CN105754955B - One plant of thiacloprid, Acetamiprid monoclonal antibody hybridoma cell strain GW and its application - Google Patents
One plant of thiacloprid, Acetamiprid monoclonal antibody hybridoma cell strain GW and its application Download PDFInfo
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- CN105754955B CN105754955B CN201610324649.8A CN201610324649A CN105754955B CN 105754955 B CN105754955 B CN 105754955B CN 201610324649 A CN201610324649 A CN 201610324649A CN 105754955 B CN105754955 B CN 105754955B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- 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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- 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
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- 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
One plant of thiacloprid, Acetamiprid monoclonal antibody hybridoma cell strain GW and its application, belong to food safety field of immunological detection.Thiacloprid of the present invention, Acetamiprid comlete antigen are uniformly mixed with equivalent QuickAntibody-Mouse 5W adjuvant, inject BALB/c mouse by leg muscle.Only, only, each immunization interval 21 days is immune with thiacloprid, Acetamiprid comlete antigen (25 μ g/ only, are free of adjuvant) impact for the last time by multiple 50 μ g/ of booster immunization by 100 μ g/ of first immunisation.Take the low IC of high-titer50The splenocyte of mouse is merged by PEG method with murine myeloma cell, is screened by Indirect cELISA and is subcloned three times, obtains a strain of hybridoma strain.The monoclonal antibody of this cell strain secretion has preferable specificity and detection sensitivity (IC to thiacloprid, Acetamiprid50Value is respectively 0.1 ng/mL, 0.4 ng/mL), it can be achieved that detection to thiacloprid, acetamiprid residue amount in water, fruits and vegetables, cereal, the immune detection for thiacloprid in food, acetamiprid residue provides condition, with practical application value.
Description
Technical field
The present invention relates to one plant of thiacloprid, Acetamiprid monoclonal antibody hybridoma cell strain GW and its applications, belong to food
Security immunization detection field.
Background technique
Thiacloprid, Acetamiprid belong to nicotinic insecticide, are the acting bodies of nAChR, at present in agricultural
It is widely used in production, thiacloprid belongs to less toxic insecticides, and Acetamiprid belongs to poisoning insecticide.This two kinds of medicines mainly act on elder brother
Worm nerve engages caudacoria, by the way that in conjunction with nicotinic acetylcholine receptor, interference insect nervous system normal conduction causes nerve logical
The obstruction in road, causes a large amount of accumulation of acetylcholine, to make insect exaltation, general spasticity, paralysis and it is dead.With relatively strong
Tag, stomach toxicity and systemic action.It is chiefly used in water fruits and vegetables etc. and prevents and kill off most of pests.There are article report, nicotinic insecticide
Exposure may have an impact the development of mammalian nervous system.Discovery nicotine is examined in food safety office of European Union (EFSA)
After insecticides are harmful to honeybee, Jin Liangnian European Union has limited use of the nicotinic insecticide medicine on some crops.
Detection thiacloprid, Acetamiprid chromatography are most widely used at present, 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 are complicated, at high cost,
Time is long, can not achieve the quick detection of a large amount of samples, thus establish fast and convenient thiacloprid, Acetamiprid detection method has
Significance.Enzyme-linked immunization (ELISA) be it is a kind of extremely efficiently, sensitive, quick detection method, to the pure of sample when detection
Spend field quick detection of less demanding and easy to operate, suitable for great amount of samples.Efficient immunological detection method is established,
The monoclonal antibody for screening high specific is important prerequisite.
Summary of the invention
The purpose of the present invention is to provide the monoclonals that a kind of thiacloprid, Acetamiprid have high specific and detection sensitivity
The preparation method of antibody hybridoma cell strain.One plant of anti-thiacloprid, Acetamiprid monoclonal antibody hybridoma cell strain are provided, by this
Cell strain preparation antibody to thiacloprid, Acetamiprid have preferably specificity and detection sensitivity, can be used to establish thiacloprid,
The immunological detection method of Acetamiprid.
Technical solution of the present invention, one plant of thiacloprid, Acetamiprid monoclonal antibody hybridoma cell strain GW have been preserved in
State's Microbiological Culture Collection administration committee common micro-organisms center, abbreviation CGMCC, deposit number are CGMCC No.12014.
Anti- thiacloprid, Acetamiprid monoclonal antibody, it is by the thiacloprid that the deposit number is CGMCC No.12014, pyridine
Worm amidine monoclonal antibody hybridoma cell strain GW secretion generates.
The application of the anti-thiacloprid, Acetamiprid monoclonal antibody, for thiacloprid in food safety detection, Acetamiprid
Analysis detection.
The preparation basic step of GW cell strain provided by the invention are as follows:
1) synthesis of haptens:
By Acetamiprid raw medicine 1 (2.2g, 10 mmol), mercaptopropionic acid (2.12 g, 2eq), Cs2CO3(32.6g, 10eq) is mixed
It is suspended from dimethyl adipate (40mL), 16 h(is reacted at 120 DEG C and are stayed overnight), it is cooled to room temperature, after tetrahydrofuran (50 mL) is added
Filtering, obtained solid crude product (35g, the easy moisture absorption) are added in a small amount of water, and prep-HPLC purifying finally obtains 70mg haptens
ATPA.It is identified and is analyzed with LC-MS analytic approach；
2) preparation of complete A antigen TPA-BSA: 5 mg ATPA, 1- ethyls-(3- dimethylaminopropyl) carbon two is weighed
Inferior amine salt hydrochlorate (EDC) 12mg, n-hydroxysuccinimide (NHS) 7mg, with the 300 anhydrous n,N-Dimethylformamide of μ L (DMF)
It dissolves (referred to as A liquid), reaction 8h is stirred at room temperature.10mg bovine serum albumin BSA is weighed, it is molten to be dissolved in 2 mL boric acid (BB) buffering
A liquid is added in B liquid by (referred to as B liquid) dropwise in room temperature condition in liquid, and room temperature reaction is overnight to get conjugate ATPA-BSA
Mixed liquor by small haptens dialysis separation comlete antigen and be not coupled, and is identified by UV absorption scan method；
3) mouse is immune: (Beijing is rich for complete A antigen TPA-BSA and equivalent QuickAntibody-Mouse 5W adjuvant
Ao Long Immune Technology Corp.) it is uniformly mixed, BALB/c mouse is injected by leg muscle.100 μ g/ of first immunisation is only, more
Only, each immunization interval 21 days uses thiacloprid, Acetamiprid complete A antigen TPA-BSA(25 to secondary 50 μ g/ of booster immunization for the last time
μ g/ only, is free of adjuvant) impact is immune；Serum titer and inhibition are detected by Indirect cELISA (ic-ELISA)；
4) cell fusion and cell strain are established: by polyethylene glycol (PEG4000) method by mouse boosting cell and mouse bone marrow cells
Oncocyte fusion detects positive cell hole using indirect competitive ELISA method, and further utilize by HAT culture medium culture
Ic-ELISA measure positive cell hole inhibitory effect, by limiting dilution assay to have the positive cell hole preferably inhibited carry out three
Secondary subclone finally screens and obtains thiacloprid, Acetamiprid monoclonal antibody hybridoma cell strain GW；
5) identification of hybridoma cell strain property: pass through ic-ELISA measurement sensitivity and specificity.
Beneficial effects of the present invention: the monoclonal antibody of cell strain secretion provided by the invention has thiacloprid, Acetamiprid
There are preferable specificity and detection sensitivity (IC50Value is respectively 0.1ng/mL, 0.4ng/mL), it can be achieved that water, fruits and vegetables, cereal
The detection of middle thiacloprid, acetamiprid residue amount, the immune detection for thiacloprid in food, acetamiprid residue provide raw material, have
There is practical 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, abbreviation CGMCC, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese Academy of Sciences microorganism are ground
Study carefully institute, deposit number is CGMCC No.12014, preservation date on January 20th, 2016.
Detailed description of the invention
Inhibition standard curve of Fig. 1 GW monoclonal antibody to thiacloprid.
Inhibition standard curve of Fig. 2 GW monoclonal antibody to Acetamiprid.
Specific embodiment
The following examples of the present invention are only used as the further explanation of the content of present invention, not as a limitation of the present invention interior
Perhaps range.Below by embodiment, the invention will be further described.
The present invention is by being immunized mouse for thiacloprid, Acetamiprid comlete antigen, and by cell fusion, HAT is selectively cultivated
Base culture screens cell conditioned medium by ic-ELISA, and having finally obtained has preferably specificity and sensitivity to thiacloprid, Acetamiprid
Monoclonal antibody hybridoma cell strain.
The preparation of 1 thiacloprid of embodiment, Acetamiprid monoclonal antibody hybridoma cell strain GW
(1) synthesis of haptens: by Acetamiprid raw medicine 1 (2.2g, 10mmol), mercaptopropionic acid (2.12g, 2eq), Cs2CO3
(32.6g, 10eq) is suspended in DMA(40mL) in, 16h(is reacted at 120 DEG C to be stayed overnight), it is cooled to room temperature, tetrahydrofuran is added
(50mL) is filtered afterwards, and obtained solid crude product (35g, the easy moisture absorption) is added in a small amount of water, and prep-HPLC purifying finally obtains 70mg
Haptens ATPA.It is identified and is analyzed with LC-MS analytic approach.
(2) synthesis of comlete antigen: weighing 5mg ATPA, EDC 12mg, NHS 7mg, (is claimed with the dissolution of 300 μ L anhydrous DMFs
For A liquid), reaction 8h is stirred at room temperature.10mg BSA is weighed, (referred to as B liquid) is dissolved in 2mL BB buffer solution, in room temperature item
A liquid is added in B liquid by part dropwise, and room temperature reaction to get conjugate ATPA-BSA mixed liquor, has been separated overnight by dialysis
Holoantigen and the small haptens not being coupled, and identified by UV absorption scan method.
(3) animal immune: the BALB/c mouse of 6~8 week old of health is selected to be immunized.Complete A antigen TPA-BSA with
Equivalent QuickAntibody-Mouse 5W adjuvant is uniformly mixed, and injects BALB/c mouse by leg muscle.First immunisation 100
μ g/ only, only, taken a blood sample, surveyed using ic-ELISA multiple 50 μ g/ of booster immunization for second immune latter 7 days by each immunization interval 21 days
Determine mice serum potency and inhibition, the mouse for selecting potency height to inhibit impacts immune, abdominal cavity note for 21 days after immune at the 4th time
It penetrates, it is desirable that punching is exempted from dosage and halved and without any adjuvant.
(4) cell fusion: after impact immune three days, according to conventional PEG(polyethylene glycol, molecular weight 4000) method into
Row cell fusion, the specific steps are as follows:
A, it plucks eyeball and takes blood, after cervical dislocation puts to death mouse, be immediately placed in 75% alcohol and sterilize, it is left to impregnate 5 min
The spleen of mouse is taken out in the right side, sterile working, is moderately ground with the rubber head of syringe and obtains splenocyte by 200 mesh cell screen clothes
Suspension is collected, and is centrifuged (1200rpm, 8min), is washed splenocyte three times with RPMI-1640 culture medium, after last time is centrifuged,
Splenocyte is diluted to certain volume, is counted, it is spare；
B, it collects SP2/0 oncocyte: 7-10 days before fusion, SP2/0 oncocyte being used and contains 10% FBS(fetal calf serum)
RPMI-1640 culture medium is in 5% CO2It is cultivated in incubator.SP2/0 oncocyte quantity is required to reach 1-4 × 10 before fusion7, protect
SP2/0 oncocyte is in logarithmic growth phase before card merges.When fusion, oncocyte is collected, RPMI-1640 basic culture solution is suspended in
In, carry out cell count；
C, fusion process 7min.The PEG 4000 of 1mL is added drop-wise in cell by 1min from slow to fast；2min, it is quiet
It sets.1mL RPMI-1640 culture medium is added dropwise in 3min and 4min in 1min；5min and 6min is added dropwise in 1min
2mL RPMI-1640 culture medium；The RPMI-1640 culture medium of 1mL is added dropwise in 7min, every 10s.Then 37 DEG C of warm bath 5min.From
The heart (800rpm, 8min) abandons supernatant, the RPMI-1640 screening and culturing liquid into 50 × HAT containing 20% fetal calf serum, 2% is resuspended
In, 96 porocyte plates are added to according to 200 holes μ L/, are placed in 37 DEG C, 5% CO2It is cultivated in incubator.
(5) cell screening and cell strain are established: carrying out RPMI-1640 screening to fused cell within the 3rd day in cell fusion
Culture solution partly changes liquid, carries out within the 5th day being carried out entirely with the RPMI-1640 transition culture solution of 100 × HT containing 20% fetal calf serum, 1%
Liquid is changed, took cell conditioned medium to be screened at the 7th day.Screen in two steps: the first step first filters out positive cell hole with ic-ELISA,
It is standard items that second step, which selects thiacloprid, Acetamiprid, carries out inhibitory effect measurement to positive cell hole with ic-ELISA.Selection pair
Thiacloprid, Acetamiprid standard items have the cell hole preferably inhibited, are subcloned using limiting dilution assay, with same method
It is detected.In triplicate, cell strain GW is obtained.
(6) preparation and identification of monoclonal antibody: taking 8-10 week old BALB/c mouse, and every mouse peritoneal injects sterile stone
Wax oil 1mL；Every mouse peritoneal injection 1 × 10 after 7 days6Hybridoma collects ascites since the 7th day, ascites is passed through
Caprylic acid-ammonium purifying.Under the conditions of meta-acid, caprylic acid can precipitate in ascites except IgG immune globulin it is ultrawhite other are miscellaneous
Albumen is then centrifuged for, and abandons precipitating；Again with the monoclonal antibody of the ammonium sulfate precipitating IgG type of equivalent saturation degree, it is centrifuged,
Supernatant is abandoned, after the dissolution of 0.01 M PBS solution (pH7.4), dialysis desalting, the monoclonal antibody for finally obtaining after purification is placed in-
20 DEG C of preservations.
6.1 coating: by coating antigen ATPA-OVA with 9.6 carbonate buffer solution of 0.05M pH the multiple proportions since 1 μ g/mL
Dilution, 100 holes μ L/, 37 DEG C of 2 h of reaction；
6.2 washings: solution in plate is inclined, and washs 3 times with cleaning solution, every time 3 min；
6.3 closings: after patting dry, 200 hole μ L/ confining liquids, 37 DEG C of reaction 2h are added.It is dried for standby after washing；
6.4 sample-addings: by antiserum since 1:1000 doubling dilution, and be added in the coating hole of each dilution, 100 μ
The hole L/, 37 DEG C of reaction 1h；Sufficiently after washing, the diluted HRP- sheep anti-mouse igg of 1:3000,100 holes μ L/, 37 DEG C of reactions are added
1h；
6.5 colour developings: ELISA Plate is taken out, and sufficiently after washing, the TMB developing solution of 100 μ L is added in every hole, and 37 DEG C are protected from light
15 min；
6.6 terminate and measure: 50 μ L terminate liquids are added to terminate reaction in every hole, and the OD in each hole is then measured with microplate reader
450 values.
With ic-ELISA measurement monoclonal antibody to the IC of thiacloprid, Acetamiprid50It 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): Na is weighed2CO31.59 g, NaHCO32.93 g, after being dissolved in a small amount of distilled water respectively
Mixing adds distilled water to mix to about 800 mL, adjusts pH value to 9.6, add distilled water to be settled to 1000 mL, 4 DEG C of storages are spare；
Phosphate buffer (PBS): 8.00g NaCl, 0.2g KCl, 0.2g KH2PO4, 2.9g Na2HPO4·12 H2O,
It is dissolved in 800mL pure water, with NaOH or HCl tune pH to 7.2~7.4, is settled to 1000mL；
PBST: the PBS containing 0.05 % polysorbas20；
TMB developing solution: A liquid: Na2HPO4 .12H218.43 g of O, 9.33 g of citric acid, pure water are settled to 1000 mL；B
Liquid: 60 mg TMB are dissolved in 100 mL ethylene glycol.A, B liquid 1:5 mixing by volume is TMB developing solution, current
Now mix.
The cross reacting rate of 1. hybridoma cell strain GW monoclonal antibody of table
。
Claims (3)
1. one plant of monoclonal cell strain GW, has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center,
Abbreviation CGMCC, deposit number are CGMCC No.12014.
2. anti-thiacloprid monoclonal antibody, it is characterised in that: its deposit number as described in claim 1 is CGMCC No.12014
Monoclonal cell strain GW secrete generate.
3. the application of anti-thiacloprid monoclonal antibody described in claim 2, it is characterised in that: for thiophene worm in food safety detection
The analysis detection of quinoline.
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Families Citing this family (5)
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| CN106867971B (en) * | 2017-04-27 | 2020-05-05 | 江南大学 | Flunixin meglumine monoclonal antibody hybridoma cell strain YY and application thereof |
| 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 |
| 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 |
| 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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