CN109112112B

CN109112112B - Anti-beta-receptor agonist cluster specific monoclonal antibody hybridoma cell strain, monoclonal antibody secreted by same and application

Info

Publication number: CN109112112B
Application number: CN201810712045.XA
Authority: CN
Inventors: 吴康; 黄芳; 胡仁莉; 原茵
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2018-07-03
Filing date: 2018-07-03
Publication date: 2021-10-22
Anticipated expiration: 2038-07-03
Also published as: CN109112112A

Abstract

The invention relates to the technical field of biology, and discloses an anti-beta-receptor agonist cluster specific monoclonal antibody hybridoma cell strain and a monoclonal antibody secreted by the same. The hybridoma cell strain is prepared from a salbutamol artificial antigen, and a monoclonal antibody secreted by the hybridoma cell strain can simultaneously identify beta-receptor agonists such as salbutamol, clenbuterol hydrochloride, terbutaline, brombutolol and sibutrol, and has cluster specificity; the prepared immunoaffinity chromatography gel is mixed with other beta-receptor agonists, such as the immunoaffinity chromatography gel of ractopamine, and is used as a machine pretreatment purifying reagent, and is combined with HPLC-FLD and LC-MS/MS detection technologies, so that various clenbuterol components in pork, pork liver and pig urine samples can be efficiently detected, and the method has high accuracy and sensitivity.

Description

Anti-beta-receptor agonist cluster specific monoclonal antibody hybridoma cell strain, monoclonal antibody secreted by same and application

Technical Field

The invention relates to the field of biotechnology, and particularly relates to an anti-beta-receptor agonist cluster specific monoclonal antibody hybridoma cell strain, a monoclonal antibody secreted by the same and application of the monoclonal antibody.

Background

Clenbuterol hydrochloride, ractopamine and salbutamol are taken as representative three main clenbuterol residual primary targets for veterinary drug monitoring and supervision by food safety supervision departments due to the fact that clenbuterol hydrochloride, ractopamine and salbutamol are caused when residual events of meat food and animal viscera are forbidden. The immunoassay technology represented by an ELISA detection kit and an immune colloidal gold test strip is widely applied to high-throughput rapid qualitative screening of the three target substance residue samples. However, the residue of the above-mentioned target must be confirmed and accurately quantified by high performance liquid chromatography with fluorescence detector (HPLC-FLD) or even liquid-mass tandem spectroscopy (LC-MS/MS). The indexes of chromatographic separation efficiency, quantitative limit, precision and the like of the instrument and equipment provided by Waters and Agilent companies can meet the actual detection requirements, but the actual detection effect is overwhelmingly dependent on the pretreatment effect of the mechanical sample introduction sample.

For the accurate quantitative analysis of the three clenbuterol extracts representing multi-component beta-receptor agonist residues, the ministry of agriculture promulgates the national standard 'detection of beta-receptor agonist residues in animal-derived food-liquid chromatography-tandem mass spectrometry detection method' in 2008 and stipulates that solid-phase extraction purification treatment sample injection detection is adopted, and the method has the following four restriction factors which seriously affect the detection effect:

firstly, the operation steps are multiple, the dosage of toxic and harmful organic solvents is large, instruments and equipment (such as a rotary evaporator, a nitrogen blowing instrument and the like) are needed, the conversion of a liquid phase dissolving system is multiple, and the consumed time is long.

Secondly, the loss rate of the target substance is high: the loss of the target object can be caused in a plurality of operation links such as biomacromolecule removal, liquid-liquid extraction/solid-phase extraction, purification of target object sample injection buffer solution redissolution and the like.

Thirdly, although the chemical structures of clenbuterol hydrochloride, ractopamine and salbutamol are phenylethylamine medicines, the physicochemical properties of the clenbuterol hydrochloride, the ractopamine and the salbutamol are greatly different, so that the unified extracting solution and extracting solution or a single solid-phase extraction column is difficult to use and the satisfactory extracting-purifying effect of each medicine can be ensured.

Fourth, the liquid-liquid extraction/solid phase extraction only has certain selectivity, and when the target object is purified, matrix interferents (especially complex matrix samples such as animal liver and kidney) with similar physicochemical properties to the target object are inevitably mixed, and if interference peaks appear in a target object retention time region, interpretation of the measured data is seriously influenced.

The great technical advantages of the immunoaffinity chromatography IAC (immunoaffinity chromatography) compared with the SPE (solid phase extraction) purification method are mainly shown as follows:

first, the target substance is easy to extract from the biological sample, the reaction system is mild, and only a little or no organic solvent is needed.

Second, the target extraction step does not require a large amount of removal of biological macromolecules from the sample (e.g., too high fat content in the sample can properly remove part of the fat to avoid affecting the specific binding of the immune beads to the target), and thus can avoid the loss of the target due to the removal of the macromolecules.

And thirdly, the selectivity to the target object is strong and is determined by the characteristic of strong specific binding reaction of antigen-antibody, and the positive sample is sharp in peak shape of each target object, definite in retention time of each component, and free of or little in interference of the impurity peak in the chromatogram after the detection of the HPLC-FLD of the on-machine sample purified by the IAC.

Fourthly, the immune affinity chromatography eluent is carefully designed and repeatedly optimized, the eluent and a sample injection buffer solution can be unified into the same buffer solution, or an organic phase (such as methanol or acetonitrile with a certain volume ratio) with a certain volume ratio is supplemented in an elution sample with a target object for direct sample injection.

Compared with the solid phase extraction column SPE (solid phase extraction) which is widely used at present, the immunoaffinity chromatography column lacks competitiveness, the use amount of the antibody is large, so that the cost is high, only the monoclonal antibody cell strain with independent intellectual property rights can greatly reduce the production cost of the purified monoclonal antibody protein, and the wide application of the monoclonal antibody protein becomes possible.

Because of its strong selectivity, it has greatly limited its application in pre-treatment of multi-component target mixed residue samples for machine testing; therefore, the preparation of the monoclonal antibody aiming at the beta-agonist multi-component cluster specificity to enlarge the target coverage of the monoclonal antibody to replace the SPE column becomes possible.

Disclosure of Invention

In view of the above, the present invention aims to provide a hybridoma cell line having a cluster specificity for a beta-receptor agonist and a monoclonal antibody generated by the hybridoma cell line, so that the monoclonal antibody generated by the hybridoma cell line has high cross-reaction to salbutamol, clenbuterol hydrochloride, terbutaline, brombuterol and sibutrol, and an immunoassay core reagent is provided for rapid screening of a multi-component mixed residue sample of the beta-receptor agonist;

the invention also aims to provide the immunoaffinity chromatography gel prepared by the monoclonal antibody and the preparation method thereof, so that the immunoaffinity chromatography gel not only has higher cross reaction rate to each beta-receptor agonist, but also has better purification treatment capability and standard increasing recovery rate in immunoaffinity chromatography detection compared with the national standard SPE method.

In order to achieve the purpose, the invention provides the following technical scheme:

the anti-beta-receptor agonist cluster specific monoclonal antibody hybridoma cell strain has a preservation number of CCTCC NO. C2016208 and is named as 1C3A, and is preserved in China center for type culture Collection at 12 months and 4 days in 2016, and the address is Wuhan university at Lojia mountain of Wuchang, Wuhan City.

According to the invention, a mouse is immunized by taking salbutamol and OVA carrier protein as immunogen, the 1C3A hybridoma cell strain is obtained by screening, the induced monoclonal antibody and a plurality of beta-receptor agonists are subjected to cross reaction rate detection, when the CR value of salbutamol is determined to be 100%, the cross reaction rates of clenbuterol hydrochloride, terbutaline, brombuterol and sibutrol are found to be higher, and the CR values are 66.8%, 57.3%, 42.1% and 121.5% respectively.

Based on the excellent technical effects, the invention provides the application of the hybridoma cell and the monoclonal antibody generated by the hybridoma cell in the preparation of beta-receptor agonist residual sample detection and/or beta-receptor agonist residual immunodetection products; wherein the beta-receptor agonist is preferably clenbuterol, and the clenbuterol is one or more of salbutamol, clenbuterol hydrochloride, terbutaline, brombuterol and sibutrol; wherein the salbutamol and the clenbuterol hydrochloride can be used as weighted target objects for key analysis.

The beta-receptor agonist residual immunoassay product is preferably an immunoaffinity chromatography gel or an immunoaffinity chromatography column pre-filled with the immunoaffinity chromatography gel.

The invention specifically provides beta-receptor agonist cluster specific immunoaffinity chromatography gel, which is obtained by secreting a hybridoma cell strain with the preservation number of CCTCC NO. C2016208 to generate a monoclonal antibody, purifying the monoclonal antibody, and chemically crosslinking the monoclonal antibody with CNBr-activated Sepharose-4B gel to form immobilized antibody protein.

Preferably, the dosage ratio of the purified monoclonal antibody protein to CNBr-activated Sepharose-4B glue is as follows: 5mg of purified monoclonal antibody protein: 1ml of CNBr-activated Sepharose-4B swelling gel.

Due to the excellent characteristics of the monoclonal antibody, the immunoaffinity chromatography gel or the immunoaffinity chromatography column (obtained by filling the immunoaffinity chromatography gel in a column bed) has excellent performance. Meanwhile, the immunoaffinity chromatography gel or immunoaffinity chromatography column has better standard-adding recovery rate and purification treatment capacity in the immunoaffinity chromatography, compared with a solid-phase extraction SPE (solid phase extraction) purification method of the national standard (beta-agonist residue detection-liquid chromatography-tandem mass spectrometry in animal-derived food announced by the Ministry of agriculture 1025-18-2008).

Meanwhile, the invention also provides a preparation method of the immunoaffinity chromatography chromatographic gel, which comprises the following steps:

step 1, obtaining monoclonal antibody protein by using hybridoma cells with the preservation number of CCTCC NO. C2016208, purifying, and dialyzing and pretreating the purified monoclonal antibody protein for later use;

performing swelling and elutriation pretreatment on CNBr-activated Sepharose-4B to obtain a treatment gel for later use;

step 2, adding the treatment gel obtained in the step 1 into the pretreated purified monoclonal antibody protein, oscillating and incubating at room temperature, centrifuging, removing supernatant, and adding NaHCO₃Continuing to perform swing incubation at room temperature by using a NaCl buffer solution, centrifuging and removing a supernatant, and washing to obtain the antibody protein cross-linked gel;

and 3, adding Tris-HCl into the antibody protein cross-linked gel in the step 2, placing the gel at room temperature for swing incubation to seal excessive activated groups which are not cross-linked with the monoclonal antibody protein in the gel, centrifuging the gel to remove supernatant, then alternately centrifuging and washing the gel by using HAC/NaCl and Tris-HCl/NaCl buffer solutions, finally suspending the cross-linked gel in the Tris-HCl-NaCl buffer solution, and storing the gel at 4 ℃ for later use to obtain the immunoaffinity chromatography gel.

The monoclonal antibody secreted and produced by the hybridoma cell strain with the preservation number of CCTCC NO. C2016208 in the immunoaffinity chromatography gel can be prepared by a conventional method in the technical field, for example, a mouse ascites sample is obtained by the hybridoma cell strain in an in vivo induction mode, and the following method can be referred to specifically:

one week before inoculating hybridoma cells, male Balb/c mice are intraperitoneally injected with 0.5mL of pristane for sensitization. Then each mouse was injected intraperitoneally with 5X 10⁵Left and right hybridoma cells. After 10 days of inoculation, the abdomen of the mouse is obviously swollen, when the mouse is in a state of dying before death, the mouse is killed, the mouse is soaked in 75% alcohol for body surface disinfection, the abdomen is opened aseptically, and ascites containing the target antibody is taken out by a dropper. And centrifuging the collected ascites to remove blood cells and plasma to obtain faint yellow ascites. Adding glycerol and 0.01mol/L PBS (pH 7.4) into ascites at a volume ratio of 50:45:5, subpackaging and storing at-20 deg.C or-80 deg.C in refrigerator.

The induced ascites sample obtained by the method is purified by a protein G resin one-step method, and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic identification results show that: the ascites purified substance only contains IgG type, two obvious protein bands containing 50Kd (antibody heavy chain) and 25Kd (antibody light chain) and no other hybrid protein bands, which shows that the hybridoma cell strain induces an ascites path to obtain high-purity monoclonal antibody protein and can meet the requirement of preparing detection antibody in immunoaffinity chromatography gel.

Preferably, the dosage ratio of the purified monoclonal antibody to CNBr-activated Sepharose-4B glue is as follows: 5mg of purified monoclonal antibody protein: 1ml of CNBr-activated Sepharose-4B swelling gel.

Preferably, the dialysis pretreatment of the purified monoclonal antibody protein is:

the purified monoclonal antibody protein is placed in a dialysis bag and put into NaHCO₃Dialyzing overnight at 4 deg.C in NaCl buffer solution, filtering with monoclonal antibody protein microporous membrane, and storing the filtrate at 4 deg.C.

Further preferably, the dialysis pretreatment of the purified monoclonal antibody protein is:

the purified monoclonal antibody protein was placed in a 7500-14000MW dialysis bag at 100-₃Dialyzing in 0.5M NaCl (pH8.3) buffer solution at 4 deg.C overnight, changing solution every 4-6 hr, filtering the dialyzed protein with 0.22 μ M microporous membrane, and storing the filtrate at 4 deg.C.

Preferably, the CNBr-activated Sepharose-4B is subjected to swelling and elutriation pretreatment as follows:

weighing CNBr-activated Sepharose 4B solid powder, adding diluted HCl for swelling, centrifuging to remove supernatant, adding diluted HCl for centrifugal washing to remove other small molecule chemical reagents in CNBr-activated Sepharose 4B gel, and then using NaHCO₃The NaCl buffer solution is centrifugally washed once for standby.

Further preferably, the CNBr-activated Sepharose-4B is subjected to swelling and elutriation pretreatment:

weighing 1g of CNBr-activated Sepharose 4B solid powder in a 50ml centrifuge tube, adding 30ml of 1mM HCl into the centrifuge tube, slightly swinging and swelling the centrifuge tube in a swinging bed at room temperature for 30min, removing supernatant at room temperature of 2000r/min for 3min, adding 30ml of 1mM HCl into the centrifuge tube, centrifugally washing the centrifuge tube by the same method for 8-10 times to sufficiently remove other small molecular chemical reagents in the gel, and then adding 30ml of 0.1M NaHCO₃The buffer solution of 0.5M NaCl (pH8.3) is washed once by centrifugation and is ready for use.

Preferably, step 2 is:

adding the pretreated and purified monoclonal antibody protein obtained in the step 1 into a 50ml centrifuge tube containing the treatment gel obtained in the step 1, covering the centrifuge tube, sealing the centrifuge tube with a parafilm, placing the centrifuge tube in a pendulum bed, incubating the centrifuge tube at room temperature for 1h in a swinging manner, discarding the supernatant at the room temperature of 2000r/min for 10min, and adding 30ml of 0.1M NaHCO into the centrifuge tube₃0.5M NaCl (pH8.3) buffer solution, placing in a swing bed, incubating at room temperature for 10min, discarding supernatant at room temperature for 10min at 2000r/min, and adding NaHCO₃And centrifugally washing the solution with NaCl buffer solution for 5-6 times to fully remove the uncrosslinked protein to obtain the antibody protein crosslinked gel.

Preferably, step 3 is:

taking the antibody protein cross-linked gel in the step 2, adding 30ml of 0.1M Tris-HCl (pH 8.0) into a centrifuge tube, placing the centrifuge tube in a swinging bed, swinging and incubating for 2h at room temperature to fully seal activated groups which are not cross-linked with the protein in the antibody protein cross-linked gel, centrifuging at room temperature of 2000r/min for 10min, discarding supernatant, alternately centrifuging and washing the centrifuge tube with 30ml of two buffers of 0.1M HAC/0.5M NaCl (pH4.0) and 30ml of 0.1M Tris-HCl/0.5M NaCl (pH 8.0) for three cycles, re-suspending the cross-linked gel in 30ml of 0.1M Tris-HCl/0.5M NaCl (pH 8.0) buffer solution, and supplementing0.02％NaN₃And storing at 4 ℃ for later use to obtain the immunoaffinity chromatography gel.

In the preparation of the immunoaffinity chromatography gel, the invention is obtained by chemically crosslinking a purified monoclonal antibody protein and a CNBr-activated Sepharose-4B gel to form a solid-phase antibody protein, and the immunoaffinity chromatography column is obtained by filling the immunoaffinity chromatography gel in a column bed. According to the detection requirement, the chromatographic column can also be added with chromatographic gel prepared by other clenbuterol, clenbuterol hydrochloride, terbutaline, brombuterol and sibutrol, such as ractopamine, and the method for preparing the chromatographic gel can refer to the preparation method before the invention.

According to the performance of the immunoaffinity chromatography chromatographic gel/column, the invention also correspondingly provides a method for detecting the beta-receptor stimulant in pork, pig urine or pig liver, and the detection is carried out by LC-MS/MS by adopting the chromatographic gel or the chromatographic column.

In a specific embodiment of the invention, an LC-MS/MS detection method for pork, pig urine or pig liver is provided for detecting salbutamol, clenbuterol hydrochloride and ractopamine, wherein the ractopamine monoclonal antibody is obtained by inducing ascites with a hybridoma cell line of patent CN104311438A, and preferably the ractopamine immunoaffinity chromatography gel prepared in patent No. 201711065894.2 is mixed with the immunoaffinity chromatography gel of the present invention.

In the sample treatment, weighing 2 g/part of homogenized pork or pork liver sample, respectively subpackaging in 50mL centrifuge tubes, adding three clenbuterol mixed standard solutions with different volumes to make the concentration of each component be 1, 5, 20ng g^-1Adding 4mL of acetone into three matrix samples (1ng/g:2g of the matrix doped with ractopamine, salbutamol and clenbuterol hydrochloride are all used as samples at 1ng/g, 5ng/g:2g of the matrix doped with ractopamine, salbutamol and clenbuterol hydrochloride are all used as samples at 5ng/g, 20ng/g:2g of the matrix doped with ractopamine, salbutamol and clenbuterol hydrochloride are all used as samples at 20 ng/g), shaking to extract a target object for 15min, centrifuging for 15min at 10000 Xg, and supernatant of the acetoneThe solution was transferred to another 15mL clean centrifuge tube; repeating the step twice to obtain 12mL of acetone supernatant, and drying by nitrogen at 50 ℃; then re-dissolving with 2mL of dilute hydrochloric acid to form the target into a hydrochloride form, and ensuring that the target exists in a water phase; adding 2mL n-hexane, reverse-phase extracting to remove fat, centrifuging at 10000 Xg for 15min, collecting water phase, and purifying with 2mol L^-1Adjusting the pH value of the solution to 7.4 by NaOH, adding PBS with the same volume, uniformly mixing, adding 1mL of water phase into an immunoaffinity chromatography gel/column (each 1.5mL of chromatography gel contains 0.5mL of chromatographic gel prepared by ractopamine monoclonal antibody);

for the swine urine sample, the swine urine is centrifuged at 10000 Xg for 10min, a plurality of 5 mL/pig urine samples are respectively taken and filled into each tube of a 15mL centrifugal tube, three clenbuterol mixed standard solutions with different volumes are added, and the standard concentration of each component is 1, 5, 20ng mL^-1(1ng/ml:2ml matrix doped with ractopamine, salbutamol, clenbuterol hydrochloride all 1ng/ml for the test sample, 5ng/ml:2ml matrix doped with ractopamine, salbutamol, clenbuterol hydrochloride all 5ng/ml for the test sample, 20ng/ml:2ml matrix doped with ractopamine, salbutamol, clenbuterol hydrochloride all 20ng/ml for the test sample); 5mL of 0.01mol L was added^-1PBS (pH 7.4) was mixed well and 1mL of urine sample was added to the immunoaffinity gel/column (0.5 mL of the gel prepared with ractopamine monoclonal antibody per 1.5mL of gel).

Mixing a sample and chromatographic column/gel, performing swing incubation for 1h at room temperature of a shaking table, centrifuging for 3min at 1000r/min, discarding the supernatant, performing centrifugal washing for 3 times by using 0.01mol/L PBS (pH 7.4), performing centrifugal washing for 3 times by using pure water, finally adding 1mL of 0.1mol/L eluent (methanol-water-ammonia water is 90:9.8:0.2) into each centrifuge tube respectively, placing the centrifuge tubes into the shaking table, shaking and incubating for 15min at room temperature, centrifuging for 3min at 1000r/min, collecting the eluted supernatants into clean 1.5mL centrifuge tubes one by one, drying the centrifuge tubes with nitrogen at 50 ℃, redissolving by using 1mL of mobile phase, filtering the filtrate, and performing detection and analysis in LC-MS/MS.

According to the technical scheme, the purified antibody protein is prepared by selecting a monoclonal antibody hybridoma cell strain source which has good affinity to salbutamol and immune cross reaction to various beta-receptor agonists, the immunoaffinity chromatography gel is prepared according to standard operation procedures of GE company by utilizing the advantages of easy acquisition, good uniformity and repeatability of the antibody protein, particularly strong affinity to a target and structural analogues of various targets, and the like, and after a chromatography gel/column product is obtained, the detection is carried out through LC-MS/MS, so that the method has better standard recovery rate and purification treatment capacity compared with a national standard SPE method.

Biological preservation information description

The classification of hybridoma cell line 1C3A for preservation was designated: hybridoma cell line 1C 3A;

the preservation unit is called as follows: china center for type culture Collection;

the preservation unit is abbreviated as: CCTCC (China center for type communication);

the address of the depository: wuhan university in Wuchang Lojia mountain;

the preservation date is as follows: 2016, 12 months, 21 days;

the preservation number is: CCTCC No. c 2016208.

Drawings

FIG. 1 is a gel electrophoresis chart of the hybridoma cell line and the hybridoma cell induced ascites and purified monoclonal antibody of patent CN 104311438A; wherein

lanes

1 and 4 are markers,

lanes

2 and 5 are salbutamol of the present invention and ractopamine ascites of the prior patent, respectively, and

lanes

3 and 6 are purified antibodies of salbutamol and ractopamine of the prior patent, respectively;

FIG. 2 shows the result of optimizing the conditions of the LC-MS/MS method according to the present invention; wherein a is a sample loading condition; b is leaching condition; c is elution conditions; d is the immunoaffinity time;

FIG. 3 shows the maximum adsorption capacity of salbutamol, clenbuterol and ractopamine in a mixed immunoaffinity chromatography column (Sal-IAC + Rac-IAC);

FIG. 4 shows an HPLC fluorescence chromatogram (salbutamol) of a sample treated with the pretreatment purification method of the present invention; wherein a and c are respectively blank pork and pork liver; b. d is the marked blank pork and pork liver;

FIG. 5 shows an HPLC fluorescence chromatogram (salbutamol) of a sample treated with SPE pretreatment clean-up; wherein e and g are respectively blank pork and pork liver; f. h is the marked blank pork and pork liver;

FIG. 6 shows an HPLC fluorescence chromatogram (ractopamine) of a sample treated with the pretreatment purification method of the invention; wherein a and c are respectively blank pork and pork liver; b. d is the marked blank pork and pork liver;

FIG. 7 shows an HPLC fluorescence chromatogram (ractopamine) of a sample treated with SPE pretreatment clean-up; wherein e and g are respectively blank pork and pork liver; f. h is the blank pork and pork liver after the labeling.

The specific implementation mode is as follows:

the invention discloses an anti-beta-receptor agonist cluster specific monoclonal antibody hybridoma cell strain, a monoclonal antibody secreted by the anti-beta-receptor agonist cluster specific monoclonal antibody hybridoma cell strain and application of the monoclonal antibody. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the hybridoma cells and monoclonal antibodies and uses thereof of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the techniques of the present invention may be practiced and used with modification, or with appropriate modification, and combination of the hybridoma cells and monoclonal antibodies and uses thereof, without departing from the spirit, scope, and spirit of the invention.

According to the test results in the specific embodiment, the invention couples the albuterol and ractopamine monoclonal antibody with CNBr activated agarose respectively to prepare the beta-receptor agonist mixed immunoaffinity chromatography gel/column, captures three beta-receptor agonists simultaneously, and is used together with LC-MS/MS equipment for quantitative determination. After the optimal conditions of sample loading, leaching, elution and the like are screened out, the standard recovery experiment is carried out on three blank pig samples, the recovery rate of each concentration of three target objects is in a reasonable range, and the relative standard deviation is lower than 4.1%; finally, the invention also carries out qualitative and quantitative comparison detection on the purification method (step before LC-MS/MS on-machine detection) of the detection method and the SPE purification method (step before LC-MS/MS on-machine detection) used in the national standard, finds that the detection method of the invention shows strong superiority in both recovery rate aspect and fluorescence chromatogram, and shows that the mixed immunoaffinity chromatography gel/column prepared by the invention can effectively remove matrix effect and can be accurately and effectively applied to practice.

The present invention provides an anti-beta-receptor agonist cluster specific monoclonal antibody hybridoma cell strain, and a monoclonal antibody secreted by the same and application thereof are further described below.

Example 1: obtaining of ascites sample containing monoclonal antibody according to the invention

1. Recovery and proliferation of hybridoma cell strain 1C3A and hybridoma cell of patent CN104311438A

Taking a freezing tube of the hybridoma cell strain from a liquid nitrogen tank, quickly melting the freezing tube in a water bath at 37 ℃, centrifuging for 5min at 600r/min, removing supernatant, adding 15% FBS/RPMI-1640 culture solution to suspend cells, supplementing the culture solution to 5ml respectively, planting the cells in a 50ml cell culture bottle, culturing in a carbon dioxide culture box, changing the culture solution after the cells grow to 30% of density, carrying out passage after the cells grow to about 60% (in logarithmic growth phase), and carrying out passage once every 2-3 days according to a ratio of 1: 3-4.

2. In vivo induction of monoclonal antibody protein and ascites acquisition

Taking 12 male BALB/c mice 8-10 weeks old 7-10 days before planting hybridoma cells in vivo, injecting 0.5ml per mouse intraperitoneally with pristane, and carefully feeding for later use after injection.

Taking the culture flask of the cells in the logarithmic growth phase, slightly beating the cell flask wall to make the cells fall off, transferring the cell suspension into a 50ml centrifuge tube after counting, centrifuging for 12min at 600r/min, discarding the culture solution, adding the serum-free RPMI-1640 with the same volume for resuspension of the cells, centrifuging again, discarding the RPMI-1640 according to the proportion of 1-2 × 10⁶0.5ml cell resuspension in serum-free RPMI-1640.

Injecting 0.5ml of the cell suspension/mouse into abdominal cavity after 7-10 days of sensitization, collecting ascites after 7-10 days, centrifuging at 1000rpm for 10min to remove cells in the abdominal water, collecting supernatant, centrifuging at 12000rpm for 30min to remove cell debris in the abdominal water, collecting supernatant, and measuring OD with ultraviolet spectrophotometer₂₈₀And OD₂₆₀Value in 1.53 XOD₂₈₀-0.71×OD₂₆₀Formula rough estimateAnd dividing the ascites into three parts, measuring the antibody titer of one part, purifying the antibody protein of the other part, and freezing and storing the rest at-20 ℃ for later use.

Example 2: the monoclonal antibody protein and the purification of the ractopamine monoclonal antibody of the hybridoma cell of the patent CN104311438A

2mL of protein G resin suspension was aspirated, transferred to a disposable PE column with a column capacity of 12mL, and then 20mL of binding buffer (3.3768G Na)₂HPO₄·12H₂O，0.0888g NaH₂PO₄·2H₂O，8.5g NaCl，1L H₂O preparation), the flow rate is 1mL min^-1And removing impurities in the suspension to precipitate the suspension. Then 4mL of ascites of the monoclonal antibody is slowly added to flow through the gel layer, protein G is the cell wall protein of group G streptococcus and can capture the Fc region of the IgG type antibody, and other subtypes can not be captured, thereby achieving the purpose of purification. After the ascites fluid completely passed through the gel layer, 100mL of binding buffer was added for washing to remove non-specific adsorption, and then 15mL of glycine buffer (0.1mol L) was added^-1pH 2.5), 100 μ L of a neutralization buffer (1mol L) was added to a 1.5mL PE centrifuge tube^-1Tris-HCl, pH 8.5), then the eluate was collected dropwise, 1mL per tube, the first 10 tubes were collected, the protein concentration was measured with the BCA kit, and the tubes of the protein solution with the highest concentration were selected and added to 0.01mol L^-1Dialyzed against PBS (pH 7.4) for one day and the dialysate was changed every 3 hours. Then, the total protein concentration was measured by BCA kit, and the purity was measured by SDS-PAGE.

FIG. 1 shows the gel electrophoresis of two antibodies,

lanes

1 and 4 for markers,

lanes

2 and 5 for salbutamol according to the invention and for ractopamine ascites of the prior art, respectively, and

lanes

3 and 6 for two purified antibodies, respectively. The purified antibody contained only the IgG type, and only bands appeared at 25 and 50 KD; as can be seen from the figure, the purity of both antibodies was higher. The concentration of the purified antibody can be determined, a BCA kit is adopted, a standard curve is made according to the standard BCA protein concentration and the corresponding absorbance, the concentration of the purified antibody can be determined by an external standard method, and the final concentrations of the albuterol and ractopamine antibodies are respectively determined1.95 and 2.23mg mL^-1。

Example 3: immunological cross-sex analysis

The invention selects 4 beta-adrenoceptor agonists of clenbuterol hydrochloride, terbutaline, brombutolol and sibutrol to carry out the detection of the cross reaction rate. Salbutamol concentration range is 0-30ng mL^-1The concentration range of the cross product is 0.1-10000ng mL^-1. Standard curves of salbutamol and other 4 kinds of cross-over substances are respectively made, and respective IC is calculated₅₀Thus, the respective CR values were determined, and the results are shown in Table 1.

TABLE 1

As can be seen from table 1, when the CR value of salbutamol is assumed to be 100%, clenbuterol hydrochloride, terbutaline, brombuterol and sibutrol are found to have a large cross-reactivity, with CR values of 66.8%, 57.3%, 42.1%, 121.5%, respectively. The four substances are very similar to the structure of the salbutamol and all have tert-butyl groups connected with nitrogen atoms, which indicates that the antigenic determinant complementary to the salbutamol monoclonal antibody screened by the invention is the tert-butyl group far away from the main structure of the salbutamol, namely, the monoclonal antibody has cluster specificity.

Example 4: monoclonal antibody protein and Sepharose^TM4B chemical crosslinking

1. Pre-crosslinking monoclonal antibody protein pretreatment

About 40mg of the monoclonal antibody protein purified in example 2 was placed in a dialysis bag (7500 + 14000MW) at 100-₃0.5M NaCl (pH8.3) solution was dialyzed overnight at 4 ℃ (changing every 4-6 h), and the dialyzed protein was filtered through 0.22 μ M microporous membrane and stored at 4 ℃ for further use.

2. CNBr-activated Sepharose 4B pretreatment before crosslinking

Weighing 1g CNBr-activated Sepharose 4B lyophilized powder in two parts respectively in 2 50ml centrifuge tubes (from corning corporation), adding 30ml 1mM HCl into each centrifuge tube, and placing in a bed at room temperatureSlightly swinging and swelling for 30min, discarding supernatant at room temperature of 2000r/min for 10min, centrifuging and washing by the same method for 8-10 times to sufficiently remove CNBr-activated Sepharose 4B and other small molecule chemical reagents, and then adding 30ml of 0.1M NaHCO into each centrifuge tube₃The precipitate was washed once by centrifugation in 0.5M NaCl (pH8.3) and the precipitate was taken for further use.

3. Chemical crosslinking of monoclonal antibody protein and Sepharose 4B

Dividing the pretreated monoclonal antibody protein solution into two equal parts, respectively adding the two equal parts into 2 50ml centrifuge tubes containing treated Sepharose 4B glue, covering the centrifuge tubes with covers, sealing the centrifuge tubes with parafilm, placing the centrifuge tubes in a rocking bed for incubation for 1h at room temperature, discarding supernatant at room temperature of 2000r/min for 10min, and taking a little from the supernatant to analyze the crosslinking effect of the monoclonal antibody protein and the Sepharose 4B by a Bradford method. Adding 30ml of 0.1M NaHCO into each centrifuge tube₃Placing the solution in 0.5M NaCl (pH8.3) in a swinging bed, incubating for 10min at room temperature in a swinging way, discarding the supernatant at room temperature of 2000r/min for 10min, repeating the steps with the crosslinking buffer solution, centrifuging and washing for 5-6 times to fully remove the uncrosslinked protein, and taking the precipitated gel.

And (3) taking the precipitation gel, adding 30ml of 0.1M Tris-HCl (pH 8.0) into each centrifuge tube, placing in a swinging bed for 2h at room temperature to fully seal activated groups which are not crosslinked with protein in the Sepharose 4B gel, centrifuging at room temperature of 2000r/min for 10min, and taking the precipitation gel.

Taking the precipitation gel, respectively using 30ml 0.1M HAC/0.5M NaCl (pH4.0) and 30ml 0.1M Tris-HCl/0.5M NaCl (pH 8.0) buffer solution to alternately centrifuge and wash for three cycles, then suspending the gel in 30ml 0.1M Tris-HCl/0.5M NaCl (pH 8.0) solution and supplementing 0.02% NaN₃Storing at 4 deg.C to obtain salbutamol immunoaffinity chromatography gel (Sal-IAC)/ractopamine immunoaffinity chromatography gel (Rac-IAC).

Example 5: the invention relates to an LC-MS/MS method for detecting salbutamol, clenbuterol hydrochloride and ractopamine in pork, pig urine or pig liver

1. Sample processing

The homogenate of pork and pork liver samples is provided by the agricultural product quality safety monitoring center in Suzhou city. Weighing 2 g/part of homogenized sample in parts, respectively loading into 50mL centrifuge tubes, adding three clenbuterol mixed standard solutions with different volumes, and standardizing each componentThe concentration is 1, 5, 20ng g^-1Respectively adding the sample into each centrifuge tube containing the sample; then adding 4 mL/tube of acetone, shaking to extract the target for 15min, centrifuging for 15min at 10000 Xg, and transferring the acetone supernatant to another 15mL clean centrifugal tube; repeating the step twice to obtain 12mL of acetone supernatant, and drying by nitrogen at 50 ℃; then re-dissolving with 2mL of dilute hydrochloric acid to form the target into a hydrochloride form, and ensuring that the target exists in a water phase; adding 2mL n-hexane, reverse-phase extracting to remove fat, centrifuging at 10000 Xg for 15min, collecting water phase, and purifying with 2mol L^-1The pH of the solution was adjusted to 7.4 with NaOH, an equal volume of PBS was added and mixed well, and 1mL of the aqueous phase was added to the mixed immunoaffinity chromatography gel (1.0mL of Sal-IAC +0.5mL of Rac-IAC).

The pig urine is also provided by the agricultural product quality safety monitoring center in Suzhou city. Firstly, pig urine is centrifuged at 10000 Xg for 10min, a plurality of 5 mL/part urine is respectively filled in each tube of a 15mL centrifuge tube, three clenbuterol mixed standard solutions with different volumes are added, and the standard concentration is 1, 5, 20ng mL^-1(ii) a 5mL of 0.01mol L was added^-1PBS (pH 7.4) was mixed well and 1mL of urine sample was added to the mixed immunoaffinity chromatography gel (1.0mL Sal-IAC +0.5mL Rac-IAC).

2. Immunoaffinity and LC-MS/MS detection

A number of 1.5mL centrifuge tubes were used, 1mL Sal-IAC and 500. mu.L Rac-IAC were added to each tube, washed 5 times with 0.01mol/L PBS (pH 7.4) and centrifuged, and the supernatant was discarded to give a final gel volume of about 150. mu.L. And transferring the sample solutions into centrifuge tubes containing the mixed immunochromatographic gel one by one, after mixing, performing swing incubation for 1h at room temperature of a shaking table, centrifuging for 3min at 1000r/min, discarding the supernatant, performing centrifugal washing for 3 times by using 0.01mol/L PBS (pH 7.4), performing centrifugal washing for 3 times by using pure water, finally adding 1mL of 0.1mol/L eluent (methanol-water-ammonia water is 90:9.8:0.2) into each centrifuge tube respectively, placing the centrifuge tubes into the shaking table, performing swing incubation for 15min at room temperature, performing centrifugal washing for 3min at 1000r/min, collecting the eluted supernatants one by one to clean 1.5mL centrifuge tubes, drying by using nitrogen at 50 ℃, re-dissolving by using 1mL of mobile phase, and performing detection analysis by using an LC-MS/MS after a filter membrane.

Example 6: the condition optimization test of the LC-MS/MS method for detecting salbutamol, clenbuterol hydrochloride and ractopamine in pork, pig urine or pig liver

The conditions of loading, washing, elution, etc. may have a large influence on the binding and dissociation of the antigen-antibody complex. In the loading step, it is desirable that the target is sufficiently completely bound to the antibody in the gel; during rinsing, non-specific adsorption is removed as much as possible to ensure the purity of the sample matrix; finally, during the elution process, the target can be completely dissociated by changing the pH of the environment. In summary, the purpose of optimizing the conditions is to improve the extraction effect and the detection sensitivity.

1. Screening of Loading and elution conditions

In this study, three different sample solutions (pure water; 0.01mol mL) were each subjected to^-1PBS；0.01mol mL^-1PBS + 10% aqueous methanol solution) and leacheate (pure water; 0.01mol mL^-1PBS + water; 0.01mol mL^-1PBS + 10% methanol water), as shown in FIGS. 2a-b, in the loading condition, the recovery rates of three targets are not greatly affected by the PBS and pure water system, but the recovery rates of three substances are greatly affected by adding a few organic reagents into the system, which may be that the organic reagents have a certain destructive effect on the antibody, so that part of the antibody on the gel is inactivated and the target cannot be completely captured; the leacheate has little influence on the extraction effect, and the recovery rate is about 90 percent. Finally, the invention selects PBS solution as the sample solution, and PBS-pure water as the leaching system.

2. Selection of eluent

Elution conditions had the greatest effect on the recovery of the three targets. The binding between antigen and antibody is the result of electrostatic force, van der waals force and hydrogen bond interaction, and in order to break the force between antigen and antibody complex, the eluent must be very acid or alkali solution, and the requirement for pH is very strict. In this study, eight eluents [ 0.1% formic acid water (pH 2.0) were examined; 0.1% formic acid water (pH 2.6); 2% HAC (pH 2.0); 60%, 80%, 100% aqueous methanol; the extraction effect of 1% ammonia methanol (pH 10.5) and methanol-water-ammonia (90-9.8-0.2, pH 10.5) showed that the methanol/water system did not sufficiently break the coupling bond between antigen and antibody as shown in FIG. 2 c. Under acidic conditions, however, the recovery of clenbuterol is not high, because its structure is slightly different from that of albuterol and ractopamine. The salbutamol and the ractopamine both contain phenolic hydroxyl and secondary amine, the phenolic hydroxyl is weakly acidic, the secondary amine is weakly alkaline, and the phenolic hydroxyl and the secondary amine can be neutralized, so that the salbutamol and the ractopamine are neutral; and the clenbuterol only contains basic groups of aniline and secondary amine, so that the clenbuterol is basic. The salbutamol monoclonal antibody is directed against secondary amine antigenic determinant, when the eluent is acidic, the salbutamol monoclonal antibody may firstly generate acid-base neutralization reaction with aniline of clenbuterol, the acidity of the eluent is reduced, and the covalent bond between the clenbuterol and the antibody cannot be broken, so that the recovery rate of the clenbuterol is generally low or even none. Therefore, only alkaline solutions can elute the three targets well. Finally, methanol-water-ammonia (90:9.8:0.2) was chosen as the optimal eluent.

3. Optimization of immunoaffinity time

Unlike the conventional immunoaffinity process, the reaction between antigen and antibody is performed in a closed system in this study. Therefore, the invention can control the immunoaffinity time and optimize the optimal combination time. The results are shown in FIG. 2d, when the time reached 45min, the three targets all reached the maximum binding with the antibody. Therefore, we finally chose 45min as the optimal immunoaffinity time.

Example 7: determination of maximum adsorption capacity of (Sal-IAC + Rac-IAC) in mixed immunoaffinity chromatography column

The maximum adsorption capacity is an important index for inspecting the performance of the immunoaffinity column; if the target substance content is too large to exceed the maximum adsorption capacity of the immunoaffinity column, the excessive target substance cannot be loaded on the column and only can flow out together with impurities, so that the result is inaccurate when the extraction recovery rate is calculated. Under the optimal optimization condition, the maximum adsorption capacity of each of salbutamol, clenbuterol hydrochloride and ractopamine is determined, so that the subsequent standard addition recovery experiment of actual samples cannot be influenced. The invention is characterized in that the concentration of 1mL is respectively 0.2, 2, 10, 25, 50 and 100ng mL^-1Salbutamol ofAdding the mixed standard solution of the clenbuterol and the ractopamine into an immunoaffinity column (Sal-IAC + Rac-IAC mixed immunoaffinity chromatography gel is added into a centrifuge tube), performing cross-linking reaction, washing, eluting, collecting eluent, processing, quantitatively determining the content of three corresponding substances at each concentration point on an upper computer, and calculating the recovery rate so as to determine the maximum column capacity of the three target substances in the immunoaffinity column.

As shown in FIG. 3, 50. mu.L of Sal-IAC + Rac-IAC mixed immunoaffinity chromatography gel adsorbed salbutamol 33ng, clenbuterol 34ng, and ractopamine 29 ng. Because the traditional immunoaffinity column is to load the chromatography gel into an empty SPE column, the sample solution slowly flows through the chromatography gel under an open system; in this study, the immunoaffinity process was performed in a closed system, which ensured that the reaction between antigen and antibody was sufficiently complete. Generally, the column volume of the immunoaffinity column is about 1mL, and in order to more clearly illustrate the rationality of the research system, the invention converts 50 μ L of chromatography gel into 1mL, so that the maximum adsorption capacities of the three substances are 660ng, 680ng and 580ng respectively, which are similar to the maximum adsorption capacity of the traditional IAC column.

Example 8: actual sample standard adding recovery rate

The actual sample labeling recovery experiment is a survey of the accuracy and precision of the immunoaffinity chromatography. The invention detects three blank actual samples in example 5, and then adds beta-receptor stimulant mixed standard solutions with different volumes into the blank sample solution to ensure that the concentration range is 0.2-20ng mL^-1A standard curve was made by LC-MS/MS and the results are shown in Table 2. In the environment of three different matrix samples, the detection limit of the three targets is obviously lower than 0.5ng g specified by the national standard^-1And r is²> 0.9946, indicating that the sensitivity of the method of the invention is high.

TABLE 2

And (3) performing a sample standard adding recovery experiment, adding clenbuterol mixed standard solutions with different volumes into the blank pig sample, detecting according to the method of the invention, and calculating the recovery rate. The results are shown in tables 3 to 5.

The recovery rates of high, medium and low labeled concentrations of each target object in each sample are averaged, and the average recovery rates can clearly show that the average recovery rates of the three target objects of salbutamol, clenbuterol and ractopamine in the pork matrix are all over 70 percent; in the pig liver and pig urine samples, the average recovery rates of salbutamol and clenbuterol are respectively about 80% and 90%, but the average recovery rate of ractopamine in the two matrixes is not very high, namely about 64%, which is probably because the immunoaffinity between the ractopamine monoclonal antibody and the hapten ractopamine is too strong, the antigen-antibody complex of the ractopamine monoclonal antibody cannot be thoroughly broken by alkaline eluent with the pH of 10.5, and the matrix of the pig liver and pig urine is more complex than that of pork, so that the standard addition recovery rate of ractopamine is lower. The relative standard deviation of the three standard-added samples is lower than 4.1 percent, which shows that the mixed immunoaffinity chromatography gel prepared by the invention can effectively remove the matrix effect of the samples and can be accurately and effectively applied to practice.

Table 3 standard recovery rate of pork (n ═ 3)

TABLE 4 recovery rate of pork liver (n ═ 3)

TABLE 5 recovery rate of swine urine (n ═ 3)

Example 9: the LC-MS/MS method for detecting salbutamol, clenbuterol hydrochloride and ractopamine in pork, pig urine or pig liver is compared with the national standard SPE

And (3) national standard SPE: MCX solid phase extraction cartridge (60mg/3mL, Waters Oasis) was used. Specifically, 3mL of methanol and 3mL of pure water are sequentially added to activate the small column, then the sample solution treated in the example 5 is added into the column, after the sample solution is drained, 3mL of pure water and 3mL of methanol are sequentially added to elute the small column, finally 3mL of 5% ammonia methanol is added to elute, the eluent is collected, the eluent is dried by nitrogen at 50 ℃ and is redissolved by 1mL of mobile phase, and the obtained solution is subjected to LC-MS/MS quantitative detection after being filtered by a membrane, and the recovery rate is calculated.

In order to verify the superiority of the detection method, a comparative experiment is carried out on the detection method and an SPE purification method used in national standard, the results are shown in tables 3-5, after three matrixes are subjected to SPE treatment after being subjected to standard addition, the average recovery rate of salbutamol 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%. By comparing the data in the table, it can be clearly seen that the recovery rate of the detection method of the present invention is higher than that of SPE in different matrixes. The SPE processed sample has low recovery rate, but can also become a national standard guiding method, because the national standard adds an isotope internal standard substance when the quantitative detection is carried out by a liquid chromatography-mass spectrometry, and an internal standard method is adopted to carry out the quantification on a target substance. However, isotopic internal standards are quite expensive, which undoubtedly adds significantly to the cost of detection. In contrast, the monoclonal antibody in the detection method of the invention can be continuously produced, and the advantages of the immunoaffinity chromatography of the invention are further reflected.

In order to more intuitively show the superiority of the detection method, the pork and pork liver sample solutions after purification treatment (step before LC-MS/MS detection) of the detection method are compared by a fluorescence chromatogram, because the clenbuterol has no fluorescence property, the salbutamol and the ractopamine are only added into the samples, and single-component qualitative analysis is carried out by HPLC-FLD, so that two targets are separately detected because of the single-component qualitative analysis of the salbutamol and the ractopamineThe optimal excitation wavelength and the emission wavelength are different, the mobile phase and the proportion thereof are different, and the invention aims to compare the pretreatment (step before LC-MS/MS detection) of the detection method with the pretreatment (step before LC-MS/MS detection) of SPE, so that other conditions are the same and optimal. In the experiment, the standard adding concentrations of the albuterol and the ractopamine are all 100ng g^-1。

4-5 are fluorescence chromatograms of salbutamol recovery with labeling, fig. 4a and c are respectively blank pork and pork liver pretreated by the detection method of the present invention, after the sample is labeled, the sample is also pretreated and purified by the detection method of the present invention, the purification effect is shown in fig. 4b and d, salbutamol appears at about 4.8 minutes, and few or no miscellaneous peaks exist on the fluorescence chromatograms; although ghost peaks appeared at 7min, this was probably a hetero-peak caused by the organic solvent used in the treatment of pork pig liver, and did not affect the qualitative analysis of salbutamol; fig. 5e and g show that the blank pork and pork liver are respectively subjected to SPE pretreatment, it can be seen that the blank sample has many and random peaks, the negative sample can detect the chromatographic peak of suspected salbutamol in 4.8 minutes, and it can be seen that the SPE method can not effectively remove the matrix effect in the sample, which has great influence on the qualitative or quantitative analysis of salbutamol residue; fig. 5f and h are fluorescence chromatograms of SPE-treated pork and pork liver, and it can be seen from the graphs that the peak area of the chromatographic peak of salbutamol is large, but actually, because impurities in the sample matrix are not cleaned and washed away, the peak area is generated in the same time period with salbutamol, and therefore, the integrated peak area is inaccurate, and quantitative determination cannot be performed.

Also, fig. 6-7 are labeled recovery chromatograms of ractopamine, concluding about the same as salbutamol. The mobile phase for detecting ractopamine is acetonitrile water, the mobile phase for detecting the ractopamine is methanol water, the polarity of acetonitrile is greater than that of methanol, and the elution capacity is stronger, so that an organic solvent in the sample treatment process does not appear in a chromatogram. FIGS. 6a, b, c, d are chromatograms of samples pre-treated by the detection method of the present invention, pre-treatment of which substantially eliminates the sample matrix, leaving the ractopamine peak pattern undisturbed at all; however, fig. 7e, f, g, and h are sample chromatograms after SPE pretreatment, and similarly, the influence of impurities on the time of ractopamine peak emergence of about 7.4 minutes, even no qualitative analysis can be performed. Through comparison of fluorescence spectrograms of the two methods, the purification effect of the pretreatment matrix of the detection method disclosed by the invention is further proved to be far better than that of SPE. Therefore, the capability of purifying the sample matrix of the detection method is far higher than that of an SPE (solid phase extraction) purification means in both quantitative detection and qualitative confirmation, so that the immunoaffinity chromatography method can be proved to be applied to purification treatment of actual samples.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. An anti-beta-receptor agonist cluster specific monoclonal antibody hybridoma cell strain which is characterized by having a preservation number of CCTCC NO. C2016208.

2. The application of the hybridoma cell strain with the preservation number of CCTCC NO. C2016208 in the detection of a beta-receptor agonist residual sample; the beta-receptor agonist is selected from one or more than two of salbutamol, clenbuterol hydrochloride, terbutaline, brombuterol and sibutrol.

3. The monoclonal antibody against beta-receptor agonist cluster specificity is characterized by being secreted and produced by a hybridoma cell strain with the preservation number of CCTCC NO. C2016208.

4. The application of the anti-beta-receptor stimulant cluster specific monoclonal antibody secreted and produced by the hybridoma cell strain with the preservation number of CCTCC NO. C2016208 in the preparation of beta-receptor stimulant residue sample detection and/or beta-receptor stimulant residue immunodetection products; the beta-receptor agonist is selected from one or more than two of salbutamol, clenbuterol hydrochloride, terbutaline, brombuterol and sibutrol.

5. The use of claim 4, wherein the immunoassay product is an immunoaffinity chromatography gel or an immunoaffinity chromatography column prepacked with the gel.

6. The beta-receptor stimulant cluster specific immunoaffinity chromatography gel is characterized in that monoclonal antibodies generated by secretion of hybridoma cell strains with the preservation number of CCTCC NO. C2016208 are purified and chemically cross-linked with CNBr-activated Sepharose-4B gel to form immobilized antibody protein, and the immobilized antibody protein is obtained.

7. The immunoaffinity chromatography gel of claim 6, wherein the purified monoclonal antibody protein and CNBr-activated Sepharose-4B gel are present in an amount ratio of: 5mg of purified monoclonal antibody protein: 1ml of CNBr-activated Sepharose-4B swelling gel.

8. A method for preparing an immunoaffinity chromatography gel of claim 6, comprising:

9. The preparation method of claim 8, wherein the dialysis pretreatment of the purified monoclonal antibody protein comprises:

the purified monoclonal antibody protein is placed in a 7500-14000MW dialysis bag and then placed in 100-14000M NaHCO₃Dialyzing in 0.5M NaCl buffer solution at 4 deg.C overnight, changing solution every 4-6h, filtering the dialyzed protein with 0.22 μ M filter membrane, and storing at 4 deg.C.

10. The preparation method of claim 8, wherein the CNBr-activated Sepharose-4B is subjected to swelling and elutriation pretreatment as follows:

11. The preparation method of claim 10, wherein the CNBr-activated Sepharose-4B is subjected to a swelling and elutriation pretreatment comprising:

weighing 1g of CNBr-activated Sepharose 4B solid powder in a 50ml centrifuge tube, adding 30ml of 1mM HCl into the centrifuge tube, slightly swinging and swelling the centrifuge tube in a swinging bed at room temperature for 30min, removing supernatant at room temperature of 2000r/min for 3min, adding 30ml of 1mM HCl into the centrifuge tube, centrifugally washing the centrifuge tube by the same method for 8-10 times to sufficiently remove other small molecular chemical reagents in the gel, and then adding 30ml of 0.1M NaHCO₃The solution is centrifuged and washed once with 0.5M NaCl buffer solution for later use.

12. The method according to claim 8, wherein the step 2 is:

adding the pretreated and purified monoclonal antibody protein obtained in the step 1 into a 50ml centrifuge tube containing the treatment gel obtained in the step 1, covering the centrifuge tube, sealing the centrifuge tube with a parafilm, placing the centrifuge tube in a pendulum bed, incubating the centrifuge tube at room temperature for 1h in a swinging manner, discarding the supernatant at the room temperature of 2000r/min for 10min, and adding 30ml of 0.1M NaHCO into the centrifuge tube₃Placing the solution in 0.5M NaCl buffer solution, incubating at room temperature for 10min, removing supernatant at room temperature for 10min at 2000r/min, and adding NaHCO₃And centrifugally washing the solution with NaCl buffer solution for 5-6 times to fully remove the uncrosslinked protein to obtain the antibody protein crosslinked gel.

13. The method according to claim 8, wherein step 3 is:

taking the antibody protein cross-linked gel in the step 2, adding 30ml of 0.1M Tris-HCl into a centrifugal tube, placing the centrifugal tube in a swinging bed, swinging and incubating for 2h at room temperature to fully seal activated groups which are not cross-linked with the protein in the antibody protein cross-linked gel, centrifuging at 2000r/min at room temperature for 10min, discarding supernatant, alternately centrifuging and washing the centrifugal tube with 30ml of 0.1M HAC/0.5M NaCl and 30ml of 0.1M Tris-HCl/0.5M NaCl buffer solution for three cycles, and then re-suspending the cross-linked gel in 30ml of 0.1M Tris-HCl/0.5M NaCl buffer solution and supplementing 0.02% NaN₃And storing at 4 ℃ for later use to obtain the immunoaffinity chromatography gel.

14. An immunoaffinity chromatography column for detecting β -receptor agonists, comprising the gel of claim 6 or 7, and a bed.

15. The column according to claim 14, further comprising a chromatography gel prepared from ractopamine monoclonal antibody.

16. A method for detecting a beta-receptor agonist in pork, pig urine or pig liver, wherein the detection is performed by LC-MS/MS using a chromatography gel according to any one of claims 6 to 7, or a chromatography column according to any one of claims 14 to 15, and the beta-receptor agonist is selected from one or more of salbutamol, clenbuterol hydrochloride, terbutaline, brombuterol and sibutrol.