CN105754954B - One plant of imidacloprid monoclonal antibody hybridoma cell strain YH5 and its application - Google Patents

One plant of imidacloprid monoclonal antibody hybridoma cell strain YH5 and its application Download PDF

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CN105754954B
CN105754954B CN201610324646.4A CN201610324646A CN105754954B CN 105754954 B CN105754954 B CN 105754954B CN 201610324646 A CN201610324646 A CN 201610324646A CN 105754954 B CN105754954 B CN 105754954B
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imidacloprid
monoclonal antibody
adjuvant
cell strain
detection
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CN105754954A (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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    • 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
    • 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
    • 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 imidacloprid monoclonal antibody hybridoma cell strain YH5 and its application, belong to food safety field of immunodetection.Imidacloprid comlete antigen of the present invention and equivalent Freund's adjuvant mixing and emulsifying are complete, and BALB/c mouse is immunized by dorsal sc injection.First immunisation (100 μ g/ are only) complete Freund's adjuvant, multiple booster immunization (50 μ g/ are only) cannots be used up full Freund's adjuvant, immune with imidacloprid comlete antigen (25 μ g/ only, are free of adjuvant) impact for the last time.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 imidacloprid50It is worth for 0.3 ng/mL) detection, it can be achieved that Determination of Imidacloprid Residue amount in water, fruits and vegetables, cereal, the immune detection for Determination of Imidacloprid Residue in food provides raw material, has practical application value.

Description

One plant of imidacloprid monoclonal antibody hybridoma cell strain YH5 and its application
Technical field
The present invention relates to one plant of imidacloprid monoclonal antibody hybridoma cell strain YH5 and its applications, belong to food safety and exempt from Epidemic disease detection field.
Background technique
Imidacloprid is a kind of efficient absorbability broad spectrum pesticide of novel nitro-methylene-type, belongs to anabasine drug, It is the acting body of nAChR, is widely used in agricultural production at present, belongs to less toxic insecticides, to honeybee Toxicity be high poison.2002, there is honeybee during using imidacloprid and largely disappear in some areas of North of Italy and France The phenomenon that mistake, anabasine drug belong to neurotoxin, and the receptor in honeybee and other insect brains can be hindered to receive mind Through signal.
The black field of Kimura (Kimura-Kuroda) et al. scientific paper being delivered on PLoS One magazine in 2012, It is proposed that the exposure of imidacloprid may have an impact the development of mammalian nervous system.Food safety office, European Union (EFSA) needle This Article analysis is found, imidacloprid may impact the nervous system in human developmental.Discovery pyrrole is examined in EFSA After worm quinoline is harmful to honeybee, Jin Liangnian European Union has limited use (" Agrow " No.665, p6) of the medicine on some crops. Foundation is necessary for quick, the accurate and sensitive detection method of imidacloprid.
Detection imidacloprid chromatography is most widely used at present, including gas chromatography (GC), high performance liquid chromatography (HPLC), gas chromatography mass spectrometry (GC-MS), LC-MS (LC-MS), supercritical fluid chromatography (SFC), Capillary Electrophoresis (CE) etc. Instrumental method, but these methods need expensive instrument, professional operator, and sample pre-treatments are complicated, at high cost, when Between it is long, can not achieve the quick detection of a large amount of samples, therefore establish fast and convenient imidacloprid detection method and be of great significance. Enzyme-linked immunization (ELISA) be it is a kind of extremely efficiently, sensitive, quick detection method, when detection not to the purity requirement of sample High and easy to operate, suitable for great amount of samples field quick detection.Efficient immunological detection method is established, Gao Te is screened Anisotropic monoclonal antibody is important prerequisite.
Summary of the invention
The object of the present invention is to provide one plant of imidacloprid monoclonal antibody hybridoma cell strains, are prepared by the cell strain anti- Body has preferably specificity and detection sensitivity to imidacloprid, can be used to establish the immunological detection method of imidacloprid.
Technical solution of the present invention, one plant of imidacloprid monoclonal antibody hybridoma cell strain YH5 have been preserved in the micro- life of China Object culture presevation administration committee common micro-organisms center, abbreviation CGMCC, deposit number are CGMCC No.12015.
Imidacloprid monoclonal antibody, it is miscellaneous by the imidacloprid monoclonal antibody that the deposit number is CGMCC No.12015 Tumor cell strain YH5 secretion is handed over to generate.
The application of the imidacloprid monoclonal antibody, the analysis detection for Determination of Imidacloprid Residue in food safety detection.
The preparation basic step of imidacloprid monoclonal antibody hybridoma cell strain YH5 provided by the invention are as follows:
1) synthesis of haptens:
By imidacloprid raw medicine 1 (2 g, 7.82 mmol), mercaptopropionic acid (1.66 g, 2 eq), Cs2CO3(25.5g, 10 eq) It is suspended in dimethyl adipate (30 mL), 16 h is reacted at 120 DEG C, be cooled to room temperature, tetrahydrofuran (50 mL) mistake afterwards is added Filter, obtained solid crude product (30g) are added in a small amount of water, and prep-HPLC purifying finally obtains 100mg haptens IMPA.With liquid matter Combination analysis method is identified and is analyzed；
2) 2.5mg IMPA, 1- ethyl-(3- dimethylaminopropyl) carbon two preparation of comlete antigen IMPA-KLH: are weighed Inferior amine salt hydrochlorate (EDC) 5mg, n-hydroxysuccinimide (NHS) 3mg, with the 300 anhydrous n,N-Dimethylformamide of μ L (DMF) It dissolves (referred to as A liquid), 8 h of reaction is stirred at room temperature.Keyhole limpet hemocyanin KLH 1mL (5mg/mL) is taken, it is slow that isometric boric acid is added It rushes solution (referred to as B liquid) A liquid is added in B liquid dropwise in room temperature condition, room temperature reaction is overnight to get conjugate IMPA- KLH mixed liquor by small haptens dialysis separation comlete antigen and be not coupled, and is reflected by UV absorption scan method It is fixed；
3) mouse is immune: after imidacloprid comlete antigen and equivalent Freund's adjuvant mixing and emulsifying, passing through dorsal sc injection Immune BALB/c mouse.First immunisation complete Freund's adjuvant, multiple booster immunization cannot be used up full Freund's adjuvant.First immunisation with It is spaced one month between second of booster immunization, is spaced 21 days between multiple booster immunization.Last time is completely anti-with imidacloprid Former (being 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 cELISA (ic-ELISA) by HAT culture medium culture, And further using the inhibitory effect of ic-ELISA measurement positive cell hole, by limiting dilution assay to there is the positive preferably inhibited Cell hole is subcloned three times, is finally screened and is obtained imidacloprid monoclonal antibody hybridoma cell strain YH5；
5) identification of hybridoma cell strain property: pass through ic-ELISA measurement sensitivity and specificity.
Imidacloprid comlete antigen and equivalent Freund's adjuvant mixing and emulsifying are complete, and it is small that BALB/c is immunized by dorsal sc injection Mouse.First immunisation (100 μ g/ are only) complete Freund's adjuvant, multiple booster immunization (50 μ g/ are only) cannot be used up full Freund's adjuvant, most It is once immune with imidacloprid comlete antigen (25 μ g/ only, are free of adjuvant) impact afterwards.Take the low IC of high-titer50The splenocyte of mouse leads to It crosses PEG method to merge with murine myeloma cell, be screened by Indirect cELISA and is subcloned three times, obtain one plant Hybridoma cell strain.The monoclonal antibody of this cell strain secretion has preferable specificity and detection sensitivity to imidacloprid (IC50It is worth for 0.3 ng/mL) detection, it can be achieved that Determination of Imidacloprid Residue amount in water, fruits and vegetables, cereal, is that imidacloprid is residual in food The immune detection stayed provides raw material, has practical application value.
Beneficial effects of the present invention: the monoclonal antibody of cell strain YH5 provided by the invention secretion, to imidacloprid have compared with Good specificity and detection sensitivity (IC50Value is 0.3 ng/mL), it can be achieved that Determination of Imidacloprid Residue amount in water, fruits and vegetables, cereal Detection, the immune detection for Determination of Imidacloprid Residue in food provide raw material, have practical application value.
Biological material specimens preservation: one plant of imidacloprid monoclonal antibody hybridoma cell strain YH5 has been preserved in the micro- life of China Object culture presevation administration committee common micro-organisms center, abbreviation CGMCC, address are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City No. 3, Institute of Microorganism, Academia Sinica, preservation date on January 20th, 2016, deposit number is CGMCC No.12015.
Detailed description of the invention
Inhibition standard curve of Fig. 1 YH5 monoclonal antibody to imidacloprid.
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 imidacloprid comlete antigen, by cell fusion, HAT selective medium culture, Cell conditioned medium is screened by ic-ELISA, having finally obtained has the preferably monoclonal of specificity and detection sensitivity anti-imidacloprid Body hybridoma cell strain.
The preparation of 1 hybridoma cell strain YH5 of embodiment
(1) synthesis of haptens: by imidacloprid raw medicine 1 (2g, 7.82mmol), mercaptopropionic acid (1.66g, 2eq), Cs2CO3 (25.5g, 10eq) is suspended in DMA(30mL) in, 16 h are reacted at 120 DEG C, are cooled to room temperature, and tetrahydrofuran (50mL) mistake afterwards is added Filter, obtained solid crude product (30g) are added in a small amount of water, and prep-HPLC purifying finally obtains 100mg haptens IMPA, with liquid matter Combination analysis method is identified.
(2) synthesis of comlete antigen: weighing 2.5mg IMPA, EDC 5mg, NHS 3mg, is dissolved with 300 μ L anhydrous DMFs Reaction 8h is stirred at room temperature in (referred to as A liquid).KLH 1mL (5 mg/mL) is taken, isometric BB solution (referred to as B liquid) is added, in room temperature A liquid is added in B liquid by condition dropwise, and room temperature reaction to get conjugate IMPA-KLH mixed liquor, is separated overnight by dialysis Comlete antigen 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.Take imidacloprid comlete antigen with After equivalent Freund's adjuvant mixing and emulsifying, BALB/c mouse is immunized by dorsal sc injection.Immune complete Freund assistant for the first time Full Freund's adjuvant is all cannotd be used up in agent later.It is spaced one month between first immunisation and second of booster immunization, multiple booster immunization Between be spaced 21 days.It takes a blood sample within 7 days after third time is immune, measures mice serum potency and inhibition using ic-ELISA, select potency The mouse that height has inhibited, impact in 21 days is immune after the fifth immunization, intraperitoneal injection, 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, 8 min), 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, collect SP2/0 cell: 7-10 days before fusion, SP2/0 oncocyte being used and contains 10% FBS(fetal calf serum) RPMI- 1640 culture mediums are in 5% CO2In incubator.SP2/0 oncocyte quantity is required to reach 1-4 × 10 before fusion7, before guaranteeing fusion SP2/0 oncocyte is in logarithmic growth phase.When fusion, oncocyte is collected, is suspended in RPMI-1640 basic culture solution, carried 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 5 min of warm bath.From The heart (800 rpm, 8 min) 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 imidacloprid, carries out inhibitory effect measurement to positive cell hole with ic-ELISA.Selection is to imidacloprid Standard items have the cell hole preferably inhibited, are subcloned using limiting dilution assay, are detected with same method.It repeats Three times, cell strain YH5 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, abandons Supernatant, after the dissolution of 0.01 M PBS solution (pH7.4), dialysis desalting, the monoclonal antibody finally obtained after purification is placed in -20 DEG C save.
6.1 coatings: by coating antigen IMPA-KLH, with 0.05M pH9.6 carbonate buffer solution, the multiple proportions since 1 μ g/mL is dilute It releases, 100 holes μ L/, 37 DEG C of reaction 2h；
6.2 washings: solution in plate is inclined, and is washed 3 times with cleaning solution, each 3min；
6.3 closings: after patting dry, 200 hole μ L/ confining liquids are added, 37 DEG C of reaction 2h are 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 reaction 1h are added；
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 instead Answer 15min；
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 the IC of ic-ELISA measurement monoclonal antibody imidacloprid50Are as follows: 0.3 ng/mL illustrates have to imidacloprid well Sensitivity can be used for the detection of imidacloprid immunoassay.
The configuration of solution: carbonate buffer solution (CBS): Na is weighed2CO31.59 g, NaHCO32.93 g, are dissolved in respectively It is mixed after a small amount of distilled water, distilled water is added to mix to about 800mL, adjusted pH value to 9.6, distilled water is added to be settled to 1000mL, 4 DEG C of storages It deposits 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 .12H2O 18.43g, citric acid 9.33g, pure water are settled to 1000mL；B liquid: 60mg TMB is dissolved in 100mL ethylene glycol.A, the 1:5 mixing by volume of B liquid is TMB, and developing solution is current existing mixed.
The cross reacting rate of table 1.YH5 monoclonal antibody
。

Claims (3)

1. one plant of imidacloprid monoclonal antibody hybridoma cell strain YH5, has been preserved in Chinese microorganism strain preservation conservator Meeting common micro-organisms center, abbreviation CGMCC, deposit number are CGMCC No.12015.
2. imidacloprid monoclonal antibody, it is characterised in that: the imidacloprid list that it is CGMCC No.12015 by the deposit number Monoclonal hybridomas cell strain YH5 secretion generates.
3. the application of imidacloprid monoclonal antibody described in claim 2, it is characterised in that: for imidacloprid in food safety detection Remaining analysis detection.
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CN109536457A (en) * 2018-11-28 2019-03-29 北京勤邦生物技术有限公司 A kind of hybridoma cell strain that secreting anti-imidacloprid monoclonal antibody and its application
CN113502272B (en) * 2021-08-08 2023-08-04 江南大学 Amaranth and carmine monoclonal antibody hybridoma cell strain and application thereof
CN114591916B (en) * 2022-03-23 2023-05-16 扬州大学 Cell strain secreting anti-imidacloprid monoclonal antibody, monoclonal antibody thereof and application thereof

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* Cited by examiner, † Cited by third party
Title
Evaluation and Validation of a Commercially Available Enzyme-Linked Immunosorbent Assay for the Neonicotinoid Insecticide Imidacloprid in Agricultural Samples;EIKI WATANABE et al;《J. Agric. Food Chem》;20040414;2756-2762 *
Monoclonal antibody-based enzyme-linked immunosorbent assay for the insecticide imidacloprid;Hee-Joo Kim et al;《Analytica Chimica Acta》;20041231;111-118 *
抗吡虫啉单克隆抗体的制备及鉴定;李刚等;《生物工程学报》;20110625;第27卷(第6期);943-951 *

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