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

One plant of alachlor, Acetochlor, pretilachlor, butachlor monoclonal antibody hybridoma cell strain GHY1 and its application, belong to food safety field of immunodetection.One plant of alachlor, Acetochlor, pretilachlor, butachlor monoclonal antibody hybridoma cell strain GHY1 of the invention, 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, by the alachlor, Acetochlor, pretilachlor, butachlor monoclonal antibody hybridoma cell strain GHY1 secretion generation that the deposit number is CGMCC No.12030.The monoclonal antibody of this cell strain secretion, there is preferable specificity and detection sensitivity to alachlor, Acetochlor, pretilachlor, butachlor, the detection to alachlor, Acetochlor, pretilachlor, butachlor residue amount in water, fruits and vegetables, cereal can be achieved, there is practical 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 food safety field of immunodetection is belonged to.
Background technique
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 related with chemical injury of crops prevention etc..Due to acetamide-group herbicides have in until higher water solubility And relatively low adsorption by soil constant, so the herbicide for being administered to paddy field easily pass through infiltration be transferred to phreatic water or Enter surface water with rainfall runoff, adverse effect can be caused to fish production and ecological environment.Wherein Acetochlor drug cost is low, It using extensive, is applied in the multiple kinds of crops such as soybean, peanut, corn, is the maximum dryland soil processing selection of usable floor area Property one of herbicide, yield and usage amount are only second to glyphosate.But Acetochlor degradation cycle is longer, in water body and soil environment It is easy to migrate, and its metabolite quinone imines has carcinogenesis.In today that environmental problem and food safety are increasingly serious, it is Facilitate the residue detection of pesticide in agricultural product, establish 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, at high cost, the time is long, can not achieve the quick detection of a large amount of samples, therefore establishes fast and convenient first grass Amine, Acetochlor, pretilachlor, butachlor detection method are of great significance.Enzyme-linked immunization (ELISA) be it is a kind of extremely efficiently, Sensitive, quick detection method, when detection, be not high and easy to operate to the purity requirement of sample, suitable for showing for great amount of samples Field quickly detection.Efficient immunological detection method is established, the monoclonal monomer for screening high specific is important prerequisite.
Summary of the invention
The object of the present invention is to provide one plant of alachlor, Acetochlor, pretilachlor, butachlor monoclonal antibody hybridoma cells Strain, the antibody prepared by the cell strain have preferably specificity and detection sensitive alachlor, Acetochlor, pretilachlor, butachlor Degree can be used to establish the immunological detection method of alachlor, Acetochlor, pretilachlor, butachlor.
Technical solution of the present invention, 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, butachlor monoclonal antibody hybridoma cell strain GHY1 secretion generate.
The application of the alachlor, Acetochlor, pretilachlor, butachlor monoclonal antibody: for first in food safety detection Careless amine, Acetochlor, pretilachlor, the analysis detection of butachlor residue.
Alachlor provided by the invention, Acetochlor, pretilachlor, the system of butachlor monoclonal antibody hybridoma cell strain GHY1 Standby basic step are as follows:
(1) synthesis of haptens:
By Acetochlor (1g, 3.7mmol), 3- mercaptopropionic acid (1.2g 11.1mmol) and potassium hydroxide (0.4g, 7.4mmol) solution in ethyl alcohol (20mL) is stirred overnight at 70 DEG C.Solution is filtered and dry by rotary evaporation.It will Product is re-dissolved in 20mL 5%NaHCO3In solution and filter.PH is adjusted to 3.0 with 6mol/L HCl, and by solution It is extracted with ethyl acetate (20mL × 3).Extraction solution is concentrated, crude product is obtained, it is purified by preparative HPLC, is obtained Haptens (130mg) AMPA.
(2) 2mg AMPA, 1- ethyl-(3- dimethylaminopropyl) carbon two preparation of complete A antigen MPA-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), reaction 8h is stirred at room temperature.Keyhole limpet hemocyanin KLH 1mL (5mg/mL) is taken, isometric borate buffer is added (BB) A liquid is added in B liquid by solution (referred to as B liquid) dropwise in room temperature condition, and room temperature reaction is overnight to get conjugate AMPA-KLH mixed liquor is scanned by small haptens dialysis separation comlete antigen and be not coupled, and by UV absorption Method identification；
(3) mouse is immune: after AMPA-KLH comlete antigen and equivalent Freund's adjuvant mixing and emulsifying, being infused by dorsal sc Penetrate immune BALB/c mouse.First immunisation complete Freund's adjuvant, multiple booster immunization cannot be used up full Freund's adjuvant.First immunisation It is spaced one month between second of booster immunization, is spaced 21 days between multiple booster immunization.Last time is complete with AMPA-KLH Holoantigen (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 alachlor, Acetochlor, pretilachlor, butachlor monoclonal antibody hybridoma Cell strain GHY1；
(5) identification of hybridoma cell strain property: pass through ic-ELISA measurement sensitivity and specificity.
Take the low IC of high-titer50The splenocyte of mouse is merged by PEG method with murine myeloma cell, by indirectly competing It strives enzyme-linked immunization screening and is subcloned three times, obtain a strain of hybridoma strain.
Beneficial effects of the present invention: the monoclonal antibody of cell strain GHY1 secretion provided by the invention, to alachlor, second grass Amine, pretilachlor, butachlor have preferable specificity and detection sensitivity (IC50Value is respectively 3 ng/mL, 3 ng/mL, 10 Ng/mL, 10 ng/mL), it can be achieved that inspection to alachlor, Acetochlor, pretilachlor, butachlor residue amount in water, fruits and vegetables, cereal It surveys, provides raw material for alachlor, Acetochlor, pretilachlor, the immune detection of butachlor residue in food, there is practical application valence 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, address are as follows: north The institute 3 of the Chaoyang District Jing Shi North Star West Road 1, Institute of Microorganism, Academia Sinica, preservation date on January 20th, 2016, preservation Number is CGMCC No.12030, and classification naming is monoclonal cell strain.
Detailed description of the invention
Fig. 1 GHY1 monoclonal antibody to alachlor, Acetochlor, pretilachlor, butachlor inhibition standard curve.
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 alachlor, Acetochlor, pretilachlor, butachlor comlete antigen, by cell fusion, HAT selective medium culture screens cell conditioned medium by ic-ELISA, has finally obtained to alachlor, Acetochlor, the third grass Amine, butachlor have the monoclonal antibody hybridoma cell strain of preferably specificity and sensitivity.
The preparation of 1 hybridoma cell strain GHY1 of embodiment
(1) synthesis of haptens: by Acetochlor (1g, 3.7mmol), 3- mercaptopropionic acid (1.2g 11.1mmol) and hydrogen-oxygen Change the solution of potassium (0.4g, 7.4mmol) in ethyl alcohol (20mL) to be stirred overnight at 70 DEG C.Solution is filtered and passes through rotation and is steamed It is dry dry.Product is re-dissolved in 20mL 5%NaHCO3In solution and filter.PH is adjusted to 3.0 with 6mol/L HCl, and Solution is extracted with ethyl acetate (20mL × 3).Extraction solution is concentrated, crude product is obtained, it is pure by preparative HPLC Change, obtains haptens (130mg) AMPA.
(2) synthesis of comlete antigen: weighing 2mg AMPA, EDC 5mg, NHS 3mg, (is claimed with the dissolution of 300 μ L anhydrous DMFs For A liquid), reaction 8h is stirred at room temperature.KLH 1mL (5 mg/mL) is taken, isometric BB solution (referred to as B liquid) is added, in room temperature item A liquid is added in B liquid by part dropwise, and room temperature reaction to get conjugate AMPA-KLH 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.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 immunization complete Freund's adjuvant all cannots be used up full Freund's adjuvant later.Between between first immunisation and second of booster immunization Every one month, it is spaced 21 days between multiple booster immunization.It takes a blood sample within 7 days after third time is immune, measures mouse blood using ic-ELISA Clear potency and inhibition, the mouse for selecting potency height to inhibit, impact in 21 days is immune after the fifth immunization, intraperitoneal injection, it is desirable that Punching is exempted from dosage and is 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 5min 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, it collects SP2/0 cell: 7-10 days before fusion, SP2/0 oncocyte being used and contains 10% FBS(fetal calf serum) RPMI-1640 culture medium is in 5% CO2In incubator.SP2/0 oncocyte quantity is required to reach 1 ~ 4 × 10 before fusion7, guarantee SP2/0 oncocyte is in logarithmic growth phase before merging.When fusion, oncocyte is collected, RPMI-1640 basic culture solution is suspended in In, carry out cell count；
C, fusion process 7min.The PEG 1500 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 drips in 1min Add 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 5 min.It is centrifuged (800 rpm, 8 min), abandons supernatant, the RPMI-1640 sieve into 50 × HAT containing 20% fetal calf serum, 2% is resuspended It selects in culture solution, is added to 96 porocyte plates according to 200 holes μ L/, is 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 alachlor, Acetochlor, pretilachlor, butachlor, carries out inhibition effect to positive cell with ic-ELISA Fruit measurement.Selection has the cell hole preferably inhibited 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 strain GHY1 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 AMPA-OVA, 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 μ L/hole confining liquid, 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 L / hole, 37 DEG C of reaction 30min；Sufficiently after washing, the diluted HRP- sheep anti-mouse igg of 1:3000,100 holes μ L/, 37 DEG C of reactions are added 30min；
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.
The IC of monoclonal antibody alachlor, Acetochlor, pretilachlor, butachlor is measured with ic-ELISA50It is respectively as follows: 3 ng/ ML, 3 ng/mL, 10 ng/mL, 10 ng/mL, illustrate there is good sensitivity to alachlor, Acetochlor, pretilachlor, butachlor, It can be used for alachlor, Acetochlor, pretilachlor, the detection of butachlor 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, B liquid is TMB developing solution by 1:5 mixing, current 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 are CGMCC No.12030.
2. alachlor, Acetochlor, pretilachlor, butachlor monoclonal antibody, it is characterised in that: it is by the deposit number The alachlor of CGMCC No. 12030, Acetochlor, pretilachlor, butachlor monoclonal antibody hybridoma cell strain GHY1 secretion produce It is raw.
3. the application of alachlor, Acetochlor described in claim 2, pretilachlor, butachlor monoclonal antibody, it is characterised in that: use 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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