CN100406116C - Method of purifying albuterol and/or clenbuterol and immune affinity chromatographic column - Google Patents
Method of purifying albuterol and/or clenbuterol and immune affinity chromatographic column Download PDFInfo
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- CN100406116C CN100406116C CNB2006100072594A CN200610007259A CN100406116C CN 100406116 C CN100406116 C CN 100406116C CN B2006100072594 A CNB2006100072594 A CN B2006100072594A CN 200610007259 A CN200610007259 A CN 200610007259A CN 100406116 C CN100406116 C CN 100406116C
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- NDAUXUAQIAJITI-UHFFFAOYSA-N albuterol Chemical compound CC(C)(C)NCC(O)C1=CC=C(O)C(CO)=C1 NDAUXUAQIAJITI-UHFFFAOYSA-N 0.000 title claims abstract description 116
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- 229960001117 clenbuterol Drugs 0.000 title claims abstract description 65
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- 238000000034 method Methods 0.000 title claims abstract description 33
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Abstract
The present invention discloses a method for purifying salbutamol and/or clenbuterol, and a special immune affinity chromatography column thereof. The immune affinity chromatography column for purifying salbutamol and/or clenbuterol loads an immune affinity adsorbent which comprises a solidoid carrier and a salbutamol polyclonal antibody or a salbutamol monoclonal antibody coupled with the solidoid carrier. The salbutamol polyclonal antibody or the salbutamol monoclonal antibody is obtained by using a salbutamol hapten and a coupler of carrier protein as immunogens. The purification method combines with a chromatography to detect the contents of salbutamol and clenbuterol in high efficiency, overcomes the defects of fewer information contents, low quantitative accuracy, low selectivity of a rationalization method and the like of a pure immunoassay technique for directly measuring a sample, and embodies the complementarity of an immunology technique and a conventional rationalization technique in an analysis mechanism.
Description
Technical Field
The invention relates to a method for purifying salbutamol and/or clenbuterol and a special kit thereof.
Background
With the development of life science, people have more and more concentrated interests in substances and changes thereof in organisms, and the analysis of biological samples becomes a necessary means for exploring and finding the mysteries of life. Due to the complex composition of biological samples, the concentration of the analyte is low, and most samples are very small, which puts higher demands on the selectivity and sensitivity of the analysis method. Immunoaffinity chromatography (IAC) is an analytical method that combines immune responses with chromatographic methods. Its high selectivity and high affinity clearly simplify the analysis process. In veterinary drug residue analysis, the simplest and most effective application mode of the IAC is used as a sample purification means of a physicochemical determination technology (such as HPLC, GC/MS), and the combined method can complement the immunological technology and the physicochemical technology in the aspects of selectivity, separation capability, speed and sensitivity and avoid the defects of direct determination of samples by an immunoassay method (such as ELISA, RIA). At present, the method is widely applied to the analysis of antibodies, hormones, polypeptides, enzymes, recombinant proteins, receptor viruses and small molecule compounds.
Salbutamol (SAL) and Clenbuterol (CL) belong to phenylethanolamine beta-agonists, are used in medical clinic, are mainly used for dilating bronchus and increasing lung ventilation, can treat bronchial asthma, obstructive pulmonary inflammation, smooth muscle spasm, shock and other symptoms, are clinically used as birth canal relaxants of cattle and horses in veterinary clinics, such as SAL for relieving asthma, and CL is used as uterine relaxant and the like, and is used as a feed additive in recent years, and is more and more illegally used in animal husbandry.
The residual detection method of salbutamol and clenbuterol is established in many countries and is prohibited from being used in animal production in most countries. Some countries implement maximum residual quantity (MRLs) limits, such as 0.5ng/g for MRLs specified in the UK and 1ng/g for MRLs specified in the Netherlands. The clear regulations in China prohibit the use of CL, SAL and preparations thereof in the feeding process of food animals.
At present, methods for detecting salbutamol and clenbuterol mainly comprise a high performance liquid chromatography (HPLC-UV), a gas-mass online method (GC-UV), a liquid-mass online method (LC-MS-MS), an enzyme-linked immunosorbent assay (ELISA) and the like. The pretreatment of the methods utilizes liquid-liquid distribution, and the conventional SPE column purification and separation have the defects of complicated treatment process, poor purification effect, much waste of organic solvent, long required time and the like in different degrees.
Disclosure of Invention
The invention aims to provide a method for purifying salbutamol and/or clenbuterol and a special immunoaffinity chromatographic column thereof.
The immunoaffinity adsorbent for purifying salbutamol and/or clenbuterol provided by the invention consists of a solid phase carrier and a salbutamol polyclonal antibody or a salbutamol monoclonal antibody coupled with the solid phase carrier; the salbutamol polyclonal antibody or the salbutamol monoclonal antibody is obtained by taking a conjugate of a salbutamol hapten and a carrier protein as an immunogen; the salbutamol hapten is prepared by completely dissolving salbutamol in methanol, then evaporating the methanol to dryness to obtain an oily substance, dissolving the oily substance in absolute ethyl alcohol, adding succinic anhydride while oscillating, and oscillating at room temperature for 3 hours to react so as to obtain a white suspension, wherein the white suspension is the hapten.
The carrier protein can be bovine serum albumin, ovalbumin and other common carrier proteins.
Salbutamol is a small molecular substance, has immunoreactivity and no immunogenicity, cannot induce an organism to generate immune response, and has immunogenicity only after being coupled with a macromolecular carrier protein. According to the invention, Salbutamol (SAL) is acylated by succinic anhydride, and a spacer arm containing 4 carbons is grafted to form a hapten, so that the characteristic structure of a salbutamol hapten determinant is highlighted, and the preparation of an antibody with high specificity for a salbutamol antigen is facilitated. Then coupling the salbutamol hapten with carrier protein by adopting a mixed anhydride method to obtain the immunogen. The combination ratio of hapten to carrier protein is too low or too high, which is unfavorable for immunity, and the combination molar ratio of hapten to Ovalbumin (OVA) and Bovine Serum Albumin (BSA) is 12: 1 and 15: 1 respectively.
The salbutamol mouse monoclonal antibody is preferably a monoclonal antibody secreted by a monoclonal hybridoma cell strain A-2-1CGMCC No.1607 with cross reaction to both salbutamol and clenbuterol.
The monoclonal hybridoma cell strain A-2-1CGMCC No.1607 with cross reaction to salbutamol and clenbuterol has been preserved in China general microbiological culture Collection center (CGMCC for short) in 2006, 2 months and 9 days.
The solid phase carrier can be cellulose, sephadex, polyacrylamide gel, porous glass, Sepharose, polyacrylamide-Sepharose and the like, and is preferably Sepharose 4B.
The immunoaffinity adsorbent can be loaded into a column to prepare an immunoaffinity chromatographic column, and the immunoaffinity chromatographic column also belongs to the protection scope of the invention.
The kit containing the immunoaffinity adsorbent or the immunoaffinity chromatography column also belongs to the protection scope of the invention.
The kit also comprises an eluent I and an eluent II; the eluent I is methanol; the eluent II is a mixed solution with the volume ratio of ethanol, deionized water and acetic acid buffer solution with pH4.0 being 80: 15: 5.
The kit also comprises a washing solution and a preservation solution; the washing solution is phosphate buffer solution with the pH value of 7.4 and the concentration of 0.01mol/L, and the phosphate buffer solution with the pH value of 7.4 and the concentration of 0.01mol/L contains 0.27g of monopotassium phosphate, 2.86g of 12-hydrated disodium hydrogen phosphate, 0.2g of potassium chloride and 8.8g of sodium chloride in 1L of water; the preservation solution is 1L solution containing potassium dihydrogen phosphate 0.27g, disodium hydrogen phosphate 12 hydrate 2.86g, potassium chloride 0.2g, sodium chloride 8.8g, and NaN30.2g，pH7.4。
The immunoaffinity adsorbent is based on immunoreaction and chromatographic reaction, is suitable for purifying salbutamol and clenbuterol from biological samples (such as animal urine), and is convenient for residue analysis. In the immunoaffinity adsorbent, the coupling rates of a salbutamol mouse monoclonal antibody and a salbutamol rabbit polyclonal antibody and Sepharose 4B activated by cyanogen bromide are 97.2 +/-1.5 percent and 96.7 +/-1.5 percent respectively, the monoclonal antibody secreted by the salbutamol polyclonal antibody or a monoclonal hybridoma cell strain A-2-1CGMCC No.1607 which has cross reaction to both salbutamol and clenbuterol has very high cross reaction to clenbuterol, the immunoaffinity chromatographic column coupled with the monoclonal antibody secreted by the monoclonal hybridoma cell strain A-2-1CGMCC No.1607 which has cross reaction to both salbutamol and clenbuterol has the capacities of 400ng/mL and 419ng/mL respectively, the absolute column capacities of 40ng/mgIgG and 42ng/mg IgG respectively, the column capacity is reduced to about 55 percent of the total column capacity after 10 times of continuous use, the shelf life was 1 year.
The method for purifying the salbutamol and/or the clenbuterol provided by the invention comprises the following steps:
1) pretreatment of a sample:
animal urine samples: diluting the sample by 10 times with a washing solution; animal tissue: adding 0.01M hydrochloric acid solution into the homogenate sample, whirling for 3 minutes, centrifuging for 10 minutes at 3000g, filtering the obtained supernatant with a 0.2um filter membrane, and diluting by 10 times with washing liquid to obtain a sample solution;
2) passing the sample solution obtained in the step 1) through an immunoaffinity chromatography column, washing with deionized water, and eluting with 3ml of eluent I and 1ml of eluent II simultaneously to obtain purified salbutamol and/or clenbuterol solution.
The immunoaffinity adsorbent has high selectivity, greatly simplifies the pretreatment process of samples, is particularly suitable for the pretreatment of salbutamol and/or clenbuterol in animal urine samples, liver samples and muscle samples, and improves the analysis quality. The high selectivity of the immunoaffinity adsorbent ensures that the detection limit of the salbutamol and/or clenbuterol analysis method mainly depends on the sampling amount, which is difficult to achieve by a simple physicochemical means; the immunoaffinity adsorbent has strong retention and concentration capacity to the components to be detected, and the retention capacity of the immunoaffinity adsorbent to the components is hardly influenced by the volume of a sample or the concentration of the components under the condition of actually measuring the sample as long as the sample adding amount does not exceed the capacity of a column. The method of the invention can provide qualitative information while purifying the components. The method has the advantages of aqueous phase operation, simple operation, good purification effect, repeated use of the immunoaffinity chromatographic column, saving of a large amount of organic solvents, and reduction of analysis cost and environmental pollution. The purification method provided by the invention combines chromatography to efficiently detect the contents of salbutamol and clenbuterol, overcomes the defects of too small information amount, poor quantitative accuracy, low selectivity of a physicochemical method and the like in a sample directly measured by a simple immunoassay technology, and embodies the complementarity of an immunological technology and a conventional physicochemical technology in an analysis mechanism.
Drawings
FIG. 1 is a chromatogram of salbutamol and clenbuterol standard
FIG. 2 is a chromatogram of the effect of salbutamol and clenbuterol in pig urine on sample purification
Detailed Description
The experimental procedures in the following examples are conventional unless otherwise specified.
EXAMPLE 1 preparation of an immunochromatographic column for purifying salbutamol and/or clenbuterol
1. Preparation of salbutamol polyclonal antiserum
Synthesis of salbutamol hapten: dissolving salbutamol in methanol completely, evaporating the methanol to dryness on a rotary evaporator to obtain oily matter, oscillating and dissolving the oily matter in 20ml of absolute ethanol, adding succinic anhydride with the same mole number as the salbutamol while oscillating, and oscillating and reacting at room temperature for 3 hours to obtain white suspension, namely the salbutamol hapten.
Preparation of immunogen: coupling salbutamol hapten with protein carrier Bovine Serum Albumin (BSA) or Ovalbumin (OVA) by a mixed anhydride method to prepare a BSA-SAL or OVA-SAL conjugate, namely immunogen.
Animal immunization: new Zealand white rabbit is used as immune animal, and the immune dose is 1 mg/ml-1BSA-SAL conjugates. New Zealand rabbits are bred for several weeks and then are inoculated for immunization, the first immunization is emulsified by 1ml of complete Freund adjuvant, and the boosting immunization is emulsified by 1ml of incomplete Freund adjuvant. The immunization is carried out for 8 times at intervals of 2 weeks, the last immunization is directly carried out by ear vein injection without adding adjuvant, blood sampling detection is carried out after 7-10d of the last immunization, carotid bleeding is carried out after the serum titer is determined, and serum is collected. The obtained salbutamol antiserum is detected by competitive ELISA, and the result shows that the salbutamol antiserum has high cross-reactivity to clenbuterol.
2. Purification of polyclonal antiserum:
the antiserum was purified by Saturated Ammonium Sulfate (SAS) and DEME cellulose ion exchange chromatography. The method comprises the following specific steps:
(1) SAS salting out: 1) 50% saturation salting out: 5ml of the rabbit antiserum prepared above is taken, added with 0.01mol/L PBS with equal amount of pH7.4 (1L solution contains 0.27g of monopotassium phosphate, 2.86g of 12-hydrated disodium hydrogen phosphate, 0.2g of potassium chloride and 8.8g of sodium chloride), mixed evenly, then gradually dropwise added with equal volume of saturated ammonium sulfate (pH7.4) solution, stirred while adding, placed at room temperature for 30min, centrifuged for 30min at 3000g, and supernatant liquid is discarded to leave precipitate. 2) 33% saturation salting out: 5ml of 0.01mol/L PBS (1L solution contains 0.27g of monopotassium phosphate, 2.86g of 12-hydrated disodium hydrogen phosphate, 0.2g of potassium chloride and 8.8g of sodium chloride) is respectively added into the precipitate obtained in the step 1) to dissolve the precipitate, saturated ammonium sulfate solution is added to reach 33% saturation degree, the mixture is stirred while adding, the mixture is placed at room temperature for 30min, and supernatant liquid is discarded to leave the precipitate. The operation was repeated 2 times. 3) Desalting: dissolving the precipitate obtained in step 2) in 0.01mol/L PBS (pH7.4 (1L solution containing potassium dihydrogen phosphate 0.27g, disodium hydrogen phosphate 12 hydrate 2.86g, potassium chloride 0.2g, and sodium chloride 8.8g), placing into a permeation bag, and suspending in 0.01mol/L PBS (pH7.4 (1L solution containing diphosphoric acid)Potassium hydrogene 0.27g, disodium hydrogenphosphate 12 hydrate 2.86g, potassium chloride 0.2g, sodium chloride 8.8g) in a beaker, and left at 4 ℃ with changing the solution 3-4 times a day with 1% BaCl2And detecting until the dialysate has no sulfate ions. 4) After dialysis, 3000g is centrifuged for 5min, and the supernatant is taken for DEME-cellulose ion exchange chromatography.
(2) DEME-cellulose ion exchange chromatography: 1) treatment of DE-52 cellulose: weighing 2g of DE-52 cellulose powder, placing the powder in a beaker filled with double distilled water, fully stirring, standing, removing redundant solution, then adding 0.5mol/LNaOH solution, uniformly stirring, carrying out suction filtration on a Buchner funnel after 1h, and fully washing the powder to be neutral by using the double distilled water. The resulting solution was treated with 0.5mol/L HCl solution in the same manner. Finally, the solution is treated once by 0.5mol/L NaOH solution and is fully washed to be neutral. 2) Balancing: soaking the DE-52 cellulose treated in the step 1) in PBS (0.01 mol/L, pH7.4, containing 0.27g of potassium dihydrogen phosphate, 2.86g of 12-hydrated disodium hydrogen phosphate, 0.2g of potassium chloride and 8.8g of sodium chloride in 1L of solution), washing thoroughly, standing, removing the excessive solution, repeating the above steps for 2-3 times until the pH of the supernatant reaches 7.4. 3) Column assembling: the equilibrated DE-52 cellulose was continuously added to the column with a dropper, continuously eluted with an eluent (1L of the solution contained 0.27g of potassium dihydrogen phosphate, 2.86g of 12-hydrated disodium hydrogen phosphate, 0.2g of potassium chloride, 8.8g of sodium chloride, pH7.4) and sufficiently eluted until the pH of the effluent was the same as that of the eluent. 4) Sample adding: the antibody obtained by SAS salting out in step (1) was diluted with 0.01mol/L of phosphate buffer solution (1L of solution containing 0.27g of potassium dihydrogen phosphate, 2.86g of 12-hydrated disodium hydrogen phosphate, 0.2g of potassium chloride, and 8.8g of sodium chloride)) having a pH of 7.4, and then slowly added along the wall of the column, the outlet of the column was opened, and the IgG diluent was allowed to flow into the column bed, and the column wall was washed with phosphate buffer solution. 5) And (3) eluting and collecting: eluent (1L solution contains 0.27g of potassium dihydrogen phosphate, 2.86g of 12-hydrated disodium hydrogen phosphate, 0.2g of potassium chloride, 8.8g of sodium chloride and pH7.4) is added, 2ml of eluent is collected in each tube, and IgG elution is detected by using 20% sulfosalicylic acid while collection. 6) The 0D values of the IgG solution at 280nm and 260nm were determined, and the result showed that the concentration of the purified rabbit polyclonal antibody was 27 mg/mL.
3. Preparation of mouse monoclonal antibody of salbutamol
Preparation of immunogen: coupling salbutamol hapten and protein carrier Bovine Serum Albumin (BSA) by adopting a mixed anhydride method to prepare BSA-SAL, namely immunogen.
Animal immunization: BALA/C mice are used as immune animals, conjugate BSA-SAL of the salbutamol hapten and a protein carrier is used as immunogen, the immune dose is 100 mu g/mouse, and the same volume of complete Freund's adjuvant is added for emulsification to carry out primary immunization. Two weeks later, the same amount of the antigen was taken and added with Freund's incomplete adjuvant, emulsified, and subjected to the second immunization. Two weeks apart, a third immunization was performed in the same manner as the second immunization. Two weeks apart, a fourth immunization was performed, in the same manner as the second immunization. BALB/C mice were boosted once more 3 days before fusion, and 100. mu.g of antigen was intraperitoneally injected without adjuvant. Before fusion, the eyeball is picked and blood is bled to obtain positive serum, then the mouse is pulled to neck and dislocated to be killed, and the spleen is taken out.
Cell fusion: splenocytes were cell fused with SP2/0 myeloma cells at a ratio of 5: 1.
Hybridoma cell cloning: and (3) screening the hybridoma cells by adopting a limiting dilution method until completely homogeneous monoclonal antibodies with cross reactivity to both salbutamol and clenbuterol and stable monoclonal hybridoma cell strains, namely the monoclonal hybridoma cell strain A-2-1CGMCC No.1607 with cross reactivity to both salbutamol and clenbuterol, are obtained. A monoclonal hybridoma cell strain A-2-1CGMCC No.1607 with cross-reactivity to both salbutamol and clenbuterol is preserved in China general microbiological culture Collection center (CGMCC for short) in 2006, 2 and 9.
Preservation of monoclonal hybridoma cells: storing at liquid nitrogen-20 deg.C, and rapidly thawing in 37 deg.C water bath.
Mass growth and purification of monoclonal antibody: injecting sterilized paraffin oil into BALB/c mouse abdominal cavity by in vivo induction method, and injecting parasardine into abdominal cavity 7-14 days laterMonoclonal hybridoma cell strain A-2-1CGMCC No. 16075X 10 with both aminol and clenbuterol having cross reactivity5-106Ascites were collected 7-10 days later.
4. Purification of monoclonal antibodies:
the monoclonal antibody is purified by an octanoic acid-saturated ammonium sulfate method. The method comprises the following specific steps:
5mL of ascites obtained by intraperitoneal injection of a monoclonal hybridoma cell strain A-2-1CGMCC No.1607 which has cross reactivity to salbutamol and clenbuterol by the method is added with 10mL of NaAc-HAc buffer solution with 0.06mol/L and pH of 4.0 and stirred uniformly, and the pH is adjusted to 7.2 by using 1mol/L NaOH solution. While stirring at room temperature, 165. mu.L of octanoic acid was added, and the mixture was stirred for 30 min. Centrifuging at 4 deg.C for 30min at 6000g, collecting supernatant, and adjusting pH to 7.2 with 1mol/L NaOH solution. Then, 15mL of a saturated ammonium sulfate solution was added under stirring at 4 ℃ so that the final mass% of ammonium sulfate was 50%, and after stirring for 30min, the mixture was centrifuged at 6000g at 4 ℃ for 30min, the supernatant was discarded, and the precipitate was suspended in 5.5mL of 0.01M PBS (1L of which contained 0.27g of potassium dihydrogenphosphate, 2.86g of disodium hydrogenphosphate 12 hydrate, 0.2g of potassium chloride, and 8.8g of sodium chloride) and pH7.2. The precipitated suspension was added to 4.5mL of saturated ammonium sulfate solution with stirring, and this was done at 4 ℃. At this time, the final concentration of ammonium sulfate was 45%, and after stirring for 30min, the mixture was centrifuged at 6000g at 4 ℃ for 30min, and the supernatant was discarded, and the precipitate was suspended in 1mL of 0.01M PBS (pH7.2PBS). And (3) putting the precipitate suspension into a dialysis bag, dialyzing with 0.01M PBS (phosphate buffer solution) with the pH value of 7.22M, and changing the solution once after 4-6 hours and 2-3 times. Measuring OD values of IgG solution at 280nm and 260nm by using an ultraviolet spectrophotometer to obtain monoclonal antibody secreted by monoclonal hybridoma cell strain A-2-1CGMCC No.1607 with cross reactivity to salbutamol and clenbuterol, and storing the purified IgG in a refrigerator at-20 ℃.
5. Preparation of immunochromatographic columns (IAC)
Preparation of a matrix: collecting cyanogen bromide activated Sepharose 4B dry powder, and placing in a container with 1.0mmol-1G of HCl3The funnel is expanded.
Preparation of antibody IgG: with 0.1mol/L NaHCO3The solution is prepared by diluting a purified rabbit polyclonal antibody or a monoclonal antibody secreted by a monoclonal hybridoma cell strain A-2-1CGMCC No.1607 with cross reactivity to salbutamol and clenbuterol to a proper concentration and adjusting the pH value of the solution to 8.4.
Coupling reaction: the expanded sol was treated with 0.1mol/L NaHCO3After the solution is balanced, the rabbit polyclonal antibody or the salbutamol monoclonal antibody solution is transferred into the rabbit polyclonal antibody or the salbutamol monoclonal antibody solution, mixed and slowly stirred for 20-24 hours at 4 ℃. The detection result of the coupling rate shows that the coupling rate of the rabbit polyclonal antibody and the Sepharose 4B activated by cyanogen bromide is 96.7 +/-1.5 percent, and the coupling rate of the salbutamol monoclonal antibody secreted by a monoclonal hybridoma cell strain A-2-1CGMCC No.1607 and the Sepharose 4B activated by the cyanogen bromide is 97.2 +/-1.5 percent, wherein the monoclonal antibody has cross reactivity to both salbutamol and clenbuterol.
Blocking of the activation site: transferring the coupled gel into Tris-HCl buffer solution with pH of 8.0 at 0.1mol/L, mixing, and slowly stirring at 4 deg.C for 2hr to block uncoupled activated site.
Washing: the gel was washed 3 times with 5 volumes of 0.1mol/L, pH4.0 acetate buffer and 0.1mol/L, pH8.0Tris-HCl buffer. After equilibration with 0.01mol/L PBS (1L solution containing 0.27g of potassium dihydrogen phosphate, 2.86g of 12-hydrated disodium hydrogen phosphate, 0.2g of potassium chloride, 8.8g of sodium chloride), pH7.4, the drained gel was transferred to a 0.01mol/L PBS containing 0.1% NaN at pH7.43Phosphate buffer (1L solution containing potassium dihydrogen phosphate 0.27g, disodium hydrogen phosphate 12 hydrate 2.86g, potassium chloride 0.2g, sodium chloride 8.8g, NaN31g) And storing at medium and 4 ℃ for later use.
Column assembling: transferring the immunoadsorbent coupled with salbutamol polyclonal antibody or salbutamol monoclonal antibody into the G-containing substrate3An immunochromatographic column (IAC column) coupled with a salbutamol polyclonal antibody or a salbutamol monoclonal antibody is prepared in a chromatographic column of a filter plate.
6. Determination of IAC column Capacity
The immunochromatographic column coupled with the salbutamol polyclonal antibody or the salbutamol monoclonal antibody prepared in step 5 is washed with 20ml of 0.01mol/L PBS (1L of solution contains 0.27g of potassium dihydrogen phosphate, 2.86g of 12-hydrated disodium hydrogen phosphate, 0.2g of potassium chloride and 8.8g of sodium chloride) with pH7.4, bubbles in the column are removed by gently shaking the IAC column up and down, and then the column is equilibrated with 10ml of 0.01mol/L PBS (1L of solution contains 0.27g of potassium dihydrogen phosphate, 2.86g of 12-hydrated disodium hydrogen phosphate, 0.2g of potassium chloride and 8.8g of sodium chloride) with pH 7.4. Will contain 100 ng/ml-1Salbutamol or clenbuterol in PBS (1L solution containing potassium dihydrogen phosphate 0.27g, disodium hydrogen phosphate 12 hydrate 2.86g, potassium chloride 0.2g, sodium chloride 8.8g) was continuously applied to the IAC column and allowed to flow out under natural gravity. When the column is saturated (salbutamol or clenbuterol concentration in the effluent is the same as the sample loading solution), 20ml of deionized water is used for washing, and interfering impurities in the extracting solution are removed. Finally, the salbutamol and the clenbuterol are eluted by a mixed solution of 3ml of methanol and 1ml of analytically pure ethanol, deionized water and acetic acid buffer solution with the pH value of 4.0 according to the volume ratio of 80: 15: 5, flow out under the natural gravity, are collected and dried, and are subjected to GC/MS determination. The dynamic column capacity and the absolute column capacity were calculated. Dynamic column capacity (dynamic column capacity) refers to the maximum absorption of the test substance per ml of immunoadsorbent (or bed volume). Absolute column capacity (specific column capacity) refers to the maximum binding capacity per mg of immobilized antibody to the test substance. The results show that the immunoaffinity chromatography column coupled with the salbutamol mouse monoclonal antibody has the capacities of 400ng/mL and 419ng/mL for the salbutamol and the clenbuterol dynamic column respectively, the absolute column capacities are 40ng/mg IgG and 42ng/mg IgG respectively,
example 2 preparation of a kit for immunochromatography column coupled with a salbutamol rabbit polyclonal antibody or a salbutamol monoclonal antibody secreted by a monoclonal hybridoma cell line A-2-1CGMCC No.1607 having cross-reactivity to both salbutamol and clenbuterol, and purification effects thereof on salbutamol and/or clenbuterol
1. Preparation of kit for purifying salbutamol and/or clenbuterol
The kit mainly comprises a kit body, an immunochromatographic column (IAC column), salbutamol, clenbuterol standard solution, washing liquid, eluent I, eluent II, preservation liquid and a sponge bracket, wherein holes and grooves are formed in the sponge bracket. A reagent bottle filled with salbutamol, clenbuterol standard solution, washing solution, eluent I, eluent II and preservation solution is arranged in the groove of the sponge bracket, and an IAC column is arranged in the hole of the sponge bracket. Wherein the immunochromatographic column is the immunochromatographic column coupled with the salbutamol polyclonal antibody or the salbutamol monoclonal antibody secreted by the monoclonal hybridoma cell strain A-2-1CGMCC No.1607 which has cross reactivity to both salbutamol and clenbuterol and is prepared in the embodiment 1.
The washing solution is phosphate buffer solution (0.01M, pH7.4), and the preparation method comprises the following steps: 1L of the solution contained potassium dihydrogen phosphate 0.27g, disodium hydrogen phosphate 12 hydrate 2.86g, potassium chloride 0.2g, and sodium chloride 8.8 g.
Eluent I is methanol of chromatographic grade.
The eluent II is a mixed solution of analytically pure ethanol, deionized water and acetic acid buffer solution with pH4.0 in a volume ratio of 80: 15: 5.
The preservation solution contains potassium dihydrogen phosphate 0.27g, disodium hydrogen phosphate 12 hydrate 2.86g, potassium chloride 0.2g, sodium chloride 8.8g, and NaN30.2g，pH7.4。
The kit of the immunochromatographic column coupled with the salbutamol polyclonal antibody or the salbutamol monoclonal antibody secreted by the monoclonal hybridoma cell strain A-2-1CGMCC No.1607 is placed at 4 ℃ for 12 months of validity.
2. Experiment on purifying effect of salbutamol and clenbuterol
The IAC purification principle is that salbutamol polyclonal antibody or salbutamol monoclonal antibody secreted by a monoclonal hybridoma cell strain A-2-1CGMCC No.1607 with cross reactivity to both salbutamol and clenbuterol is coupled with inert matrix to prepare immunoadsorbent, and the immunoadsorbent is packed into a column. When the mixture containing the salbutamol and/or clenbuterol to be detected flows through the IAC column, the immobilized antibody selectively binds the salbutamol and/or clenbuterol, other unidentified sample impurities flow out of the IAC column without being blocked, after washing, the antigen-antibody complex is dissociated and eluted, and the salbutamol and/or clenbuterol is purified or separated. The IAC column can be reused after regeneration treatment.
Treatment of the detection sample: taking 2g (mL) of a non-contacted salbutamol and clenbuterol pig urine sample, respectively adding a salbutamol standard substance and a clenbuterol standard substance to ensure that the final concentrations of the salbutamol and the clenbuterol are both 10 mu g/mL, and diluting by 10 times through the washing liquid in the kit to obtain a urine sample solution;
taking a sample of a pig muscle and pig liver homogenate which is not contacted with salbutamol and clenbuterol, respectively adding a salbutamol standard substance and a clenbuterol standard substance to ensure that the final concentrations of the salbutamol and the clenbuterol are both 10 mu g/kg, respectively adding 10ml of 0.01M hydrochloric acid solution, whirling for 3 minutes, centrifuging for 10 minutes at 3000g, taking supernate, and filtering with a 0.20 mu M filter membrane to obtain a filtrate, namely a sample solution.
An immunochromatography column coupled with a salbutamol polyclonal antibody or a salbutamol monoclonal antibody secreted by a monoclonal hybridoma cell strain A-2-1CGMCC No.1607 is balanced to room temperature, and is washed by 20ml of washing liquid in the kit, and then the sample solutions pass through the column and are washed by 20ml of deionized water, so that impurities which are not specifically adsorbed are removed. Eluting with 3ml of eluent I in the above kit and 1ml of eluent II in the above kit, and eluting at 45 deg.C with N2Blowing the mixture under air flow, and then carrying out GC/MS analysis to determine the content of the salbutamol and the clenbuterol by taking the standard product (10 mu g/kg) of the salbutamol and the clenbuterol as a control. The IAC column was stored in a refrigerator at 4 ℃ in equilibrium with 20ml of the stock solution for future use. The determination result shows that the IAC is used for purifying the sample, does not interfere the chromatographic peak of the medicament, can be completely separated, and shows that the prepared IAC has extremely small nonspecific adsorption, wherein the immune chromatographic column coupled with the salbutamol monoclonal antibody secreted by the hybridoma cell strain A-2-1CGMCC No.1607 is added with salbutamol and clenbuterol with the concentration of 10 mu g/kgThe purification effect of the standard pig urine sample is shown in fig. 1 and 2.
Claims (2)
1. A kit for purifying salbutamol and/or clenbuterol comprises an immunoaffinity chromatographic column loaded with an immunoaffinity adsorbent, an eluent I, an eluent II, a washing solution and a preservation solution; wherein,
the immunoaffinity adsorbent consists of a solid phase carrier and a salbutamol monoclonal antibody coupled with the solid phase carrier; the solid phase carrier is Sepharose 4B activated by cyanogen bromide, and the monoclonal antibody is secreted by a monoclonal hybridoma cell strain A-2-1CGMCC No. 1607;
the eluent I is methanol;
the eluent II is a mixed solution of ethanol, water and acetic acid buffer solution with the pH value of 4.0 according to the volume portion ratio of 80: 15: 5;
the washing solution is a phosphate buffer solution with the pH value of 7.4 and the mol/L of 0.01, and the phosphate buffer solution is an aqueous solution containing 0.27g of monopotassium phosphate, 2.86g of 12-hydrated disodium phosphate, 0.2g of potassium chloride and 8.8g of sodium chloride in 1L;
the preservation solution contains potassium dihydrogen phosphate 0.27g, disodium hydrogen phosphate 12 hydrate 2.86g, potassium chloride 0.2g, sodium chloride 8.8g, and NaN in 1L30.2g of aqueous solution, pH 7.4.
2. A method for purifying salbutamol and/or clenbuterol using the kit for purifying salbutamol and/or clenbuterol according to claim 1, comprising the steps of:
1) pretreatment of a sample:
animal urine samples: diluting the sample by 10 times with a washing solution; animal tissue: adding 0.01M hydrochloric acid solution into homogenate of an animal tissue sample, whirling for 3 minutes, centrifuging for 10 minutes at 3000g, filtering the obtained supernatant through a filter membrane of 0.2um, and diluting by 10 times with washing liquid to obtain a sample solution;
2) passing the sample solution obtained in the step 1) through an immunoaffinity chromatography column, washing with deionized water, and eluting with 3ml of eluent I and 1ml of eluent II simultaneously to obtain purified salbutamol and/or clenbuterol solution.
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