CN101880325A - Monoclonal antibody for detecting imidacloprid pesticide residue - Google Patents
Monoclonal antibody for detecting imidacloprid pesticide residue Download PDFInfo
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Abstract
The invention relates to an imidacloprid pesticide against monoclonal antibody and a preparation method thereof, and belongs to the technical field of biology. The preparation method comprises the following steps of: immunizing a BALB/c mouse by using a coupling substance of immune hapten 1-[6-(2-carboxyethylsulfenyl-3-pyridine) methyl]-N-nitro-2-imidazoline imine and bovine serum albumin, preparing hybrid tumor cells from spleen cells and myeloma cells Sp2/0 of the immunized mouse by the hybrid tumor technology, and obtaining hybrid tumor strains 2F11/A9 capable of stably secreting the imidacloprid pesticide against monoclonal antibody. By effectiveness verification, the antibody can be used for sensitive and quick detection of imidacloprid residues in agricultural production environments and agricultural products. The preparation technique for the imidacloprid pesticide against monoclonal antibody is simple and feasible, does not need special instruments and equipment in the whole preparation process of the antibody, and is easy for scale production in factories.
Description
(1) technical field
The present invention relates to anti-pesticide imidacloprid monoclonal antibody and preparation method thereof, belong to biological technical field.Be exclusively used in residual sensitivity, the rapid detection of Provado in agricultural-food and the agriculture production environment, be specially adapted to batch samples and detect.
(2) background technology
Provado (imidacloprid), 1-(6-chloro-3-picolyl)-N-nitro-2-tetrahydroglyoxaline imines belongs to novel nicotinamide insecticide, and moderate toxicity makes the insect paralysis by acting on the intravital acetylcholinesterase acceptor of insect, and is final dead.Provado can the effectively preventing aphid, plant hopper, and Agricultural pests such as leafhopper have obtained using widely in agriculture production.Because a large amount of uses of this agricultural chemicals in agriculture production have in recent years not only caused pollution to environment, and HUMAN HEALTH and some non-target organisms have been brought serious threat, there is relevant report to show, Provado has stronger toxic action to honeybee and silkworm, and the people also toxicity symptoms such as dizziness, vomiting can occur if eat by mistake accidentally after containing the residual food of imidacloprid pesticide.Therefore, it is very urgent to set up the residual detection method of Provado.
At present vapor-phase chromatography (GC) and high performance liquid chromatography (HPLC) are adopted in the retention analysis of imidacloprid pesticide mostly, these methods must be used expensive plant and instrument, to the sample pre-treatments complexity, operator must pass through professional training, so these methods also are not suitable for the batch screening of sample, agricultural product security are supervised work so just for food safety regulator and have brought many inconvenience.
Since eighties of last century has been started agricultural chemicals immune quantitative analytical technology the seventies, the pesticide residue immuno analytical method has obtained development fast, American Chemical Society classifies immune analysis method three big pillars of chemical pesticide retention analysis as with vapor-phase chromatography, high performance liquid chromatography, is the competitive and challenging ultramicron detection technique of tool of 21 century.The immuno analytical method of pesticide residue is based on antibody and small molecules agricultural chemicals specificity bonded analytical technology to be measured, this analytical technology has highly sensitive, high specificity, be easy to stdn, be fit to advantages such as large vol sample analysis, and this method does not need the expensive experimental instrument, and is easy and simple to handle, generally do not need test sample is done complicated pre-treatment.The foreign study person has reported the immune method for detecting residue of Multiple Pesticides the nineties successively in eighties of last century, the research of domestic this respect is later, just began to have the correlative study personnel to pay attention in recent years and be engaged in relevant research, do not see relevant report for the imidacloprid pesticide method for preparing monoclonal antibody is domestic.
(3) summary of the invention
Technical problem the purpose of this invention is to provide a kind of monoclonal antibody that can be used for the imidacloprid pesticide residue detection and preparation method thereof, by synthetic immunizing antigen, immunity BALB/c mouse, monoclonal antibody by hybridoma technology preparation energy specific recognition imidacloprid pesticide can be used for the residual sensitive rapid detection of Provado in agricultural-food and the agriculture production environment.
Technical scheme
1, anti-imidacloprid pesticide monoclonal antibody specific is produced by hybridoma cell strain 2F11/A9, is IgG3 type antibody.
2, anti-imidacloprid pesticide MONOCLONAL ANTIBODIES SPECIFIC FOR method comprises:
(1) the imidacloprid pesticide artificial antigen is synthetic:
With immune haptens 1-[6-(2-carboxyethylthio-3-pyridine) methyl]-N-nitro-2-tetrahydroglyoxaline imines, be called for short H1, with bovine serum albumin BSA coupling, obtain Provado artificial immunogen H1-BSA; To wrap by haptens 1-(6-chloro-3-picolyl)-3-carboxylic propyl group-N-nitro-2-tetrahydroglyoxaline imines, be called for short H2,, obtain envelope antigen H2-OVA with ovalbumin OVA coupling;
(2) mouse immune:
With the H1-BSA for preparing as female BALB/c mouse in immunogen immune 6-8 age in week, the immunogen consumption 100 μ g of each every mouse, immunity is four times altogether, first immunisation is by isopyknic immunogen and Freund's complete adjuvant emulsification, abdominal injection, later four immunity are with isopyknic Freund's incomplete adjuvant and immunogen emulsification; For the first time with 3 weeks of interval of immunity for the second time, 2 weeks of interval later on;
From for the third time, each immunity one week of back, the caudal vein blood sampling, with H2-OVA as coating antigen, detect tiring of antibody with non-competing indirect elisa method: be cushioned liquid dilution bag by H2-OVA with the carbonate bag, every hole adds 50 μ L in the 96 hole enzyme plates, hatches 2 hours in 37 ℃, wash 5 times with coating buffer in the enzyme plate to the greatest extent, and with the phosphate buffered saline buffer that contains volume ratio 0.05% polysorbas20;
Gelatin is mixed with 1% confining liquid with phosphate buffered saline buffer dissolving, and every hole adds 100 μ L in the 96 hole enzyme plates, hatches 1.5 hours for 37 ℃, with confining liquid in the enzyme plate to the greatest extent, and washs 5 times with the phosphate buffered saline buffer that contains 0.05% polysorbas20;
Mice serum is diluted with the phosphate buffered saline buffer that contains 0.05% polysorbas20, every hole adds 50 μ L in the 96 hole enzyme plates, use simultaneously without mice immunized serum negative control is set, hatched 0.5 hour for 37 ℃, wash 5 times with reaction solution in the enzyme plate to the greatest extent, and with the phosphate buffered saline buffer that contains 0.05% polysorbas20;
Sheep anti-mouse antibody with the phosphate buffered saline buffer dilution horseradish peroxidase-labeled that contains 0.05% polysorbas20, every hole adds 50 μ L in the 96 hole enzyme plates, hatched 0.5 hour for 37 ℃, reaction solution in the enzyme plate is use up, and wash 5 times with the phosphate buffered saline buffer that contains 0.05% polysorbas20;
Now join tetramethyl benzidine colour developing liquid with substrate buffer solution, every hole adds 50 μ L in the 96 hole enzyme plates, 37 ℃ of dark place reaction 10min;
Every hole adds 2mol/L sulfuric acid 25 μ L, termination reaction in the 96 hole enzyme plates;
Enzyme plate after the color development stopping is measured the single wavelength light absorption value in each hole immediately under 450nm;
(3) cytogamy
Selection detects the highest BALB/c mouse of tiring through indirect non-competing ELISA method, get spleen, the Sp2/0 myeloma cell of splenocyte and logarithmic phase is prepared hybridoma by hybridoma technology, 37 ℃, volume ratio 5% CO2gas incubator was cultivated after 7-10 days, merged hole supernatant screening hybridoma by detecting;
(4) hybridoma screening
Merge the hole supernatant, adopt indirect non-competing ELISA method to carry out preliminary screening, its method is:
Be cushioned liquid dilution coating antigen H2-OVA with the carbonate bag, every hole adds 50 μ L in the 96 hole enzyme plates, hatches 2 hours in 37 ℃, with coating buffer in the enzyme plate to the greatest extent, and washs 5 times with the phosphate buffered saline buffer that contains 0.05% polysorbas20;
Gelatin is mixed with 1% confining liquid with phosphate buffered saline buffer dissolving, and every hole adds 100 μ L in the 96 hole enzyme plates, hatches 1.5 hours for 37 ℃, with confining liquid in the enzyme plate to the greatest extent, and washs 5 times with the phosphate buffered saline buffer that contains 0.05% polysorbas20;
Immune serum is diluted with the phosphate buffered saline buffer that contains 0.05% polysorbas20, every hole adds 50 μ L in the 96 hole enzyme plates, with the supernatant of fused cell not as negative control, with the positive contrast of the serum of immune mouse, hatched 0.5 hour for 37 ℃, wash 5 times with reaction solution in the enzyme plate to the greatest extent, and with the phosphate buffered saline buffer that contains 0.05% polysorbas20;
Sheep anti-mouse antibody with the phosphate buffered saline buffer dilution horseradish peroxidase-labeled that contains 0.05% polysorbas20, every hole adds 50 μ L in the 96 hole enzyme plates, hatched 0.5 hour for 37 ℃, reaction solution in the enzyme plate is use up, and wash 5 times with the phosphate buffered saline buffer that contains 0.05% polysorbas20;
Now join tetramethyl benzidine colour developing liquid with substrate buffer solution, every hole adds 50 μ L in the 96 hole enzyme plates, 37 ℃ of dark place reaction 10min;
Every hole adds 2mol/L sulfuric acid 25 μ L, color development stopping in the 96 hole enzyme plates;
Enzyme plate after the color development stopping is measured the single wavelength light absorption value in each hole immediately under 450nm;
When having the reading of the supernatant of fused cell to be equal to or greater than 2.1 times of negative control reading, such fusion hole is positive hole;
In order to determine that further obtaining the secreted antibody in positive hole is at imidacloprid pesticide, again by the screening of competition indirect elisa method.Its method is:
Be cushioned liquid dilution coating antigen H2-OVA with the carbonate bag, every hole adds 50 μ L in the 96 hole enzyme plates, hatches 2 hours in 37 ℃, with coating buffer in the enzyme plate to the greatest extent, and washs 5 times with the phosphate buffered saline buffer that contains 0.05% polysorbas20;
Gelatin is mixed with 1% confining liquid with phosphate buffered saline buffer dissolving, and every hole adds 100 μ L in the 96 hole enzyme plates, hatches 1.5 hours for 37 ℃, with confining liquid in the enzyme plate to the greatest extent, and washs 5 times with the phosphate buffered saline buffer that contains 0.05% polysorbas20;
Prepare the imidacloprid pesticide standard substance of a series of concentration, acetone solution, after 20 times of the phosphate buffered saline buffer dilutions that contains 0.05% polysorbas20, with cells and supernatant equal-volume thorough mixing, be added in the enzyme plate with every hole 50 μ L, as negative control, hatched 0.5 hour for 37 ℃ with the supernatant of fused cell not and the phosphate buffered saline buffer equal-volume mixed solution that contains 0.05% polysorbas20, wash 5 times with reaction solution in the enzyme plate to the greatest extent, and with the phosphate buffered saline buffer that contains 0.05% polysorbas20;
Sheep anti-mouse antibody with the phosphate buffered saline buffer dilution horseradish peroxidase-labeled that contains 0.05% polysorbas20, every hole adds 50 μ L in the 96 hole enzyme plates, hatched 0.5 hour for 37 ℃, reaction solution in the enzyme plate is use up, and wash 5 times with the phosphate buffered saline buffer that contains 0.05% polysorbas20;
Now join tetramethyl benzidine colour developing liquid with substrate buffer solution, every hole adds 50 μ L in the 96 hole enzyme plates, 37 ℃ of dark place reaction 10min;
Every hole adds 2mol/L sulfuric acid 25 μ L in the 96 hole enzyme plates, color development stopping, and the enzyme plate after the color development stopping is measured the single wavelength light absorption value in each hole immediately under 450nm, and it is B that the positive control light absorption value is made in the hole that does not contain agricultural chemicals with only adding the cell culture supernatant
0, the hole light absorption value that adds agricultural chemicals is B, if B＜B
0, the reaction of prepared antibody capable and agricultural chemicals then is described, such hole is positive hole, and subclone is carried out by limiting dilution assay in positive hole, and its hybridoma excretory supernatant is made monoclonal antibody.
The hybridoma cell strain 2F11/A9 that produces said monoclonal antibody is preserved in Chinese typical culture collection center on May 19th, 2010, and deposit number is CCTCC NO:C201047.
Beneficial effect
Compare with existing detection technique, its advantage and positively effect show:
Practicality is good: traditional analysis of agricultural drugs is mainly carried out in the laboratory, its pre-treatment process is loaded down with trivial details, analysis speed is slow, cost is high, the a large amount of organic solvents that use easily cause environmental pollution, and breadboard instrumentation degree and peopleware are had relatively high expectations, can not satisfy in the Agricultural Products Trade demands quick, easy, accurate, a large amount of detections.And advantage such as the ELISA that we provide (Enzyme Linked Immunoadsorbent Assay) detection method has (doing detection with the trace analysis plate that seals only needed get final product in 1.5-2 hour) simple to operate, quick, analysis cost is low, the analysis capacity is big, safe and reliable, generally do not need expensive instrument, can simplify even save the pre-treatment process in a large number, level professional technology to the user of service is less demanding, popularize easily and promote, be specially adapted to pattern detection in enormous quantities and field monitoring.
Antigen and antibody good stability: this method synthetic immunogen and coating antigen have stability preferably, and-20 ℃ of refrigerators can be deposited at least and not influence its immunogenicity in 5 years.Hybridoma is frozen to be kept 3 years at least in liquid nitrogen, and the stability of antibody is also relatively good, and antibody purified-20 ℃ refrigerator can be deposited 2 years at least.
Specificity is good: the monoclonal antibody of the resulting anti-Provado of this method can specific identification imidacloprid pesticide, to the almost not identification of other agricultural chemicals.
Highly sensitive: detectability will be lower than other conventional detection methods, can reach 1.08 μ g/L.
The present invention has obtained the hybridoma cell strain of the anti-imidacloprid pesticide monoclonal antibody of energy stably excreting specificity on a large amount of previous work bases such as artificial chemosynthesis antigen, immune mouse, cytogamy, screening.The anti-imidacloprid pesticide monoclonal antibody of specificity based on preparation, with conjugate H2-OVA as detecting antigen, by optimizing detection architecture, set up one and overlapped the residual enzyme-linked immunity of sensitive imidacloprid pesticide (ELISA) detection method more, for the exploitation of imidacloprid pesticide residue detection test kit provides the foundation, for the important instrument that provides is provided environmental safety.
(4) embodiment
1, the synthetic method of imidacloprid pesticide immunogen H1-BSA is:
(1) imidacloprid pesticide immunity haptens 1-[6-(2-carboxyethylthio-3-pyridine) methyl]-the N-nitro-(molecular formula is 2-tetrahydroglyoxaline imines: C
12H
15O
4N
5S, structural formula is:
) synthetic: adopt Zhu state such as thoughts grade to be published in method synthetic compound 1-[6-(2-carboxyethylthio-3-pyridine) methyl on the academic journals " Scientia Agricultura Sinica "]-N-nitro-2-tetrahydroglyoxaline imines (H1).This compound is used as imidacloprid pesticide immunity haptens by us.The particular location of the research paper that contains the above-claimed cpd synthetic method on " Scientia Agricultura Sinica " that people such as Zhu Guonian deliver be 2005 the 38th the volume the 511st to 515 page.
(2) imidacloprid pesticide immunogen H1-BSA's is synthetic: add imidacloprid pesticide immunity haptens H1 0.026g and N-hydroxy-succinamide 0.018g in the reaction flask, add N again, dinethylformamide 0.3mL dissolving; Take by weighing the 0.04g dicyclohexylcarbodiimide, use 0.2mL N, dinethylformamide dissolving, and slowly being added drop-wise in the reaction flask is reflected under the magnetic agitation room temperature and carried out 12 hours.React whole liquid centrifugal 5 minutes, get in the bovine serum albumin (BSA) that supernatant joins 2mL 15mg/mL lentamente, be reflected under the magnetic agitation stirring at room 6 hours through 8000r/min.After question response is finished, reaction solution is placed in the dialysis tubing, in 0.02mol/L, 4 ℃ are stirred dialysis in the phosphoric acid buffer of pH 6.8, change dialyzate one time in per four hours, dialyse altogether 60 hours.After dialysis finished, with the liquid in the dialysis tubing through 8000r/min centrifugal 5 minutes, the gained supernatant liquor was imidacloprid pesticide immunogen H1-BSA.Gained immunogen H1-BSA is taken out packing to be stored in-20 ℃ of refrigerators standby.
2, the synthetic method of imidacloprid pesticide envelope antigen H2-OVA is:
(1) (6-chloro-3-picolyl)-3-carboxylic propyl group-N-nitro-(molecular formula is 2-tetrahydroglyoxaline imines the imidacloprid pesticide bag: C by haptens 1-
13H
16O
4N
5Cl, structural formula is:
) synthetic:
Take by weighing Provado 1.03g (4mmol) in the 100ml there-necked flask, dissolve with 15mL DMF, under 0 ℃ condition, add NaH (0.1032g while stirring, 4.3mmol), react after 1 hour, in reaction solution, drip 4-bromo-butyric acid ethyl ester (2.23g, 10mmol), continue at room temperature to stir 12 hours, reactant is poured in the 80mL water then, the pH value of regulator solution is between the 6-7, (2 * 80mL) extractions are used anhydrous sodium sulfate drying after the washing of organic layer process to mixture, and concentrating under reduced pressure gets crude product then with chloroform.Silica gel column chromatography (cyclohexane/ethyl acetate 2: 7 (v/v)) pure product.Pure product hydrolysis in acidic solution (2mol/L HCl that 1.71g (4.6mmol) is obtained by above-mentioned steps, 60-70 ℃, 4h) adjust pH value to 6 with 1mol/L NaOH solution afterwards, with ethyl acetate extraction (2 * 50mL), anhydrous sodium sulfate drying, concentrating under reduced pressure gets crude product then.Silica gel column chromatography (ethyl acetate/methanol 4: 1 (v/v)) 1-(6-chloro-3-picolyl)-3-carboxylic propyl group-N-nitro-pure product of 2-tetrahydroglyoxaline imines (H2).
(2) imidacloprid pesticide immunogen H2-OVA's is synthetic: add the imidacloprid pesticide bag in the reaction flask by haptens H2 0.026g and N-hydroxy-succinamide 0.0115g, add N again, dinethylformamide 0.3mL dissolving; Take by weighing the 0.0248g dicyclohexylcarbodiimide, use 0.2mL N, dinethylformamide dissolving, and slowly being added drop-wise in the reaction flask is reflected under the magnetic agitation room temperature and carried out 12 hours.React whole liquid centrifugal 5 minutes, get in the ovalbumin (OVA) that supernatant joins 2mL 10mg/mL lentamente, be reflected under the magnetic agitation stirring at room 6 hours through 8000r/min.After question response is finished, reaction solution is placed in the dialysis tubing, in 0.02mol/L, 4 ℃ are stirred dialysis in the phosphoric acid buffer of pH 6.8, change dialyzate one time in per four hours, dialyse altogether 60 hours.After dialysis finished, with the liquid in the dialysis tubing through 8000r/min centrifugal 5 minutes, the gained supernatant liquor was imidacloprid pesticide immunogen H2-OVA.Gained immunogen H2-OVA is taken out packing to be stored in-20 ℃ of refrigerators standby.
3, monoclonal antibody validation verification:
Reclaim the validity of experimental verification antibody in the imidacloprid pesticide residue detection by adding.In quantity of sample, add a certain amount of standard sample of pesticide, by adding extracting solution the Provado in the sample is extracted, through being used for elisa assay after the dilution, through obtaining the content of agricultural chemicals in the sample after the formula conversion, and then calculate recovery rate, if the rate of recovery between 80%-120%, thinks that then this antibody can be used for Provado residue detection in this sample.
Embodiment 1: detect the imidacloprid pesticide residual quantity in the tap water sample
ELISA reaction system underlying condition
H2-OVA encrusting substance 1.82 μ g/mL every hole in the trace analysis plate adds 50 μ L, monoclonal antibody is diluted 32 times as working concentration, 1% gelatin is made closure, acetone content is 5% in the antigen antibody reaction liquid (phosphate buffered saline buffer that contains 0.05% polysorbas20), the pH value is 7.5, ionic strength is 0.8mol/L, every hole 50 μ L in the trace analysis plate.
Testing sample is prepared
Tap water sample: Provado standard substance net weight 250mg joined in the 1mL acetone soln dissolve, be mixed with the Provado acetone soln of 250mg/mL, measure tap water 1L, add the Provado acetone soln of 1 μ L 250mg/mL, be mixed with the Provado tap water sample of 250 μ g/L.
Adopt the ELISA detection method that above sample is detected, operate as follows:
(1) bag quilt
H2-OVA is as encrusting substance, and 1.82 μ g/mL every hole in the trace analysis plate adds 50 μ L, hatches 2 hours for 37 ℃, and evacuation to contain the phosphate buffered saline buffer washing 5 times and the inversion of 0.05% polysorbas20, pats dry on thieving paper;
(2) sealing
1% gelatin is made closure, and the every hole 100 μ L of trace analysis plate were hatched 1.5 hours for 37 ℃, and evacuation to contain the phosphate buffered saline buffer washing 5 times and the inversion of 0.05% polysorbas20, pats dry on thieving paper;
(3) add the mixed solution of antibody and testing sample
Getting testing sample 100 μ L is added in the 900 μ L response matrix and is mixed with liquid to be measured, dilute 16 times cells and supernatant and liquid to be measured and the phosphate buffered saline buffer equal-volume thorough mixing that contains 0.05% polysorbas20, be added in the trace analysis plate with every hole 50 μ L, hatched 0.5 hour for 37 ℃, evacuation, to contain the phosphate buffered saline buffer washing 5 times and the inversion of 0.05% polysorbas20, on thieving paper, pat dry;
(4) add ELIAS secondary antibody
With the phosphate buffered saline buffer that contains 0.05% polysorbas20 of pH 7.4,0.15mol/L with 20000 times of the sheep anti-mouse antibody dilutions of commercial horseradish peroxidase-labeled, the every hole of trace analysis plate adds 50 μ L, hatched 0.5 hour for 37 ℃, evacuation, to contain the phosphate buffered saline buffer washing 5 times and the inversion of 0.05% polysorbas20, on thieving paper, pat dry.
(5) colour developing
The every hole of trace analysis plate adds the tetramethyl benzidine substrate solution of 50 μ L, 37 ℃ of color reactions 10 minutes.
(6) termination reaction and reading
The sulfuric acid that adds 2mol/L, the every hole of trace analysis plate add the reaction of 25 μ L color development stopping, and light absorption value, B read in the 450nm place on the enzyme connection instrument
0=0.805, testing sample light absorption value B=0.193;
(7) data processing
Calculate combination rate B/B
0=23.975%, substitution formula 1
Logit (B/B
0)=In[(B/B
0)/(1-B/B
0)] formula 1
Obtain Logit (B/B
0)=-1.154
For the following detection equation of people:
Logit (B/B
0)+3.7732LogC-2.9435=0 formula 2
Wherein C is the Provado concentration that records in the trace analysis plate hole, tries to achieve C=12.189 μ g/L.Provado residual quantity (X represents with concentration) can be tried to achieve by formula 3 in the testing sample:
X=10 * 2C formula 3
Try to achieve sample to be tested Provado residual quantity concentration X=243.8 μ g/L, its rate of recovery (representing with Y) can be tried to achieve by formula 4:
Rate of recovery Y (%)=actual detected concentration/reality is added concentration * 100% formula 4
Trying to achieve the rate of recovery is 97.5%, illustrates that this antibody can be used for the Provado residual quantity of water sample in the testing environment.
Embodiment 2: detect the imidacloprid pesticide residual quantity in the rice sample
ELISA reaction system underlying condition is with embodiment 1.
Testing sample is prepared
Take by weighing the 1g rice, grind into powder, with concentration is that Provado standardized solution (acetone solution) 1mL of 250 μ g/L adds in the sample of 1g, then the Provado content in the rice sample is 250 μ g/kg, concussion (60r/min) 2 hours, leave standstill several minutes after concussion finishes, get supernatant 100 μ L and be added to 900 μ L and contain in the phosphate buffered saline buffer of 0.05% polysorbas20 and be mixed with liquid to be measured.
Adopt the ELISA detection method that above sample is detected, operate as follows:
Step (1), (2) are with embodiment 1.
(3) add the mixed solution of antibody and testing sample
After diluting 16 times cells and supernatant and liquid to be measured and containing the phosphate buffered saline buffer equal-volume thorough mixing of 0.05% polysorbas20, be added in the trace analysis plate with every hole 50 μ L, hatched 0.5 hour for 37 ℃, evacuation, to contain the phosphate buffered saline buffer washing 5 times and the inversion of 0.05% polysorbas20, on thieving paper, pat dry;
Step (4), (5), (6) are with embodiment 1;
Step reads light absorption value, B in (6)
0=0.857, testing sample light absorption value B=0.190;
(7) data processing
Calculate combination rate B/B
0=22.184%, substitution formula 1
Logit (B/B
0)=In[(B/B
0)/(1-B/B
0)] formula 1
Obtain Logit (B/B
0)=-1.255
For the following detection equation of people:
Logit (B/B
0)+3.7732LogC-2.9435=0 formula 2
Wherein C is the Provado concentration that records in the trace analysis plate hole, tries to achieve C=12.963 μ g/kg.Provado residual quantity (X represents with concentration) can be tried to achieve by formula 3 in the testing sample:
X=10 * 2C formula 3
Try to achieve sample to be tested Provado residual quantity concentration X=259.3 μ g/kg, its rate of recovery (representing with Y) can be tried to achieve by formula 4:
Rate of recovery Y (%)=actual detected concentration/reality is added concentration * 100% formula 4
Trying to achieve the rate of recovery is 103.7%, illustrates that this antibody can be used for detecting the Provado residual quantity of agricultural-food rice.
Claims (3)
1. anti-imidacloprid pesticide monoclonal antibody specific is produced by hybridoma cell strain 2F11/A9, is IgG3 type antibody.
2. the described anti-imidacloprid pesticide MONOCLONAL ANTIBODIES SPECIFIC FOR method of claim 1 comprises:
(1) the imidacloprid pesticide artificial antigen is synthetic:
With immune haptens 1-[6-(2-carboxyethylthio-3-pyridine) methyl]-N-nitro-2-tetrahydroglyoxaline imines, be called for short H1, with bovine serum albumin BSA coupling, obtain Provado artificial immunogen H1-BSA; To wrap by haptens 1-(6-chloro-3-picolyl)-3-carboxylic propyl group-N-nitro-2-tetrahydroglyoxaline imines, be called for short H2,, obtain envelope antigen H2-OVA with ovalbumin OVA coupling;
(2) mouse immune:
With the H1-BSA for preparing as female BALB/c mouse in immunogen immune 6-8 age in week, the immunogen consumption 100 μ g of each every mouse, immunity is four times altogether, first immunisation is by isopyknic immunogen and Freund's complete adjuvant emulsification, abdominal injection, later four immunity are with isopyknic Freund's incomplete adjuvant and immunogen emulsification; For the first time with 3 weeks of interval of immunity for the second time, 2 weeks of interval later on;
From for the third time, each immunity one week of back, the caudal vein blood sampling, with H2-OVA as coating antigen, detect tiring of antibody with non-competing indirect elisa method: be cushioned liquid dilution bag by H2-OVA with the carbonate bag, every hole adds 50 μ L in the 96 hole enzyme plates, hatches 2 hours in 37 ℃, wash 5 times with coating buffer in the enzyme plate to the greatest extent, and with the phosphate buffered saline buffer that contains volume ratio 0.05% polysorbas20;
Gelatin is mixed with 1% confining liquid with phosphate buffered saline buffer dissolving, and every hole adds 100 μ L in the 96 hole enzyme plates, hatches 1.5 hours for 37 ℃, with confining liquid in the enzyme plate to the greatest extent, and washs 5 times with the phosphate buffered saline buffer that contains 0.05% polysorbas20;
Mice serum is diluted with the phosphate buffered saline buffer that contains 0.05% polysorbas20, every hole adds 50 μ L in the 96 hole enzyme plates, use simultaneously without mice immunized serum negative control is set, hatched 0.5 hour for 37 ℃, wash 5 times with reaction solution in the enzyme plate to the greatest extent, and with the phosphate buffered saline buffer that contains 0.05% polysorbas20;
Sheep anti-mouse antibody with the phosphate buffered saline buffer dilution horseradish peroxidase-labeled that contains 0.05% polysorbas20, every hole adds 50 μ L in the 96 hole enzyme plates, hatched 0.5 hour for 37 ℃, reaction solution in the enzyme plate is use up, and wash 5 times with the phosphate buffered saline buffer that contains 0.05% polysorbas20;
Now join tetramethyl benzidine colour developing liquid with substrate buffer solution, every hole adds 50 μ L in the 96 hole enzyme plates, 37 ℃ of dark place reaction 10min;
Every hole adds 2mol/L sulfuric acid 25 μ L, termination reaction in the 96 hole enzyme plates;
Enzyme plate after the color development stopping is measured the single wavelength light absorption value in each hole immediately under 450nm;
(3) cytogamy
Selection detects the highest BALB/c mouse of tiring through indirect non-competing ELISA method, get spleen, the Sp2/0 myeloma cell of splenocyte and logarithmic phase is prepared hybridoma by hybridoma technology, 37 ℃, volume ratio 5% CO2gas incubator was cultivated after 7-10 days, merged hole supernatant screening hybridoma by detecting;
(4) hybridoma screening
Merge the hole supernatant, adopt indirect non-competing ELISA method to carry out preliminary screening, its method is:
Be cushioned liquid dilution coating antigen H2-OVA with the carbonate bag, every hole adds 50 μ L in the 96 hole enzyme plates, hatches 2 hours in 37 ℃, with coating buffer in the enzyme plate to the greatest extent, and washs 5 times with the phosphate buffered saline buffer that contains 0.05% polysorbas20;
Gelatin is mixed with 1% confining liquid with phosphate buffered saline buffer dissolving, and every hole adds 100 μ L in the 96 hole enzyme plates, hatches 1.5 hours for 37 ℃, with confining liquid in the enzyme plate to the greatest extent, and washs 5 times with the phosphate buffered saline buffer that contains 0.05% polysorbas20;
Immune serum is diluted with the phosphate buffered saline buffer that contains 0.05% polysorbas20, every hole adds 50 μ L in the 96 hole enzyme plates, with the supernatant of fused cell not as negative control, with the positive contrast of the serum of immune mouse, hatched 0.5 hour for 37 ℃, wash 5 times with reaction solution in the enzyme plate to the greatest extent, and with the phosphate buffered saline buffer that contains 0.05% polysorbas20;
Sheep anti-mouse antibody with the phosphate buffered saline buffer dilution horseradish peroxidase-labeled that contains 0.05% polysorbas20, every hole adds 50 μ L in the 96 hole enzyme plates, hatched 0.5 hour for 37 ℃, reaction solution in the enzyme plate is use up, and wash 5 times with the phosphate buffered saline buffer that contains 0.05% polysorbas20;
Now join tetramethyl benzidine colour developing liquid with substrate buffer solution, every hole adds 50 μ L in the 96 hole enzyme plates, 37 ℃ of dark place reaction 10min;
Every hole adds 2mol/L sulfuric acid 25 μ L, color development stopping in the 96 hole enzyme plates;
Enzyme plate after the color development stopping is measured the single wavelength light absorption value in each hole immediately under 450nm;
When having the reading of the supernatant of fused cell to be equal to or greater than 2.1 times of negative control reading, such fusion hole is positive hole;
In order to determine that further obtaining the secreted antibody in positive hole is at imidacloprid pesticide, again by the screening of competition indirect elisa method.Its method is:
Be cushioned liquid dilution coating antigen H2-OVA with the carbonate bag, every hole adds 50 μ L in the 96 hole enzyme plates, hatches 2 hours in 37 ℃, with coating buffer in the enzyme plate to the greatest extent, and washs 5 times with the phosphate buffered saline buffer that contains 0.05% polysorbas20;
Gelatin is mixed with 1% confining liquid with phosphate buffered saline buffer dissolving, and every hole adds 100 μ L in the 96 hole enzyme plates, hatches 1.5 hours for 37 ℃, with confining liquid in the enzyme plate to the greatest extent, and washs 5 times with the phosphate buffered saline buffer that contains 0.05% polysorbas20;
Prepare the imidacloprid pesticide standard substance of a series of concentration, acetone solution, after 20 times of the phosphate buffered saline buffer dilutions that contains 0.05% polysorbas20, with cells and supernatant equal-volume thorough mixing, be added in the enzyme plate with every hole 50 μ L, as negative control, hatched 0.5 hour for 37 ℃ with the supernatant of fused cell not and the phosphate buffered saline buffer equal-volume mixed solution that contains 0.05% polysorbas20, wash 5 times with reaction solution in the enzyme plate to the greatest extent, and with the phosphate buffered saline buffer that contains 0.05% polysorbas20;
Sheep anti-mouse antibody with the phosphate buffered saline buffer dilution horseradish peroxidase-labeled that contains 0.05% polysorbas20, every hole adds 50 μ L in the 96 hole enzyme plates, hatched 0.5 hour for 37 ℃, reaction solution in the enzyme plate is use up, and wash 5 times with the phosphate buffered saline buffer that contains 0.05% polysorbas20;
Now join tetramethyl benzidine colour developing liquid with substrate buffer solution, every hole adds 50 μ L in the 96 hole enzyme plates, 37 ℃ of dark place reaction 10min;
Every hole adds 2mol/L sulfuric acid 25 μ L in the 96 hole enzyme plates, color development stopping, and the enzyme plate after the color development stopping is measured the single wavelength light absorption value in each hole immediately under 450nm, and it is B that the positive control light absorption value is made in the hole that does not contain agricultural chemicals with only adding the cell culture supernatant
0, the hole light absorption value that adds agricultural chemicals is B, if B＜B
0, the reaction of prepared antibody capable and agricultural chemicals then is described, such hole is positive hole, and subclone is carried out by limiting dilution assay in positive hole, and its hybridoma excretory supernatant is made monoclonal antibody.
3. produce claim 1 or 2 described hybridoma cell strain 2F11/A9 and be preserved in Chinese typical culture collection center on May 19th, 2010, deposit number is CCTCC NO:C201047.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102507930A (en) * | 2011-11-07 | 2012-06-20 | 石洪波 | Kit for quickly detecting trace imidacloprid residue |
| CN103102415A (en) * | 2012-07-11 | 2013-05-15 | 南京农业大学 | Bispecific monoclonai antibody capable of simultaneously detecting imidacloprid and parathion-methyl |
| CN104101712A (en) * | 2013-04-10 | 2014-10-15 | 北京勤邦生物技术有限公司 | Imidacloprid detection ELISA (enzyme linked immunosorbent assay) kit and application thereof |
| CN104311669A (en) * | 2014-09-24 | 2015-01-28 | 江苏省农业科学院 | Hybridoma cell strain FQ-E7, imidacloprid-resistant monoclonal antibody produced by hybridoma cell strain FQ-E7 and use of imidacloprid-resistant monoclonal antibody |
| CN105137081A (en) * | 2015-04-28 | 2015-12-09 | 南京农业大学 | Monoclonal antibody-based imidacloprid detection test paper strip |
| CN105301233A (en) * | 2015-08-21 | 2016-02-03 | 贵州勤邦食品安全科学技术有限公司 | Enzyme linked immunosorbent assay kit for detecting imidacloprid residue in tea leaves and application method of enzyme linked immunosorbent assay kit |
| CN105510576A (en) * | 2014-10-17 | 2016-04-20 | 镇江亿特生物科技发展有限公司 | Chemiluminescence enzyme-linked immunoassay for detecting imidacloprid |
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| CN115246818A (en) * | 2022-05-25 | 2022-10-28 | 中国医学科学院药用植物研究所 | Hapten, artificial antigen and antibody for detecting imidacloprid as well as preparation methods and applications thereof |
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| CN102507930A (en) * | 2011-11-07 | 2012-06-20 | 石洪波 | Kit for quickly detecting trace imidacloprid residue |
| CN103102415A (en) * | 2012-07-11 | 2013-05-15 | 南京农业大学 | Bispecific monoclonai antibody capable of simultaneously detecting imidacloprid and parathion-methyl |
| CN103102415B (en) * | 2012-07-11 | 2014-07-30 | 南京农业大学 | Bispecific monoclonai antibody capable of simultaneously detecting imidacloprid and parathion-methyl |
| CN104101712A (en) * | 2013-04-10 | 2014-10-15 | 北京勤邦生物技术有限公司 | Imidacloprid detection ELISA (enzyme linked immunosorbent assay) kit and application thereof |
| CN104311669A (en) * | 2014-09-24 | 2015-01-28 | 江苏省农业科学院 | Hybridoma cell strain FQ-E7, imidacloprid-resistant monoclonal antibody produced by hybridoma cell strain FQ-E7 and use of imidacloprid-resistant monoclonal antibody |
| CN105510576A (en) * | 2014-10-17 | 2016-04-20 | 镇江亿特生物科技发展有限公司 | Chemiluminescence enzyme-linked immunoassay for detecting imidacloprid |
| CN105137081A (en) * | 2015-04-28 | 2015-12-09 | 南京农业大学 | Monoclonal antibody-based imidacloprid detection test paper strip |
| CN105301233A (en) * | 2015-08-21 | 2016-02-03 | 贵州勤邦食品安全科学技术有限公司 | Enzyme linked immunosorbent assay kit for detecting imidacloprid residue in tea leaves and application method of enzyme linked immunosorbent assay kit |
| CN105754955A (en) * | 2016-05-17 | 2016-07-13 | 江南大学 | Thiacloprid and acetamiprid monoclonal antibody hybridoma cell strain GW and application thereof |
| CN105754955B (en) * | 2016-05-17 | 2019-02-15 | 江南大学 | One plant of thiacloprid, Acetamiprid monoclonal antibody hybridoma cell strain GW and its application |
| CN115246818A (en) * | 2022-05-25 | 2022-10-28 | 中国医学科学院药用植物研究所 | Hapten, artificial antigen and antibody for detecting imidacloprid as well as preparation methods and applications thereof |
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