CN106929478A - One plant of anti-Mabuterol monoclonal antibody hybridoma cell strain GZ03 and its application - Google Patents

One plant of anti-Mabuterol monoclonal antibody hybridoma cell strain GZ03 and its application Download PDF

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CN106929478A
CN106929478A CN201710280909.0A CN201710280909A CN106929478A CN 106929478 A CN106929478 A CN 106929478A CN 201710280909 A CN201710280909 A CN 201710280909A CN 106929478 A CN106929478 A CN 106929478A
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mabuterol
monoclonal antibody
hybridoma cell
cell strain
antibody hybridoma
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CN106929478B (en
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匡华
刘睿
胥传来
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
    • G01N33/94—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

The invention discloses one plant of anti-Mabuterol monoclonal antibody hybridoma cell strain GZ03 and its application, belong to food security field of immunological detection.Anti- Mabuterol monoclonal antibody hybridoma cell strain GZ03 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.13091.The monoclonal antibody of this cell line secretion, has preferably specificity and detection sensitivity to Mabuterol（IC50It is 0.41ng/mL to be worth）, can be used for the residue detection of Mabuterol in food.

Description

One plant of anti-Mabuterol monoclonal antibody hybridoma cell strain GZ03 and its application
Technical field
The anti-Mabuterol monoclonal antibody hybridoma cell strain GZ03 of one plant of the present invention and its application, belong to food security and exempt from Epidemiology detection field, is related to the anti-Mabuterol monoclonal antibody of hybridoma cell strain GZ03 and its generation.
Background technology
β-adrenoreceptor excitant is the feed addictive that a class can promote lean meat to grow, including hydrochloric acid Ke Lunte Sieve, bromine Boot sieve, Mabuterol, salbutamol, Ractopamine, Dopamine hydrochloride, bricalin, phenolethanolamine A, class 15 kinds of Boot sieve, hydrochloric acid Zilpaterol, clorprenaline hydrochloride, western Boot sieve, tartaric acid Afromoterol, formoterol fumarate etc. Medicine.Wherein clenobuterol hydrochloride is used as feed addictive earliest, and the scope of application is also the most extensive.Mabuterol（Mab）'s Chemical constitution and Clenbuterol（cl）It is closely similar, it is more likely that the substitute as Clenbuterol.β-adrenoreceptor is excited This kind of medicine of agent medically has lax bronchial smooth muscle, reduces vasopermeability, the effect that performance is relievingd asthma, it is also possible to plus Cardiac stimulant myotility, accelerates the rhythm of the heart, improves the excitability of heart, if this kind of medicine is added in feed, can be notable The Lean Gain of the livestocks such as pig, ox, sheep is improved, so that body weight is also increased.And this kind of pharmaceutical properties are stable, not fragile It is bad, can be detrimental to health by intake of taking food, cause obvious physiological-toxicity, such as palpitaition, tremble, n and V, dizziness Dizzy, oversensitive, vasodilation, palpitate quickly, particularly there is more obvious harm to three-high patient and hyperthyroid patient, It is serious it could even be possible to causing death.Accordingly legislation is forbidded strictly to use for numerous countries and regions such as European Union, the U.S., China Beta-2-agonists are used as animal feed additive.The Ministry of Agriculture of China has also successively promulgated No. 176, No. 193, No. 235, No. 1519 etc. one Series bulletin, must not clearly propose the adding as animal feed and its drinking water using 15 kinds of β-adrenoreceptor excitants of the above Plus agent.
The method of detection bromine Boot sieve, Clenbuterol and Mabuterol is mainly gas chromatography at present（GC）, liquid phase color Spectrometry（HPLC）, the side such as AAS, molecular imprinting method, Capillary Electrophoresis, gas chromatography mass spectrometry method, electrochemical immunosensor Method, but these methods exist cumbersome, take, the shortcomings of expense is somewhat expensive, it is impossible to realize the quick detection of a large amount of samples. Hence set up a kind of fast and convenient detection method significant.ELISA（ELISA）It is a kind of extremely efficient, quick Sense, quick detection method, purity requirement during detection to sample is not high and easy to operate, it is adaptable to the scene of great amount of samples Quick detection.But obtaining high specific and highly sensitive monoclonal monomer is the premise of immunology detection, wherein manually resisting Former synthesis is a wherein important step.
The content of the invention
It is an object of the invention to provide a kind of to monoclonal of the Mabuterol with preferably specificity and detection sensitivity The preparation method of antibody hybridoma cell strain.
The present invention provides a kind of anti-Mabuterol monoclonal antibody hybridoma cell strain, the antibody pair prepared by the cell line Mabuterol has preferably specificity and detection sensitivity, can be used to set up the immunological detection method of Mabuterol.
Technical scheme：One plant of anti-Mabuterol monoclonal antibody hybridoma cell strain GZ03, in being preserved in State's Microbiological Culture Collection administration committee common micro-organisms center, abbreviation CGMCC, deposit number is CGMCC No.13091.
Anti- Mabuterol monoclonal antibody, it is the anti-Mabuterol Dan Ke of CGMCC No.13091 by the deposit number Grand antibody hybridoma cell strain GZ03 secretions are produced.
The application of anti-Mabuterol monoclonal antibody, can be used for the residue detection of Mabuterol in food.
The preparation basic step of cell line GZ03 that the present invention is provided is：
1）The synthesis of immunogene：15.62 mg Mabuterols are weighed in vial, the 1 M HCl solutions of 135 μ L are added, 1 mL ultra-pure waters are added to being completely dissolved, 4 DEG C of min of precooling 30；30% NaNO of 13 μ L is added in the solution2Solution, ice The h of water-bath 1, obtains activating solution；Three parts of equivalent KLH protein 10s mg are diluted with CB buffer solutions；By activating solution according to volume ratio The ︰ 3 of 1 ︰ 2 are slowly dropped in each part carrier protein KLH respectively, and coupling protein builds immunogene.Side is added dropwise activating solution, side regulation pH To 9.0, until reaction solution darkens, yellow stops that activating solution is added dropwise；After 4 DEG C of ice bath reaction 4h, separated by dialysing and resisted completely Small molecule that is former and not being coupled, and identified by UV absorption scan method；
2）The synthesis of coating antigen：Because salbutamol and Mabuterol all have tert-butylamine based structures, so this experiment is using different Source coating detection, claims the mg of salbutamol 100（0.42 mmol）It is dissolved in absolute ethyl alcohol；Again plus 5.1 mg DMAP（4- dimethyl Aminopyridine）With 0.5 mL anhydrous pyridines, 63 mg succinyl oxides are divided into 5 equal portions, add one every 0.5 h by the reaction of 4 DEG C of ice baths Part in reaction solution system, 5 h are reacted under ice bath environment while stirring；Filtering reacting liquid, white precipitate material is collected, and uses second Alcohol washs white depositions, and drying obtains compound A.9.4 mg compounds A are weighed in brown vial, adds 400 μ L's The pH 4.7 of dry DMF and 600 μ L, the dissolving of 0.1 M MES cushioning liquid；15.9 mg EDC and 9.5 mg NHS solids are added Enter in the reaction bulb of the compound A to after dissolving, stir 6~8 h, obtain activating solution；Weigh bovine serum albumin BSA and be dissolved in CB In solution, activating solution is added in protein solution, pH to 9.0 is adjusted with 1 M NaOH solutions, room temperature reaction is overnight.Entirely Process lucifuge is carried out.The small haptens for separating comlete antigen and not being coupled by dialysing, and by UV absorption scanning side Method is identified；
3）Mouse it is immune：After Mabuterol comlete antigen and equivalent Freund's adjuvant mixing and emulsifying, exempted from by dorsal sc injection Epidemic disease BALB/c mouse.First time immune complete Freund's adjuvant, all cannot be used up full Freund's adjuvant afterwards.Between head exempts to exempt from reinforcement Interval one month, reinforcement is spaced 21 days between exempting from.Last time uses Mabuterol comlete antigen（Without adjuvant）Impact is immune；It is logical Cross indirect ELISA detection serum titer and suppression；
4）Cell fusion is set up with cell line：By polyethylene glycol（PEG 1500）Method is thin by mouse boosting cell and mouse myeloma Born of the same parents are merged, and by HAT medium cultures, positive cell hole are detected using indirect ELISA, and further with indirect competition ELISA method determines the inhibition of positive cell hole, by limiting dilution assay to there is the positive cell hole for preferably suppressing to carry out three Secondary subclone, final screening obtains hybridoma cell strain GZ03；
5）The identification of hybridoma cell strain GZ03 properties：Sensitivity and specificity are determined by ELISA method.
Beneficial effects of the present invention：（1）The anti-Mabuterol cell strain of monoclonal antibody GZ03 that the present invention is obtained, to horse cloth Special sieve has preferable detection sensitivity and specificity（IC50It is 0.41ng/mL to be worth）, can be used for the residual of Mabuterol in food Detection；（2）A kind of thinking of new hapten synthesis, is detected with different primordial covering, obtains the good monoclonal of sensitivity Antibody cell strain.
Biological material specimens preservation：Monoclonal cell strain GZ03, has been preserved in Chinese microorganism strain preservation conservator Can common micro-organisms center, abbreviation CGMCC, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese Academy of Sciences microorganism Research institute, deposit number is CGMCC No.13091, preservation date on October 31st, 2016.
Brief description of the drawings
Fig. 1 Mabuterols immunogene synthesizes ultraviolet qualification figure.
The suppression standard curve of the monoclonal antibody of Fig. 2 GZ03 secretions.
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 is by the way that by Mabuterol comlete antigen immune mouse, by cell fusion, HAT selective mediums are trained Support, cell conditioned medium is screened by indirect ELISA and indirect competitive ELISA, having finally given has preferable specificity to Mabuterol With the monoclonal antibody hybridoma cell strain of sensitivity.
Embodiment 1：The preparation of hybridoma cell strain GZ03
1st, the synthesis of comlete antigen
7.81 mg Mabuterols are weighed in vial, the 1 M HCl solutions of 68 μ L are added, 1 mL ultra-pure waters is added extremely It is completely dissolved, 4 DEG C of min of precooling 30；30% NaNO of 13 μ L is added in the solution2Solution, ice-water bath reacts 1 h, is lived Change liquid；KLH protein 10s mg is diluted with CB buffer solutions；Activating solution is slowly dropped in carrier protein KLH, side is added dropwise activating solution, Side adjusts pH to 9.0, until reaction solution darkens, yellow stops that activating solution is added dropwise；After 4 DEG C of ice bath reaction 4h, by dialysis point From comlete antigen and the small molecule not being coupled, and identified by UV absorption scan method.
2nd, animal immune
The BALB/C mice of 6~8 week old of health is selected to be immunized.Mabuterol comlete antigen is taken to be mixed with equivalent Freund's adjuvant After closing emulsification, BALB/c mouse is immunized by dorsal sc injection.Immune complete Freund's adjuvant, all cannots be used up afterwards for the first time Full Freund's adjuvant.Head exempt from reinforcement exempt between be spaced one month, reinforcement exempt between be spaced 21 days.Three exempt to take a blood sample for latter 7 days, between use Connect competitive ELISA method and determine mice serum potency and suppression, select the potency mouse for having suppressed high, impact within 18 days after exempting from four It is immune, do not use adjuvant, intraperitoneal injection.
3rd, cell fusion
After impact is immune 3 days, according to conventional PEG（Polyethylene glycol, molecular weight is 1500）Method carries out cell fusion, specific step It is rapid as follows：（1）It is aseptic to take mouse spleen, grind and obtain splenocyte suspension by 200 mesh cell screen clothes, and carry out cytometer Number；（2）SP2/0 cells are collected, is suspended in RPMI-1640 basic culture solutions, carry out cell count；（3）By splenocyte and SP2/0 cells mix according to the ratio than 1 ︰ 10 of counting, with 50% PEG fusions after centrifugation, the min of time 1, afterwards according to from slow To fast, RPMI-1640 basic culture solutions are added, the RPMI- containing 20% hyclone, 2% 50 × HAT is suspended in after centrifugation In 1640 screening and culturing liquid, 96 porocyte culture plates are added to, are placed in 37 DEG C, 5%CO2Incubator in cultivate.
4th, cell screening and cell line are set up carries out RPMI-1640 screening trainings on the 3rd day in cell fusion to fused cell Nutrient solution partly changes liquid, carries out within the 5th day being changed entirely with the RPMI-1640 transition nutrient solutions containing 20% hyclone, 1% 100 × HT Liquid, took cell conditioned medium and is screened at the 7th day.Screening is in two steps：The first step first filters out positive cell hole with indirect ELISA, Second step is standard items from Mabuterol, and inhibition measure is carried out to positive cell with indirect competitive ELISA.Selection is to horse Boot sieve standard items have the cell hole of preferable suppression, are subcloned using limiting dilution assay, are detected with same method. In triplicate, cell line GZ03 is obtained.
5th, the preparation of monoclonal antibody and identification
Take 8~10 week old BALB/c mouses, every mL of mouse peritoneal injection paraffin oil 1；Every mouse peritoneal injection 1 after 7 days × 106Hybridoma GZ03, collected ascites since the 7th day, ascites was purified by caprylic acid-ammonium, the monoclonal antibody of acquisition It is placed in -20 DEG C of preservations.
Using indirect competitive ELISA, the IC of monoclonal antibody Mabuterol is determined50For：0.41ng/mL, illustrates to horse cloth Special sieve has good sensitivity, can be used for the detection of Mabuterol immunoassay.
6th, antibody application
Hybridoma cell strain GZ03 is added back by the ELISA that monoclonal antibody prepared by internal ascites is applied to Mabuterol Acceptance test, comprises the following steps that：
（1）Coating：By coating antigen sal-BSA with 0.05M pH9.6 carbonate buffer solutions doubling dilution, 100 since 2 μ g/mL μ L/ holes, 37 DEG C of reaction 2h；
（2）Washing：Solution in plate is inclined, is dried, and washed with cleaning solution 3 times, each 3min；
（3）Closing：After patting dry, 200 μ L/ holes confining liquids, 37 DEG C of reaction 2h, dry for standby after washing are added；
（4）Sample-adding：PBS solution and standard solution are added in plate after the drying, antiserum is opened in 50 μ L/ holes from 1 ︰ 1000 Beginning doubling dilution, and be added in the coating hole of each dilution factor, 50 μ L/ holes, 37 DEG C of 30 min of reaction；Fully after washing, add The HRP- sheep anti-mouse iggs of the dilutions of 1 ︰ 3000,100 μ L/ holes, 37 DEG C of 30 min of reaction；
（5）Colour developing：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；
（6）Terminate and determine：50 μ L 2M H are added per hole2SO4Then terminate liquid determines each hole with terminating reaction with ELIASA OD450Value；
（7）As a result interpretation：With OD4502.1 times of value more than or equal to negative control hole（That is P/N >=2.1）Corresponding serum is most Highly diluted multiple is the ELISA potency of serum.
The configuration of solution：
Carbonate buffer solution（CBS）：Weigh Na2CO31.59g, NaHCO32.93g, mixes after a small amount of distilled water is dissolved in respectively, Plus distilled water is mixed to about 800mL, pH value to 9.6, plus distilled water is adjusted to be settled to 1000mL, 4 DEG C of storages are standby；
Phosphate buffer（PBS）：8.00 g NaCl, 0.2 g KCl, 0.2 g KH2PO4, 2.9 g Na2HPO4·12 H2O, is dissolved in 800 mL pure water, adjusts pH to 7.2~7.4 with NaOH or HCl, is settled to 1000 mL；
PBST：PBS containing 0.05% Tween-20；
TMB nitrite ions：A liquid：Na2HPO4·12H2O 18.43g, citric acid 9.33g, pure water is settled to 1000 mL；B liquid：60 Mg TMB are dissolved in 100 mL ethylene glycol；The mixing of 1 ︰ 5 by volume of A, B liquid is TMB nitrite ions, is now mixed with existing.
It is only in sum presently preferred embodiments of the present invention, not for limiting practical range of the invention.It is i.e. all The equivalence changes made according to the content of scope of the present invention patent and modification, all should be technology category of the invention.

Claims (3)

1. one plant of anti-Mabuterol monoclonal antibody hybridoma cell strain GZ03, has been preserved in Chinese microorganism strain preservation management Committee's common micro-organisms center, abbreviation CGMCC, deposit number is CGMCC No.13091.
2. anti-Mabuterol monoclonal antibody, it is characterised in that：It is the anti-horse of CGMCC No.13091 by the deposit number The strain GZ03 secretions of Boot sieve monoclonal antibody hybridoma cell are produced.
3. the application of anti-Mabuterol monoclonal antibody described in claim 2, it is characterised in that：Can be used for Mabuterol in food Residue detection.
CN201710280909.0A 2017-04-26 2017-04-26 Marbuterol-resistant monoclonal antibody hybridoma cell strain GZ03 and application thereof Active CN106929478B (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0338507A2 (en) * 1988-04-21 1989-10-25 Basotherm GmbH Use of mabuterol and related compounds for intraocular pressure adjustment
CN103149352A (en) * 2013-02-05 2013-06-12 江西中德生物工程有限公司 Mabuterol colloidal gold test strip and preparation method thereof
CN103884790A (en) * 2014-03-21 2014-06-25 烟台杰科检测服务有限公司 Method for determining multiresidue of veterinary drugs in animal-derived foods

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0338507A2 (en) * 1988-04-21 1989-10-25 Basotherm GmbH Use of mabuterol and related compounds for intraocular pressure adjustment
CN103149352A (en) * 2013-02-05 2013-06-12 江西中德生物工程有限公司 Mabuterol colloidal gold test strip and preparation method thereof
CN103884790A (en) * 2014-03-21 2014-06-25 烟台杰科检测服务有限公司 Method for determining multiresidue of veterinary drugs in animal-derived foods

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LIQIANG LIU 等: "structure-specific hapten design for the screening of highly sensitive and specific monoclonal antibody to salbutamol", 《ANALYTICAL METHODS》 *
苏丽芳 等: "苯乙醇胺A半抗原、抗原及其制备方法的建立", 《中国兽医杂志》 *

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