CN106947742B - One plant of paclobutrazol monoclonal antibody hybridoma cell strain CS12-1 and its application - Google Patents
One plant of paclobutrazol monoclonal antibody hybridoma cell strain CS12-1 and its application Download PDFInfo
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- CN106947742B CN106947742B CN201611233572.XA CN201611233572A CN106947742B CN 106947742 B CN106947742 B CN 106947742B CN 201611233572 A CN201611233572 A CN 201611233572A CN 106947742 B CN106947742 B CN 106947742B
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- 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 paclobutrazol monoclonal antibody hybridoma cell strain CS12-1 and its application, belong to food safety field of immunodetection.Paclobutrazol monoclonal antibody hybridoma cell strain CS12-1 of the invention, has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, abbreviation CGMCC, and deposit number is CGMCC No.13082.Paclobutrazol monoclonal antibody is secreted by the paclobutrazol monoclonal antibody hybridoma cell strain CS12-1 that the deposit number is CGMCC No.13082 and is generated.The application of the paclobutrazol monoclonal antibody, for the remaining analysis detection of paclobutrazol in food safety detection.The monoclonal antibody of this cell strain secretion has preferable specificity and detection sensitivity (IC to paclobutrazol50Value is 5 ng/mL), raw material is provided for the remaining immune detection of paclobutrazol in food, there is practical application value.
Description
Technical field
The present invention relates to one plant of paclobutrazol monoclonal antibody hybridoma cell strain CS12-1 and its applications, belong to food safety
Field of immunodetection.
Background technique
Paclobutrazol has as a kind of plant growth regulator for inhibiting class and delays plant growth, inhibits stem elongation, contracting
Between pipe nipple, promote bud differentiation, increase plant stress-resistance performance, improve the functions such as crop yield, be widely used in rice, wheat,
The production estimations such as peanut, fruit tree, tobacco, rape, soybean.Paclobutrazol is easily absorbed in the soil, and the residence time is long, may be right
Non-targeted object generates harm, and many European Union member countries have disabled, and many countries have also made stringent limitation to paclobutrazol, and China exists
To most tight limitation≤0.2mg/kg of paclobutrazol on agricultural product.
The case where in order to use paclobutrazol in effective Supervision food, it is good to need to find a species specificity, high sensitivity
Measuring method, it is superfluous to isolate and purify process and detection method such as thin layer chromatography, gas chromatography, liquid chromatography etc. at present
Long, sensitivity is low, in addition chaff interferent is more in food, it is difficult to obtain accurate result.Therefore fast and convenient paclobutrazol detection is established
Method is of great significance.Enzyme-linked immunization (ELISA) be it is a kind of extremely efficiently, sensitive, quick detection method, when detection pair
The purity requirement of sample is not high and easy to operate, the field quick detection suitable for great amount of samples.Establish efficient immunology
Detection method, the monoclonal antibody for screening high specific is important prerequisite.
Summary of the invention
The object of the present invention is to provide one plant of paclobutrazol monoclonal antibody hybridoma cell strains, are prepared by the cell strain anti-
Body has preferably specificity and detection sensitivity to paclobutrazol, can be used to establish the immunological detection method of paclobutrazol.
Technical solution of the present invention, one plant of paclobutrazol monoclonal antibody hybridoma cell strain CS12-1, has been preserved in China
Microbiological Culture Collection administration committee common micro-organisms center, abbreviation CGMCC, deposit number are CGMCC No.13082.
Paclobutrazol monoclonal antibody, it is miscellaneous by the paclobutrazol monoclonal antibody that the deposit number is CGMCC No.13082
Tumor cell strain CS12-1 secretion is handed over to generate.
The application of the paclobutrazol monoclonal antibody, for the remaining analysis detection of paclobutrazol in food safety detection.
The preparation basic step of paclobutrazol monoclonal antibody hybridoma cell strain CS12-1 provided by the invention are as follows:
1) synthesis of haptens:
Synthetic route is as follows:
I paclobutrazol 5g(17.06mmol of Weigh Compound) and 4- bromo-butyric acid benzyl ester 13.16g(51.19mmol) be dissolved in
In dimethyl sulfoxide 50mL, potassium hydroxide 1.05g(18.77mmol is added), then plus water 30mL it is stirred overnight at 90 DEG C, uses
Ethyl acetate extraction.Organic layer is washed with brine, and is dried and concentrated, and crude product is obtained.Crude product is purified by preparative-HPLC,
Obtain compound ii 800mg；II 600mg(1.25mmol of Weigh Compound) be dissolved in tetrahydrofuran 3mL and water 2mL mixing it is molten
In liquid, lithium hydroxide 131.5mg(3.13mmol is added), and 2h is stirred at room temperature.Reaction solution is extracted with ethyl acetate.
Organic layer is washed with brine, is dried and concentrated, crude product is obtained.By preparative HPLC purification of crude product, haptens is obtained
PBBA 200mg；
2) PBBA 4.3mg, dicyclohexylcarbodiimide 3mg, N- hydroxyl amber the preparation of comlete antigen PBBA-KLH: are weighed
Reaction 4-5h is stirred at room temperature with the anhydrous n,N-Dimethylformamide dissolution (referred to as A liquid) of 300 μ L in amber acid imide 2mg.Take keyhole
Isometric borate buffer solution (referred to as B liquid) is added, in room temperature condition, dropwise in hemocyanin KLH 1.47mL (6.8mg/mL)
A liquid is added in B liquid, room temperature reaction overnight to get conjugate PBBA-KLH mixed liquor, by dialysis separation comlete antigen and
The small haptens not being coupled, and identified by UV absorption scan method；
3) mouse is immune: after PBBA-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 PBBA-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 paclobutrazol monoclonal antibody hybridoma cell strain CS12-1；
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 CS12-1 secretion provided by the invention has paclobutrazol
There are preferable specificity and detection sensitivity (IC50Value is 5 ng/mL), it can be achieved that paclobutrazol residual quantity in water, fruits and vegetables, cereal
Detection, provide raw material for the remaining immune detection of paclobutrazol in food, have practical application value.
Biological material specimens preservation: one plant of paclobutrazol monoclonal antibody hybridoma cell strain CS12-1 has been preserved in China
Microbiological Culture Collection administration committee common micro-organisms center, abbreviation CGMCC, address are as follows: BeiChen West Road, Chaoyang District, BeiJing City 1
Number institute 3, Institute of Microorganism, Academia Sinica, preservation date on October 31st, 2016, classification naming is monoclonal cell strain,
Deposit number is CGMCC No.13082.
Detailed description of the invention
Inhibition standard curve of Fig. 1 CS12-1 monoclonal antibody to paclobutrazol.
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 paclobutrazol comlete antigen, by cell fusion, HAT selective medium culture,
Cell conditioned medium is screened by ic-ELISA, having finally obtained has preferably specificity and the monoclonal antibody of sensitivity miscellaneous paclobutrazol
Hand over tumor cell strain.
The preparation of 1 hybridoma cell strain CS12-1 of embodiment
(1) synthesis of haptens: paclobutrazol 5g(17.06mmol is weighed) and 4- bromo-butyric acid benzyl ester 13.16g
(51.19mmol) is dissolved in dimethyl sulfoxide 50mL, and potassium hydroxide 1.05g(18.77mmol is added), it is stirred at 90 DEG C
Then plus water 30mL night is extracted with ethyl acetate.Organic layer is washed with brine, and is dried and concentrated, and crude product is obtained.Crude product is logical
Preparative-HPLC purifying is crossed, compound A 800mg is obtained；Weigh Compound A 600mg(1.25mmol) it is dissolved in tetrahydrofuran
In 3mL and water 2mL mixed solution, lithium hydroxide 131.5mg(3.13mmol is added), and 2h is stirred at room temperature.Reaction solution
It is extracted with ethyl acetate.Organic layer is washed with brine, is dried and concentrated, crude product is obtained.It is thick by preparative HPLC purifying
Product obtains haptens PBBA 200mg.
(2) PBBA 4.3mg, dicyclohexylcarbodiimide 3mg, n-hydroxysuccinimide the synthesis of comlete antigen: are weighed
Reaction 4-5h is stirred at room temperature with the anhydrous n,N-Dimethylformamide dissolution (referred to as A liquid) of 300 μ L in 2mg.Take keyhole limpet hemocyanin
KLH 1.47mL (6.8mg/mL) is added isometric borate buffer solution (referred to as B liquid) and A liquid is added dropwise in room temperature condition
Into B liquid, room temperature reaction is not coupled by dialysis separation comlete antigen and overnight to get conjugate PBBA-KLH mixed liquor
Small haptens, 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 paclobutrazol 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, impregnate 5 min
The spleen of mouse is taken out in left and right, sterile working, and it is thin to obtain spleen with the grinding of the rubber head of syringe appropriateness and by 200 mesh cell screen clothes
Born of the same parents' suspension is collected, and is centrifuged (1200rpm, 8 min), is washed splenocyte three times with RPMI-1640 culture medium, and last time is centrifuged
Afterwards, splenocyte is diluted to certain volume, 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% CO2It is cultivated in incubator.SP2/0 oncocyte quantity is required to reach (1 ~ 4) × 10 before fusion7,
Guarantee that SP2/0 oncocyte is in logarithmic growth phase before merging.When fusion, oncocyte is collected, is suspended in the culture of the basis RPMI-1640
In liquid, cell count is carried out；
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 screening into 50 × HAT containing 20% fetal calf serum, 2% is resuspended
In culture solution, 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 paclobutrazol, carries out inhibitory effect measurement to positive cell with ic-ELISA.Selection is to paclobutrazol mark
Quasi- product have the cell hole preferably inhibited, are subcloned using limiting dilution assay, are detected with same method.Repeat three
It is secondary, obtain cell strain CS12-1.
(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 PBBA-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；
PBBA-BSA preparation is as follows:
A, PBBA 2.5mg, 1- ethyl-(3- dimethylaminopropyl) carbodiimide hydrochloride of step (1) preparation are weighed
3.3mg, n-hydroxysuccinimide 2mg are dissolved with the anhydrous n,N-Dimethylformamide of 300 μ L and the ultrapure water mixed liquid of 100 μ L,
Reaction 4-6h is stirred at room temperature in referred to as A liquid；Weighing 10mg chicken ovalbumin OVA, PBBA and OVA molar ratio is 30 ︰ 1, dissolution
In 4mL borate buffer solution, referred to as B liquid；A liquid is added in B liquid dropwise at room temperature, room temperature reaction is stayed overnight, i.e.,
Obtain conjugate PBBA-OVA mixed liquor；
B, it dialyses: taking the bag filter of 10cm, 5min is boiled in boiling water, then rinse 3min with 60 DEG C of deionized water, protected
There are spare in 4 DEG C of deionized waters；Conjugate PBBA-OVA mixed liquor is put into bag filter and dialyses 3 in the PBS of 0.01mol/L
It, changes liquid three times a day to get coating antigen PBBA-OVA；
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
The hole L/, 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
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 paclobutrazol50For 5 ng/mL, illustrate to have paclobutrazol sensitive well
Degree can be used for the detection of paclobutrazol 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 1 ︰ 5 mixing by volume is TMB developing solution, current existing mixed.
Claims (3)
1. one plant of paclobutrazol monoclonal antibody hybridoma cell strain CS12-1 has been preserved in Chinese microorganism strain preservation management committee
Member can common micro-organisms center, abbreviation CGMCC, classification naming is monoclonal cell strain, the deposit date is on October 31st, 2016,
Deposit number is CGMCC No.13082.
2. paclobutrazol monoclonal antibody, it is characterised in that: the paclobutrazol list that it is CGMCC No.13082 by the deposit number
Monoclonal hybridomas cell strain CS12-1 secretion generates.
3. the application of paclobutrazol monoclonal antibody described in claim 2, it is characterised in that: for paclobutrazol in food safety detection
Remaining analysis detection.
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| CN107267465A (en) * | 2017-07-13 | 2017-10-20 | 江南大学 | One plant of Procalcitonin monoclonal antibody hybridoma cell strain CS12 1 and its application |
| CN107653229A (en) * | 2017-11-02 | 2018-02-02 | 江南大学 | A kind of strain of bisphenol S monoclonal antibody hybridoma cell and its application |
| CN108517317A (en) * | 2018-04-04 | 2018-09-11 | 江南大学 | A kind of anti-Clorprenaline monoclonal antibody hybridoma cell strain and its application |
| CN109022366A (en) * | 2018-08-16 | 2018-12-18 | 江南大学 | One plant of hybridoma cell strain for secreting anti-levamisol monoclonal antibody and its application |
| CN108866009B (en) * | 2018-08-16 | 2019-10-01 | 江南大学 | One plant of metalaxyl monoclonal antibody hybridoma cell strain and its application |
| CN114480296B (en) * | 2022-01-28 | 2023-09-15 | 北京壹拾智检生物科技有限公司 | Hybridoma cell strain, monoclonal antibody, detection kit and detection method |
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