CN106867971A - One plant of flunixin meglumine monoclonal antibody hybridoma cell strain YY and its application - Google Patents
One plant of flunixin meglumine monoclonal antibody hybridoma cell strain YY and its application Download PDFInfo
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- CN106867971A CN106867971A CN201710285176.XA CN201710285176A CN106867971A CN 106867971 A CN106867971 A CN 106867971A CN 201710285176 A CN201710285176 A CN 201710285176A CN 106867971 A CN106867971 A CN 106867971A
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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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- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
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Abstract
One plant of flunixin meglumine monoclonal antibody hybridoma cell strain YY and its application, belong to food security field of immunodetection.Flunixin meglumine monoclonal antibody hybridoma cell strain YY of the present 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.13096.The monoclonal antibody of this cell line secretion, has preferably specificity and detection sensitivity to flunixin meglumine（IC50It is 0.24 ng/mL to be worth）, for the immune detection of the pungent meglumine residual of Fluorine in Foods Buddhist nun provides raw material, with actual application value.
Description
Technical field
The present invention relates to one plant of flunixin meglumine monoclonal antibody hybridoma cell strain YY and its application, belong to food peace
Panimmunity detection field.
Background technology
Flunixin meglumine (flunixin meglumine, FM) is a kind of new, nonsteroidal animal specific solution
Hot analgesic, wherein, flunixin（Flunixin, molecular formula C14H11F3N2O2）It is the special class I non-steroid class of structure
Anti-inflammatory and antalgic alexipyretic, meglumine（Meglumine, molecular formula C7H17NO5）The effect of main stabilization agent, the two is with fixation
Ratio（The flunixin molecule of 1: 1, i.e., one combines a meglumine molecule）Occur, as FM.It is usually used in delaying on veterinary clinic
Solve the internal organ angina of horse, the pain that muscle and skeletal disorders cause and anti-inflammatory；The acute inflammation that the various diseases infection of ox causes
Control, such as founder, arthritis can also be used for the auxiliary treatment of sow mammitis, hysteritis and agalactia syndrome in addition.
The flunixin meglumine that Qilu Animal Health Products Co., Ltd. independently develops and declares is on June 1st, 2007 by the Ministry of Agriculture batch
Standard is national three classes novel chiral synthon, and certificate number is（2007）Novel chiral synthon demonstrate,proves word 23,24.EMEA（European drug administration）Rule
Determine limitation of the flunixin meglumine in ox muscle, fat, liver and renal tissue and be respectively 20,30,300 and 100 μ g/
kg；The domestic animal that U.S. FDA is ratified in April, 2002 specifies it in cattle liver and muscle with flunixin meglumine file
Residue limits are respectively 125 and 25 μ g/kg.For the situation on effective Supervision veterinary clinic using flunixin meglumine,
Need to find a species specificity is good, sensitivity is high assay method, and detection method such as TLC, gas-chromatography at present
Method, liquid chromatography etc., isolate and purify process tedious, and sensitivity is low, adds chaff interference many, it is difficult to obtain accurate result.Therefore
Set up fast and convenient flunixin meglumine detection method significant.ELISA（ELISA）It is a kind of extremely high
Effect, sensitive, quick detection method, purity requirement during detection to sample is not high and easy to operate, it is adaptable to great amount of samples
Field quick detection.Efficient immunological detection method is set up, the monoclonal antibody for screening high specific is important prerequisite.
The content of the invention
It is an object of the invention to provide one plant of flunixin meglumine monoclonal antibody hybridoma cell strain, by the cell line system
Standby antibody has preferably specificity and detection sensitivity to flunixin meglumine, can be used to set up exempting from for flunixin meglumine
Epidemiology detection method.
Technical scheme：One plant of flunixin meglumine monoclonal antibody hybridoma cell strain YY, in being preserved in
State's Microbiological Culture Collection administration committee common micro-organisms center, abbreviation CGMCC, deposit number is CGMCC No.13096.
Flunixin meglumine monoclonal antibody, it is the flunixin meglumine of CGMCC No.13096 by the deposit number
Monoclonal antibody hybridoma cell strain YY secretions are produced.
The application of the flunixin meglumine monoclonal antibody, for flunixin meglumine residual in food safety detection
Analysis detection.
The present invention provide flunixin meglumine monoclonal antibody hybridoma cell strain YY preparation basic step be：
1）The preparation of comlete antigen：
The preparation of immunogene FM-BSA：2.2mg FM are weighed, 1- ethyl-carbodiimide hydrochlorides 2.6mg, N- hydroxysuccinimidyl acyl is sub-
Amine 1.6mg, is dissolved with the anhydrous DMFs of 400 μ L, and reaction 6-8h is stirred at room temperature（Referred to as A liquid）.Take cow's serum egg
White BSA 10mg（FM is 30 ︰ 1 with BSA mol ratios）, dissolved with 4mL borate buffer solutions（Referred to as B liquid）.In room temperature condition, by
Drop A liquid is added in B liquid, room temperature reaction overnight, obtains final product conjugate FM-BSA mixed liquors, by dialyse separate comlete antigen and
The small haptens not being coupled.The preparation of coating antigen FM-OVA：Weigh 1.6mg FM, 1- ethyl-carbodiimide hydrochlorides
1.9mg, N-hydroxy-succinamide 1.2mg, are dissolved with the anhydrous DMFs of 300 μ L, and 6~8h of reaction is stirred at room temperature
（Referred to as A liquid）.Weigh 5mg chicken ovalbumins OVA（FM is 30 ︰ 1 with OVA mol ratios）, it is dissolved in 2mL borate buffer solutions
In, at ambient temperature, dropwise A liquid being added in B liquid, room temperature reaction overnight, obtains final product conjugate FM-OVA mixed liquors, passes through
Dialysis separates coating antigen and the small haptens not being coupled；
2）Mouse it is immune：It is immune by dorsal sc injection after FM-BSA comlete antigens and equivalent Freund's adjuvant mixing and emulsifying
BALB/c mouse.First immunisation（100 μ g/ are only）With complete Freund's adjuvant, multiple booster immunization（50 μ g/ are only）Cannot be used up full Freund
Adjuvant.It is spaced 28 days between first immunisation and second booster immunization, is spaced 21 days between multiple booster immunization.Last time is used
The spurt of FM-BSA comlete antigens is immune（25 μ g/, without adjuvant）；By Indirect cELISA（ic-ELISA）Detection
Serum titer and suppression；Take the low IC of high-titer50The splenocyte of mouse, is merged by PEG methods with murine myeloma cell, is passed through
Indirect cELISA is screened and three subclones, obtains strain of hybridoma strain；
3）Cell fusion is set up with cell line：By polyethylene glycol（PEG 4000）Method is thin by mouse boosting cell and mouse myeloma
Born of the same parents are merged, by HAT medium cultures, using Indirect cELISA（ic-ELISA）Detection positive cell hole, goes forward side by side
One step determines the inhibition of positive cell hole using ic-ELISA, by limiting dilution assay to there is the positive cell for preferably suppressing
Hole carries out three subclones, and final screening obtains flunixin meglumine monoclonal antibody hybridoma cell strain YY；
4）The identification of hybridoma cell strain property：Sensitivity and specificity are determined by ic-ELISA.
Beneficial effects of the present invention：The monoclonal antibody of the cell line YY secretions that the present invention is provided, to flunixin meglumine
With preferable specificity and detection sensitivity（IC50It is 0.24 ng/mL to be worth）, it is the immune of flunixin meglumine residual for animals
Detection provides raw material, with actual application value.
Biological material specimens preservation：One plant of monoclonal cell strain YY, has been preserved in Chinese microorganism strain preservation management committee
Member's meeting common micro-organisms center, abbreviation CGMCC, address is：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the Chinese Academy of Sciences is micro-
Biological study institute, deposit number is CGMCC No.13096, preservation date on October 31st, 2016.
Brief description of the drawings
Suppression standard curve of the monoclonal antibody of Fig. 1 YY secretions to flunixin meglumine.
Specific embodiment
The following examples of the present invention only further illustrating as present invention, it is impossible to as in restriction of the invention
Perhaps scope.Below by embodiment, the invention will be further described.
The present invention by by flunixin meglumine comlete antigen immune mouse, by cell fusion, HAT selective mediums
Culture, cell conditioned medium is screened by ic-ELISA, and having finally given has preferably specific and sensitivity to flunixin meglumine
Monoclonal antibody hybridoma cell strain.
The preparation of the hybridoma cell strain YY of embodiment 1
（1）The preparation of comlete antigen：Weigh 2.2mg FM, 1- ethyl-carbodiimide hydrochloride 2.6mg, N-hydroxy-succinamide
1.6mg, is dissolved with the anhydrous DMFs of 400 μ L, and reaction 6-8h is stirred at room temperature（Referred to as A liquid）.Take bovine serum albumin
BSA 10mg（FM is 30 ︰ 1 with BSA mol ratios）, dissolved with 4mL borate buffer solutions（Referred to as B liquid）.In room temperature condition, dropwise
A liquid is added in B liquid, room temperature reaction overnight, obtains final product conjugate FM-BSA mixed liquors, by dialysis separation comlete antigen and not
The small haptens of coupling.The preparation of coating antigen FM-OVA：1.6mg FM, 1- ethyl-carbodiimide hydrochloride 1.9mg are weighed,
N-hydroxy-succinamide 1.2mg, is dissolved with the anhydrous DMFs of 300 μ L, and 6~8h of reaction is stirred at room temperature（Referred to as A
Liquid）.Weigh 5mg chicken ovalbumins OVA（FM is 30 ︰ 1 with OVA mol ratios）, it is dissolved in 2mL borate buffer solutions, in room
Under the conditions of temperature, dropwise A liquid is added in B liquid, room temperature reaction overnight, obtains final product conjugate FM-OVA mixed liquors, by dialysis point
From coating antigen and the small haptens not being coupled.
（2）Animal immune：The BALB/c mouse of 6~8 week old of health is selected to be immunized.Take flunixin meglumine complete
After antigen and equivalent Freund's adjuvant mixing and emulsifying, BALB/c mouse is immunized by dorsal sc injection.It is immune for the first time to use complete
Freund's adjuvant, all cannot be used up full Freund's adjuvant afterwards.It is spaced 28 days between first immunisation and second booster immunization, is repeatedly strengthened
It is spaced 21 days between immune.Take a blood sample within 7 days after third time is immune, mice serum potency and suppression, selection are determined using ic-ELISA
The potency mouse for having suppressed high, intraperitoneal injection, it is desirable to punching exempt from dosage halve and be free of immune in the 5th immune spurt in latter 21 days
Any adjuvant.
（3）Cell fusion：After spurt is immune three days, according to conventional PEG（Polyethylene glycol, molecular weight is 4000）Method is entered
Row cell fusion, comprises the following steps that：
A, pluck eyeball and take blood, after cervical dislocation puts to death mouse, be immediately placed in 75% alcohol and sterilize, 5 min of immersion or so,
The spleen of mouse is taken out in sterile working, is moderately ground and is obtained splenocyte by 200 mesh cell screen clothes with the glue head of syringe and hanged
Liquid, collects, centrifugation（1200rpm, 8 min）, splenocyte is washed with RPMI-1640 culture mediums three times, after last time is centrifuged, will
Splenocyte is diluted to certain volume, counts, standby；
B, collection SP2/0 cells：Fusion is first 7~10 days, by SP2/0 oncocytes with containing 10% FBS（Hyclone）
RPMI-1640 culture mediums are in 5% CO2Cultivated in incubator.Require that SP2/0 oncocyte quantity reaches before fusion（1~ 4）×
107, it is ensured that SP2/0 oncocytes are in exponential phase before fusion.During fusion, oncocyte is collected, be suspended in RPMI-1640 bases
In nutrient solution, cell count is carried out；
C, fusion process 7min.1min, the PEG 4000 of 1mL is added drop-wise in cell from slow to fast；2min, stands.
3min and 4min, is added dropwise 1mL RPMI-1640 culture mediums in 1min；5min and 6min, is added dropwise in 1min
2mL RPMI-1640 culture mediums；7min, the RPMI-1640 culture mediums of 1mL are added dropwise per 10s.Then 37 DEG C of warm bath 5
min.Centrifugation（800 rpm, 8 min）, abandon supernatant, it is resuspended enter the RPMI-1640 sieves containing 20% hyclone, 2% 50 × HAT
Select in nutrient solution, 96 porocyte plates are added to according to 200 μ L/ holes, be placed in 37 DEG C, 5% CO2Cultivated in incubator.
（4）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, taking cell conditioned medium at the 7th day is screened.Screening is in two steps：The first step first filters out positive cell hole with ic-ELISA,
Second step is standard items from flunixin meglumine, and inhibition measure is carried out to positive cell with ic-ELISA.Selection is to fluorine
The pungent meglumine standard items of Buddhist nun have the cell hole of preferable suppression, are subcloned using limiting dilution assay, are entered with same method
Row detection.In triplicate, cell line YY is obtained.
（5）The preparation of monoclonal antibody and identification：8~10 week old BALB/c mouses are taken, every mouse peritoneal injection is aseptic
Paraffin oil 1mL；Every mouse peritoneal injection 1 × 10 after 7 days6Hybridoma, collected ascites since the 7th day, and ascites is led to
Cross caprylic acid-ammonium purifying.Under the conditions of meta-acid, caprylic acid can be precipitated in ascites except ultrawhite other of IgG immune globulins
Foreign protein, is then centrifuged for, and abandons precipitation；The monoclonal antibody of IgG types is precipitated with the ammonium sulfate of equivalent saturation degree again, is centrifuged,
Supernatant is abandoned, with 0.01 M PBS solutions（pH7.4）After dissolving, dialysis desalting, the monoclonal antibody for finally giving after purification is placed in-
20 DEG C of preservations.
5.1 coatings：By coating antigen FM-OVA with 0.05M pH9.6 carbonate buffer solutions the doubling dilution since 1 μ g/mL,
100 μ L/ holes, 37 DEG C of reaction 2h；
5.2 washings：Solution in plate is inclined, and is washed with cleaning solution 3 times, each 3min；
5.3 closings：After patting dry, 200 μ L/hole confining liquid, 37 DEG C of reaction 2h are added.Dry for standby after washing；
5.4 sample-addings：By antiserum since 1 ︰ 1000 doubling dilution, and be added in the coating hole of each dilution factor, 100 μ L/ holes,
37 DEG C of reaction 30min；Fully after washing, the HRP- sheep anti-mouse iggs of the dilutions of 1 ︰ 3000,100 μ L/ holes, 37 DEG C of reactions are added
30min；
5.5 colour developings：ELISA Plate is taken out, fully after washing, the TMB nitrite ions of 100 μ L, 37 DEG C of lucifuge reactions is added per hole
15min；
5.6 terminate and determine：50 μ L2M H are added per hole2SO4Then terminate liquid determines each hole with terminating reaction with ELIASA
OD 450Value.
The IC of monoclonal antibody flunixin meglumine is determined with ic-ELISA50It is 0.24 ng/mL, illustrates to Flunixin Portugal
Methylamine has good sensitivity, can be used for the detection of flunixin meglumine immunoassay.
The configuration of solution：Carbonate buffer solution（CBS）：Weigh Na2CO31.59 g, NaHCO32.93 g, are dissolved in respectively
Mix after a small amount of distilled water, plus distilled water is mixed to about 800mL, adjusts pH value to 9.6, plus distilled water to be 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 % Tween-20s；
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.The mixing of 1 ︰ 5 by volume of A, B liquid is TMB nitrite ions, is now mixed with existing.
Claims (3)
1. one plant of flunixin meglumine monoclonal antibody hybridoma cell strain YY, has been preserved in Chinese microorganism strain preservation management
Committee's common micro-organisms center, abbreviation CGMCC, deposit number is CGMCC No.13096.
2. flunixin meglumine monoclonal antibody, it is characterised in that：It is the fluorine of CGMCC No.13096 by the deposit number
The pungent meglumine monoclonal antibody hybridoma cell strain YY secretions of Buddhist nun are produced.
3. the application of flunixin meglumine monoclonal antibody described in claim 2, it is characterised in that：For in food safety detection
The analysis detection of flunixin meglumine residual.
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| CN201710285176.XA CN106867971B (en) | 2017-04-27 | 2017-04-27 | Flunixin meglumine monoclonal antibody hybridoma cell strain YY and application thereof |
| PCT/CN2018/081847 WO2018196573A1 (en) | 2017-04-27 | 2018-04-04 | Flunixin meglumine monoclonal antibody hybridoma cell strain yy and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018196573A1 (en) * | 2017-04-27 | 2018-11-01 | 江南大学 | Flunixin meglumine monoclonal antibody hybridoma cell strain yy and application thereof |
| CN108866009A (en) * | 2018-08-16 | 2018-11-23 | 江南大学 | One plant of metalaxyl monoclonal antibody hybridoma cell strain and its application |
| CN114316027A (en) * | 2020-10-10 | 2022-04-12 | 中国农业大学 | Flunixin artificial antigen and preparation method and application thereof |
| CN114774368A (en) * | 2022-05-16 | 2022-07-22 | 江南大学 | Hybridoma cell strain secreting flumioxazin-resistant monoclonal antibody and application thereof |
| CN118373916A (en) * | 2024-06-26 | 2024-07-23 | 北京纳百生物科技有限公司 | Flunixin meglumine monoclonal antibody, detection test strip and application thereof |
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| CN105754955A (en) * | 2016-05-17 | 2016-07-13 | 江南大学 | Thiacloprid and acetamiprid monoclonal antibody hybridoma cell strain GW and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106867971B (en) * | 2017-04-27 | 2020-05-05 | 江南大学 | Flunixin meglumine monoclonal antibody hybridoma cell strain YY and application thereof |
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| CN105754955A (en) * | 2016-05-17 | 2016-07-13 | 江南大学 | Thiacloprid and acetamiprid monoclonal antibody hybridoma cell strain GW and application thereof |
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| BELAL, F. F.等: "Micellar high performance liquid chromatographic determination of flunixin meglumine in bulk, pharmaceutical dosage forms, bovine liver and kidney", 《ANALYTICAL CHEMISTRY RESEARCH》 * |
| LU LIN ET AL.: "Development of IC-ELISA and immunochromatographic strip assay for the detection of flunixin meglumine in milk", 《FOOD AND AGRICULTURAL IMMUNOLOGY》 * |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018196573A1 (en) * | 2017-04-27 | 2018-11-01 | 江南大学 | Flunixin meglumine monoclonal antibody hybridoma cell strain yy and application thereof |
| CN108866009A (en) * | 2018-08-16 | 2018-11-23 | 江南大学 | One plant of metalaxyl monoclonal antibody hybridoma cell strain and its application |
| CN114316027A (en) * | 2020-10-10 | 2022-04-12 | 中国农业大学 | Flunixin artificial antigen and preparation method and application thereof |
| CN114316027B (en) * | 2020-10-10 | 2023-09-05 | 中国农业大学 | Fluoronii Xin Rengong antigen and preparation method and application thereof |
| CN114774368A (en) * | 2022-05-16 | 2022-07-22 | 江南大学 | Hybridoma cell strain secreting flumioxazin-resistant monoclonal antibody and application thereof |
| CN118373916A (en) * | 2024-06-26 | 2024-07-23 | 北京纳百生物科技有限公司 | Flunixin meglumine monoclonal antibody, detection test strip and application thereof |
| CN118373916B (en) * | 2024-06-26 | 2024-08-16 | 北京纳百生物科技有限公司 | Flunixin meglumine monoclonal antibody, detection test strip and application thereof |
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| WO2018196573A1 (en) | 2018-11-01 |
| CN106867971B (en) | 2020-05-05 |
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