CN103102415B - Bispecific monoclonai antibody capable of simultaneously detecting imidacloprid and parathion-methyl - Google Patents
Bispecific monoclonai antibody capable of simultaneously detecting imidacloprid and parathion-methyl Download PDFInfo
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- CN103102415B CN103102415B CN201210238321.6A CN201210238321A CN103102415B CN 103102415 B CN103102415 B CN 103102415B CN 201210238321 A CN201210238321 A CN 201210238321A CN 103102415 B CN103102415 B CN 103102415B
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Abstract
The invention relates to a bispecific monoclonai antibody capable of simultaneously recognizing imidacloprid and parathion-methyl pesticides, and a preparation method thereof, belonging to the field of biological technology. The bispecific monoclonai antibody FQ-D6 which can be stably secreted and simultaneously resist imidacloprid and parathion-methyl pesticides is prepared by fusing hybridoma cell capable of secreting anti-parathion-methyl monoclonal antibody and an imidacloprid antigen-immunized rat splenocyte cell through a hydridizatio-hybridoma technique. Proved by effectiveness verification, the antibody can be used for fast and sensitively detecting residuals of the parathion-methyl and the imidacloprid in agricultural production environment and agricultural products. The preparation technology of the bispecific monoclonai antibody capable of simultaneously resisting imidacloprid and parathion-methyl pesticides is simple, convenient and feasible, the whole preparation process of the antibody is not required for special instruments and equipment, thereby being easy for factorization scale production.
Description
(1) technical field
The present invention relates to be applied to detect Provado and parathion-methyl bispecific monoclonal antibody and preparation method simultaneously, belong to biological technical field.Be exclusively used in Provado in agricultural-food and agriculture production environment, residual sensitive, the rapid detection of parathion-methyl, be specially adapted to batch samples and detect.
(2) background technology
Provado (imidacloprid, 1-(6-chloro-3-pyridylmethyl)-N-nitro-2-tetrahydroglyoxaline imines) and parathion-methyl (Parathion-methyl, O, O-dimethyl-O-(4-nitrophenyl) thiophosphatephosphorothioate) be the sterilant being widely used in agriculture production, Provado belongs to moderate toxicity novel nicotinamide insecticide, and parathion-methyl belongs to high malicious organophosphate insecticides.What at present, to these two kinds of agricultural chemicals, the residue detection in agricultural-food adopted is vapor-phase chromatography (GC) and high performance liquid chromatography (HPLC); These two kinds of pesticide residue immune analysis methods are also had to relevant report, but simultaneously for the many method for detecting residue of immunity of these two kinds of agricultural chemicals, have not yet to see report.
Pesticide multi-residues immuno analytical method mainly contains following three kinds: 1., by multiple hapten conjugation to carrier proteins, immune animal obtains antiserum(antisera) and carries out pesticide multi-residues analysis; 2. extract the public structure of a series of agricultural chemicals, design universal hapten, immune animal obtains monoclonal antibody for pesticide multi-residues analysis; 3. bi-specific antibody method.First method makes it on producing, be subject to certain limitation because being merely able to obtain polyclonal antibody; The prepared monoclonal antibody of second method is merely able to identify a class agricultural chemicals, for different types of agricultural chemicals, can not detect simultaneously; And bi-specific antibody has solved this problem well, bi-specific antibody has two different antigen binding sites because of it, and reaches the object that simultaneously detects different sorts agricultural chemicals.
Prepare bi-specific antibody and mainly contain following three kinds of methods: chemical crosslink technique, hybrid-hybridoma technique and genetic engineering antibody technology.The present invention utilizes parathion-methyl hybridoma and the technology that the BALB/C mice splenocyte that is subject to Provado immunogen immune merges, and prepares the secretion trisome hybridoma of anti-Provado-parathion-methyl monoclonal antibody simultaneously.
(3) summary of the invention
Technical problem the object of this invention is to provide a kind of can be used for detecting parathion-methyl and the residual bispecific monoclonal antibody of imidacloprid pesticide and preparation method thereof simultaneously.By the hybridoma of the anti-parathion-methyl monoclonal antibody of secretion and the strategy that the BALB/C mice splenocyte that is subject to Provado immunogen immune merges are prepared to the trisome hybridoma that can secrete anti-parathion-methyl-Provado bispecific antibody.
Technical scheme
1. anti-parathion-methyl and Provado bispecific monoclonal antibody, produced by trisome hybridoma cell strain FQ-D6, is IgG2b-IgG1 type, and light chain of antibody is kappa chain.
2. the preparation method of anti-parathion-methyl and Provado bispecific monoclonal antibody, comprising:
(1) secrete the HGPRT deficient hybridoma cell strain screening of anti-parathion-methyl monoclonal antibody
The hybridoma cell strain of the anti-parathion-methyl antibody of secretion is placed in to 5ug/ml8-AG substratum and cultivates, in culturing process, improve gradually 8-AG concentration in substratum, finally make cell strain can adapt in the substratum of the 8-AG of 50ug/ml and grow; Utilize afterwards HAT selective d MEM substratum to screen, by cell, whether survive the enzyme in identification of cell whether to lack, under deletion condition, clone and enlarged culturing, and cell cryopreservation is standby.
(2) be subject to the preparation of Provado immunogen immune BALB/C mice splenocyte
Exempted from by Provado immunogen 5, serum titer reached more than 1: 32000 BALB/C mice booster immunization after 3 days, and eye socket is got blood and prepared serum, the positive control during as trisome filtering hybridoma; Draw neck dislocation to put to death the mouse after bloodletting, and in 75% alcohol body surface sterilization 10 minutes, move into subsequently in Bechtop, be positioned in sterile petri dish, left abdomen upward; Utilize aseptic operation tweezer and eye scissors to take out mouse spleen, with DMEM basic medium, wash gently, peel off as far as possible spleen fat and reticular tissue around; Spleen is placed in 120 object nylon filter bag, and puts into the culture dish that another fills DMEM basic culture solution; Spleen in nylon filter bag is punctured and constantly extruding, splenocyte is discharged in DMEM basic culture solution completely; Splenocyte suspension is transferred in 50ml centrifuge tube, and centrifugal 10 molecules of 1000rpm, abandon supernatant, and precipitation is washed once again and suspended afterwards with about 30mlDMEM nutrient solution, standby
(3) cytogamy
The HGPRT deficient hybridoma cell strain of splenocyte and the good anti-parathion-methyl antibody of secretion of growth conditions is prepared to trisome hybridoma by hybrid-hybridoma technique.And in 37 ℃, volume ratio 5% CO2gas incubator is cultivated, after 7 days, by non-competing ELISA, detect and merge hole supernatant screening hybridoma.
(4) screening of trisome hybridoma
To merging porocyte supernatant, adopt indirect non-competing ELISA method to carry out preliminary screening, method is:
With the coated damping fluid of carbonate, dilute respectively parathion-methyl and Provado coating antigen, in 96 hole enzyme plates, every hole adds 50ul, and overnight incubation at 4 ℃ by coating buffer in enzyme plate to the greatest extent, and is washed 3 times with the phosphate buffered saline buffer containing 0.05% polysorbas20; Gelatin is configured to 1% confining liquid with ultrapure water, in 96 hole enzyme plates, every hole adds 200ul, hatches 1.5 hours for 37 ℃, by confining liquid in enzyme plate to the greatest extent, and washs 5 times with the phosphate buffered saline buffer containing 0.05% polysorbas20
In 96 hole enzyme plates, every hole adds the fusion porocyte supernatant to be measured that 50ul collects; To with the phosphate buffered saline buffer containing 0.05% polysorbas20, diluted by immune serum, in 96 hole enzyme plates, every hole adds 50ul as positive control; In 96 hole enzyme plates every hole add 50ul not fused cell supernatant as negative control.Hatch 1 hour for 37 ℃, by reaction solution in enzyme plate to the greatest extent, and wash 5 times with the phosphate buffered saline buffer containing 0.05% polysorbas20;
Goat anti-mouse antibody by the phosphate buffered saline buffer dilution horseradish peroxidase-labeled containing 0.05% polysorbas20, in 96 hole enzyme plates, every hole adds 50ul, at 37 ℃, hatch 45 minutes, by reaction solution in enzyme plate to the greatest extent, and wash 5 times with the phosphate buffered saline buffer containing 0.05% polysorbas20;
With Trisodium Citrate substrate buffer solution, now join tetramethyl benzidine nitrite ion, in 96 hole enzyme plates, every hole adds 50ul, under 37 ℃ of dark conditions, reacts 10 minutes;
In 96 hole enzyme plates, every hole adds 2mol/L sulfuric acid 25ul, color development stopping;
Enzyme plate after color development stopping is measured the single wavelength light absorption value in each hole immediately under 450nm;
Under two kinds of coating antigens, while having the supernatant liquor reading of fused cell to be equal to or to be greater than 2.1 times of negative control reading, such fusion hole is positive hole;
In order further to determine that screening the secreted antibody in positive hole is the bispecific monoclonal antibody of identifying parathion-methyl and Provado simultaneously, then screen by indirect competitive ELISA method, method is:
With the coated damping fluid of carbonate, dilute respectively parathion-methyl, Provado coating antigen, in 96 hole enzyme plates, every hole adds 50ul, and overnight incubation at 4 ℃ by coating buffer in enzyme plate to the greatest extent, and is washed 5 times with the phosphate buffered saline buffer containing 0.05% polysorbas20;
Gelatin is configured to 1% confining liquid with ultrapure water, in 96 hole enzyme plates, every hole adds 200ul, hatches 1.5 hours for 37 ℃, by confining liquid in enzyme plate to the greatest extent, and washs 5 times with the phosphate buffered saline buffer containing 0.05% polysorbas20;
Configure respectively parathion-methyl and the imidacloprid pesticide standard substance methanol solution of a series of concentration, with after 20 times of the phosphate buffered saline buffer dilutions containing 0.05% polysorbas20, fully mix with cell culture supernatant equal-volume; Two kinds of agricultural chemicals-cell conditioned medium mixed solutions are added in enzyme plate with every hole 50ul; Think fused cell supernatant and contain 0.05% polysorbas20 phosphate buffered saline buffer equal-volume mixed solution as negative control; Hatch 1 hour for 37 ℃, by reaction solution in enzyme plate to the greatest extent, and wash 5 times with the phosphate buffered saline buffer containing 0.05% polysorbas20;
Goat anti-mouse antibody by the phosphate buffered saline buffer dilution horseradish peroxidase-labeled containing 0.05% polysorbas20, in 96 hole enzyme plates, every hole adds 50ul, at 37 ℃, hatch 45 minutes, by reaction solution in enzyme plate to the greatest extent, and wash 5 times with the phosphate buffered saline buffer containing 0.05% polysorbas20;
With Trisodium Citrate substrate buffer solution, now join tetramethyl benzidine nitrite ion, in 96 hole enzyme plates, every hole adds 50ul, under 37 ℃ of dark conditions, reacts 10 minutes;
In 96 hole enzyme plates, every hole adds 2mol/L sulfuric acid 25ul, color development stopping; After color development stopping, enzyme plate is measured the single wavelength light absorption value in each hole immediately under 450nm.Under setting parathion-methyl coating antigen is coated, the reading that does not contain the cell conditioned medium of agricultural chemicals is B
01, the cell conditioned medium reading that contains parathion-methyl agricultural chemicals is B
11, the cell conditioned medium liquid reading that contains imidacloprid pesticide is B
12; Under Provado coating antigen is coated, the reading that does not contain the cell conditioned medium of agricultural chemicals is B
02, the cell conditioned medium reading that contains parathion-methyl agricultural chemicals is B
21, the cell conditioned medium liquid reading that contains imidacloprid pesticide is B
22.When there is following phenomenon: B simultaneously
11< B
01, B
12=B
01, B
21< B
02, B
22=B
02, just can assert that the antibody of its secretion is bispecific monoclonal antibody, and utilize limiting dilution assay to carry out subclone to cell, the supernatant of its secretion is made bispecific monoclonal antibody.
The hybridoma cell strain Classification And Nomenclature that produces above-mentioned bispecific monoclonal antibody is: hybridoma cell strain FQ-D6; On July 10th, 2012 is preserved in Chinese Typical Representative culture collection center (China, Wuhan, Wuhan University), and deposit number is: CCTCC NO:C201274
Beneficial effect
Practicality is good: traditional analysis of agricultural drugs is mainly carried out in laboratory, its pretreatment process is loaded down with trivial details, analysis speed is slow, cost is high, the a large amount of organic solvents that simultaneously use easily cause environmental pollution, and higher to the instrumentation degree in laboratory and personnel specialty competency profiling, can not meet in Agricultural Products Trade requirements quick, easy, accurate, a large amount of detections.And we provide, take the advantages such as antigen antibody reaction is easy and simple to handle, quick as basic ELISA (Enzyme Linked Immunoadsorbent Assay) method has, analysis cost is low, safe and reliable, generally do not need expensive instrument, can simplify in a large number sample pretreatment process, professional technique to user of service is less demanding, easily universal and popularization, is specially adapted to pattern detection in enormous quantities and Site Detection.
Sensing range is wide: the bispecific monoclonal antibody that utilizes hybrid-hybridoma technique to prepare, there is the characteristic that simultaneously detects two class pesticide structures, and compare conventional monoclonal antibody, sensing range is wider, and application prospect is better.
Antibody good stability: the hybridoma that this method obtains at least can be preserved 3 years under liquid nitrogen condition, purified antibody can be deposited 2 years under-20 ℃ of conditions
Accompanying drawing explanation
(1) standard working curve of Provado, parathion-methyl
Embodiment
Provado, parathion-methyl residues detection in embodiment 1 tap water sample
ELISA reaction system underlying condition
Provado coating antigen concentration is 4.88ug/ml, and ascites extension rate is 2000 times; Parathion-methyl coating antigen concentration is 2.3ug/ml, and ascites extension rate is 4000 times.1% gelatin is made confining liquid, and in antigen antibody reaction liquid (containing the phosphate buffered saline buffer of 0.05% polysorbas20), methanol content is 5%, pH value is 8.5, ionic strength is 0.8mol/L, every hole 50ul in trace analysis plate.
Testing sample is prepared
Tap water sample: Provado and parathion-methyl agricultural chemicals are made into the methanol solution of different concns, Provado concentration is respectively 6,12ug/ml, and parathion-methyl concentration is respectively 0.64,1.28ug/ml.By above agricultural chemicals methanol solution, with after 20 times of tap water dilutions, pesticide concentration is respectively: Provado 300,600ng/ml, parathion-methyl 32,64ng/ml.
Adopt ELISA detection method to detect above sample, operate as follows:
(1) coated
Provado coating antigen concentration is 4.88ug/ml, and parathion-methyl coating antigen concentration is 2.3ug/ml, and in 96 hole enzyme plates, every hole adds 50ul, 4 ℃ of overnight incubation, to the greatest extent,
(2) sealing
1% gelatin is made closure, and in 96 hole enzyme plates, every hole adds 200ul, hatches 1.5 hours for 37 ℃, to the greatest extent, with the phosphate buffered saline buffer washing containing 0.05% polysorbas20 5 times, and pats dry;
(3) application of sample
By the Provado of above-mentioned preparation, parathion-methyl solution, be to add in enzyme plate after the secreted antibody-solutions equal-volume of CCTCC NO:C201274 trisome hybridoma mixes with deposit number, in 96 hole enzyme plates, every hole adds 50ul, hatch 1 hour for 37 ℃, wash 5 times by reaction solution in enzyme plate to the greatest extent, and with the phosphate buffered saline buffer that contains 0.05% polysorbas20;
(4) add ELIAS secondary antibody
Goat anti-mouse antibody by the phosphate buffered saline buffer dilution horseradish peroxidase-labeled containing 0.05% polysorbas20, in 96 hole enzyme plates, every hole adds 50ul, at 37 ℃, hatch 45 minutes, by reaction solution in enzyme plate to the greatest extent, and wash 5 times with the phosphate buffered saline buffer containing 0.05% polysorbas20;
(5) data processing
By obtained combination rate (B/B
0) in substitution Fig. 1 in the typical curve of Provado, parathion-methyl, obtain the concentration of Provado and parathion-methyl, obtained concentration data is multiplied by multiple 2.And compare with adding concentration, obtaining the interpolation rate of recovery of Provado in tap water is 97%-104%, the interpolation rate of recovery of parathion-methyl is 87%-99%.
Provado, parathion-methyl residues detection in embodiment 2 pond water samples
ELISA reaction system underlying condition is with embodiment 1
Testing sample is prepared
Pond water sample: the pond water of collecting is centrifugal, get supernatant liquor and test.Provado and parathion-methyl agricultural chemicals are made into the methanol solution of different concns, and Provado concentration is respectively 6,12ug/ml, and parathion-methyl concentration is respectively 0.64,1.28ug/ml.By above agricultural chemicals methanol solution, with after 20 times of pond water dilutions, pesticide concentration is respectively: Provado 300,600ng/ml, parathion-methyl 32,64ng/ml.
Adopt ELISA detection method to detect above sample, operate as follows:
Step (1), (2) are with embodiment 1
(3) application of sample
After being mixed with antibody-solutions equal-volume, the Provado of above-mentioned preparation, parathion-methyl solution adds in enzyme plate, in 96 hole enzyme plates, every hole adds 50ul, hatch 1 hour for 37 ℃, by reaction solution in enzyme plate to the greatest extent, and wash 5 times with the phosphate buffered saline buffer containing 0.05% polysorbas20;
(4) add ELIAS secondary antibody
Goat anti-mouse antibody by the phosphate buffered saline buffer dilution horseradish peroxidase-labeled containing 0.05% polysorbas20, in 96 hole enzyme plates, every hole adds 50ul, at 37 ℃, hatch 45 minutes, by reaction solution in enzyme plate to the greatest extent, and wash 5 times with the phosphate buffered saline buffer containing 0.05% polysorbas20;
(5) data processing
By obtained combination rate (B/B
0) in substitution Fig. 1 in the typical curve of Provado, parathion-methyl, obtain the concentration of Provado and parathion-methyl, and obtained concentration numerical value is multiplied by multiple 2.And compare with adding concentration, obtaining the interpolation rate of recovery of Provado in pond water is 102%-104%, the interpolation rate of recovery of parathion-methyl is 102%-104%.
Claims (2)
1. detect a bispecific monoclonal antibody for Provado and parathion-methyl, the trisome hybridoma cell strain FQ-D6 that is CCTCC NO:C201274 by deposit number produces simultaneously, is IgG2b-IgG1 type, and light chain of antibody is kappa chain.
2. produce the hybridoma cell strain FQ-D6 that simultaneously detects the bispecific monoclonal antibody of Provado and parathion-methyl claimed in claim 1, on July 10th, 2012, be preserved in Chinese Typical Representative culture collection center, deposit number is CCTCC NO:C201274.
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| CN103308697B (en) * | 2013-06-09 | 2014-11-26 | 卫生部北京医院 | Kit used for detecting natural bispecific antibody resistant to HCV (hepatitis C virus) coded non-structural proteins NS3 and NS5 |
| CN107305213A (en) * | 2016-04-25 | 2017-10-31 | 赵芳 | A kind of method and kit for detecting organophosphate and carbamate pesticide |
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