CN105754955A - Thiacloprid and acetamiprid monoclonal antibody hybridoma cell strain GW and application thereof - Google Patents

Thiacloprid and acetamiprid monoclonal antibody hybridoma cell strain GW and application thereof Download PDF

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CN105754955A
CN105754955A CN201610324649.8A CN201610324649A CN105754955A CN 105754955 A CN105754955 A CN 105754955A CN 201610324649 A CN201610324649 A CN 201610324649A CN 105754955 A CN105754955 A CN 105754955A
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thiacloprid
acetamiprid
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cell strain
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CN105754955B (en
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胥传来
姚蕾珺
匡华
徐丽广
马伟
刘丽强
吴晓玲
宋珊珊
胡拥明
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Jiangnan University
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
    • G01N2430/10—Insecticides

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Abstract

The invention discloses a thiacloprid and acetamiprid monoclonal antibody hybridoma cell strain GW and application thereof, and belongs to the field of food security immunodetection. Thiacloprid and acetamiprid complete antigen of the strain is uniformly mixed with an equal amount of QuickAntibody-Mouse 5W adjuvant, and is injected to BALB/c mice through leg muscle. The dosage is 100mu g/mouse for the first time of immunization, the dosage is 50[mu]g/mouse for multiple times of intensified immunization, the immunization is implemented at an interval of 21 days, and thiacloprid and acetamiprid complete antigen (25[mu]g/mouse, without adjuvant) is adopted for immunization impact for the last time. Splenocyte of high-potency low IC50 mice is taken and fused with mouse myeloma cells by using a PEG method, indirect competitive inhibition enzyme-linked immunosorbent assay is adopted for screening, and three times of subcloning is implemented, so as to obtain the hybridoma cell strain. A monoclonal antibody secreted from the cell strain has relatively good specificity and detection sensitivity (the IC50 values are 0.1ng/mL and 0.4ng/mL respectively) for thiacloprid and acetamiprid, detection on the residual amounts of thiacloprid and acetamiprid in water, fruits and vegetables and cereals can be achieved, conditions are provided for immunodetection on thiacloprid and acetamiprid residues in food can be provided, and practical use values can be made.

Description

One strain thiacloprid, Acetamiprid monoclonal antibody hybridoma cell strain GW and application thereof
Technical field
The present invention relates to a strain thiacloprid, Acetamiprid monoclonal antibody hybridoma cell strain GW and application thereof, belong to food Security immunization detection field.
Background technology
Thiacloprid, Acetamiprid all belong to nicotinic insecticide, are the acting bodies of nAChR, at present in agricultural Being widely used in production, thiacloprid belongs to lower toxicity insecticide, and Acetamiprid belongs to poisoning insecticide.These two kinds of medicines mainly act on elder brother Worm nerve engages caudacoria, by being combined, interference insect nervous system normal conduction with nAChR, causes neural logical The obstruction in road, causes a large amount of accumulation of acetylcholine so that insecticide exaltation, general spasticity, paralysis and dead.Have stronger Tag, stomach toxicity and systemic action.It is used for water fruits and vegetables etc. and prevents and kill off most of insect.Article is had to report, nicotinic insecticide Exposure may produce impact to the growth of mammalian nervous system.Examine in food safety office of European Union (EFSA) and find nicotine After Apis is harmful to by insecticides, Jin Liangnian European Union has limited the use on some crops of the nicotinic insecticide medicine.
Detect thiacloprid at present, Acetamiprid chromatography is most widely used, including gas chromatography (GC), high-efficient liquid phase color Spectrometry (HPLC), gas chromatography mass spectrometry (GC-MS), LC-MS (LC-MS), supercritical fluid chromatography (SFC), capillary electrophoresis (CE) Deng instrumental method, but these methods need expensive instrument, professional operator, and sample pre-treatments complicated, cost is high, Time is long, it is impossible to realize the quick detection of a large amount of sample, hence sets up fast and convenient thiacloprid, Acetamiprid detection method has Significance.Euzymelinked immunosorbent assay (ELISA) (ELISA) is one detection method the most efficient, sensitive, quick, pure to sample during detection Spend less demanding and easy and simple to handle, it is adaptable to the field quick detection of great amount of samples.Set up efficient immunological detection method, The monoclonal antibody of screening high specific is important prerequisite.
Summary of the invention
It is an object of the invention to provide a kind of thiacloprid, Acetamiprid has high specific and the monoclonal of detection sensitivity The preparation method of antibody hybridoma cell strain.There is provided an anti-thiacloprid of strain, Acetamiprid monoclonal antibody hybridoma cell strain, by this Antibody prepared by cell strain has preferable specificity and detection sensitivity to thiacloprid, Acetamiprid, can be used to set up thiacloprid, The immunological detection method of Acetamiprid.
Technical scheme, a strain thiacloprid, Acetamiprid monoclonal antibody hybridoma cell strain GW, in being preserved in State's Microbiological Culture Collection administration committee common micro-organisms center, is called for short CGMCC, and deposit number is CGMCC No.12014.
Anti-thiacloprid, Acetamiprid monoclonal antibody, it is the thiacloprid of CGMCC No.12014, pyridine by described deposit number Worm amidine monoclonal antibody hybridoma cell strain GW secretes generation.
Described anti-thiacloprid, the application of Acetamiprid monoclonal antibody, thiacloprid, Acetamiprid in food safety detection Analyze detection.
The preparation basic step of the GW cell strain that the present invention provides is:
1) haptenic synthesis:
By former for Acetamiprid medicine 1 (2.2g, 10 mmol), mercaptopropionic acid (2.12 g, 2eq), Cs2CO3(32.6g, 10eq) suspendible In dimethyl adipate (40mL), react 16 h(at 120 DEG C overnight), it is cooled to room temperature, adds oxolane (50 mL) mistake afterwards Filter, gained solid crude product (35g, the easy moisture absorption) adds in a small amount of water, and prep-HPLC purification finally obtains 70mg hapten ATPA. Identify by LC-MS analytic process and analyze；
2) preparation of complete A antigen TPA-BSA: weigh 5 mg ATPA, 1-ethyl-(3-dimethylaminopropyl) carbodiimide Hydrochlorate (EDC) 12mg, N-hydroxy-succinamide (NHS) 7mg, dissolve with the anhydrous DMF of 300 μ L (DMF) (referred to as A liquid), is stirred at room temperature reaction 8h.Weigh 10mg bovine serum albumin BSA, be dissolved in 2 mL boric acid (BB) buffer solution (referred to as B liquid), at room temperature condition, dropwise joins in B liquid by A liquid, and room temperature reaction overnight, obtains conjugate ATPA-BSA mixing Liquid, is separated complete antigen and the small haptens of non-coupling by dialysis, and is identified by uv absorption scan method；
3) immunity of mice: complete A antigen TPA-BSA and equivalent QuickAntibody-Mouse 5W adjuvant (Beijing Bo Aolong Immune Technology Corp.) mix homogeneously, inject BALB/c mouse by leg muscle.First immunisation 100 μ g/ only, repeatedly adds Only, each immunization interval 21 days, for the last time with thiacloprid, Acetamiprid complete A antigen TPA-BSA(25 μ g/ for the most immune 50 μ g/ Only, without adjuvant) impact immunity；By Indirect cELISA (ic-ELISA) detection serum titer and suppression；
4) cell merges with cell strain foundation: by Polyethylene Glycol (PEG4000) method by thin to mouse boosting cell and mouse myeloma Born of the same parents are merged, and by HAT culture medium culturing, utilize indirect competitive ELISA method to detect positive cell hole, and further with ic- ELISA measures the inhibition in positive cell hole, by limiting dilution assay to there being the positive cell hole preferably suppressed to carry out three times Sub-clone, final screening obtains thiacloprid, Acetamiprid monoclonal antibody hybridoma cell strain GW；
5) qualification of hybridoma cell strain character: measure sensitivity and specificity by ic-ELISA.
Beneficial effects of the present invention: the monoclonal antibody of the cell strain secretion that the present invention provides, to thiacloprid, Acetamiprid tool There are preferable specificity and detection sensitivity (IC50Value is respectively 0.1ng/mL, 0.4ng/mL), can realize water, fruit and vegerable, corn Middle thiacloprid, the detection of acetamiprid residue amount, provide raw material for thiacloprid, the immune detection of acetamiprid residue in food, tool There is actual application value.
Biological material specimens preservation: monoclonal cell strain GW, has been preserved in China Committee for Culture Collection of Microorganisms Common micro-organisms center, is called for short CGMCC, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese Academy of Sciences's microorganism is ground Studying carefully institute, deposit number is CGMCC No.12014, preservation date on January 20th, 2016.
Accompanying drawing explanation
Fig. 1. the GW monoclonal antibody suppression standard curve to thiacloprid.
Fig. 2. the GW monoclonal antibody suppression standard curve to Acetamiprid.
Detailed description of the invention
The following examples of the present invention are only used as further illustrating of present invention, it is impossible to as in the restriction of the present invention Perhaps scope.Below by embodiment, the invention will be further described.
The present invention is by by thiacloprid, Acetamiprid complete antigen immune mouse, merging by cell, and HAT selectivity is cultivated Base is cultivated, and screens cell conditioned medium by ic-ELISA, has finally given and thiacloprid, Acetamiprid are had preferable specificity and sensitivity Monoclonal antibody hybridoma cell strain.
Embodiment 1 thiacloprid, the preparation of Acetamiprid monoclonal antibody hybridoma cell strain GW
(1) haptenic synthesis: by former for Acetamiprid medicine 1 (2.2g, 10mmol), mercaptopropionic acid (2.12g, 2eq), Cs2CO3 (32.6g, 10eq) is suspended in DMA(40mL) in, react 16h(at 120 DEG C overnight), it is cooled to room temperature, adds oxolane (50mL) filtering after, gained solid crude product (35g, the easy moisture absorption) adds in a small amount of water, and prep-HPLC purification finally obtains 70mg Hapten ATPA.Identify by LC-MS analytic process and analyze.
(2) synthesis of complete antigen: weigh 5mg ATPA, EDC 12mg, NHS 7mg, dissolves by 300 μ L dry DMF and (claims For A liquid), reaction 8h is stirred at room temperature.Weigh 10mg BSA, be dissolved in 2mL BB buffer solution (referred to as B liquid), at room temperature bar Part, dropwise joins in B liquid by A liquid, and room temperature reaction overnight, obtains conjugate ATPA-BSA mixed liquor, has been separated by dialysis Holoantigen and the small haptens of non-coupling, and identified by uv absorption scan method.
(3) animal immune: select the BALB/c mouse of 6～8 healthy week old to carry out immunity.Complete A antigen TPA-BSA with Equivalent QuickAntibody-Mouse 5W adjuvant mix homogeneously, injects BALB/c mouse by leg muscle.First immunisation 100 Only, repeatedly booster immunization 50 μ g/ is only, each immunization interval 21 days for μ g/, and second time immunity blood sampling in latter 7 days uses ic-ELISA to survey Determining mice serum titer and suppression, select the mice that titer height has suppressed, in the 4th immunity impact immunity in latter 21 days, abdominal cavity is noted Penetrate, it is desirable to punching is exempted from dosage and halved and without any adjuvant.
(4) cell merges: after impact immunity three days, according to conventional PEG(Polyethylene Glycol, molecular weight is 4000) method enters Row cell merges, and specifically comprises the following steps that
A, pluck eyeball and take blood, after cervical dislocation puts to death mice, be immediately placed in 75% ethanol sterilization, soak about 5 min, nothing The spleen of mice is taken out in bacterium operation, grinds by the glue head appropriateness of syringe and obtains splenocyte suspension by 200 mesh cell screen clothes, Collect, centrifugal (1200rpm, 8min), wash splenocyte three times by RPMI-1640 culture medium, after being centrifuged for the last time, spleen is thin Born of the same parents are diluted to certain volume, counting, standby；
B, collection SP2/0 oncocyte: merge first 7-10 days, by SP2/0 oncocyte with containing 10% FBS(hyclone) RPMI- 1640 culture medium are at 5% CO2Incubator is cultivated.Require before fusion that SP2/0 oncocyte quantity reaches 1-4 × 107, it is ensured that merge Front SP2/0 oncocyte is in exponential phase.During fusion, collect oncocyte, be suspended in RPMI-1640 basic culture solution, enter Row cell counting；
C, fusion process 7min.1min, is added drop-wise in cell from slow to fast by the PEG 4000 of 1mL；2min, stands.The 3min and 4min, drips 1mL RPMI-1640 culture medium in 1min；5min and 6min, drips 2mL in 1min RPMI-1640 culture medium；The RPMI-1640 culture medium of 7min, every 10s dropping 1mL.Then 37 DEG C of temperature bath 5min.Centrifugal (800rpm, 8min), abandons supernatant, resuspended enter containing 20% hyclone, the 50 × HAT of 2% RPMI-1640 screening and culturing liquid in, It is added to 96 porocyte plates according to 200 μ L/ holes, is placed in 37 DEG C, 5% CO2Incubator is cultivated.
(5) cell screening is set up with cell strain: fused cell was carried out RPMI-1640 screening in the 3rd day what cell merged Culture fluid partly changes liquid, within the 5th day, carries out carrying out entirely with the RPMI-1640 transition culture fluid containing 20% hyclone, the 100 × HT of 1% Change liquid, took cell conditioned medium at the 7th day and screen.Screen in two steps: the first step first filters out positive cell hole with ic-ELISA, Second step selects thiacloprid, Acetamiprid to be standard substance, with ic-ELISA, positive cell hole is carried out inhibition mensuration.It is right to select Thiacloprid, Acetamiprid standard substance all have the cell hole of preferably suppression, use limiting dilution assay to carry out sub-clone, by same method Detect.In triplicate, it is thus achieved that cell strain GW.
(6) preparation of monoclonal antibody and qualification: take 8-10 week old BALB/c mouse, every mouse peritoneal injects aseptic stone Wax oil 1mL；Every mouse peritoneal injection 1 × 10 after 7 days6Hybridoma, started to collect ascites from the 7th day, ascites is passed through Caprylic acid-ammonium purification.Under the conditions of meta-acid, caprylic acid can precipitate in ascites except ultrawhite other of IgG immune globulin are miscellaneous Albumen, is then centrifuged for, and abandons precipitation；It is again by the monoclonal antibody of the ammonium sulfate precipitation IgG type of equivalent saturation, centrifugal, Abandon supernatant, with 0.01 M PBS solution (pH7.4) dissolve after, dialysis desalting, finally give monoclonal antibody after purification be placed in- 20 DEG C of preservations.
6.1 are coated: from 1 g/mL, coating antigen ATPA-OVA 0.05M pH 9.6 carbonate buffer solution is started multiple proportions Dilution, 100 μ L/ holes, 37 DEG C of reaction 2 h；
6.2 washings: solution in plate is inclined, and wash 3 times with cleaning mixture, each 3 min；
6.3 close: after patting dry, and add 200 μ L/ hole confining liquids, 37 DEG C of reaction 2h.Washing post-drying is standby；
6.4 sample-addings: antiserum is started doubling dilution from 1:1000, and join each dilution be coated in hole, 100 μ L/ holes, 37 DEG C of reaction 1h；Fully after washing, add the HRP-sheep anti-mouse igg of 1:3000 dilution, 100 μ L/ holes, 37 DEG C of reaction 1h；
After 6.5 colour developings: ELISA Plate taken out, fully washing, every hole adds the TMB nitrite ion of 100 μ L, 37 DEG C of lucifuge reactions 15 min；
6.6 terminate and measure: every hole adds 50 μ L stop buffers to terminate reaction, then with the OD 450 in each hole of microplate reader mensuration Value.
Monoclonal antibody is measured to thiacloprid, the IC of Acetamiprid with ic-ELISA50It is respectively as follows: 0.1 ng/mL, 0.4ng/ ML, illustrates that antibody has good sensitivity to thiacloprid, Acetamiprid, can be used for thiacloprid, the detection of Acetamiprid immunoassay.
The configuration of solution:
Carbonate buffer solution (CBS): weigh Na2CO31.59 g, NaHCO32.93 g, mixed after being dissolved in a small amount of distilled water respectively Closing, add distilled water and mix to about 800 mL, adjust pH value to 9.6, add distilled water and be settled to 1000 mL, 4 DEG C of storages are standby；
Phosphate buffer (PBS): 8.00g NaCl, 0.2g KCl, 0.2g KH2PO4, 2.9g Na2HPO4·12 H2O, molten In 800mL pure water, adjust pH to 7.2～7.4 with NaOH or HCl, be settled to 1000mL；
PBST: containing the PBS of 0.05 % polysorbas20；
TMB nitrite ion: A liquid: Na2HPO4 .12H2O 18.43 g, citric acid 9.33 g, pure water is settled to 1000 mL；B liquid: 60 Mg TMB is dissolved in 100 mL ethylene glycol.A, B liquid 1:5 by volume mixing is TMB nitrite ion, existing use
Now mix.
The cross reacting rate of table 1. hybridoma cell strain GW monoclonal antibody
。

Claims (3)

1. a strain thiacloprid, Acetamiprid monoclonal antibody hybridoma cell strain GW, be preserved in Chinese microorganism strain preservation pipe Reason committee's common micro-organisms center, is called for short CGMCC, and deposit number is CGMCC No.12014.
The most anti-thiacloprid, Acetamiprid monoclonal antibody, it is characterised in that: it is CGMCC No.12014's by described deposit number Thiacloprid, Acetamiprid monoclonal antibody hybridoma cell strain GW secrete generation.
3. anti-thiacloprid, the application of Acetamiprid monoclonal antibody described in claim 2, it is characterised in that: examine for food safety The analysis detection of thiacloprid, Acetamiprid in survey.
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CN106867971A (en) * 2017-04-27 2017-06-20 江南大学 One plant of flunixin meglumine monoclonal antibody hybridoma cell strain YY and its application
CN107652365A (en) * 2017-09-12 2018-02-02 杨蕾 A kind of method for preparing four kinds of general antigen of chlorination nicotinic insecticide
CN109061146A (en) * 2018-09-21 2018-12-21 中国烟草总公司郑州烟草研究院 A kind of test strips and its preparation method and application detecting Acetamiprid
CN109991419A (en) * 2019-03-18 2019-07-09 浙江大学 Nicotinic insecticide thiacloprid method for detecting residue based on SPR technique
CN112111010A (en) * 2020-07-28 2020-12-22 浙江大学 Variable region sequence of specific anti-thiacloprid antibody and recombinant complete antibody thereof

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CN107652365A (en) * 2017-09-12 2018-02-02 杨蕾 A kind of method for preparing four kinds of general antigen of chlorination nicotinic insecticide
CN109061146A (en) * 2018-09-21 2018-12-21 中国烟草总公司郑州烟草研究院 A kind of test strips and its preparation method and application detecting Acetamiprid
CN109061146B (en) * 2018-09-21 2021-07-16 中国烟草总公司郑州烟草研究院 Test strip for detecting acetamiprid and preparation method and application thereof
CN109991419A (en) * 2019-03-18 2019-07-09 浙江大学 Nicotinic insecticide thiacloprid method for detecting residue based on SPR technique
CN112111010A (en) * 2020-07-28 2020-12-22 浙江大学 Variable region sequence of specific anti-thiacloprid antibody and recombinant complete antibody thereof
CN112111010B (en) * 2020-07-28 2022-04-19 浙江大学 Variable region sequence of specific anti-thiacloprid antibody and recombinant complete antibody thereof

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