residues in agricultural product and food, not only residual in cropsGetting worse is stayed, and can be shifted by approach such as metabolism into environment, potential threat is formed to human health, it can be influencedBrain development, acts thyroid gland system, causes the reduction of blood Triiodothyronine, and excess ingestion may cause spasmAnd dizziness, especially children's health is endangered larger.
In view of this, use of the developed country to chlopyrifos has carried out stringent regulation, and chlopyrifos is continuously improved in agricultureResidue limits in product.European Union is 0.05mg/kg to the highest limitation of spinach Chlorpyrifos；The country of chlopyrifos is marked by JapanAccurate most stringent, in the residual limit standard table of agriculture that its in April, 2002 announces, the chlopyrifos limitation of its spinach is 0.01mg/kg；StateBorder Codex Committee on Food (CAC) is 0.05mg/kg to spinach Chlorpyrifos Residue limitation.Maximum residue limit country of China markStandard is：Grain 0.1mg/kg；The operatic circle, leaf vegetables 1mg/kg；Cottonseed oil 0.05mg/kg.
Therefore, in order to prevent and tackle environment caused by chlopyrifos residue and food pollution problem, it is necessary to establish a kind ofSensitive, easy, quick chlopyrifos detection method.The residue detection of chlopyrifos mainly has high performance liquid chromatography at present(HPLC), gas chromatographymass spectrum (GC-MS).Expensive instrument and special technical staff, sample are needed using these analysis methodsPretreatment process is complex and expensive, time-consuming length, it is difficult to meets the needs that a large amount of samples and field sample quickly detect.It is enzyme-linked to exempt fromEpidemic disease adsorption analysis method (ELISA) have the characteristics that it is easy quick, special it is sensitive, sample capacity is big, analysis cost is low, can simplifySample purification step even is saved, unique advantage, Neng Gougeng are shown in great amount of samples and the quick selective mechanisms of field samplesMeet that the developments such as China's food enterprise, government function supervision department detect work, great development potentiality well.
The content of the invention
It is an object of the invention to provide a kind of simple in structure, easy to use, cheap, portable for poisoning with poisonThe enzyme linked immunological kit of tick detection, and provide a kind of efficient, accurate, simplicity, suitable for the qualitative, quantitative of high-volume screening sampleDetection method.
Kit of the present invention, it includes：It is coated with the ELISA Plate, Dursban monoclonal antibody, enzyme of chlopyrifos coupled antigenMark antiantibody, chlopyrifos standard solution, substrate nitrite ion, terminate liquid, cleaning solution, redissolution liquid；The chlopyrifos coupling is anti-Original is to be obtained by chlopyrifos haptens with carrier protein couplet, and the carrier protein is mouse haemocyanin, thyroprotein, ox bloodPure albumen, rabbit serum proteins, human albumin, ovalbumin, hemocyanin or fibrinogen, the chlopyrifos haptensIt is that to carry out nucleophilic displacement of fluorine with the chlorine of neighbouring nitrogen-atoms on pyridine ring with aminobutyric acid in alkaline conditions by chlopyrifos active compound anti-It should obtain, molecular structural formula is：
The Dursban monoclonal antibody is prepared using chlopyrifos coupled antigen as immunogene.
The antiantibody is sheep anti mouse antiantibody.
The marker enzyme of the enzyme mark antiantibody is horseradish peroxidase；The antiantibody of enzyme mark is to use glutaraldehyde methodOr marker enzyme and antiantibody are coupled by Over-voltage protection.
In order to be more convenient on-site supervision and great amount of samples examination, the kit further includes chlopyrifos standard solution, bottomThing nitrite ion, terminate liquid, cleaning solution, redissolution liquid.
6 bottles of the chlopyrifos standard solution, concentration are respectively 0 μ g/L, 0.6 μ g/L, 1.8 μ g/L, 5.4 μ g/L, 16.2 μG/L and 48.6 μ g/L.
The substrate nitrite ion is made of substrate solution A liquid and substrate solution B liquid, and A liquid is hydrogen peroxide or urea peroxide, B liquidFor o-phenylenediamine or tetramethyl benzidine, the terminate liquid is the sulfuric acid solution of 1~2mol/L.
The cleaning solution is preferably that pH value is 7.4, contains 0.5%~1.0% Tween-20,0.01 ‰~0.03 ‰ nitrineChange 0.1~0.3mol/L phosphate buffers of sodium.
The liquid that redissolves is preferably the 0.1mol/L phosphate buffers that pH value is 7.0.
Used coating buffer solution is the 0.05mol/L carbonate that pH value is 9.6 wherein in ELISA Plate preparation processBuffer solution, confining liquid are that pH value is 7.1~7.5,0.1~0.3mol/L phosphate buffers containing 1%~3% casein.
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 hole100 μ L, 37 DEG C of lucifuges are incubated 2h or 4 DEG C overnight, and liquid in hole of inclining, is washed 2 times, each 30s, pats dry, then with cleaning solution150~200 μ L confining liquids are added in every hole, 37 DEG C of lucifuges are incubated 1~2h, and liquid pats dry in hole of inclining, and aluminium film is used after dryVacuum sealing preserves.
The present invention testing principle be：
The pre-coated chlopyrifos coupled antigen on capillary strip, after adding sample solution or standard solution, adds and poisons with poisonTick monoclonal antibody solution, coated chlopyrifos coupled antigen competition Dursban monoclonal on the chlopyrifos and ELISA Plate in sampleAntibody, adds enzyme mark antiantibody and is amplified effect, developed the color with nitrite ion, the content of sample absorbance and chlopyrifos is in negativeCorrelation, the residual quantity of sample Chlorpyrifos is relatively can obtain with standard curve；At the same time according to the depth of color on ELISA Plate, withThe comparison of the standard solution color of series concentration can judgement sample Chlorpyrifos Residue amount roughly concentration range.
Present invention also offers a kind of method using above-mentioned enzyme linked immunological kit detection chlopyrifos, it includes step：
(1) Sample pretreatment；
(2) it is detected with kit；
(3) testing result is analyzed.
The enzyme linked immunological kit of present invention detection chlopyrifos is mainly qualitatively or quantitatively examined using indirect competitive ELISA methodThe content of this Chlorpyrifos of test sample；Pre-treatment requirement to sample is low, and Sample pretreatment process is simple, can quickly detect at the same time bigBatch sample；Main agents are provided in the form of working solution, and the method for inspection is convenient and easy, have specific height, high sensitivity, essenceThe features such as exactness is high, accuracy is high.The enzyme linked immunological kit of the present invention, simple in structure, easy to use, cheap, carryingConvenient, detection method efficiently, it is accurate, easy, suitable for the qualitative and quantitative analysis of high-volume screening sample.
Brief description of the drawings
Fig. 1：Chlopyrifos hapten synthesis route map
Fig. 2：Chlopyrifos haptens hydrogen nuclear magnetic resonance spectrogram
Fig. 3：Kit standard curve map
Embodiment
With reference to specific embodiment, the present invention is further explained.It is to be understood that these embodiments are merely to illustrate thisInvention, and be not used to limit the scope of the invention.
The preparation of 1 reagent constituents of embodiment
1st, the synthesis (synthetic route is shown in attached drawing 1) and identification of chlopyrifos haptens
1.0g chlopyrifos active compound 30mL ethanol dissolves, and is hydrogenated with sodium oxide molybdena 0.41g, adds aminobutyric acid 0.35g, back flow reaction3h, detection, raw material total overall reaction are completed.Ethanol is evaporated, adds water-ethyl acetate extraction, water phase, adjusting pH value to 3, ethyl acetateExtraction, organic phase are evaporated, and are recrystallized with dichloromethane and n-hexane, that is, are obtained chlopyrifos haptens product.
Above-mentioned haptens is taken to be identified through nuclear magnetic resonance spectroscopy, the result is shown in attached drawing 2.Chemical shift is at 11.0ppm in collection of illustrative platesCarboxyl signal peak, the methylene signals peak at 2.30ppm, 1.89ppm and 3.35ppm, it was demonstrated that chlopyrifos haptens structure is justReally.
2nd, the synthesis and identification of chlopyrifos coupled antigen
It is prepared by immunogene --- and chlopyrifos haptens obtains immunogene with bovine serum albumin(BSA) (BSA) coupling.
9mg haptens is taken, is dissolved in 1mL n,N-Dimethylformamide (DMF)；Take 30mg carbodiimides (EDC) andN-hydroxysuccinimide (NHS) is added in haptens solution after fully being dissolved with 0.2mL water, stirs 24h at room temperature, you canTo reaction solution A；BSA 50mg are weighed, is allowed to be substantially dissolved in 3.8mL CB (pH9.0), reaction solution A is slowly added to dropwiseInto protein solution, and 24h is stirred at room temperature；With 4 DEG C of dialysis 3d of 0.01mol/L PBS, 3 dialyzates are changed daily；PointDress, saves backup in -20 DEG C.
It is prepared by coating antigen --- and chlopyrifos haptens obtains immunogene with ovalbumin (OVA) coupling.
9mg haptens is taken, is dissolved in 1mL DMF；30mg EDC and NHS is taken to add half after fully being dissolved with 0.2mL waterIn antigenic solution, 24h is stirred at room temperature, you can obtains reaction solution A；OVA 50mg are weighed, are allowed to be substantially dissolved in 3.8mL CB(pH9.0) in, reaction solution A is slowly added into protein solution dropwise, and stir 24h at room temperature；With 0.01mol/L PBS4 DEG C of dialysis 3d, change 3 dialyzates daily；Packing, saves backup in -20 DEG C.
In the ratio of haptens, carrier protein and coupled product used in synthesis chlopyrifos coupled antigen reaction, carry out ultraviolet(200nm~400nm) sweep measuring, by compare three respectively the light absorption value of 260nm and 280nm calculate its combine than.It is evenJoin thing chlopyrifos hapten-carrier albumen maximum absorption band with chlopyrifos haptens, carrier protein maximum absorption band compared withThere occurs obvious change, the synthesis for showing chlopyrifos hapten-carrier albumen is successful.
3rd, the preparation of Dursban monoclonal antibody
(1) acquisition of hybridoma
1) first immunisation：Chlopyrifos haptens-BSA conjugate (immunogene) and the Freund's complete adjuvant of equivalent is fully newbornChange, the Balb/c mouse of 6 week old, every 0.2mL is subcutaneously injected；
2) booster immunization is twice：Since first immunisation, booster immunization once, is replaced with not formula Freund's incomplete adjuvant every two weeksFreund's complete adjuvant, method and the same first immunisation of dosage；
3) potency and suppression are surveyed in eyeground vein blood sampling to last time booster immunization after a week, have suppression and potency reaches 1:Following final immunization is carried out when more than 10000：Intraperitoneal injection is not added with the immunogen solution 0.1mL of any adjuvant, is put to death after three daysMouse, takes its spleen to be merged with myeloma cell；
4) using indirect competitive enzyme-linked immunosorbent analysis method measure cell supernatant, positive hole is screened.Utilize limiting dilutionMethod carries out cloning to positive hole, obtains and establishes the hybridoma cell strain of stably excreting Dursban monoclonal antibody, takes and be inCell suspension is made with frozen stock solution in the hybridoma of exponential phase, is sub-packed in cryopreservation tube, is preserved for a long time in liquid nitrogen.
(2) preparation of monoclonal antibody
1) cell recovery：Dursban monoclonal antibody hybridoma cell strain cryopreservation tube is taken out, is immediately placed in 37 DEG C of water-bathsSpeed is melted, and after centrifugation removes frozen stock solution, moves into culture culture in glassware；
2) ascites and antibody purification are prepared：Using method is induced in vivo, by Balb/c mouse (8 week old) Intraperitoneal injection sterilizing stoneOnly, pneumoretroperitoneum injects hybridoma 5 × 10 to wax oil 0.5mL/ within 7 days5A/only, gather ascites after 7 days.With octanoic acid-saturation sulfuric acidAmmonium method is purified, and obtains Dursban monoclonal antibody solution (- 20 DEG C of preservations).
(3) measure of antibody titer
Potency with indirect competitive ELISA method measure antibody is 1:(100000~150000).
Indirect competitive ELISA method：With chlopyrifos haptens-OVA conjugate coated elisa plates, chlopyrifos standard items are addedThe sheep anti mouse antiantibody solution of solution, Dursban monoclonal antibody solution and horseradish peroxidase-labeled, 25 DEG C of reactions30min, pours out liquid in hole, is washed 3~5 times with cleaning solution, is patted dry with blotting paper；Add substrate nitrite ion, 25 DEG C of reactionsAfter 15min, add terminate liquid and terminate reaction；Setting microplate reader is measured at wavelength 450nm per hole absorbance.
(4) measure of monoclonal antibody specificity
Antibody specificity refers to the ability of its homospecificity antigen binding and the ratio with such antigen-analogues abilityCompared with common cross reacting rate is as evaluation criterion.Cross reaction is smaller, and the specificity of antibody is then higher.
This experiment is by chlopyrifos, chlorpyrifos-methyl, basudin, parathion-methyl, fenifrothion, malathion, parathionIt is serially diluted, carries out indirect competitive ELISA with monoclonal antibody respectively, make standard curve, analysis obtains IC50, then pressFollowing formula calculates cross reacting rate：
The cross reacting rate of each analog of the results show is：Chlopyrifos 100%, chlorpyrifos-methyl 7.08%, basudin ＜1%th, parathion-methyl ＜ 1%, fenifrothion ＜ 1%, malathion ＜ 1%, parathion ＜ 1%.Antibody of the present invention is to methylOther pesticide no cross reactions such as chlopyrifos, basudin, parathion-methyl, fenifrothion, malathion, parathion, just forChlopyrifos has specific binding.
4th, the preparation of sheep anti mouse antiantibody
Using sheep as immune animal, using mouse source antibody as immunogen immune pathogen-free domestic sheep, sheep anti mouse antiantibody is obtained.
5th, the preparation of enzyme mark antiantibody
Sheep anti mouse antiantibody is coupled with horseradish peroxidase (HRP) using the Over-voltage protection after improvement.PassIt is 4 that the Over-voltage protection of system, which requires the molar concentration rate of enzyme and antibody in reaction system,:1, since horseradish peroxidase is strongMany sites with antibody binding are produced under oxidation, the horseradish peroxidase molecule so activated act as connecting each pointThe bridge of son, reduces the enzymatic activity of enzyme marker, makes to be mixed with many condensates in the conjugate of preparation.Asked to solve thisTopic, we are improved traditional method, i.e.,：
(1) closed process of amino is eliminated, because the amino that can produce the connection of itself amino is actually seldom；
(2) molar concentration ratio of horseradish peroxidase and antibody is reduced to 2:1, the method after improvement is than traditional sideMethod is easy, and the loss to enzymatic activity is reduced.
6th, the preparation of ELISA Plate
Coating antigen (chlopyrifos haptens-OVA conjugates) is diluted to 20 μ g/mL with coating buffer solution, 100 are added per holeμ L, 37 DEG C of lucifuges are incubated 2h, and liquid in hole of inclining, is washed 2 times, each 30s, pats dry, and is then added in every hole with cleaning solution200 μ L confining liquids, 37 DEG C of lucifuges are incubated 2h, and liquid pats dry in hole of inclining, and are preserved after dry with aluminium film vacuum sealing.
Embodiment 2 detects the establishment of the enzyme linked immunological kit of chlopyrifos
The enzyme linked immunological kit of detection chlopyrifos is set up, makes that it includes following components：
(1) it is coated with the ELISA Plate of chlopyrifos coupled antigen；
(2) 6 bottles of chlopyrifos standard solution, concentration are respectively 0 μ g/L, 0.6 μ g/L, 1.8 μ g/L, 5.4 μ g/L, 16.2 μG/L and 48.6 μ g/L；
(3) Dursban monoclonal antibody working solution；
(4) the sheep anti mouse antiantibody of horseradish peroxidase-labeled is used；
(5) substrate nitrite ion is made of A liquid and B liquid, and A liquid is urea peroxide, and B liquid is tetramethyl benzidine；
(6) terminate liquid is 2mol/L sulfuric acid；
(7) cleaning solution is that pH value is 7.4, contains 0.5%~1.0% Tween-20,0.01 ‰~0.03 ‰ sodium azide0.1~0.3mol/L phosphate buffers；
(8) it is the 0.1mol/L phosphate buffers that pH value is 7.0 to redissolve liquid.
The detection of 3 veterinary antibiotics of embodiment and tealeaves sample Chlorpyrifos
1st, Sample pretreatment
1.0g ± 0.05g samples are weighed into 10mL polystyrene centrifuge tubes, 5mL methanol are added, with vortex mixed instrument whirlpoolDynamic 2min, mixes；(20~25 DEG C) centrifugation 5min of 3000g room temperatures, take 50 μ L supernatant liquors into 2mL polystyrene centrifuge tubes,Add 950 μ L and redissolve liquid, whirling motion 20s；50 μ L are taken to be used to analyze.
2nd, detected with kit
Chlopyrifos standard solution is added into the ELISA Plate micropore for be coated with chlopyrifos coupled antigen or through pre-treatment50 μ L/ holes of sample solution, then add 50 μ L/ holes of Dursban monoclonal antibody working solution, and gently vibration mixes, with cover board membrane cover25 DEG C of lucifuge reaction 30min of plate postposition；Liquid in hole is poured out, 250 μ L cleaning solutions are added per hole and are fully washed 4~5 times, between eachEvery 10s, patted dry with blotting paper；The 100 μ L/ holes of sheep anti mouse antiantibody of horseradish peroxidase-labeled are added, gently vibration is mixedIt is even, with 25 DEG C of lucifuge reaction 30min of cover board membrane cover plate postposition, take out and repeat board-washing step；Substrate nitrite ion A liquid mistakes are added per hole50 μ L of urea, substrate nitrite ion B liquid tetramethyl benzidine (TMB) 50 μ L are aoxidized, gently vibration mixes, with cover board membrane cover plate postposition 25DEG C lucifuge colour developing 15min, 50 μ L of terminate liquid 2mol/L sulfuric acid are added per hole, and gently vibration mixes, and is set in microplate reader wavelengthAt 450nm, measure per hole absorbance (OD values).
3rd, Analysis of test results
With the absorbance values (B) divided by first standard solution (0 of the standard solution of each concentration obtainedStandard) absorbance (B0) multiplied by with 100%, obtain percentage absorbance.With pair of chlopyrifos standard concentration (μ g/L)Numerical value is X-axis, and percentage absorbance is Y-axis, draws standard curve, as shown in Figure 3.Sample solution is calculated with same methodPercentage absorbance, the chlopyrifos content of each corresponding sample can then be read from standard curve.
The definite experiment of 4 chlopyrifos enzyme linked immunological kit technical parameter of embodiment
1st, kit sensitivity and test limit
Conventionally assay kit sensitivity, kit standard curve minimum point are 0.6 μ g/L, standard curveScope is 0.6~48.6 μ g/L, IC50(50% inhibition concentration) domain of walker is 1.0~2.0 μ g/L；To blank Chinese cabbage, apple,Each 20 parts of tealeaves sample is detected, and the concentration corresponding to each percentage absorbance is found from standard curve, with 20 parts of samplesThe average value of concentration represents test limit plus 3 times of standard deviations, as a result obtains test limit of this method to veterinary antibiotics, tealeaves sampleFor 100 μ g/kg.2nd, sample preci-sion and accuracy is tested
It is inclined with the testing result relative standard of a certain concentration samples of replication using the rate of recovery as accuracy estimating indexPoor (RSD%) is used as precision evaluation index.Calculation formula is：The rate of recovery (%)=actual measured value/theoretical value × 100%,The wherein addition concentration of theoretical value sample；Relative standard deviation RSD%=SD/X × 100%, wherein SD are standard deviation, XFor the average value of determination data.
By 100 μ g/kg, 200 μ g/kg, 400 μ g/kg, tri- concentration chlopyrifos to blank Chinese cabbage, apple, tealeaves sample intoRow addition recycling measure, each sample do 4 it is parallel, be measured with three batches of different kits, calculate the average recycling of sampleRate and precision result see the table below.
1 precision of table and accuracy test
Blank Chinese cabbage, apple, tealeaves sample are added with the chlopyrifos of 100,200,400 tri- concentration of μ g/kg, put downThe equal rate of recovery is between 70%~110%；Batch in, batch between relative standard deviation be respectively less than 15%.
3rd, stabilization of kit is tested
Kit preservation condition is 2~8 DEG C, by the measure of 12 months, the maximum absorbance value (zero standard) of kit,50% inhibition concentration, chlopyrifos 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, the results showed thatThe kit indices comply fully with requirement.Situation is freezed in view of kit, it is cold that kit is put into -20 DEG C of refrigeratorsFreeze 7 days, measurement result also indicates that kit indices are completely normal.It can show that kit can be at 2~8 DEG C from result aboveAt least preserve more than 12 months.