CN106995800A - One plant of alachlor, Acetochlor, pretilachlor, butachlor monoclonal antibody hybridoma cell strain GHY1 and its application - Google Patents

One plant of alachlor, Acetochlor, pretilachlor, butachlor monoclonal antibody hybridoma cell strain GHY1 and its application Download PDF

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CN106995800A
CN106995800A CN201610967802.9A CN201610967802A CN106995800A CN 106995800 A CN106995800 A CN 106995800A CN 201610967802 A CN201610967802 A CN 201610967802A CN 106995800 A CN106995800 A CN 106995800A
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acetochlor
butachlor
pretilachlor
alachlor
monoclonal antibody
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CN106995800B (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

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  • Food Science & Technology (AREA)
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Abstract

One plant of alachlor, Acetochlor, pretilachlor, butachlor monoclonal antibody hybridoma cell strain GHY1 and its application, belong to food security field of immunodetection.One plant of alachlor of the present invention, Acetochlor, pretilachlor, butachlor monoclonal antibody hybridoma cell strain GHY1, have been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, abbreviation CGMCC, and deposit number is CGMCC No.12030.The monoclonal antibody, is produced by the alachlor that the deposit number is CGMCC No. 12030, Acetochlor, pretilachlor, the strain GHY1 secretions of butachlor monoclonal antibody hybridoma cell.The monoclonal antibody of this cell line secretion, there is preferably specificity and detection sensitivity to alachlor, Acetochlor, pretilachlor, butachlor, can be achieved to alachlor in water, fruits and vegetables, cereal, Acetochlor, pretilachlor, butachlor residue amount detection, with actual application value.

Description

One plant of alachlor, Acetochlor, pretilachlor, butachlor monoclonal antibody hybridoma cell Strain GHY1 and its application
Technical field
The present invention relates to one plant of alachlor, Acetochlor, pretilachlor, butachlor monoclonal antibody hybridoma cell strain GHY1 and It is applied, and belongs to food security field of immunodetection.
Background technology
Alachlor, Acetochlor, pretilachlor, butachlor are all the acetamide-group herbicides for producing and using extensively, are directed not only to The monitoring of environmental pollution, it is also relevant with chemical injury of crops prevention etc..Higher water solubility is waited until in having due to acetamide-group herbicides And relatively low adsorption by soil constant, so be administered to rice terrace herbicide easily by infiltration be transferred to phreatic water or Enter surface water with rainfall runoff, fish production and ecological environment can be had undesirable effect.Wherein Acetochlor drug cost is low, Using extensive, applied in the multiple kinds of crops such as soybean, peanut, corn, be the maximum dryland soil processing selection of usable floor area Property one of herbicide, yield is only second to glyphosate with usage amount.But Acetochlor degradation cycle is longer, in water body and soil environment Easily migration, and its metabolite quinone imines has carcinogenesis.In today that environmental problem and food security are increasingly serious, it is Facilitate the residue detection of agricultural product Pesticides, set up for alachlor, Acetochlor, pretilachlor, butachlor quick, accurate and Sensitive detection method is necessary.
Detection alachlor, Acetochlor, pretilachlor, butachlor chromatography are 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), the instrumental method of Capillary Electrophoresis (CE) etc., but these methods need expensive instrument, professional operator, and sample Product complex pretreatment, cost is high, and the time is long, it is impossible to realize the quick detection of a large amount of samples, therefore sets up fast and convenient first grass Amine, Acetochlor, pretilachlor, butachlor detection method are significant.ELISA（ELISA）Be it is a kind of extremely efficiently, Sensitive, quick detection method is not during detection high and easy to operate to the purity requirement of sample, it is adaptable to which that great amount of samples shows Field quick detection.Efficient immunological detection method is set up, the monoclonal monomer of screening high specific is important prerequisite.
The content of the invention
It is an object of the invention to provide one plant of alachlor, Acetochlor, pretilachlor, butachlor monoclonal antibody hybridoma cell Strain, the antibody prepared by the cell line has preferably specificity to alachlor, Acetochlor, pretilachlor, butachlor and detected sensitive Degree, can be for setting up the immunological detection method of alachlor, Acetochlor, pretilachlor, butachlor.
Technical scheme, one plant of alachlor, Acetochlor, pretilachlor, butachlor monoclonal antibody hybridoma cell Strain GHY1, has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, abbreviation CGMCC, deposit number For CGMCC No.12030.
The alachlor, Acetochlor, pretilachlor, butachlor monoclonal antibody, it is CGMCC No. by the deposit number 12030 alachlor, Acetochlor, pretilachlor, the strain GHY1 secretions of butachlor monoclonal antibody hybridoma cell are produced.
The alachlor, Acetochlor, pretilachlor, the application of butachlor monoclonal antibody：For first in food safety detection Careless amine, Acetochlor, pretilachlor, the analysis detection of butachlor residue.
Alachlor that the present invention is provided, Acetochlor, pretilachlor, butachlor monoclonal antibody hybridoma cell strain GHY1 system It is for basic step：
（1）The synthesis of haptens：
By Acetochlor（1g, 3.7mmol）, 3- mercaptopropionic acids（1.2g 11.1mmol）And potassium hydroxide（0.4g, 7.4mmol） Ethanol（20mL）In solution be stirred overnight at 70 DEG C.Solution is filtered and dried by rotary evaporation.Product is redissolved In 20mL 5%NaHCO3In solution and filter.PH is adjusted to 3.0 with 6mol/L HCl, and by solution ethyl acetate （20mL×3）Extraction.Extraction solution is concentrated, crude product is obtained, it is purified by preparation HPLC, obtain haptens （130mg）AMPA.
（2）Complete A antigen MPA-KLH preparation：Weigh 2mg AMPA, 1- ethyls-（3- dimethylaminopropyls）Carbon two Inferior amine salt hydrochlorate（EDC）5mg, n-hydroxysuccinimide（NHS）3mg, with the 300 anhydrous DMFs of μ L（DMF） Dissolving (is referred to as A liquid), and reaction 8h is stirred at room temperature.Keyhole limpet hemocyanin KLH 1mL (5mg/mL) are taken, isometric borate buffer is added （BB）Solution（Referred to as B liquid）, in room temperature condition, A liquid is added in B liquid dropwise, room temperature reaction is stayed overnight, produces conjugate AMPA-KLH mixed liquors, the small haptens for separating comlete antigen by dialysing and not being coupled, and scanned by UV absorption Method is identified；
（3）Mouse it is immune：AMPA-KLH comlete antigens by dorsal sc injection with after equivalent Freund's adjuvant mixing and emulsifying, being exempted from Epidemic disease BALB/c mouse.First immunisation complete Freund's adjuvant, multiple booster immunization cannots be used up full Freund's adjuvant.First immunisation and the It is spaced one month, is spaced 21 days between multiple booster immunization between secondary booster immunization.Last time is resisted completely with AMPA-KLH It is former（Without adjuvant）Impact is immune；Pass through Indirect cELISA（ic-ELISA）Detect serum titer and suppression；
（4）Cell fusion is set up with cell line：Pass through polyethylene glycol（PEG4000）Method is thin by mouse boosting cell and mouse myeloma Born of the same parents are merged, and by HAT medium cultures, utilize Indirect cELISA（ic-ELISA）Positive cell hole is detected, is gone forward side by side One step determines the inhibition of positive cell hole, the positive cell preferably suppressed to having by limiting dilution assay using ic-ELISA Hole carries out three subclones, and final screening obtains alachlor, Acetochlor, pretilachlor, butachlor monoclonal antibody hybridoma cell Strain GHY1；
（5）The identification of hybridoma cell strain property：Sensitivity and specificity are determined by ic-ELISA.
Take the low IC of high-titer50The splenocyte of mouse, is merged by PEG methods with murine myeloma cell, by indirectly competing ELISA screening and three subclones are striven, strain of hybridoma strain is obtained.
Beneficial effects of the present invention：The monoclonal antibody for the cell line GHY1 secretions that the present invention is provided, to alachlor, second grass Amine, pretilachlor, butachlor have preferably specificity and detection sensitivity（IC50Value is respectively 3 ng/mL, 3 ng/mL, 10 ng/mL、10 ng/mL）, can be achieved to alachlor in water, fruits and vegetables, cereal, Acetochlor, pretilachlor, butachlor residue amount inspection Survey, raw material is provided for alachlor, Acetochlor, pretilachlor, the immune detection of butachlor residue in food, with practical application valency Value.
Biological material specimens preservation：One plant of alachlor, Acetochlor, pretilachlor, butachlor monoclonal antibody hybridoma cell Strain GHY1, has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, abbreviation CGMCC, and address is：North The institute 3 of Jing Shi Chaoyang Districts North Star West Road 1, Institute of Microorganism, Academia Sinica, preservation date on January 20th, 2016, preservation Numbering is CGMCC No.12030, and Classification And Nomenclature is monoclonal cell strain.
Brief description of the drawings
Fig. 1 GHY1 monoclonal antibodies to alachlor, Acetochlor, pretilachlor, butachlor suppression standard curve.
Embodiment
The following examples of the present invention only further illustrating as present invention, it is impossible in the restriction as the present invention Perhaps scope.Below by embodiment, the invention will be further described.
The present invention by alachlor, Acetochlor, pretilachlor, butachlor comlete antigen by being immunized mouse, by cell fusion, HAT selective medium cultures, cell conditioned medium is screened by ic-ELISA, has been finally given to alachlor, Acetochlor, the third grass Amine, butachlor have preferably specificity and the strain of the monoclonal antibody hybridoma cell of sensitivity.
The hybridoma cell strain GHY1 of embodiment 1 preparation
（1）The synthesis of haptens：By Acetochlor（1g, 3.7mmol）, 3- mercaptopropionic acids（1.2g 11.1mmol）And potassium hydroxide （0.4g, 7.4mmol）In ethanol（20mL）In solution be stirred overnight at 70 DEG C.Solution is filtered and done by rotary evaporation It is dry.Product is re-dissolved in 20mL 5%NaHCO3In solution and filter.PH is adjusted to 3.0 with 6mol/L HCl, and will be molten Liquid ethyl acetate（20mL×3）Extraction.Extraction solution is concentrated, crude product is obtained, it is purified by preparation HPLC, Obtain haptens（130mg）AMPA.
（2）The synthesis of comlete antigen：2mg AMPA, EDC 5mg, NHS 3mg are weighed, is dissolved and (claimed with 300 μ L dry DMFs For A liquid), reaction 8h is stirred at room temperature.KLH 1mL (5 mg/mL) are taken, isometric BB solution is added（Referred to as B liquid）, in room temperature bar A liquid, is added in B liquid, room temperature reaction is stayed overnight by part dropwise, produces conjugate AMPA-KLH mixed liquors, has been separated 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.Take alachlor, Acetochlor, third After careless amine, butachlor comlete antigen and equivalent Freund's adjuvant mixing and emulsifying, BALB/c mouse is immunized by dorsal sc injection.The Primary immune response complete Freund's adjuvant, all cannots be used up full Freund's adjuvant afterwards.Between between first immunisation and second of booster immunization Every one month, it is spaced 21 days between multiple booster immunization.Take a blood sample within 7 days after third time is immune, mouse blood is determined using ic-ELISA Clear potency and suppression, the mouse that selection potency height has suppressed are immune in the 5th immune impact in latter 21 days, intraperitoneal injection, it is desirable to Punching is exempted from dosage and halved and without any adjuvant.
（4）Cell fusion：After impact is immune three days, according to conventional PEG（Polyethylene glycol, molecular weight is 4000）Method is entered Row cell fusion, is comprised the following steps that：
A, pluck eyeball and take blood, cervical dislocation is put to death after mouse, is immediately placed in 75% alcohol and is sterilized, immersion 5min or so, The spleen of mouse is taken out in sterile working, is moderately ground with the glue head of syringe and is obtained splenocyte by 200 mesh cell screen clothes and hanged Liquid, is collected, centrifugation（1200rpm, 8 min）, splenocyte is washed with RPMI-1640 culture mediums three times, will after last time is centrifuged Splenocyte is diluted to certain volume, counts, standby；
B, collection SP2/0 cells：7-10 days before fusion, SP2/0 oncocytes are used and contain 10% FBS（Hyclone）RPMI- 1640 culture mediums are in 5% CO2In incubator.Require that SP2/0 oncocyte quantity reaches 1 ~ 4 × 10 before fusion7, it is ensured that before fusion SP2/0 oncocytes are in exponential phase.During fusion, oncocyte is collected, is suspended in RPMI-1640 basic culture solutions, carried out Cell count；
C, fusion process 7min.1min, 1mL PEG 1500 is added drop-wise in cell from slow to fast；2min, stands；The 3min and 4min, is added dropwise 1mL RPMI-1640 culture mediums in 1min；5min and 6min, is added dropwise 2mL in 1min RPMI-1640 culture mediums；7min, 1mL RPMI-1640 culture mediums are added dropwise per 10s.Then 37 DEG C of min of warm bath 5. Centrifugation（800 rpm, 8 min）, supernatant is abandoned, is resuspended into the RPMI-1640 screening and culturings containing 20% hyclone, 2% 50 × HAT In liquid, 96 porocyte plates are added to according to 200 μ L/ holes, 37 DEG C, 5% CO are placed in2Cultivated in incubator.
（5）Cell screening is set up with cell line：RPMI-1640 screenings were carried out to fused cell in the 3rd day in cell fusion Nutrient solution partly changes liquid, carries out within the 5th day being carried out entirely with the RPMI-1640 transition nutrient solution containing 20% hyclone, 1% 100 × HT Liquid is changed, took cell conditioned medium to be screened at the 7th day.Screening is in two steps：The first step first filters out positive cell hole with ic-ELISA, Second step is standard items from alachlor, Acetochlor, pretilachlor, butachlor, and suppression effect is carried out to positive cell with ic-ELISA Fruit determines.Selection has the cell hole preferably suppressed to alachlor, Acetochlor, pretilachlor, butachlor standard items, using limited dilute Interpretation of the law is subcloned, and is detected with same method.In triplicate, cell line GHY1 is obtained.
（6）The preparation and identification of monoclonal antibody：Take 8-10 week old BALB/c mouses, the sterile stone of every mouse peritoneal injection Wax oil 1mL；Every mouse peritoneal injection 1 × 10 after 7 days6Hybridoma, ascites was collected since the 7th day, ascites is passed through Caprylic acid-ammonium is purified.Under the conditions of meta-acid, caprylic acid can precipitate in ascites except IgG immune globulins it is ultrawhite other are miscellaneous Albumen, is then centrifuged for, and abandons precipitation；The monoclonal antibody of IgG types is precipitated with the ammonium sulfate of equivalent saturation degree again, centrifugation is abandoned Supernatant, with 0.01 M PBS solutions（pH7.4）After dissolving, dialysis desalting, the monoclonal antibody finally given after purification is placed in -20 DEG C preserve.
6.1 coating：By coating antigen AMPA-OVA, with 0.05M pH9.6 carbonate buffer solutions, the multiple proportions since 1 μ g/mL is dilute Release, 100 μ L/ holes, 37 DEG C of reaction 2h；
6.2 washing：Solution in plate is inclined, and washed 3 times with cleaning solution, each 3min；
6.3 closing：After patting dry, 200 μ L/hole confining liquid, 37 DEG C of reaction 2h are added.Dry for standby after washing；
6.4 sample-adding：By antiserum from 1:1000 start doubling dilution, and are added in the coating hole of each dilution factor, and 100 μ L/ Hole, 37 DEG C of reaction 30min；Fully after washing, 1 is added:The HRP- sheep anti-mouse iggs of 3000 dilutions, 100 μ L/ holes, 37 DEG C of reactions 30min；
6.5 colour developing：ELISA Plate is taken out, fully after washing, 100 μ L TMB nitrite ions, 37 DEG C of lucifuge reactions are added per hole 15min；
6.6 terminate and determine：50 μ L terminate liquids are added per hole with terminating reaction, the OD 450 in each hole is then determined with ELIASA Value.
The IC of monoclonal antibody alachlor, Acetochlor, pretilachlor, butachlor is determined with ic-ELISA50Respectively：3 ng/ ML, 3 ng/mL, 10 ng/mL, 10 ng/mL, illustrate there is good sensitivity to alachlor, Acetochlor, pretilachlor, butachlor, Available for alachlor, Acetochlor, pretilachlor, the detection of butachlor immunoassay.
The configuration of solution：Carbonate buffer solution（CBS）：Weigh Na2CO31.59 g, NaHCO32.93 g, are dissolved in respectively Mixed after a small amount of distilled water, plus distilled water is mixed to about 800mL, tune pH value to 9.6, plus distilled water are settled to 1000mL, 4 DEG C of storages Deposit standby；
Phosphate buffer（PBS）：8.00g NaCl, 0.2g KCl, 0.2g KH2PO4, 2.9g Na2HPO4·12 H2O, it is molten In 800mL pure water, adjust pH to 7.2~7.4 with NaOH or HCl, be settled to 1000mL；
PBST：PBS containing 0.05 % polysorbas20s；
TMB nitrite ions：A liquid：Na2HPO4 .12H2O 18.43g, citric acid 9.33g, pure water is settled to 1000mL；B liquid：60mg TMB is dissolved in 100mL ethylene glycol.A, B liquid press 1:5 mixing are TMB nitrite ions, now with existing mixed.

Claims (3)

1. one plant of alachlor, Acetochlor, pretilachlor, butachlor monoclonal antibody hybridoma cell strain GHY1, have been preserved in China Microbiological Culture Collection administration committee common micro-organisms center, abbreviation CGMCC, deposit number is CGMCC No.12030.
2. alachlor, Acetochlor, pretilachlor, butachlor monoclonal antibody, it is characterised in that：It is by the deposit number CGMCC No. 12030 alachlor, Acetochlor, pretilachlor, the strain GHY1 secretion productions of butachlor monoclonal antibody hybridoma cell It is raw.
3. alachlor, Acetochlor, pretilachlor, the application of butachlor monoclonal antibody described in claim 2, it is characterised in that：With The analysis detection of alachlor, Acetochlor, pretilachlor, butachlor residue in food safety detection.
CN201610967802.9A 2016-11-01 2016-11-01 One plant of alachlor, Acetochlor, pretilachlor, butachlor monoclonal antibody hybridoma cell strain GHY1 and its application Active CN106995800B (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005035893A (en) * 2003-07-15 2005-02-10 Horiba Biotechnology Co Ltd Alachlor hapten and antibody against alachlor and immunoassay using the same
CN2847296Y (en) * 2005-12-06 2006-12-13 万积成 Colloidal gold test peper for quick detecting alachlor, acetochlor and butachlor residue
CN105572367A (en) * 2014-10-13 2016-05-11 江苏维赛科技生物发展有限公司 Chemiluminescent enzyme-linked immunoassay method for detecting pretilachlor in rice

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005035893A (en) * 2003-07-15 2005-02-10 Horiba Biotechnology Co Ltd Alachlor hapten and antibody against alachlor and immunoassay using the same
CN2847296Y (en) * 2005-12-06 2006-12-13 万积成 Colloidal gold test peper for quick detecting alachlor, acetochlor and butachlor residue
CN105572367A (en) * 2014-10-13 2016-05-11 江苏维赛科技生物发展有限公司 Chemiluminescent enzyme-linked immunoassay method for detecting pretilachlor in rice

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