CN109112112A - Monoclonal antibody and the application of a kind of anti-beta-receptor agonist group-specific monoclonal antibody hybridoma cell strain and its secretion - Google Patents
Monoclonal antibody and the application of a kind of anti-beta-receptor agonist group-specific monoclonal antibody hybridoma cell strain and its secretion Download PDFInfo
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- 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
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- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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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
The present invention relates to field of biotechnology, the monoclonal antibody of a kind of anti-beta-receptor agonist group-specific monoclonal antibody hybridoma cell strain and its secretion is disclosed.Hybridoma cell strain of the present invention is prepared by salbutamol artificial antigen, and the monoclonal antibody of secretion can identify the beta-receptors agonist such as salbutamol, clenobuterol hydrochloride, Terbutaline, bromine Boot sieve and Cimbuterol simultaneously, has group-specific；Thus immunoaffinity chromatography glue and other beta-receptor agonists are prepared, pre-treatment purification reagent is surveyed as machine such as the immunoaffinity chromatography glue mixing of Ractopamine and combines a variety of clenbuterol hydrochloride ingredients that can efficiently detect in pork, pork liver and pig urine samples with HPLC-FLD, LC-MS/MS detection technique, has higher accuracy, sensitivity.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of anti-beta-receptor agonist group-specific monoclonal antibody is miscellaneous
Hand over monoclonal antibody and the application of tumor cell strain and its secretion.
Background technique
It is to represent three kinds of main clenbuterol hydrochlorides in meat product and move with clenobuterol hydrochloride, Ractopamine, salbutamol
Object internal organ residual event, which remains incessant after repeated prohibition, to happen occasionally because of the primary mesh of residue of veterinary drug for forming food safety Regulation department Monitoring and supervision
Mark object.It is widely used to using ELISA detection kit and immune colloidal gold test strip as the immunoassay technology of representative above-mentioned
The high-throughput fast qualitative screening of three kinds of object residual samples.But confirmation is remained to above-mentioned object and accurate quantification must
It must be through high performance liquid chromatography with fluorescence detector (HPLC-FLD) even liquid-matter series connection spectrum (LC-MS/MS) measurement.With Waters
Actually detected need can be met in indexs such as chromatographic isolation efficiency, quantitative limit, precision by providing above-mentioned instrument and equipment with Agilent company
It asks, but actually detected effect overwhelming depends on machine and surveys sample introduction sample pre-treatment effect.
For above-mentioned three kinds of clenbuterol hydrochlorides be represent multicomponent beta-receptor agonist remain accurate quantification analysis, the Ministry of Agriculture in
Disclose within 2008 national standard " beta-receptor agonist residue detection-liquid chromatography-tandem mass spectrometry detection in animal derived food
Method " and provide to handle sample detection using Solid phase extraction, there are following four big restraining factors to seriously affect detection effect for the method
Fruit:
The first, operating procedure is more, poisonous and harmful consumption of organic solvent is big, needing instrument and equipment, more (such as Rotary Evaporators, nitrogen are blown
Instrument etc.), liquid phase dissolved system transform is more, time-consuming.
The second, object loss late is high: large biological molecule removal, liquid-liquid extraction/Solid Phase Extraction, purify object into
Object loss is likely to cause in multiple operation links such as sample buffer redissolution.
Although third, clenobuterol hydrochloride, Ractopamine, salbutamol belong to phenyl ethylamine class medicine in chemical structure
Object, but three's physicochemical property has larger difference, is difficult with unified extracting solution and extract liquor or single solid-phase extraction column and guarantees every
Kind drug, which can obtain, is satisfied with extraction-clean-up effect.
4th, the only certain selectivity of liquid-liquid extraction/Solid Phase Extraction, is being purified to object simultaneously, is unavoidably being mixed into
With matrix interference object similar in object physicochemical property (the especially matrix such as Animal Liver, kidney complex samples), Interference Peaks are as occurred
The interpretation of machine measured data can be seriously affected in object retention time region.
Immunoaffinity chromatography chromatography IAC (Immunoaffinity chromatography) is compared to SPE (solid
Phase extraction) purification method its huge technical advantage is mainly shown as:
The first, object extracts simply from biological sample, and reaction system is mild, it is only necessary to which minute quantity does not use even organic
Solvent.
The second, (fat content is excessively high in such as sample without large biological molecule in a large amount of removal samples for object extraction step
Partial fat suitably can be removed and object is specifically bound in order to avoid influencing Immunobead), therefore can avoid removing because of macromolecular
Target portion is caused to lose.
Third, to the powerful selectivity of object, specifically bind response characteristic by force by Ag-Ab and determined, it is positive
Each object peak shape is sharp in chromatogram after upper press proof product HPLC-FLD that sample is purified through IAC measurement, each component retention time
Clearly, without miscellaneous peak or rarely, miscellaneous peak is interfered.
4th, optimize to the well-designed of immunoaffinity chromatography eluent and repeatedly, it can be by eluent and sample introduction buffer
It is unified into same buffer, or adds organic phase (such as certain proportion of certain volume ratio in the eluted sample with object
Methanol or acetonitrile) direct injected.
Compared to now widely used solid-phase extraction column SPE (solid phase extraction), affine in immunity layer
Analysis column, which is lacked competitiveness, is that antibody dosage is big and causes high cost, and only possessing independent intellectual property rights monoclonal antibody cell strain-can pole
It is big to reduce purified monoclonal antibody protein production cost, it is allowed to extensive use and is possibly realized.
It is mixed before residual sample machine is surveyed in processing just because of its strong selectivity strongly limits it in multicomponent object
Application；Therefore preparation expands its object covering surface substitution SPE column for beta-2-agonists multicomponent group-specific monoclonal antibody and becomes
It may.
Summary of the invention
Resist for beta-receptor agonist with group-specific monoclonal in view of this, the purpose of the present invention is to provide one kind
Body hybridoma cell strain and its monoclonal antibody of generation, so that the monoclonal antibody that the hybridoma cell strain generates is simultaneously to husky butylamine
Alcohol, clenobuterol hydrochloride, Terbutaline, bromine Boot sieve and Cimbuterol have higher cross reaction, are that beta-receptor agonist is more
Component mixing residual sample rapid screening provides immune detection core reagent；
Another object of the present invention is that providing a kind of immunoaffinity chromatography prepared using said monoclonal antibody
Chromatography glue and preparation method thereof, so that the immunoaffinity chromatography chromatography glue not only has biggish intersection to each beta-receptor agonist
Reactivity, and can have more preferably purified treatment ability and mark-on than national standard SPE approach in immune affinity chromatographic detection
The rate of recovery.
To achieve the above object, the invention provides the following technical scheme:
Anti- beta-receptor agonist group-specific monoclonal antibody hybridoma cell strain, deposit number CCTCC
NO.C2016208 is named as 1C3A, and is preserved in China typical culture collection center on December 4th, 2016, and address is force
In the Luo Jia Shan Wuhan University of the Wuchang Han Shi.
The present invention combines OVA carrier protein as immunogen immune mouse using salbutamol, obtains by screening above-mentioned
1C3A hybridoma cell strain, the monoclonal antibody induced and a variety of beta-receptor agonists carry out the detection of cross reacting rate, husky when assert
When the CR value of butylamine alcohol is 100%, discovery clenobuterol hydrochloride, Terbutaline, bromine Boot sieve and Cimbuterol cross reacting rate
Larger, CR value is respectively 66.8%, 57.3%, 42.1%, 121.5%.
Based on above-mentioned excellent technical effect, the present invention provides the hybridoma and its monoclonal antibodies of generation
The application in pattern detection and/or the residual immune detection product preparation of beta-receptor agonist is remained in beta-receptor agonist；Wherein,
The beta-receptor agonist is preferably clenbuterol hydrochloride, and the clenbuterol hydrochloride is selected from salbutamol, clenobuterol hydrochloride, Terbutaline, bromine
One or more of Boot sieve and Cimbuterol；Wherein salbutamol, clenobuterol hydrochloride can be used as weight object
Carry out selective analysis.
Beta-receptor agonist of the present invention residual immune detection product is preferably immunoaffinity chromatography chromatography glue or thus
The immune affinity chromatography chromatographic column of pre-fill.
The present invention specifically provides a kind of beta-receptor agonist group-specific immunoaffinity chromatography chromatography glue, by deposit number
CCTCC NO.C2016208 hybridoma cell strain secretion generate monoclonal antibody it is purified again with CNBr-activated
Sepharose-4B gelatinization is cross-linked to form immobilized antibody albumen and obtains.
Preferably, the amount ratio of the purified monoclonal antibody albumen and CNBr-activated Sepharose-4B glue: 5mg
Purified monoclonal antibody albumen: 1ml CNBr-activated Sepharose-4B swelling gum.
By the excellent characteristics that monoclonal antibody of the present invention has, so that immunoaffinity chromatography of the present invention
Chromatography glue or immune affinity chromatography chromatographic column (filling in column bed by immunoaffinity chromatography chromatography mucilage binding to obtain) are same with preferably
Performance.Meanwhile the immunoaffinity chromatography chromatography glue or immune affinity chromatography chromatographic column be in immunoaffinity chromatography chromatography, phase
Than national standard (beta-2-agonists residue detection-liquid chromatography-tandem matter in 1025 bulletin -18-2008 animal derived food of the Ministry of Agriculture
Spectrometry) Solid Phase Extraction SPE (solid phase extraction) purification method have more preferably recovery of standard addition and purification
Processing capacity.
Meanwhile the present invention also provides the preparation methods of the immunoaffinity chromatography chromatography glue, comprising:
Step 1 obtains monoclonal antibody protein with the hybridoma of deposit number CCTCC NO.C2016208 and carries out
Purifying, monoclonal antibody albumen carries out dialysis pretreatment after purification, spare；
Acquisition processing glue is spare after CNBr-activated Sepharose-4B is swollen, eluriates pretreatment；
Step 2 handles glue to by being added in pretreated purified monoclonal antibody albumen described in step 1, room temperature lower swing is incubated for,
It is then centrifuged for abandoning supernatant, NaHCO is added3/ NaCl buffer solution continues to swing incubation at room temperature, and supernatant is abandoned in centrifugation, and washing obtains
Obtain antibody protein cross-linked rubber；
Step 3 takes antibody protein cross-linked rubber addition Tris-HCl in step 2 to be placed in the incubation of room temperature lower swing to close the glue
In not with the excess activation group of monoclonal antibody protein-crosslinking, centrifugation abandon supernatant, then with HAC/NaCl with bis- kinds of Tris-HCl/NaCl
The washing of buffer alternating centrifugal, is finally resuspended in Tris-HCl-NaCl buffer for cross-linked rubber, saves backup, that is, obtain in 4 DEG C
The immunoaffinity chromatography chromatography glue.
It is produced in immunoaffinity chromatography glue of the present invention by deposit number for the secretion of CCTCC NO.C2016208 hybridoma cell strain
Raw monoclonal antibody can be used the art conventional method and be prepared, and such as pass through the side of inducing in vivo with hybridoma cell strain
Formula obtains mouse ascites sample, specifically can refer to following method:
In inoculation hybridoma the last week, 0.5mL norphytane sensitization first is injected to male Balb/c mouse peritoneal.So
Every mouse peritoneal injection 5 × 10 afterwards5The hybridoma of left and right.After inoculation 10 days, mouse web portion obvious tumefaction, at mouse
It when dying state on one's deathbed frequently, is put to death, is immersed in body surface in 75% alcohol and sterilizes, it is sterile to split abdomen, abdomen is taken out with dropper
Water contains target antibody in ascites.The ascites gathered is centrifuged, haemocyte and blood plasma is removed, obtains flaxen ascites.It is past
Glycerol and 0.01mol/L PBS (pH 7.4), volume ratio 50:45:5 are added in ascites, packing is placed in -20 DEG C or -80 DEG C of ice
It is saved in case.
The present invention obtains according to the method described above induces ascites sample by protein G resin one-step method purified monoclonal antibody egg
It is white, SDS-PAGE electroresis appraisal as the result is shown: ascites purified contains only IgG type, (resists containing 50Kd (heavy chain of antibody) and 25Kd
Body light chain) two obvious protein bands without other foreign protein bands show that ascites approach is induced by the hybridoma cell strain to be obtained
High-purity monoclonal antibody albumen is obtained, can satisfy the detection antibody demand in immunoaffinity chromatography glue for preparing.
Preferably, the amount ratio of the purified monoclonal antibody and CNBr-activated Sepharose-4B glue: 5mg purifying
Monoclonal antibody albumen: 1ml CNBr-activated Sepharose-4B swelling gum.
Preferably, the dialysis of the purified monoclonal antibody albumen pre-processes are as follows:
Purified monoclonal antibody albumen is placed in bag filter, in NaHCO34 DEG C of dialysed overnights in/NaCl buffer solution, it is single after dialysis
Anti- albumen filtering with microporous membrane, filtrate save backup in 4 DEG C.
It is further preferred that the purified monoclonal antibody albumen carries out dialysis pretreatment are as follows:
Purified monoclonal antibody albumen is placed in 7500-14000MW bag filter, in 100-200 × 0.1M NaHCO3/0.5M NaCl
(pH8.3) 4 DEG C of dialysed overnights in buffer solution, every 4-6h are changed the liquid once, 0.22 μM of filtering with microporous membrane of albumen after dialysis, filter
Liquid is saved backup in 4 DEG C.
Preferably, described be swollen CNBr-activated Sepharose-4B, eluriate pretreatment are as follows:
It weighs CNBr-activated Sepharose 4B solid powder and dilute HCl swelling is added, centrifugation is abandoned supernatant, is added
Dilute HCl centrifuge washing is to remove in CNBr-activated Sepharose 4B glue after other small-molecule chemical reagents, then uses
NaHCO3/ NaCl buffer solution centrifuge washing is primary, spare.
It is further preferred that described be swollen CNBr-activated Sepharose-4B, eluriate pretreatment are as follows:
1g CNBr-activated Sepharose 4B solid powder is weighed in 50ml centrifuge tube, centrifuge tube adds 30ml
1mM HCl room temperature gently swings swelling 30min in swing-bed, and room temperature 2000r/min 3min abandons supernatant, and centrifuge tube adds 30ml
1mM HCl repeats sufficiently to remove in above-mentioned glue after other small-molecule chemical reagents for 8-10 times with same method centrifuge washing, then
With 30ml 0.1M NaHCO3/ 0.5M NaCl (pH8.3) buffer solution centrifuge washing is primary, spare.
Preferably, step 2 are as follows:
Pretreatment purified monoclonal antibody albumen described in step 1 is added into containing the 50ml centrifuge tube for handling glue described in step 1, lid
Good lid is simultaneously sealed with parafilm film, is placed in room temperature in swing-bed and is swung incubation 1h, and room temperature 2000r/min 10min abandons supernatant, from
Heart pipe adds 30ml 0.1M NaHCO3/ 0.5M NaCl (pH8.3) buffer solution is placed in room temperature in swing-bed and swings incubation 10min,
Room temperature 2000r/min 10min abandons supernatant, then uses NaHCO3It is not handed over sufficiently removing for/NaCl buffer solution centrifuge washing 5-6 times
The albumen of connection obtains antibody protein cross-linked rubber.
Preferably, step 3 are as follows:
Step 2 antibody protein cross-linked rubber is taken, centrifuge tube is added 30ml 0.1M Tris-HCl (pH 8.0) and is placed in swing-bed
Room temperature swing be incubated for 2h in adequate closure antibody protein cross-linked rubber not with the activated group of protein-crosslinking, room temperature 2000r/min
It is centrifuged 10min and abandons supernatant, centrifuge tube uses 30ml 0.1M HAC/0.5M NaCl (pH4.0) and 30ml 0.1M Tris- respectively
After two kinds of buffer alternating centrifugals of HCl/0.5M NaCl (pH 8.0) wash three circulations, cross-linked rubber is resuspended in 30ml
0.1M Tris-HCl/0.5M NaCl (pH 8.0) buffer adds 0.02%NaN3, saved backup in 4 DEG C, i.e., described in acquisition
Immunoaffinity chromatography chromatography glue.
In the preparation of immunoaffinity chromatography chromatography glue, the present invention passes through purified monoclonal antibody albumen and CNBr-activated
Sepharose-4B gelatinization is cross-linked to form immobilized antibody albumen and obtains, and the immune affinity chromatography chromatographic column is by institute
It states immunoaffinity chromatography chromatography glue and is filled in column bed and obtain.According to the needs of detection, the chromatographic column can also be added and be removed sand
Other clenbuterol hydrochloride monoclonal antibodies preparation except butylamine alcohol, clenobuterol hydrochloride, Terbutaline, bromine Boot sieve and Cimbuterol
Chromatography glue, the method for such as Ractopamine, preparation chromatography glue can refer to preparation method before the present invention.
According to the performance of above-mentioned immunoaffinity chromatography chromatography glue/column, the present invention is also corresponding to provide a kind of detection pork, pig
The method of beta-receptor agonist is detected using the chromatography glue or the chromatographic column by LC-MS/MS in urine or pork liver.
In the specific embodiment of the invention, the LC-MS/MS detection method for pork, pig urine or pork liver is provided, is used
To detect salbutamol, clenobuterol hydrochloride and Ractopamine, wherein Ractopamine monoclonal antibody uses patent
Hybridoma cell strain induction ascites in CN104311438A obtains, and preferably using in Chinese patent 201711065894.2
The Ractopamine immunoaffinity chromatography glue of preparation is mixed with immunoaffinity chromatography chromatography glue of the present invention.
In sample treatment, weighs several portions of 2g/ portions of porks to homogenize or Pig Liver is sub-packed in 50mL centrifuge tube respectively
In, three kinds of clenbuterol hydrochloride mixed standard solutions of different volumes are added, make 1,5,20ng g of each component concentration-1In three parts of matrix samples
(Ractopamine, salbutamol, the clenobuterol hydrochloride mixed in 1ng/g:2g matrix is that 1ng/g makees sample, 5ng/ in this
Ractopamine, salbutamol, the clenobuterol hydrochloride mixed in g:2g matrix is that 5ng/g makees sample, 20ng/g:2g base
Ractopamine, salbutamol, the clenobuterol hydrochloride mixed in matter is that 20ng/g makees sample), then 4mL is added in every pipe
Acetone, mechanical shaking extraction object 15min, 10000 × g are centrifuged 15min, by acetone supernatant move to another 15mL it is clean from
In heart pipe；It repeats this step twice, obtains 12mL acetone supernatant, 50 DEG C are dried with nitrogen；It is redissolved later with 2mL dilute hydrochloric acid, makes mesh
Mark the form that object forms hydrochloride, it is ensured that it is present in water phase；Add 2mL n-hexane reversed phase extraction degreasing, 10000 × g
After being centrifuged 15min, water phase is collected, with 2mol L-1NaOH adjust pH value of solution to 7.4, it is equal to add isometric PBS mixing
It is even, take 1mL water phase to be added in immunoaffinity chromatography glue/column (the monoclonal of Ractopamine containing 0.5mL in every 1.5ml chromatography glue
The chromatography glue of Antibody preparation)；
And for pig urine samples, pig urine is centrifuged 10min at 10000 × g, part 5mL/ parts of pig urine of fetching is sub-packed in
In each pipes of 15mL centrifuge tube, three kinds of clenbuterol hydrochloride mixed standard solutions of different volumes are added, make each component spiked levels 1,5,
20ng mL-1(Ractopamine, salbutamol, the clenobuterol hydrochloride mixed in 1ng/ml:2ml matrix is that 1ng/ml is tried
Sample, Ractopamine, salbutamol, the clenobuterol hydrochloride mixed in 5ng/ml:2ml matrix is that 5ng/ml makees sample,
Ractopamine, salbutamol, the clenobuterol hydrochloride mixed in 20ng/ml:2ml matrix is that 20ng/ml makees sample)；Again
5mL 0.01mol L is added-1PBS (pH 7.4) is uniformly mixed, and takes 1mL urine sample to be added in immunoaffinity chromatography glue/column (every
The chromatography glue that in 1.5ml chromatography glue prepared by ractopamine monoclonal antibody containing 0.5mL).
It being swung after sample and chromatographic column/glue mixing in shaking table room temperature and is incubated for 1h, 1000r/min is centrifuged 3min, supernatant is abandoned,
With 0.01mol/L PBS (pH 7.4) centrifuge washing 3 times, then with pure water centrifuge washing 3 times, in last every centrifuge tube respectively plus
1mL 0.1mol/L eluent (methanol-water-ammonium hydroxide=90:9.8:0.2) is placed in shaking table room temperature and shakes incubation 15min,
1000r/min is centrifuged 3min, and elution supernatant is corresponded and is collected into clean 1.5mL centrifuge tube, 50 DEG C of nitrogen are blown
It is dry, it is redissolved with 1mL mobile phase, enters in LC-MS/MS after filter membrane and test and analyze.
From the above technical scheme, the present invention chooses to salbutamol affinity and exempts to a variety of beta-receptor agonists
The good monoclonal antibody hybridoma cell strain source of epidemic disease cross reaction prepares its antibody purification albumen, using the antibody protein it is easy acquisition,
Good homogeneity, repeatability, especially to object and plurality of target object analogue powerful affinity the advantages that, and press
GE company standard operating instruction prepares immunoaffinity chromatography glue, after obtaining chromatography glue/column product, is examined by LC-MS/MS
It surveys, has more preferably recovery of standard addition and purified treatment ability compared with national standard SPE approach.
Biological deposits information explanation
The classification naming of hybridoma cell strain 1C3A for preservation are as follows: hybridoma cell strain 1C3A；
Depositary institution's full name: China typical culture collection center；
Depositary institution's abbreviation: CCTCC；
Depositary institution address: in the Wuhan University of wuchang, wuhan Luo Jia Shan；
Preservation date: on December 4th, 2016；
Deposit number: CCTCC NO.C2016208.
Detailed description of the invention
Fig. 1 show hybridoma cell strain of the present invention and patent CN104311438A hybridoma induces ascites
With the gel electrophoresis figure of purified monoclonal antibody；Wherein, swimming lane 1 and 4 is marker, and swimming lane 2 and 5 is salbutamol of the present invention respectively and shows
There is patent Ractopamine ascites, swimming lane 3 and 6 is the antibody of salbutamol and existing patent Ractopamine after purification respectively；
Fig. 2 show LC-MS/MS method condition optimizing result of the present invention；Wherein, (a) is loading condition；It (b) is elution
Condition；It (c) is elution requirement；It (d) is the affine in immunity time；
Fig. 3 show salbutamol, Clenbuterol and the Ractopamine (Sal-IAC+ in mixed immunity affinity column
Rac-IAC the maximum adsorption capacity in)；
Fig. 4 show the HPLC fluorescence chromatogram (salbutamol) of the sample handled through pre-treatment purification method of the present invention；
Wherein, (a), (c) are blank pork and pork liver respectively；(b), (d) is the blank pork and pork liver after mark-on；
Fig. 5 show the HPLC fluorescence chromatogram (salbutamol) of the sample handled through SPE pre-treatment purification method；Its
In, (e), (g) be blank pork and pork liver respectively；(f), (h) is the blank pork and pork liver after mark-on；
Fig. 6 show HPLC fluorescence chromatogram (the Lake DOPA of the sample handled through pre-treatment purification method of the present invention
Amine)；Wherein, (a), (c) are blank pork and pork liver respectively；(b), (d) is the blank pork and pork liver after mark-on；
Fig. 7 show the HPLC fluorescence chromatogram (Ractopamine) of the sample handled through SPE pre-treatment purification method；Its
In, (e), (g) be blank pork and pork liver respectively；(f), (h) is the blank pork and pork liver after mark-on.
Specific embodiment:
The invention discloses a kind of anti-beta-receptor agonist group-specific monoclonal antibody hybridoma cell strain and its secretions
Monoclonal antibody and application, those skilled in the art can use for reference present disclosure, be suitably modified realization of process parameters.In particular
, all similar substitutions and modifications are apparent to those skilled in the art, they are considered as being included in
The present invention.Hybridoma and its monoclonal antibody and application of the invention is described by preferred embodiment, related personnel
Can obviously the content of present invention not departed from, hybridoma as described herein and its monoclonal antibody and application are being carried out in spirit and scope
Change or appropriate changes and combinations, carry out implementation and application the technology of the present invention.
In conjunction with the test result in specific embodiment it is found that the present invention resists salbutamol and Ractopamine monoclonal
Agarose of the body respectively with CNBr activation is coupled, and is prepared for beta-receptor agonist mixed immunity affinity chromatography glue/column, is captured simultaneously
Three kinds of beta-receptor agonists, and quantitative determined with the combination of LC-MS/MS equipment.The present invention filter out optimal loading,
After the conditions such as elution, elution, recovery testu, the rate of recovery of three kinds of each concentration of object have been carried out to three kinds of blank pig samples
In the reasonable scope, relative standard deviation is lower than 4.1%；The last present invention is also by the purification method (LC- of the detection method
Step before the upper machine testing of MS/MS) with national standard in provide the SPE purification method (step before the upper machine testing of LC-MS/MS used
Suddenly the detection of qualitative and quantitative contrast has been done), discovery is either in terms of the rate of recovery or on fluorescence chromatogram, inspection of the invention
Survey method has all shown powerful superiority, shows that mixed immunity affinity chromatography glue/column can be effective prepared by the present invention
Removal matrix effect, can accurately and effectively be applied in practice.
Below just a kind of anti-beta-receptor agonist group-specific monoclonal antibody hybridoma cell strain provided by the invention and
Its monoclonal antibody secreted and application are described further.
Embodiment 1: the acquisition of the sample of ascites containing monoclonal antibody of the present invention
1, the recovery and proliferation of hybridoma cell strain 1C3A and patent CN104311438A hybridoma
The cryopreservation tube that hybridoma cell strain is taken from liquid nitrogen container melts rapidly in 37 DEG C of water-baths, 600r/min centrifugation
5min, abandon supernatant, add 15%FBS/RPMI-1640 culture solution suspension cell, after respectively adding above-mentioned culture solution to 5ml, plantation in
In 50ml Tissue Culture Flask, carbon dioxide incubator culture is set, grows to that 30% density is later half to change liquid to cell, is grown to cell
60% or so (being in logarithmic growth phase) passage, it is primary by 1:3-4 passage every 2-3 days.
2, it induces in monoclonal antibody proteosome and is obtained with ascites
7-10d before plantation hybridoma in vivo, takes 12 8-10 week old male BALB/c mouse by 0.5ml/ abdominal cavity
Norphytane is injected, is raised meticulously after injection spare.
The above-mentioned culture bottle in logarithmic growth phase cell is taken, gently patting cell bottle wall makes cell detachment, will after counting
Cell suspension is gone in 50ml centrifuge tube, and 600r/min is centrifuged 12min, is abandoned culture solution, is added isometric serum-free RPMI-1640
Cell is resuspended, then is centrifuged primary abandoning RPMI-1640, by 1-2 × 106It is standby that cell is resuspended in/0.5ml serum-free RPMI-1640
With.
After sensitization 7-10 days, the above-mentioned cell suspension of 0.5ml/above-mentioned sensitization mouse is only injected intraperitoneally acquired abdomen after 7~10 days
Water, 1000rpm are centrifuged 10min to remove cell in ascites, take 12000rpm centrifugation 30min after supernatant thin in ascites to remove
Born of the same parents' fragment, then supernatant is taken, ultraviolet specrophotometer surveys OD respectively280And OD260Value, by 1.53 × OD280-0.71×OD260Formula
Its total protein concentration of rough estimate, and above-mentioned ascites is divided into three parts, one point is measured with antibody titer, and one point for the pure of antibody protein
Change, remaining -20 DEG C freeze it is spare.
Embodiment 2: the Lake of monoclonal antibody protein and patent CN104311438A hybridoma of the present invention
The purifying of dopamine monoclonal antibody
2mL protein G resin suspension is drawn, is transferred them in the disposable PE column that column capacity is 12mL, later
20mL combination buffer (3.3768g Na is added2HPO4·12H2O, 0.0888g NaH2PO4·2H2O, 8.5g NaCl, 1L
H2O is prepared), flow velocity is 1mL min-1, the impurity in suspension is removed, suspension is precipitated.Then it is slowly added to 4mL monoclonal antibody
Ascites makes it slowly flow through gel layer, and protein G is the cell wall protein of G group streptococcus, can capture the Fc of IgG type antibody
Region, and other hypotypes can not capture, to achieve the purpose that purifying.Gel layer is flowed completely through to ascites, 100mL is added and combines
Buffer washing, removes non-specific adsorption, and 15mL glycine buffer (0.1mol L is added later-1, pH=2.5) and elution, in advance
100 μ L neutralization buffer (1mol L are first added in 1.5mL PE centrifuge tube-1Tris-HCl, pH=8.5), it receives dropwise later
Collect eluent, every pipe 1mL collects preceding 10 pipe, surveys protein concentration with BCA kit, it is molten to select the highest several tubulins of concentration
Liquid, in 0.01mol L-1It dialyses one day in PBS (pH 7.4) solution, a dialyzate was changed every 3 hours.Pass through again later
BCA kit surveys total protein concentration, and SDS-PAGE surveys purity.
It is the gel electrophoresis figure of two kinds of antibody shown in Fig. 1, swimming lane 1 and 4 is marker, and swimming lane 2 and 5 is the present invention respectively
Salbutamol and existing patent Ractopamine ascites, swimming lane 3 and 6 are two kinds of antibody after purification respectively.The antibody of purifying is only
Containing IgG type, only band can occur on 25 and 50 KD；It is seen that the purity of two kinds of antibody is higher.After purification
Antibody can determine concentration, using BCA kit, standard curve be made with corresponding absorbance with standard BCA protein concentration, by outer
Mark method can measure antibody concentration after purification, and salbutamol and Anti-ractopamine antibody ultimate density are respectively 1.95 and 2.23mg
mL-1。
Embodiment 3: immunological cross analysis
The present invention selected clenobuterol hydrochloride, Terbutaline, bromine Boot sieve, Cimbuterol, this 4 kinds of beta-adrenalines by
The detection of body agonist progress cross reacting rate.Salbutamol concentration range 0-30ng mL-1, intersecting object concentration range is 0.1-
10000ng mL-1.The standard curve for making salbutamol and other 4 kinds of intersections objects respectively, calculates respective IC50, to ask
Respective CR value is obtained, the results are shown in Table 1.
Table 1
As can be seen from Table 1, when the CR value for assert salbutamol is 100%, clenobuterol hydrochloride, Te Buta are found
Woods, bromine Boot sieve and Cimbuterol cross reacting rate are very big, and CR value is respectively 66.8%, 57.3%, 42.1%, 121.5%.And
These four substances and salbutamol structure are very similar, all have the tert-butyl being connected with nitrogen-atoms, show that the present invention screens
Salbutamol monoclonal antibody, the antigenic determinant being complementary are the tert-butyl far from salbutamol main structure, that is,
It says, this monoclonal antibody has group-specific.
Embodiment 4: monoclonal antibody protein and SepharoseTM4B chemical crosslinking
1, monoclonal antibody protein pretreatment before being crosslinked
2 monoclonal antibody purification albumen of about 40mg embodiment is taken to be placed in bag filter (7500-14000MW), in 100-
200×0.1M NaHCO3/ 0.5M NaCl (pH 8.3) 4 DEG C of dialysed overnights of solution (every 4-6h is changed the liquid once), albumen after dialysis
With 0.22 μM of filtering with microporous membrane, is saved backup in 4 DEG C after filtering
2, CNBr-activated Sepharose 4B pretreatment before being crosslinked
Two parts of 1g CNBr-activated Sepharose 4B freeze-dried powders are weighed respectively in 2 50ml centrifuge tubes
(corning Products), every centrifuge tube add 30ml 1mM HCl room temperature gently to swing swelling 30min, room temperature in swing-bed
2000r/min 10min abandons supernatant, repeats 8-10 sufficiently removal CNBr-activated Sepharose with method centrifuge washing
After other small-molecule chemical reagents of 4B, every centrifuge tube uses 30ml 0.1M NaHCO again3/ 0.5M NaCl (pH 8.3) centrifugation is washed
It washs once, takes precipitating glue spare.
3, monoclonal antibody albumen and Sepharose 4B are chemically crosslinked
Above-mentioned pretreated monoclonal antibody protein liquid is divided into 2 that two equal portions are separately added into the Sepharose 4B glue containing processing
It in 50ml centrifuge tube, covers lid and is sealed with parafilm film, be placed in room temperature in swing-bed and swing incubation 1h, room temperature 2000r/min
10min abandons supernatant, takes from the supernatant a little with Bradford method analysis monoclonal antibody albumen and Sepharose 4B cross-linking effect.
Every centrifuge tube adds 30ml 0.1M NaHCO3/ 0.5M NaCl (pH 8.3) solution is placed in room temperature in swing-bed and swings incubation
10min, room temperature 2000r/min 10min abandon supernatant, then repeat the above steps centrifuge washing 5-6 times with the cross-linking buffer to fill
The albumen for dividing removal uncrosslinked, takes precipitating glue.
Above-mentioned precipitating glue is taken, every centrifuge tube is added 30ml 0.1M Tris-HCl (pH 8.0) and is placed in room temperature pendulum in swing-bed
It is dynamic be incubated for 2h in adequate closure Sepharose 4B glue not with the activated group of protein-crosslinking, room temperature 2000r/min centrifugation
10min takes precipitating glue.
Precipitating glue is taken, uses 30ml 0.1M HAC/0.5M NaCl (pH4.0) and 30ml 0.1M Tris-HCl/ respectively
After two kinds of buffer alternating centrifugals of 0.5M NaCl (pH 8.0) wash three circulations, glue is resuspended in 30ml 0.1M Tris-
HCl/0.5M NaCl (pH 8.0) liquid adds 0.02% NaN3, saved backup in 4 DEG C, obtain salbutamol affine in immunity layer
It analyses glue (Sal-IAC)/Ractopamine immunoaffinity chromatography glue (Rac-IAC).
Embodiment 5: salbutamol, clenobuterol hydrochloride and Ractopamine in present invention detection pork, pig urine or pork liver
LC-MS/MS method
1, sample treatment
The homogenate of pork Pig Liver is provided by agricultural product quality and safety monitoring center of Suzhou City.Weigh several parts 2g/ parts
Three kinds of clenbuterol hydrochloride mixed standard solutions of different volumes are added in being sub-packed in each pipe of 50mL centrifuge tube in the sample of matter, make each
Component spiked levels are 1,5,20ng g-1Each centrifuge tube containing sample is made an addition to respectively；Then 4mL/ pipe acetone is added, oscillation mentions
Object 15min, 10000 × g is taken to be centrifuged 15min, acetone supernatant is moved in the clean centrifuge tube of another 15mL；Repeat this
Step twice, obtains 12mL acetone supernatant, and 50 DEG C are dried with nitrogen；It is redissolved later with 2mL dilute hydrochloric acid, object is made to form hydrochloride
Form, it is ensured that it is present in water phase；2mL n-hexane reversed phase extraction degreasing is added, after 10000 × g is centrifuged 15min, is received
Collect water phase, with 2mol L-1NaOH adjust pH value of solution to 7.4, add isometric PBS and be uniformly mixed, 1mL water phase is taken to add
Enter into mixed immunity affinity chromatography glue (1.0mL Sal-IAC+0.5mL Rac-IAC).
Pig urine is provided by agricultural product quality and safety monitoring center of Suzhou City.First by pig urine at 10000 × g from
Heart 10min, part 5mL/ parts of urine of access are sub-packed in each pipe of 15mL centrifuge tube, and three kinds of clenbuterol hydrochloride hybrid standards of different volumes are added
Solution makes 1,5,20ng mL of spiked levels-1；Add 5mL 0.01mol L-1PBS (pH 7.4) is uniformly mixed, and takes 1mL
Urine sample is added in mixed immunity affinity chromatography glue (1.0mL Sal-IAC+0.5mL Rac-IAC).
2, affine in immunity and LC-MS/MS detection
Several 1.5mL centrifuge tubes are taken, every pipe is separately added into 1mL Sal-IAC and 500 μ L Rac-IAC, uses 0.01mol/L
It is centrifuged after PBS (pH 7.4) centrifuge washing 5 times, final colloid product is about 150 μ L after discarding supernatant.It is again that sample solution one is a pair of
It should move into the centrifuge tube of the immunochromatography glue containing mixing, be swung after mixing in shaking table room temperature and be incubated for 1h, 1000r/min centrifugation
3min abandons supernatant, last often fragmented with 0.01mol/L PBS (pH 7.4) centrifuge washing 3 times, then with pure water centrifuge washing 3 times
Add 1mL 0.1mol/L eluent (methanol-water-ammonium hydroxide=90:9.8:0.2) respectively in heart pipe, is placed in the shake of shaking table room temperature and incubates
15min is educated, 1000r/min is centrifuged 3min, and elution supernatant is corresponded and is collected into clean 1.5mL centrifuge tube, 50 DEG C
It is dried with nitrogen, is redissolved with 1mL mobile phase, enter in LC-MS/MS after filter membrane and test and analyze.
Embodiment 6: salbutamol, clenobuterol hydrochloride and Ractopamine in present invention detection pork, pig urine or pork liver
LC-MS/MS method condition optimizing test
The conditions such as loading, elution, elution may combination to antigen-antibody complex and dissociation generate large effect.
In loading step, perfect condition is desirable to object can be sufficiently completely in chromatography glue in conjunction with antibody；And in elution,
Non-specific adsorption should be removed, as far as possible to ensure the pure of sample substrate；Finally during elution, by changing environment
PH object can be made all to disintegrate down.In short, the purpose of the optimization of condition is to improve detection to improve effect of extracting
Sensitivity.
1, the screening of loading and elution condition
In our current research, respectively to three kinds of different sample solution (pure water；0.01mol mL-1PBS； 0.01mol mL- 1PBS+10% methanol aqueous solution) and leacheate (pure water；0.01mol mL-1PBS+ water；0.01mol mL-1PBS+10% methanol
Water) experiment screening has been carried out, as a result as shown in Fig. 2 (a-b), in loading condition, PBS and pure aquatic system are to three kinds of objects
The rate of recovery influences less, but a little organic reagent is added in system, and the rate of recovery of three kinds of substances receives large effect,
This may be that organic reagent generates certain destruction to antibody, so that antibody a part inactivates on glue, can not capture completely
Object；And leacheate influences less effect of extracting, the rate of recovery is 90% or so.Finally, the present invention selects PBS solution to make
For sample solution, PBS- pure water is as elution system.
2, the selection of eluent
Elution requirement influences the rate of recovery of three kinds of objects maximum.Combination between antigen-antibody is electrostatic force, model
De Huali and interaction of hydrogen bond as a result, in order to break the active force between antigen antibody complex, eluent must be pole
The solution of acid or pole alkali, the requirement to pH are very harsh.In this research, eight kinds of eluents [0.1% formic acid water (pH has been investigated
2.0)；0.1% formic acid water (pH 2.6)；2%HAC (pH 2.0)；60%, 80%, 100% methanol aqueous solution；1% ammonium hydroxide methanol
(pH 10.5), methanol-water-ammonium hydroxide (90-9.8-0.2, pH 10.5)] effect of extracting, as a result as shown in Fig. 2 (c), methanol/
Aqueous systems can not be sufficiently destroyed the coupling key between antigen and antibody.And in acid condition, the rate of recovery of Clenbuterol is not
Height, the reason is that its structure and husky butylamine and Ractopamine have a little difference.Salbutamol, Ractopamine all contain phenolic hydroxyl group
And secondary amine, phenolic hydroxyl group show faintly acid, secondary amine shows alkalescent, and the two can neutralize, therefore salbutamol and Ractopamine are to be in neutrality
's；And Clenbuterol contains only basic group aniline and secondary amine, therefore in alkalinity.Resist since salbutamol monoclonal antibody is directed to secondary amine
Former determinant first may occur acid-base neutralization reaction with the aniline of Clenbuterol, reduce elution when eluent is acid
The acidity of liquid cannot then break the covalent bond between Clenbuterol and antibody, cause the Clenbuterol rate of recovery it is universal it is relatively low even
No.Therefore only alkaline solution, could elute well three kinds of objects.Final choice methanol-water-ammonium hydroxide (90:
9.8:0.2) it is used as optimal eluent.
3, the optimization of affine in immunity time
Different from previous traditional affine in immunity process, in this research, antigen with reacting for antibody is closed at one
System in carry out.Therefore, the present invention can control the affine in immunity time, the optimal binding time of optimization.Knot
Shown in fruit such as Fig. 2 (d), when the time reaching 45min, three kinds of objects and antibody reach maximum combination.Therefore, I
Finally selected 45min be the optimal affine in immunity time.
Embodiment 7: the measurement of (Sal-IAC+Rac-IAC) maximum adsorption capacity in mixed immunity affinity column of the present invention
Maximum adsorption capacity is an important indicator for investigating affine in immunity column performance；If object content is excessive, surpass
Cross immune affinity column maximal absorptive capacity, then excessive object will be unable to be supported on pillar, it can only be flowed out together with impurity,
When calculating recovery of extraction, so that result is inaccurate.Under optimal optimal conditions, salbutamol, clenobuterol hydrochloride are determined
With the respective maximum adsorption capacity of Ractopamine, subsequent actual sample recovery testu just will not influence.The present invention passes through
It is respectively 0.2,2,10,25,50,100ng mL by 1mL concentration-1Salbutamol, Clenbuterol, Ractopamine hybrid standard
Solution is added in affine in immunity " column " (in centrifuge tube plus Sal-IAC+Rac-IAC mixed immunity affinity chromatography glue), and crosslinking is anti-
It answers, is washed out, elutes, finally collect eluent, upper machine quantitative determines corresponding three kinds of substances in each concentration point after processing
Content, calculate the rate of recovery, maximum column capacity of three kinds of objects in the affine in immunity " column " can determine with this.
As shown in figure 3, salbutamol can be adsorbed on the Sal-IAC+Rac-IAC mixed immunity affinity chromatography glue of 50 μ L
33ng, Clenbuterol 34ng, Ractopamine 29ng.Since traditional immune affinity column is will to chromatograph mucilage binding to enter empty SPE column
In, so that sample solution is slowly flowed across chromatography glue under open system；And in this research, affine in immunity process is in closed system
It carries out, which ensures that the reactions between antigen-antibody can sufficiently completely.The column volume of usual immune affinity column is about
1mL, in order to illustrate that the chromatography glue of 50 μ L is converted into 1mL by the reasonability of this research system, the present invention vividerly, then three kinds of objects
The maximum adsorption capacity of matter is respectively 660 ng, 680ng, 580ng, and the maximum adsorption capacity with traditional IAC column is similar.
Embodiment 8: actual sample recovery of standard addition
Actual sample recovery testu is the investigation to immunoaffinity chromatography accuracy and precision.The present invention is to reality
It applies 5 three kinds of blank actual samples of example to be detected, the beta-receptor agonist of different volumes is added in blank sample solution later
Mixed standard solution makes its concentration range in 0.2-20ng mL-1, standard curve is made by LC-MS/MS, as a result such as 2 institute of table
Show.For three kinds of objects in the environment of three kinds of different substrates samples, detection limit is significantly lower than the 0.5ng g of national regulations-1, and
And r2> 0.9946 shows that the sensitivity of method of the invention is very high.
Table 2
Sample recovery testu is carried out, the clenbuterol hydrochloride mixed standard solution of different volumes is added in blank pig sample, presses
It is detected according to the method for the present invention, calculates the rate of recovery.As a result as shown in Table 3-5.
The present invention equalizes the rate of recovery of every kind of object high, normal, basic three kinds of spiked levels in each sample, by flat
The equal rate of recovery it is clear that in pork matrix, salbutamol, three kinds of objects of Clenbuterol and Ractopamine it is flat
The equal rate of recovery is 70% or more；And in pork liver and pig urine samples, the average recovery rate of salbutamol and Clenbuterol is distinguished
It is 80% and 90% or so, but Ractopamine, in both matrix, it is 64% left that average recovery rate, which is not very high,
The right side, this may be the pH because the affine in immunity power between ractopamine monoclonal antibody and haptens Ractopamine is too strong
10.5 alkaline eluant cannot thoroughly break its antigen antibody complex, and the matrix of pork liver pig urine is more more multiple than pork
It is miscellaneous, therefore cause the recovery of standard addition of Ractopamine lower.The relative standard deviation of three kinds of mark-on samples is lower than 4.1%, shows
Mixed immunity affinity chromatography glue prepared by the present invention can effectively remove the matrix effect of sample, can accurately and effectively answer
It uses in practice.
3 pork recovery of standard addition (n=3) of table
4 pork liver recovery of standard addition (n=3) of table
5 pig of table urinates recovery of standard addition (n=3)
Embodiment 9: salbutamol, clenobuterol hydrochloride and Ractopamine in present invention detection pork, pig urine or pork liver
LC-MS/MS method and national standard SPE comparison
National standard SPE: it uses MCX solid phase extraction column (60mg/3mL, Waters Oasis).Concrete operations are, successively plus
Enter 3mL methanol and 3mL pure water activation pillar, the sample solution handled well in embodiment 5 is added in column later, to sample solution
It drains off, 3mL pure water is successively added and 3mL methanol elutes pillar, is eventually adding the elution of 5% ammonium hydroxide methanol of 3mL, collects elution
Liquid, 50 DEG C are dried with nitrogen, and are redissolved with 1mL mobile phase, and quantitative detection in LC-MS/MS is entered after filter membrane, calculate the rate of recovery.
For the superiority for verifying detection method, the SPE purification method that it is used with national regulations is compared
Experiment, as a result as shown in Table 3-5, is handled through SPE after three kinds of matrix mark-ons, and salbutamol average recovery rate is not higher than 65%,
The average recovery rate of Clenbuterol is less than 62%, and the average recovery rate of Ractopamine is below 47%.Pass through data pair in table
Than, it can be clearly seen that, detection method, in different substrates, recovery of standard addition is above SPE.And the sample of SPE processing
The product rate of recovery is lower, but can also become national standard guidance method, this is because national standard is passing through LC-MS chromatograph quantitative detection
When, it joined Isotopic Internal Standard object, object quantified using internal standard method.But Isotopic Internal Standard price lattice are very
Expensive, this undoubtedly considerably increases testing cost.And the monoclonal antibody in detection method be can be continually
Production, in contrast, the advantage of immunoaffinity chromatography of the present invention further embodies out.
In order to more intuitively show the superiority of detection method, after the purified treatment of detection method
Pork, the Pig Liver solution of (step before LC-MS/MS detection) have done fluorescence chromatogram comparison, because Clenbuterol is without glimmering
Light property, therefore salbutamol and Ractopamine are only added in the sample, one-component qualitative analysis is carried out by HPLC-FLD, it
So two kinds of objects of separate detection, be because the optimal excitation wavelength and launch wavelength of salbutamol and Ractopamine not
Identical, mobile phase and its ratio are also different, and the purpose of the present invention is to detection method pre-treatment, (LC-MS/MS is detected
Preceding step) and SPE pre-treatment (LC-MS/MS detection before step) compare, thus other conditions all should it is identical and
It is optimal.In experiment, the spiked levels of salbutamol and Ractopamine are 100ng g-1。
Fig. 4-5 is the mark-on reclaims fluorescence chromatogram of salbutamol, and Fig. 4 (a), (c) are through detection method respectively
The blank pork and pork liver of pre-treatment, after sample mark-on, the same pre-treatment using detection method is purified, clean-up effect
As shown in Fig. 4 (b), (d), salbutamol about in 4.8 minutes appearances, fluorescence chromatogram seldom miscellaneous peak even without；Although
There is ghost peak appearance at 7min, but this may be miscellaneous peak caused by the organic solvent used when handling pork pork liver, have no effect on
Qualitative analysis to salbutamol；And Fig. 5 (e), (g) are blank pork and pork liver through SPE pre-treatment respectively, it can be seen that
Miscellaneous peak is more and random in blank sample, and negative sample can detect the chromatographic peak of doubtful salbutamol at 4.8 minutes, it is seen that SPE
Method can not effectively remove the matrix effect in sample, this all causes the qualitative or quantitative analysis of salbutamolum residue
Tremendous influence；Fig. 5 (f), (h) are then the pork handled through SPE, the fluorescence chromatogram of pork liver, it can be seen from the figure that husky fourth
The chromatographic peak peak area of amine alcohol is very big, but is washed away because having impurity not to be cleaned in sample substrate in fact, with salbutamol
In same period appearance, thus integrate the peak area come out be it is inaccurate, can not be quantitative determined.
Equally, Fig. 6-7 is the mark-on reclaims chromatogram of Ractopamine, and conclusion is roughly the same with salbutamol.Due to inspection
The mobile phase for surveying Ractopamine is acetonitrile water, and the mobile phase of salbutamol is methanol-water, and the polarity of acetonitrile is greater than methanol, elution
Ability is stronger, therefore the organic solvent in sample handling processes does not occur in chromatogram.Fig. 6 (a), (b), (c), (d) be through
The sample chromatogram figure of detection method pre-treatment, detection method pre-treatment substantially eliminate sample substrate, Lay
Gram dopamine peak type is at all interference-free；But Fig. 7 (e), (f), (g), (h) are the sample chromatogram figures through SPE pre-treatment, equally
It is influenced at Ractopamine appearance time about 7.4 minutes by impurity, or even can not all carry out qualitative analysis.Pass through two kinds
The fluorescence chromatogram of method compares, and can further prove, detection method pre-treatment matrix clean-up effect is far superior to
SPE.Therefore, either quantitative detection or qualitative confirmation, the ability of the purification sample substrate of detection method are all remote high
In SPE purification means, thus provable immunoaffinity chromatography of the invention, can be applied to the purified treatment of actual sample
In.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (17)
1. anti-beta-receptor agonist group-specific monoclonal antibody hybridoma cell strain, which is characterized in that deposit number CCTCC
NO.C2016208。
2. the hybridoma cell strain that deposit number is CCTCC NO.C2016208 remains in pattern detection in beta-receptor agonist
Using.
3. applying according to claim 2, which is characterized in that the beta-receptor agonist is clenbuterol hydrochloride.
4. anti-beta-receptor agonist group-specific monoclonal antibody, which is characterized in that by deposit number be CCTCC
The hybridoma cell strain of NO.C2016208, which is secreted, to be generated.
5. the anti-beta-receptor agonist cluster that the hybridoma cell strain secretion that deposit number is CCTCC NO.C2016208 generates is special
Property monoclonal antibody beta-receptor agonist remain pattern detection and/or beta-receptor agonist residual immune detection product preparation in
Application.
6. applying according to claim 5, which is characterized in that the immune detection product be immunoaffinity chromatography chromatography glue or
By the immune affinity chromatography chromatographic column of the glue pre-fill.
7. a kind of beta-receptor agonist group-specific immunoaffinity chromatography chromatography glue, which is characterized in that by deposit number CCTCC
NO.C2016208 hybridoma cell strain secretion generate monoclonal antibody it is purified again with CNBr-activated Sepharose-4B glue
Chemical crosslinking forms immobilized antibody albumen and obtains.
8. immunoaffinity chromatography chromatography glue according to claim 7, which is characterized in that the purified monoclonal antibody albumen and CNBr-
The amount ratio of activated Sepharose-4B glue: 5mg purified monoclonal antibody albumen: 1ml CNBr-activated
Sepharose-4B swelling gum.
9. the preparation method of immunoaffinity chromatography chromatography glue described in claim 7 characterized by comprising
Step 1 obtains monoclonal antibody protein with the hybridoma of deposit number CCTCC NO.C2016208 and carries out pure
Change, monoclonal antibody albumen carries out dialysis pretreatment after purification, spare；
Acquisition processing glue is spare after CNBr-activated Sepharose-4B is swollen, eluriates pretreatment；
Step 2 handles glue to by being added in pretreated purified monoclonal antibody albumen described in step 1, room temperature lower swing is incubated for, then
Supernatant is abandoned in centrifugation, and NaHCO is added3/ NaCl buffer solution continues to swing incubation at room temperature, and supernatant is abandoned in centrifugation, and washing is resisted
Body protein cross-linked rubber；
Step 3 takes antibody protein cross-linked rubber addition Tris-HCl in step 2 to be placed in the incubation of room temperature lower swing to close in the glue not
With the excess activation group of monoclonal antibody protein-crosslinking, supernatant is abandoned in centrifugation, then with HAC/NaCl and bis- kinds of Tris-HCl/NaCl bufferings
The washing of liquid alternating centrifugal, is finally resuspended in Tris-HCl-NaCl buffer for cross-linked rubber, saves backup in 4 DEG C, i.e., described in acquisition
Immunoaffinity chromatography chromatography glue.
10. preparation method according to claim 9, which is characterized in that the albumen of monoclonal antibody after purification carries out dialysis pretreatment
Are as follows:
Monoclonal antibody albumen is placed in 7500-14000MW bag filter after purification, then is placed in 100-200 × 0.1M NaHCO3/0.5M
4 DEG C of dialysed overnights in NaCl (pH8.3) buffer solution, every 4-6h are changed the liquid once, after dialysis albumen in 0.22 μM of membrane filtration, and
It is saved backup in 4 DEG C.
11. preparation method according to claim 9, which is characterized in that described by CNBr-activated Sepharose-4B
It is swollen, eluriates pretreatment are as follows:
It weighs CNBr-activated Sepharose 4B solid powder and dilute HCl swelling is added, centrifugation abandons supernatant, dilute HCl is added
Centrifuge washing is to remove in CNBr-activated Sepharose 4B glue after other small-molecule chemical reagents, then uses NaHCO3/
NaCl buffer solution centrifuge washing is primary, spare.
12. preparation method according to claim 11, which is characterized in that described by CNBr-activated Sepharose-
4B is swollen, eluriates pretreatment are as follows:
1g CNBr-activated Sepharose 4B solid powder is weighed in 50ml centrifuge tube, centrifuge tube adds 30ml 1mM
HCl room temperature gently swings swelling 30min in swing-bed, and room temperature 2000r/min 3min abandons supernatant, and centrifuge tube adds 30ml 1mM
HCl repeats sufficiently to remove in above-mentioned glue after other small-molecule chemical reagents for 8-10 times with same method centrifuge washing, then uses
30ml 0.1M NaHCO3/ 0.5M NaCl (pH8.3) buffer solution centrifuge washing is primary, spare.
13. preparation method according to claim 9, which is characterized in that step 2 are as follows:
Pretreatment purified monoclonal antibody albumen described in step 1 is added into containing the 50ml centrifuge tube for handling glue described in step 1, covers lid simultaneously
It is sealed with parafilm film, is placed in room temperature in swing-bed and swings incubation 1h, room temperature 2000r/min 10min abandons supernatant, and centrifuge tube adds
30ml 0.1M NaHCO3/ 0.5M NaCl (pH8.3) buffer solution is placed in room temperature in swing-bed and swings incubation 10min, room temperature
2000r/min 10min abandons supernatant, then uses NaHCO3/ NaCl buffer solution centrifuge washing 5-6 times uncrosslinked sufficiently to remove
Albumen obtains antibody protein cross-linked rubber.
14. preparation method according to claim 9, which is characterized in that step 3 are as follows:
Step 2 antibody protein cross-linked rubber is taken, centrifuge tube is added 30ml 0.1M Tris-HCl (pH 8.0) and is placed in room temperature in swing-bed
Swing be incubated for 2h in adequate closure antibody protein cross-linked rubber not with the activated group of protein-crosslinking, room temperature 2000r/min centrifugation
10min abandons supernatant, and centrifuge tube uses 30ml 0.1M HAC/0.5M NaCl (pH4.0) and 30ml 0.1M Tris-HCl/ respectively
After two kinds of buffer alternating centrifugals of 0.5M NaCl (pH 8.0) wash three circulations, cross-linked rubber is resuspended in 30ml 0.1M
Tris-HCl/0.5M NaCl (pH 8.0) buffer adds 0.02%NaN3, saved backup in 4 DEG C, that is, obtain the immune parent
With thin layer chromatography glue.
15. a kind of immune affinity chromatography chromatographic column for detecting beta-receptor agonist, which is characterized in that including claim 7 or 8 institutes
State chromatography glue and column bed.
16. chromatographic column according to claim 15, which is characterized in that further include being prepared by ractopamine monoclonal antibody
Chromatography glue
17. a kind of method of beta-receptor agonist in detection pork, pig urine or pork liver, which is characterized in that use claim 7-8
Any one of any one chromatography glue or claim the 15-16 chromatographic column, are detected by LC-MS/MS.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810712045.XA CN109112112B (en) | 2018-07-03 | 2018-07-03 | Anti-beta-receptor agonist cluster specific monoclonal antibody hybridoma cell strain, monoclonal antibody secreted by same and application |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810712045.XA CN109112112B (en) | 2018-07-03 | 2018-07-03 | Anti-beta-receptor agonist cluster specific monoclonal antibody hybridoma cell strain, monoclonal antibody secreted by same and application |
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| Publication Number | Publication Date |
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| CN112239751A (en) * | 2020-10-13 | 2021-01-19 | 苏州大学 | Florfenicol/thiamphenicol monoclonal antibody hybridoma cell strain, monoclonal antibody secreted by same and application |
| CN113372448A (en) * | 2021-05-19 | 2021-09-10 | 新乡学院 | anti-CIB single-chain antibody and screening method and application thereof |
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| CN112239751A (en) * | 2020-10-13 | 2021-01-19 | 苏州大学 | Florfenicol/thiamphenicol monoclonal antibody hybridoma cell strain, monoclonal antibody secreted by same and application |
| CN113372448A (en) * | 2021-05-19 | 2021-09-10 | 新乡学院 | anti-CIB single-chain antibody and screening method and application thereof |
| CN113372448B (en) * | 2021-05-19 | 2022-09-23 | 新乡学院 | anti-CIB single-chain antibody and screening method and application thereof |
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