CN113960305B - Immunomagnetic bead for clenbuterol Luo Fuji purification and preparation method and application thereof - Google Patents
Immunomagnetic bead for clenbuterol Luo Fuji purification and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to an immunomagnetic bead for purifying clenbuterol Luo Fuji, a preparation method and application thereof, wherein the immunomagnetic bead is prepared by taking carboxyl magnetic beads as a carrier and clenbuterol monoclonal antibodies as recognition intermediates through the processes of activation, coupling, washing and sealing, and can efficiently capture and enrich clenbuterol in a detection sample by incubating the immunomagnetic beads coupled with the clenbuterol monoclonal antibodies in a proper buffer solution under a certain condition. The immunomagnetic beads for purifying the clenbuterol Luo Fuji have the advantages of concentrating the concentration of the clenbuterol in a sample to be detected, improving the lower limit of detection, eliminating impurity interference, improving the detection accuracy and reliability, and reducing the sample processing time to achieve rapid detection.
Description
Technical Field
The invention relates to an enrichment purification treatment process of clenbuterol Luo Yangben, in particular to an immunomagnetic bead for purifying clenbuterol Luo Fuji, and a preparation method and application thereof.
Background
Clenbuterol (CL), also known as dichloramine, amicrotin, and kechuansu, is a type of beta 2 -adrenergic receptor agonistsIs also a derivative of catecholamine, and has the chemical name of alpha- (tert-butylamino) methyl-4-amino 3, 5-dichlorobenzyl alcohol and the molecular formula of C 12 H 18 Cl 2 N 2 O, the molecular weight of which is 277.22, clenbuterol Luo Chang is white or white-like crystalline powder, has no stink smell and little bitter, is easily soluble in water, can be dissolved in ethanol, is extremely slightly soluble in acetone or chloroform, and is almost insoluble in diethyl ether or benzene.
Because CL has obvious effects of promoting growth and improving lean meat percentage in animal production, the abuse problem in the domestic and foreign breeding industries is driven to be very serious by the benefit, and because CL is continuously used in excess in production and has long elimination half-life in vivo, drug accumulation is extremely easy to cause in animal tissues. Since the discovery of toxic and side effects of clenbuterol in the middle of 80 s in the last century, thousands or even tens of thousands of people have been poisoned worldwide by eating animal-derived foods with residual clenbuterol. The livestock product with clenbuterol left is used by people and then has the symptoms of muscle excitability enhancement, and the other people have the symptoms of muscle tremors and gastrocnemius spasm of severe patients after poisoning; at the same time, myocardial contraction is enhanced, and heart rate is accelerated; after eating poisoning, the hypertension patient leads to the rise of blood pressure, the expansion of microcirculation blood vessel, the compression and stimulation of peripheral nerves, and the headache and dizziness are caused; serious diseases such as canceration, fetal teratogenesis and the like can be caused after the pregnant woman is poisoned.
The 47 th JECFA conference in 1996 passed the specification on the maximum residual limit criteria of clenbuterol, namely that the maximum residual limit (maximum residue limit, MRL) of clenbuterol in horses, cattle muscle and fat was 0.2. Mu.g/kg, MRLs in kidneys, liver was 0.6. Mu.g/kg, and 0.05. Mu.g/L in cow's milk, and the recommended detection method was gas chromatography-mass spectrometry (gas chromatography-mass spectrometry, GC-MS). In 2003, the 26 th International Commission on food Council (Codex Alimentarius Commission, CAC) resolution passed this standard and has been adopted to date. The "affirmative list regime" (Positive List System) was implemented in japan 2006, and a new residual limit criterion was implemented. The "tentative standard" is applied to the clenbuterol residual standard, namely, the CAC standard is referred to, but the standard is clearly defined in the liver, kidney, muscle and fat of pigs, and the clenbuterol cannot be detected. The maximum residual limit standard (EC 2377/90) of clenbuterol published by European Union is lower than the CAC standard, and the maximum residual limit in the muscle of horses and cattle is 0.1 mug/kg, and the maximum residual limit in viscera is 0.5 mug/kg. The EU was under the regulations of 12 months of the European Union EC 16129/07, aiming at making lower levels of MRL and abrogating the current standards, and the EU has more stringent requirements on clenbuterol residues (Table 1) and higher. The chinese department of agriculture in 1999 in the notice of release of the highest residual quantity of veterinary drugs in animal food (farm animal hair No. 1999) specified that MRLs of clenbuterol in horses, cattle muscle, viscera, kidneys and milk were 0.1, 0.5, 0.05 μg/kg. However, since 12 months in 2002, the highest limit of veterinary drug residue in animal food was revised in Japanese patent application No. 235, and the farmer's grazing incidence No. 1999 [ 17 ] was abolished, and the new bulletin prescribes that clenbuterol Luo Zaisuo was undetectable in animal food.
The main extraction methods of clenbuterol in the food at present comprise an organic solvent extraction method, an immunoaffinity chromatography method and the like. The organic solvent extraction method is the most common extraction method at present, but the extraction of the organic solvent has the defects of complex process, high toxicity, long required time and the like, and other fluorescent substances, pigments and structural analogues possibly exist in the final extract, so that the detection result is interfered; the immunoaffinity chromatography improves the purifying effect of the sample, and simultaneously can obviously reduce the use of toxic and harmful reagents, but the pretreatment of the sample is more complex, the used equipment is expensive, the technical requirements on operators are higher, and the method is not suitable for large-scale sample detection and large-scale popularization and application.
The immunomagnetic bead separation technique (Immunomagnetic bead-based separation, IMS) is a new immunological technique developed in recent years. The immune magnetic beads (Immunomagnetic bead, IMB) can be combined with active protein antibodies and attracted by magnets, after treatment, the antibodies can be combined on the magnetic beads to form antibody carriers, after the antibodies on the magnetic beads are combined with specific antigen substances, antigen-antibody-magnetic bead immune complexes are formed, and the complexes mechanically move under the action of magnetic force to separate the complexes from other substances, so that the purpose of separating specific antigens is achieved. Immune Magnetic Beads (IMB) are a platform, can be applied to all fields working by utilizing the antigen-antibody combination principle, and have achieved remarkable results in aspects of medical and biological bone marrow transplantation, stem cell separation, organelles, cancer cells, hormones, pathogenic bacteria, target objects and the like. In recent years, IMB is widely applied to the separation and detection of target objects in food, water, biological samples, environmental samples and other specimens by virtue of high sensitivity and specificity, and has good development and application prospects.
The IMB can effectively collect and concentrate a small amount of target substances in a large amount of samples, can avoid the defects existing in the extraction of an organic solvent extraction method and an immunoaffinity chromatography, has the advantages of high separation rate, high specificity, high stability, no pollution, no toxicity, simple operation, small sample consumption, no damage to the structure of the target substances and the like, and can directly produce the effect of enriching, separating and purifying the target substances in 2-5 hours without adding a plurality of purification steps to remove the impurities compared with the conventional method. And the antibody coupled to the magnetic beads can accurately identify the characteristic groups on the target object, so that the omission factor is reduced, and the potential safety hazard is reduced.
The invention optimizes the preparation process and application conditions of the immune magnetic beads capable of enriching the clenbuterol by utilizing the coupling principle of the magnetic beads and the antibodies, greatly improves the enrichment capacity and detection sensitivity of the clenbuterol, and can further improve the efficiency of rapidly detecting the clenbuterol by applying the immune magnetic beads to the separation and enrichment of the clenbuterol in animal urine, animal tissues and animal serum by virtue of the characteristics of rapidness, convenience and low price of the immune magnetic beads.
Disclosure of Invention
The invention aims to make up the defects in the prior art, and provides an immunomagnetic bead for purifying clenbuterol Luo Fuji, a preparation method and application thereof, so as to solve the technical problems of complex purification and separation operation, low separation efficiency and large potential safety hazard of a clenbuterol sample.
In order to achieve the aim of the invention, the invention adopts the following technical scheme: providing an immunomagnetic bead for purifying clenbuterol Luo Fuji, wherein the immunomagnetic bead is coupled with a clenbuterol monoclonal antibody; the clenbuterol monoclonal antibody is obtained by immunizing a white mouse with a clenbuterol artificial antigen; the clenbuterol artificial antigen is a conjugate of a clenbuterol hapten and a carrier protein.
The clenbuterol hapten is prepared by the following method:
dissolving 2.21g of 2-amino-1- (4-amino-3, 5-dichlorophenyl) ethanol in 20ml of N, N-dimethylformamide, adding 1.86g of carbonyldiimidazole, adding 0.5ml of triethylamine, stirring for 2 hours at room temperature, adding 1.72g of succinic acid monoazide hydrochloride, stirring for 4 hours at room temperature, stopping the reaction, adding 80ml of water, adding 1mol/L hydrochloric acid to adjust the pH value to 6, adding 100ml of ethyl acetate for 3, extracting for three times, merging organic phases, evaporating to dryness, and eluting and separating by using a silica gel column and 1, 2-dichloroethane/methanol (v/v, 10/1) to obtain 2.76g of carboxyl-type clenbuterol hapten product, wherein the yield is 72.8%.
The invention also provides a preparation method of the immune magnetic bead, which takes carboxyl magnetic beads with the particle diameter of 2.8 mu m as a carrier, wherein the tail ends of the carboxyl magnetic beads are provided with reactive carboxyl groups, and after the carboxyl magnetic beads are subjected to combined treatment by an activator EDC-NHS, the activated magnetic beads can be coupled with a clenbuterol monoclonal antibody to prepare the immune magnetic bead capable of enriching and purifying clenbuterol;
the preparation method of the immunomagnetic beads is specifically described as follows:
(1) Cleaning: taking 100 mu L of carboxyl magnetic beads in a centrifuge tube, washing with 100 mu L of MES solution containing 0.05% Tween-20 and having pH of 5.0 and 25mmol/L for 2 times, and removing the supernatant after magnetic separation;
(2) Activating: 50mmol/L EDC and NHS solutions are respectively prepared by using MES solutions stored at 4 ℃ and with pH of 5.0 and 25 mmol/L; adding 50 mu L of newly prepared EDC and NHS solution into a centrifuge tube filled with magnetic beads respectively, mixing uniformly by vortex, activating at room temperature for 30min, removing supernatant after magnetic separation, and washing with (1) MES solution for 2-3 times;
(3) Coupling: dissolving the clenbuterol monoclonal antibody into 60 mu L of MES solution with pH of 5.0 and 25mmol/L, regulating the total volume to 100 mu L by using the MES solution, gently adding the MES solution into activated magnetic beads, and coupling for 2 hours at room temperature for 30min or at 4 ℃, wherein the magnetic beads are kept in a uniformly mixed state;
(4) Closing: removing the supernatant after magnetic separation, adding 100 mu L of TRIS solution with pH of 7.4, and reacting for 15min to seal the magnetic beads;
(5) And (3) preserving: removing supernatant after magnetic separation, washing the blocked magnetic beads with 100 μL TRIS solution containing 0.1% -0.3% Bovine Serum Albumin (BSA) and 0.1% Tween-20 for 3-5 times, and redissolving the magnetic beads in solution containing 0.1% -0.5% BSA, 0.01% -0.1% Tween-20 and 0.02% NaN 3 Is preserved at 2-8deg.C for standby.
The invention also provides application of the immunomagnetic beads in enrichment and purification of clenbuterol.
Experiments prove that the immune magnetic beads for purifying the clenbuterol Luo Fuji have high enrichment rate and recovery rate of the clenbuterol Luo You, the enrichment rate of the clenbuterol is 50ng/mg (50 ng of the clenbuterol can be captured by each milligram of the immune magnetic beads), and the recovery rate of the clenbuterol is more than 85%. The immunomagnetic beads for purifying the clenbuterol Luo Fuji can efficiently, accurately, reliably, quickly and simply enrich the clenbuterol in a sample to be detected, so that the immunomagnetic beads can be further applied to detection of chemical analysis instruments (LC-MS/MS, liquid chromatography tandem mass spectrometry), detection of an enzyme-linked immunosorbent assay and quick test of colloidal gold test strips, and have the advantages of concentrating the concentration of the clenbuterol in the sample to be detected, improving the lower limit of detection, eliminating impurity interference, improving the accuracy and reliability of detection and reducing the sample processing time to achieve quick detection.
Drawings
FIG. 1 is a schematic diagram of the synthesis route of clenbuterol hapten
Detailed Description
The invention is described in further detail below with reference to the drawings and the specific examples. The experimental methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are commercially available.
EXAMPLE 1 preparation of immunomagnetic beads for purification of clenbuterol Luo Fuji
The example shows the preparation of conjugate of clenbuterol monoclonal antibody and carboxyl-containing immunomagnetic bead as immunomagnetic bead for enriching and purifying clenbuterol. The method comprises the following steps:
1. preparation of clenbuterol monoclonal antibody
1. Synthesis of clenbuterol hapten
Dissolving 2.21g of 2-amino-1- (4-amino-3, 5-dichlorophenyl) ethanol in 20ml of N, N-dimethylformamide, adding 1.86g of carbonyldiimidazole, adding 0.5ml of triethylamine, stirring for 2 hours at room temperature, adding 1.72g of succinic acid monoazide hydrochloride, stirring for 4 hours at room temperature, stopping the reaction, adding 80ml of water, adding 1mol/L hydrochloric acid to adjust the pH value to 6, adding 100ml of ethyl acetate for 3, extracting for three times, merging organic phases, evaporating to dryness, and eluting and separating by using a silica gel column and 1, 2-dichloroethane/methanol (v/v, 10/1) to obtain 2.76g of carboxyl-type clenbuterol hapten product, wherein the yield is 72.8%.
The clenbuterol intermediate 2-amino-1- (4-amino-3, 5-dichlorophenyl) ethanol is taken as a starting material, reacted with carbonyl diimidazole and reacted with succinic acid monoazide to obtain a carboxyl hapten product.
2. Synthesis of clenbuterol artificial antigen
(1) Synthesis of immunogens
Taking 14.14mg of carboxyl-clenbuterol hapten product, adding 1ml of DMSO for dissolving and clarifying, adding 0.1ml of triethylamine, adding 11 microlitres of isobutyl chloroformate, fully dissolving and uniformly mixing, and reacting for 2 hours at room temperature to obtain hapten activating solution A; taking 50mg of Bovine Serum Albumin (BSA), adding 0.1M CB9.1 ml to dissolve to obtain solution B, dropwise adding solution A into solution B, reacting at room temperature for 6h, stopping the reaction, dialyzing with 0.02M PBS, purifying for 3 days, changing the solution 3 times a day, centrifuging and packaging to obtain clenbuterol-BSA conjugate, namely the immunogen.
(2) Synthesis of coating Material
Taking 15.4mg of carboxyl-clenbuterol hapten product, adding 1ml of DMF to dissolve and clarify, adding 8.2mg of NHS and 15.5mg of EDC, fully dissolving and uniformly mixing, and reacting for 2 hours at room temperature to obtain hapten activating solution A; taking 100mg of Ovalbumin (OVA), adding 0.1M CB9.1 ml for dissolution to obtain solution B, dropwise adding the solution A into the solution B, reacting at room temperature for 6h, stopping the reaction, dialyzing and purifying with 0.02M PBS for 3 days, changing the solution 3 times a day, centrifuging and packaging to obtain the clenbuterol-OVA conjugate, namely the coating antigen.
3. Preparation of clenbuterol monoclonal antibody
(1) Acquisition of hybridoma cells
1) Primary immunization: fully emulsifying clenbuterol hapten-BSA conjugate (immunogen) with an equal amount of Freund's complete adjuvant, and subcutaneously injecting 0.2mL each of 6-week-old Balb/c mice;
2) Boosting was performed twice: boosting once every two weeks from the primary immunization, replacing Freund's complete adjuvant with Freund's incomplete adjuvant, and performing the same method and dosage as the primary immunization;
3) The last immunization is carried out for one week, the fundus vein blood sampling is tested for potency and inhibition, and the following last immunization is carried out when the potency reaches more than 1:10000: injecting 0.1mL of an immunogen solution without any adjuvant into the abdominal cavity, and killing the mice after three days, and taking the spleen to fuse with myeloma cells;
4) And (3) measuring cell supernatant by adopting an indirect competitive ELISA method, and screening positive holes. Cloning the positive hole by using a limiting dilution method to obtain and establish a hybridoma cell strain which stably secretes the clenbuterol monoclonal antibody, taking the hybridoma cells in the logarithmic growth phase, preparing cell suspension by using a freezing solution, subpackaging the cell suspension in a freezing tube, and preserving the cell suspension in liquid nitrogen for a long time.
(2) Preparation of monoclonal antibodies
1) Cell resuscitation: taking out the frozen tube of the clenbuterol monoclonal antibody hybridoma cell strain, immediately putting the frozen tube into a water bath at 37 ℃ for medium-speed thawing, centrifuging to remove frozen solution, and transferring the frozen solution into a culture flask for culture;
2) Preparation of ascites and antibody purification: balb/c mice (8 weeks old) were intraperitoneally injected with sterilized paraffin oil 0.5 mL/mouse by in vivo induction, and after 7 days, hybridoma cells were intraperitoneally injected 5X 10 5 Ascites were collected after 7 days. Purification was performed by octanoic acid-saturated ammonium sulfate to obtain a solution of clenbuterol monoclonal antibody (-20 ℃ C. Preservation).
(3) Determination of monoclonal antibody titers
The antibody titer was 1 (100000 ～ 150000) as determined by competition ELISA.
Competition ELISA method: coating an ELISA plate with a clenbuterol artificial antigen, adding a clenbuterol standard substance solution and a clenbuterol monoclonal antibody marked by horseradish peroxidase, reacting for 10min at 25 ℃, pouring out liquid in a hole, washing for 3-5 times with a PBST washing liquid, and beating to dry with absorbent paper; adding a substrate color development solution, reacting for 5min at 25 ℃, and adding a stop solution to stop the reaction; the absorbance value of each well was measured at a wavelength of 450nm by setting a microplate reader.
(4) Determination of monoclonal antibody specificity
Antibody specificity refers to the comparison of its ability to bind to a specific antigen with the ability to bind to an analog of that antigen, with the cross-reactivity being commonly used as an evaluation criterion. The smaller the cross-reaction, the higher the specificity of the antibody.
The experiment comprises the steps of serial dilution of clenbuterol, ractopamine, salbutamol, tobuterol, clenbuterol Pan Te, bromobuterol, bromoclobuterol, ma Pente, marbuterol, tertbutyline, clenbuterol, pranoputerol, chlorpanacoline, simatrol, hydroxymethyl clenbuterol, zilpaterol, clenbuterol and the like, respectively performing competition ELISA with monoclonal antibodies, preparing a standard curve, and analyzing to obtain the IC 50 The cross-reactivity was then calculated as follows:
the results show that the cross-reactivity of each analogue is: clenbuterol 100%, ractopamine 1%, salbutamol 1%, sibuterol 1%, tobuterol 1%, clenbuterol Pan Te 1%, bromobuterol 1%, bromochlorobuterol 1%, ma Pente% 1%, marbuterol 1%, terbutaline 1%, clenbuterol 1%, pabuterol 1%, chloropropanerol 1%, clenbuterol 1%, simaroline 1%, hydroxymethyl clenbuterol 1%, zipraline 1%, and clenbuterol 1%. The antibody of the invention has no cross reaction to other targets such as ractopamine, salbutamol, bubuterol, tobuterol, clenbuterol Pan Te, bromobuterol, bromoclobuterol, ma Pente, marbuterol, tertbutyline, clenbuterol, etc., and is only specifically combined with clenbuterol Luo You.
2. Preparation of immunomagnetic beads
The process uses carboxyl magnetic beads with the particle size of 2.8 mu m as a carrier, the tail ends of the carboxyl magnetic beads are provided with reactive carboxyl groups, and after the carboxyl magnetic beads are combined and treated by an activating agent EDC-NHS, the activated magnetic beads can be coupled with a clenbuterol monoclonal antibody to prepare the immune magnetic beads capable of enriching and purifying clenbuterol. The method comprises the following specific steps:
(1) Cleaning: 100. Mu.L of carboxyl magnetic beads (purchased from DYNAL, particle size of 2.8 μm, content of 0.15 eq/g) were taken in a centrifuge tube, washed 2 times with 100. Mu.L of MES solution of pH5.0 containing 0.05% Tween-20, 25mmol/L, and the supernatant was removed after magnetic separation;
(2) Activating: 50mmol/L EDC and NHS solutions are respectively prepared by using MES solutions stored at 4 ℃ and with pH of 5.0 and 25 mmol/L; adding 50 mu L of newly prepared EDC and NHS solution into a centrifuge tube filled with magnetic beads respectively, mixing uniformly by vortex, activating at room temperature for 30min, removing supernatant after magnetic separation, and washing with (1) MES solution for 2-3 times;
(3) Coupling: dissolving the clenbuterol monoclonal antibody into 60 mu L of MES solution with pH of 5.0 and 25mmol/L, regulating the total volume to 100 mu L by using the MES solution, gently adding the MES solution into activated magnetic beads, and coupling for 2 hours at room temperature for 30min or at 4 ℃, wherein the magnetic beads are kept in a uniformly mixed state;
(4) Closing: removing the supernatant after magnetic separation, adding 100 mu L of TRIS solution with pH of 7.4, and reacting for 15min to seal the magnetic beads;
(5) And (3) preserving: removing supernatant after magnetic separation, washing the blocked magnetic beads 3-5 times with 100 μl TRIS solution containing 0.1% -0.3% BSA and 0.1% Tween-20, removing supernatant after magnetic separation, redissolving the magnetic beads in a solution containing 0.1% -0.5% BSA, 0.01% -0.1% Tween-20 and 0.02% NaN 3 Is preserved at 2-8deg.C in TRIS solution (concentration of 10 mg/mL)And storing for standby.
Example 2 detection of the Properties of immunomagnetic beads
0.1mL (concentration of 10 mg/mL) of the clenbuterol-enriched immunomagnetic beads prepared in example 1 was taken in a 10mL centrifuge tube, the beads were rinsed 2 times with 5mL deionized water, and the supernatant was removed after magnetic separation; then adding 1mL of sample to be tested (the solution of the clenbuterol with the concentration of 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL and 90ng/mL is prepared by PBS buffer solution for the standard clenbuterol respectively and is taken as a blank sample to be tested), uniformly mixing, capturing at 25 ℃ for 20min, and uniformly mixing the magnetic beads for 5min; after magnetic separation, the supernatant was removed and the beads were rinsed 2 times with 5mL deionized water to remove interfering impurities. And finally adding 1mL of methanol for eluting, collecting eluent, and detecting the content of clenbuterol in the sample to be detected by using an LC-MS/MS method according to a liquid chromatography tandem mass spectrometry method for detecting the residual amounts of various beta-receptor agonists in the GB/T22286-2008 animal-derived food. The results are shown in Table 1.
TABLE 1 results of detection of the characteristics of immunomagnetic beads
The results show that: (1) eluting the blank sample to be detected by using an eluent, and detecting no clenbuterol; (2) when the standard amount is between 10ng and 50ng, clenbuterol eluted by the eluent is respectively 9.16ng, 17.31ng, 26.73ng, 37.36ng and 46.10ng, and the recovery rate is more than 85 percent; (3) when the standard amount is more than 50ng, the clenbuterol Luo Quxiang on the immunomagnetic bead coupling is saturated, and the recovery rate is lowered instead. The enrichment rate of the immune magnetic beads for purifying the clenbuterol Luo Fuji to the clenbuterol is 50ng/mg (50 ng of clenbuterol can be captured by each milligram of the immune magnetic beads), and the recovery rate of the clenbuterol is more than 85%.
EXAMPLE 3 methods of Using immunomagnetic beads
1. Sample pretreatment
(1) Animal urine sample
A50. Mu.L clear urine sample is taken for direct measurement (e.g., turbidity of the urine sample must be measured by filtration or centrifugation at 3000g at room temperature (20-25 ℃ C./68-77 ℃ F.) for 10min until clear), and samples that are not used should be stored frozen.
(2) Animal tissue (muscle, liver) sample
Weighing 2.0 g+/-0.05 g of homogenized tissue sample into a 50mL polystyrene centrifuge tube;
5mL of deionized water and 1mL of 0.1M hydrochloric acid solution were added, respectively, and vortexed with a vortexing apparatus for 5min, and 3000g was centrifuged at room temperature (20-25 ℃ C./68-77 ℃ F.) for 5min; removing 1mL of supernatant (if suspended matters are avoided on the upper layer), adding 0.5M sodium hydroxide solution until the pH value of the sample solution is 7-8.5, (generally adding 20 mu L of liver, 50 mu L of pork, 30 mu L of beef and mutton, otherwise, adjusting with sodium hydroxide or hydrochloric acid), adding 500 mu L of washing working solution, whirling for 30s with a whirler, and uniformly mixing; 50. Mu.L was taken for analysis.
(3) Animal bovine serum sample
Washing working solution for the centrifuged bovine serum according to the weight ratio of 1:1, diluting the mixture in volume ratio, and uniformly mixing the mixture by using an oscillator; water bath at 80deg.C (176 deg.F) for 5min; 50. Mu.L was taken for analysis.
2. Immunomagnetic bead capture
Taking 0.2ml of clenbuterol immunomagnetic beads in a 10ml centrifuge tube, rinsing the magnetic beads with 5ml of deionized water for 2 times, and separating the washing liquid by a magnetic separation frame each time (standing for 3min on the magnetic separation frame each time to ensure that the magnetic beads are all adsorbed); adding 5ml of the processed sample into the rinsed clenbuterol immunomagnetic beads, uniformly mixing, and uniformly mixing the magnetic beads at 25 ℃ for 20min and 5min; the immunomagnetic beads were separated by a magnetic separation rack, and rinsed with deionized water 2 times 5ml of immunomagnetic beads each time (the same method as immunomagnetic beads).
3. Immunomagnetic bead elution
Washing the immunomagnetic beads with 1ml of methanol, uniformly mixing, standing for 1min, separating the immunomagnetic beads by using a magnetic separation frame, and recovering methanol liquid to obtain the enriched and purified clenbuterol sample, which can be applied to chemical analysis instrument detection (LC-MS/MS, liquid chromatography tandem mass spectrometry), enzyme-linked immunosorbent assay or rapid test of colloidal gold test strips.
Claims (5)
1. An immunomagnetic bead for clenbuterol Luo Fuji purification, characterized in that: the immune magnetic beads are coupled with clenbuterol monoclonal antibodies; the clenbuterol monoclonal antibody is obtained by immunizing a white mouse with a clenbuterol artificial antigen; the clenbuterol artificial antigen is a conjugate of a clenbuterol hapten and a carrier protein, and the clenbuterol hapten is prepared by the following method:
2.21g of 2-amino-1- (4-amino-3, 5-dichlorophenyl) ethanol is taken, 20ml of N, N-dimethylformamide is added for dissolution, 1.86g of carbonyl diimidazole is added, 0.5ml of triethylamine is added, stirring is carried out at room temperature for 2h, 1.72g of succinic acid monoazide hydrochloride is added, stirring is carried out at room temperature for 4h, the reaction is stopped, 80ml of water is added, 1mol/L hydrochloric acid is added for regulating the pH value to 6, 100ml of ethyl acetate is added for three times, the extraction is carried out, the organic phases are combined, the mixture is evaporated to dryness, and a silica gel column is added, wherein the volume ratio of the mixture is 10:1, 2-dichloroethane/methanol elution and separation to obtain 2.76g of carboxyl-clenbuterol hapten product with a yield of 72.8%;
the molecular structural formula of the clenbuterol hapten is as follows:
。
2. the immunomagnetic bead of claim 1, wherein: the clenbuterol artificial antigen is prepared by the following method:
taking a carboxyl-clenbuterol hapten product 14.14 and mg, adding 1ml of DMSO to dissolve and clarify, adding 0.1ml of triethylamine and 11 microlitres of isobutyl chloroformate, fully dissolving and uniformly mixing, and reacting for 2 hours at room temperature to obtain hapten activating solution A; taking 50mg of bovine serum albumin BSA, adding 0.1mol/L carbonate buffer solution CB 4ml with pH of 9.1 for dissolution to obtain solution B, dropwise adding solution A into solution B, reacting at room temperature for 6 hours, stopping the reaction, dialyzing and purifying for 3 days by 0.02mol/L PBS, changing the solution 3 times a day, centrifuging and split charging to obtain clenbuterol-BSA conjugate, namely immunogen;
taking a carboxyl-clenbuterol hapten product 15.4 and mg, adding 1ml of DMF to dissolve and clarify, adding 8.2mg of NHS and 15.5mg of EDC, fully dissolving and uniformly mixing, and reacting for 2 hours at room temperature to obtain hapten activating solution A; taking 100mg of ovalbumin OVA, adding 0.1mol/L carbonate buffer solution CB 9ml with pH of 9.1 for dissolution to obtain solution B, dropwise adding the solution A into the solution B, reacting at room temperature for 6 hours, stopping the reaction, dialyzing and purifying with 0.02mol/L PBS for 3 days, changing the solution 3 times a day, centrifuging and split charging to obtain clenbuterol-OVA conjugate, namely the coating source.
3. A method of preparing the immunomagnetic bead of claim 1, comprising: carboxyl magnetic beads with the particle size of 2.8 mu m are used as carriers, the tail ends of the carboxyl magnetic beads are provided with reactive carboxyl groups, and after the combination treatment of an activating agent EDC-NHS, the activated magnetic beads can be coupled with a clenbuterol monoclonal antibody to prepare the immune magnetic beads capable of enriching and purifying the clenbuterol.
4. The method for preparing immunomagnetic beads according to claim 3, wherein: the method specifically comprises the following steps:
(1) Cleaning: taking 100 mu L of carboxyl magnetic beads in a centrifuge tube, washing with 100 mu L of MES solution containing 0.05% Tween-20 and having pH of 5.0 and 25mmol/L for 2 times, and removing the supernatant after magnetic separation;
(2) Activating: 50mmol/L EDC and NHS solutions are respectively prepared by using MES solutions stored at 4 ℃ and with pH of 5.0 and 25 mmol/L; adding 50 mu L of newly prepared EDC and NHS solution into a centrifuge tube filled with magnetic beads respectively, mixing uniformly by vortex, activating at room temperature for 30min, removing supernatant after magnetic separation, and washing with (1) MES solution for 2-3 times;
(3) Coupling: dissolving the clenbuterol monoclonal antibody into 60 mu L of MES solution with pH of 5.0 and 25mmol/L, regulating the total volume to 100 mu L by using the MES solution, gently adding the MES solution into activated magnetic beads, and coupling for 2 hours at room temperature for 30min or at 4 ℃, wherein the magnetic beads are kept in a uniformly mixed state;
(4) Closing: removing the supernatant after magnetic separation, adding 100 mu L of TRIS solution with pH of 7.4, and reacting for 15min to seal the magnetic beads;
(5) And (3) preserving: removing supernatant after magnetic separation, washing the blocked magnetic beads 3-5 times with 100 μl of TRIS solution containing 0.1% -0.3% bovine serum albumin and 0.1% Tween-20, removing supernatant after magnetic separationRe-dissolving the magnetic beads in a solution containing 0.1-0.5% of bovine serum albumin, 0.01-0.1% of Tween-20 and 0.02% of NaN 3 Is preserved at 2-8deg.C for standby.
5. Use of the immunomagnetic beads according to claim 1 for enriching and purifying clenbuterol.
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