Background technology
Imidacloprid (IMI) is nicotinoids ultra high efficiency pesticide, has wide spectrum, efficient, low toxicity, low-residual, and insect is difficult for producing resistance, to features such as people, animal, plant and natural enemies securities, and have tag, the multiple action such as stomach toxicity and interior suction.
At present, containing the Pesticidal products of IMI, in more than 140 of more than 120 country, planting registration on crops uses, range of application relates to grain, economy, fruit tree, tealeaves and vegetables etc. more than 60 and plants crop, and controlling object relates to 7 order 50 various pests such as Homoptera, Semiptera, Thysanoptera, Diptera, coleoptera, Lepidoptera.
A large amount of uses of IMI have not only caused and have had a strong impact on environment, and livestock and poultry, because of the long-term edible residual plant of IMI that contains, make its enrichment in vivo simultaneously, finally by food chain, enter human body, are detrimental to health.Worked up at present the maximum residue limit standard (MRLs) of IMI in multiple agricultural product both at home and abroad.European Union's rehearing in 2008 and revised the MRLs of IMI in nearly 400 kinds of agricultural product, wherein the MRL in most of cereal, fruit, vegetables is 0.05 mg/kg.In U.S.'s regulation cereal, vegetables, the MRL of IMI is 0.05 mg/kg, and in poultry meat product, the MRL of IMI is 0.3 mg/kg, is 0.05 mg/kg in poultry prod.In the food of Japan's execution in 29 days Mays in 2006, agriculture chemical residues " Positive List System " (Positive List System) is more harsh to the regulation of the MRLs of IMI in poultry, poultry product, wherein the different tissues of different animals all has corresponding MRLs, in most of musculature, be 0.02 mg/kg, in liver, be 0.05 mg/kg, in fat, be 0.3 mg/kg, to not working out the agricultural chemicals of MRLs, its content in food is carried out " uniform limit ", can not surpass 0.01 mg/kg without exception.The domestic MRL to IMI in agricultural product only has regulation roughly, in cereal, vegetables, is no more than 0.5 mg/kg, see on the whole also lag behind aspect the MRLs formulation of China IMI in food external.
From bibliographical information, about the residual detection method of IMI mainly concentrates on high performance liquid chromatography (HPLC), vapor-phase chromatography (GC), electrochemical methods (EA) and four aspects of immunoassay (ELISA), be more prone to chromatographic technique and immunoassay technology.Although chromatography is current generally acknowledged medicament residue detection method, this class methods ubiquity that required instrument is expensive, instrumentation technical requirement is high, sample pre-treatments is loaded down with trivial details and analyze length consuming time, testing cost high, be unsuitable for the problems such as on-the-spot detection in real time.And the complexity of medicament residue immunoassay technology in residual sample testing process, speed, sensitivity, accuracy, specificity, in the unit interval, the comparative advantages of the aspect such as sample analysis capacity, testing cost and instrumental method are outstanding, all deficiencies that instrumental method shows have almost been overcome, in Safety of Food Quality detects, obtained in recent years application more and more widely, and by people, approved gradually.
In today that environmental protection and agricultural product security cry are improved day by day, effective detection means of Residual Pesticides in Farm Produce cause close important.Along with the regulation increasingly stringent to IMI MRLs in agricultural product in the world, for adapting to better foreign trade and the protection people's health, in developing food products, the residual Fast Detection Technique of IMI is imperative.Not only detectability is low for the immunological detection method of residue, high specificity, and easy and simple to handle, detection speed is fast, testing cost is low, be very easy to promote, and is the study hotspot that current medicament residue detects.Although the external bibliographical information of the existing residual immunology detection of IMI, all exists that residual sample pre-treatment program is complicated, expense is high, detectability is higher, can not meet the problems such as MRLs of IMI in food in the world to some extent.Therefore, finding a more effective method to address these problems, is pendulum major issue urgently to be resolved hurrily in face of us.
Summary of the invention
The object of the present invention is to provide a kind of enzyme linked immunological kit that can detect Determination of Imidacloprid Residue amount in vegetables, fruit sample, and a kind of efficient, accurate, easy, qualitative and quantitative analysis method of being suitable for batch samples screening is provided.
Kit of the present invention, it comprises: the ELISA Plate, specific antibody, enzyme labeling thing, Imidacloprid standard solution, substrate nitrite ion, stop buffer, cleansing solution, the redissolution liquid that are coated with coating antigen, described coating antigen is Imidacloprid coupled antigen, and described enzyme labeling thing is enzyme labeling antiantibody.
Described Imidacloprid coupled antigen is to be obtained by Imidacloprid haptens and carrier protein couplet, described Imidacloprid haptens is to be obtained by redox reaction, and described carrier protein can be mouse haemocyanin, thyroprotein, bovine serum albumin(BSA), rabbit anteserum albumen, human albumin, ovalbumin, hemocyanin or fibrinogen.
Described Imidacloprid specific antibody is to using Imidacloprid coupled antigen to prepare as immunogene, and described Imidacloprid specific antibody can be Imidacloprid monoclonal antibody or Imidacloprid polyclonal antibody, wherein preferred Imidacloprid monoclonal antibody.
Described antiantibody is sheep anti mouse antiantibody or goat-anti rabbit antiantibody, wherein preferred sheep anti mouse antiantibody.
The marker enzyme of described enzyme labeling thing is that horseradish peroxidase or bacterium are extracted alkaline phosphatase, wherein preferred horseradish peroxidase; The antiantibody of enzyme labeling adopts glutaraldehyde method or sodium periodate method that marker enzyme and antiantibody are carried out to coupling and obtains.
For more convenient on-site supervision and great amount of samples examination, described kit also comprises Imidacloprid standard solution, substrate nitrite ion, stop buffer, cleansing solution, redissolution liquid.
6 bottles of described Imidacloprid standard solutions, 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.
When marker enzyme is horseradish peroxidase, described substrate nitrite ion is comprised of substrate solution A liquid and substrate solution B liquid, A liquid is hydrogen peroxide or urea peroxide, and B liquid is o-phenylenediamine or tetramethyl benzidine, the sulfuric acid that described stop buffer is 1 ~ 2mol/L or hydrochloride buffer; When marker enzyme is bacterium extraction alkaline phosphatase, described substrate nitrite ion is to nitro phosphate buffer, and described stop buffer is 1 ~ 2mol/L sodium hydroxide solution.
It is 7.4 that described cleansing solution is preferably pH value, the phosphate buffer that contains 0.5% ~ 1.0% Tween-20,0.01 ‰ ~ 0.03 ‰ sodium azide antiseptic, 0.1 ~ 0.3mol/L, and described number percent is percent weight in volume.
Described redissolution liquid is preferably that pH value is 7.0, the phosphate buffer of 0.02mol/L, and described number percent is percent weight in volume.
Wherein in ELISA Plate preparation process, coated damping fluid used is that pH value is 9.6, the carbonate buffer solution of 0.05mol/L, confining liquid is that pH value is 7.1 ~ 7.5, the phosphate buffer that contains 1% ~ 3% casein, 0.1 ~ 0.3mol/L, and described number percent is percent weight in volume.
In the present invention, the preparation process of ELISA Plate is: with coated damping fluid, coating antigen is diluted to 20 μ g/ml, every hole adds 100 μ l, 37 ℃ of lucifuges are hatched 2h or 4 ℃ and are spent the night, and liquid in the hole of inclining, with cleansing solution washing 2 times, each 30s, pat dry, then in every hole, add 150 ~ 200 μ l confining liquids, 37 ℃ of lucifuges are hatched 1 ~ 2h, the liquid in hole that inclines pats dry, and preserves after dry with the vacuum seal of aluminium film.
Detection principle of the present invention is:
Pre-coated Imidacloprid coupled antigen on capillary strip, add after sample solution or standard solution, add again Imidacloprid specific antibody solution, in Imidacloprid in sample and ELISA Plate, coated Imidacloprid coupled antigen is competed anti-Imidacloprid specific antibody, add enzyme labeling antiantibody to carry out amplification, with nitrite ion colour developing, the content of sample absorbance and Imidacloprid is negative correlation, relatively can obtain the residual quantity of Imidacloprid in sample with typical curve; Simultaneously according to the depth of color in ELISA Plate, with the comparison of the standard solution color of the series concentration concentration range of Determination of Imidacloprid Residue amount in judgement sample roughly.
The present invention also provides a kind of method that above-mentioned enzyme linked immunological kit detects Imidacloprid of applying, and it comprises step:
(1) sample pre-treatments;
(2) with kit, detect;
(3) analyzing and testing result.
The enzyme linked immunological kit that the present invention detects Imidacloprid mainly adopts competitive ELISA method qualitative or quantitatively detect the content of Imidacloprid in sample; Pre-treatment requirement to sample is low, and sample pretreatment process is simple, simultaneously fast detecting batch samples; Main agents provides with the form of working fluid, and the method for inspection is convenient and easy, has that specificity is high, highly sensitive, degree of accuracy is high, accuracy high.Enzyme linked immunological kit of the present invention, simple in structure, easy to use, low price, carrying convenience, detection method be efficient, accurate, easy, be suitable for the qualitative, quantitative of batch samples screening.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only for the present invention is described, and be not used for limiting the scope of the invention.
The preparation of embodiment 1 reagent constituents
1, the haptenic preparation of Imidacloprid
In bis-mouthfuls of bottles of 100ml, add Imidacloprid 0.25g, the dimethyl formamide of 1ml (DMF), the ethanol of hydration tin chloride 0.75g and 10ml, nitrogen purge, 65 ℃ of reaction 40min of temperature control, reactant liquor shows brown, and thin-layer chromatography (TLC) detection reaction is complete.Process: be cooled to room temperature, add ethyl acetate 50ml, add saturated sodium bicarbonate aqueous solution and be adjusted to alkalescent, salt out in a large number, ethyl acetate 40ml * 2 time extraction turbid solution, merges organic phase, salt water washing, anhydrous sodium sulfate drying, solvent evaporated, obtains solid product 0.22g.
Get above-mentioned product through nuclear magnetic resonance hydrogen spectruming determining, as shown in Figure 2, the peak of 4.7ppm left and right is two methylene signals peaks on imidazoline ring, and the peak of 5.0ppm left and right is benzyl signal peak, 7.2-8.7ppm is amino and aromatic ring mixed signal peak, illustrates that target haptens synthesizes successfully.
2, the preparation of antigen
Immunogene preparation---Imidacloprid haptens and bovine serum albumin(BSA) (BSA) coupling obtains immunogene.
Get 7mg haptens, be dissolved in 1ml DMF; Get glutaraldehyde water solution 0.1ml and add in haptens solution, under room temperature, stir 24h, can obtain reactant liquor A; Take bovine serum albumin(BSA) (BSA) 30mg, make it to be fully dissolved in 2.7ml 0.1mol/L carbonate buffer solution (CB) (pH 9.6), reactant liquor A is dropwise slowly added drop-wise in protein solution, and stir 24h under room temperature, sodium borohydride aqueous solution 0.2ml reduction reaction 4h with 5mol/L, with 4 ℃ of dialysis 3d of 0.01mol/L phosphate buffer (PBS), change dislysate every day 3 times, to remove unreacted small-molecule substance; Packing, saves backup in-20 ℃.
Coating antigen preparation---Imidacloprid haptens and ovalbumin (OVA) coupling obtains immunogene.
Get 7mg haptens, be dissolved in 1ml DMF; Get glutaraldehyde water solution 0.1ml and add in haptens solution, under room temperature, stir 24h, can obtain reactant liquor A; Take OVA 30mg, make it to be fully dissolved in 2.7ml 0.1mol/L CB(pH 9.6) in, reactant liquor A is dropwise slowly added drop-wise in protein solution, and stir 24h under room temperature, sodium borohydride aqueous solution 0.2ml reduction reaction 4h with 5M, with 4 ℃ of dialysis 3d of 0.01mol/L PBS, change dislysate every day 3 times, to remove unreacted small-molecule substance; Packing, saves backup in-20 ℃.
3, the preparation of Imidacloprid monoclonal antibody
Animal immune: the immunogene that above-mentioned steps is 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's fusion, adopt indirect competitive ELISA to measure cell conditioned medium liquid, screen positive hole.Utilize limiting dilution assay to carry out cloning to positive hole, until obtain secreting the hybridoma cell strain of Imidacloprid monoclonal antibody.
Cell cryopreservation and recovery: monoclonal hybridoma strain is made to 1 * 10 with cryopreserving liquid6the cell suspension of individual/ml is preserved for a long time in liquid nitrogen.During recovery, take out cryopreservation tube, put into immediately 37 ℃ of water-bath middling speeds and melt, after centrifugal removal cryopreserving liquid, move in culture flask and cultivate.
The production of monoclonal antibody and purifying: Balb/c mouse peritoneal is only injected to sterilizing paraffin oil 0.5ml/, and within 7 days, pneumoretroperitoneum is injected stable monoclonal hybridoma strain 5 * 105individual/only, after 7 days, to gather ascites.By sad-saturated ammonium sulfate method, carry out ascites and purify ,-20 ℃ of preservations.
4, the preparation of sheep anti mouse antiantibody
Take sheep as immune animal, take mouse source antibody as immunogen immune pathogen-free domestic sheep, obtain sheep anti mouse antiantibody.
5, the preparation of enzyme labeling antiantibody
Adopt the sodium periodate method after improvement to carry out coupling sheep anti mouse antiantibody and horseradish peroxidase (HRP).In traditional sodium periodate method requirement reaction system, the molar concentration rate of enzyme and antibody is 4:1, because horseradish peroxidase produces many sites of being combined with antibody under strong oxidation, the horseradish peroxidase molecule of activation has served as the bridge that connects each molecule like this, reduce the enzymatic activity of enzyme labeling thing, in the conjugate that makes to prepare, be mixed with many condensates.In order to address this problem, we improve traditional method, that is:
(1) saved amino closed process, because can produce self amino amino reality connecting seldom;
(2) the volumetric molar concentration ratio that reduces horseradish peroxidase and antibody is to 2:1, and the method after improvement is easier than traditional method, and the loss of enzymatic activity is reduced.
6, the preparation of ELISA Plate
With coated damping fluid, coating antigen is diluted to 20 μ g/ml, every hole adds 100 μ l, 37 ℃ of lucifuges are hatched 2h, and liquid in the hole of inclining, with cleansing solution washing 2 times, each 30s, pat dry, then in every hole, add 200 μ l confining liquids, 37 ℃ of lucifuges are hatched 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 Imidacloprid
Set up the enzyme linked immunological kit that detects Imidacloprid, make it comprise following component:
(1) ELISA Plate of coated Imidacloprid coupled antigen;
(2) Imidacloprid standard solution is 6 bottles, and 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) Imidacloprid monoclonal antibody working fluid;
(4) use the sheep anti mouse antiantibody of horseradish peroxidase-labeled;
(5) substrate nitrite ion is comprised 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 that pH value is 7.4, the phosphate buffer that contains 0.5% ~ 1.0% Tween-20,0.01 ‰ ~ 0.03 ‰ sodium azide antiseptic, 0.1 ~ 0.3mol/L, and described number percent is percent weight in volume;
(8) redissolving liquid is that pH value is 7.0, the phosphate buffer of 0.02mol/L, and described number percent is percent weight in volume.
The detection of Imidacloprid in embodiment 3 vegetables, fruit sample
1, sample pre-treatments
With homogenizer homogeneous sample; Take the equal pledge of 2.0 ± 0.05g to 50ml polystyrene centrifuge tube, add respectively 2ml 2mol/L sodium hydroxide solution, 10ml ethyl acetate, with vortex instrument vortex 5min, more than 3000g, the centrifugal 5min of room temperature (20-25 ℃); Pipette 1ml upper organic phase to the clean glass test tube of 15ml, under (50-60 ℃) water-bath nitrogen/air stream, dry up; Add 1ml redissolution working fluid, whirling motion 3min; Get 50ul for analyzing.
2, with kit, detect
To being coated with, in the ELISA Plate micropore of Imidacloprid coupled antigen, add Imidacloprid standard solution or through the sample solution 50 μ l/ holes of pre-treatment, add again Imidacloprid monoclonal antibody 50 μ l/ holes, vibration mixes gently, with reacting 30min in the rearmounted 25 ℃ of lucifuge environment of cover plate membrane cover plate; Pour out liquid in hole, every hole adds 250 μ l cleansing solutions fully to wash 4 ~ 5 times, and every minor tick 10s, pats dry with thieving paper; The sheep anti mouse antiantibody 100 μ l/ holes that add horseradish peroxidase-labeled, vibration mixes gently, with reacting 30min in the rearmounted 25 ℃ of lucifuge environment of cover plate membrane cover plate, takes out and washes plate; Every hole adds substrate nitrite ion A liquid hydrogen peroxide 50 μ l, substrate nitrite ion B liquid tetramethyl benzidine (TMB) 50 μ l, vibration mixes gently, with the rearmounted 25 ℃ of lucifuges colour developing of cover plate membrane cover plate 15min, every hole adds stop buffer 2mol/L sulfuric acid 50 μ l, vibration mixes gently, by microplate reader wavelength set, at 450nm place, measures every hole absorbance (OD value).
3, Analysis of test results
The mean light absorbency value (diplopore) of standard items or sample is divided by the mean light absorbency value of first standard items (0 standard), then is multiplied by 100%, obtains the percentage absorbance of standard items or sample.The standard items percentage absorptance of take is ordinate, and the Imidacloprid standard items concentration (ppb) of take is horizontal ordinate, drawing standard curve map.By in the percentage absorptance substitution typical curve of sample, from typical curve, read the corresponding concentration of sample, be multiplied by its corresponding extension rate and be Imidacloprid actual concentrations in sample.
Definite test of embodiment 4 Imidacloprid technical parameters
1, kit sensitivity and detectability
According to conventional method, measure kit sensitivity, kit typical curve minimum point is 4 μ g/L, and the scope of typical curve is 4 ~ 324 μ g/L, IC50(50% inhibition concentration) domain of walker is 10 ~ 16 μ g/L; 20 duplicate samples are detected, from typical curve, find the concentration corresponding to each percentage absorbance, with the mean value of 20 parts of concentration of specimens, add that 3 times of standard deviations represent detectability, result obtains the method the detection of sample is limited to 20 μ g/kg.
2, sample preci-sion and accuracy test
Using the recovery as accuracy estimating index, and the testing result relative standard deviation (RSD%) of a certain concentration sample of replication is as precision evaluation index.Computing formula is: the recovery (%)=practical measurement value/theoretical value * 100%, the interpolation concentration that wherein theoretical value is sample; Relative standard deviation RSD%=SD/X * 100%, wherein SD is standard deviation, the mean value that X is determination data.
By 100 μ g/kg, 300 μ g/kg, tri-concentration Imidacloprids of 500 μ g/kg, respectively vegetables and fruit sample are added to recovery mensuration, each sample do 4 parallel, with three batches of different reagent, measure, average recovery rate and the precision of calculation sample the results are shown in following table.
Table 1 vegetables precision and accuracy test
Table 2 fruit precision and accuracy test
Imidacloprid with tri-concentration of 100,300,500 μ g/kg adds vegetables and fruit sample respectively, and average recovery rate is between 83.5% ~ 117.9%; Batch in, batch between relative standard deviation be all less than 10%.
3, kit stability test
Kit preservation condition is 2 ~ 8 ℃, and through the mensuration of 12 months, the maximum absorbance value (zero standard) of kit, 50% inhibition concentration, Imidacloprid added practical measurement value all within normal range.Consideration, in transportation and use procedure, has improper preservation condition and occurs, kit is placed 7 days under 37 ℃ of preservation conditions, carries out accelerated deterioration experiment, and result shows that this kit indices meets the requirements completely.Consider that the freezing situation of kit occurs, kit is put into-20 ℃ of refrigerator freezings 7 days, measurement result also shows that kit indices is completely normal.From above result, can show that kit can at least preserve more than 12 months at 2 ~ 8 ℃.