CN100406115C - Method of purifying alpha corn gibberellol and its metabolic substance and immune affinity chromatographic column - Google Patents
Method of purifying alpha corn gibberellol and its metabolic substance and immune affinity chromatographic column Download PDFInfo
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- CN100406115C CN100406115C CNB2006100072397A CN200610007239A CN100406115C CN 100406115 C CN100406115 C CN 100406115C CN B2006100072397 A CNB2006100072397 A CN B2006100072397A CN 200610007239 A CN200610007239 A CN 200610007239A CN 100406115 C CN100406115 C CN 100406115C
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- zearalanol
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Abstract
The present invention discloses a method for purifying alpha-zeranol and metabolites thereof, and an immune affinity chromatography column. The special immune affinity chromatography column for purifying alpha-zeranol and/or beta-zeranol and/or zearanol and/or alpha-zearalenol loads with an immune affinity adsorbent which comprises a solidoid carrier and an alpha-zeranol monoclonal antibody coupled with the solidoid carrier, wherein the alpha-zeranol monoclonal antibody is obtained by using alpha-zeranol and carrier protein as couplers. The purification method combines with a chromatography to detect the contents of alpha-zeranol, beta-zeranol, zearanol and alpha-zearalenol in high efficiency, and 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.
Description
Technical Field
The invention relates to a method for purifying alpha-zearalanol and metabolites thereof and a special immunoaffinity chromatographic column thereof, in particular to a method for purifying alpha-zearalanol and/or beta-zearalanol and/or zearalenone and/or alpha-zearalenol and a special immunoaffinity chromatographic column 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 components of the biological sample, the concentration of the substance to be detected is low, and most of samples are few, which puts higher requirements on the selectivity and the sensitivity of the analysis method. Immunoaffinity chromatography (IAC) is used for veterinary residue analysis, and its high selectivity and high affinity undoubtedly simplify the decontamination process. The combination of GC and HPLC can make immunological technique and physicochemical technique complementary in selectivity, separating ability, speed and sensitivity, and avoid many defects of immunoassay (such as ELISA, RIA) for directly determining samples. At present, the method is widely applied to the analysis of antibodies, hormones, polypeptides, enzymes, recombinant proteins, receptor viruses and small molecule compounds.
Alpha-corn gibberellin, beta-corn gibberellin, corn gibberellin and alpha-corn gibberellin alcohol belong to triptolide non-steroidal hormones, alpha-corn gibberellin alcohol is used as a growth promoter applied to ruminants and has the function of promoting protein synthesis, and beta-corn gibberellin and corn gibberellin are metabolites of the alpha-corn gibberellin alcohol. Alpha-zearalenol is a metabolite of zearalenone. Alpha-zearalol, beta-zearalol, zearalenone and alpha-zearalenol have weak estrogenic effects, and its residues in animal tissues can affect human health, cause sexual dysfunction in humans in mild cases, affect normal development of secondary sex characteristics in severe cases, and may be carcinogenic under external conditions. Meanwhile, after the zearalanol is discharged out of the animal body, secondary pollution and environmental pollution can be caused by drinking water and food, and the European Union in 1998 definitely prohibits the application of hormone medicines to livestock production. China also banned its use for all food animals in 2002, and it was not detected in all food tissues.
At present, methods for detecting alpha-zearalanol, beta-zearalanol, zearalenone and alpha-zearalenol mainly comprise High Performance Liquid Chromatography (HPLC), gas-mass spectrometry (GC-MS), liquid-mass spectrometry (LC-MS), enzyme-linked immunosorbent assay (ELISA) and the like. The pretreatment of the methods mostly 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. The immunoaffinity technology is a new technology applied in the analysis field in 90 years, but the simultaneous purification of alpha-Zearalanol (ZER), beta-Zearalanol (TAL), Zearalenone (ZEN) and alpha-zearalenol (alpha-ZOL) in a matrix by using an immunoaffinity column is not reported, and the immunoaffinity column is not sold in a commercial IAC column.
Disclosure of Invention
The invention aims to provide a method for purifying alpha-zearalanol and/or beta-zearalanol and/or zearalenone and/or alpha-zearalenol and a special immunoaffinity adsorbent thereof.
The immunoaffinity adsorbent for purifying alpha-zearalol and/or beta-zearalol and/or zearalenone and/or alpha-zearalenol, which is provided by the invention, consists of a solid phase carrier and an alpha-zearalenol monoclonal antibody which is coupled with the solid phase carrier and has cross reaction on the beta-zearalol, the zearalenone and the alpha-zearalenol; the alpha-zearalanol monoclonal antibody which has cross reaction on beta-zearalanol, zearalenone and alpha-zearalenol is obtained by taking a conjugate of alpha-zearalanol hapten and carrier protein as an immunogen; the alpha-corn gibberellin hapten is ZER-7-hemisuccinate obtained by reacting alpha-corn gibberellin with succinic anhydride, and the Zer-7-hemisuccinate is the alpha-corn gibberellin hapten.
The carrier protein can be bovine serum albumin, ovalbumin and other common carrier proteins.
Alpha-zearalanol is a small molecular substance, has immunoreactivity, has no immunogenicity, cannot induce an organism to generate immune response, and has immunogenicity only after being coupled with a macromolecular carrier protein. The invention makes alpha-corn gibberellin react with succinic anhydride to form hapten, thus highlighting the characteristic structure of alpha-corn gibberellin hapten determinant, and then the alpha-corn gibberellin is coupled with carrier protein by adopting a mixed anhydride method to obtain immunogen. The combination ratio of the alpha-zearalanol hapten to the carrier protein is too low or too high, which is unfavorable for immunity, and the combination molar ratio of the alpha-zearalanol hapten to Ovalbumin (OVA) and Bovine Serum Albumin (BSA) is 8: 1 and 10: 1 respectively.
The alpha-zearalanol monoclonal antibody is preferably an alpha-zearalanol murine monoclonal antibody.
The alpha-zearalanol mouse monoclonal antibody is preferably a monoclonal antibody secreted by a monoclonal hybridoma cell strain A-1-1CGMCC No.1603, which has cross reaction on alpha-zearalanol, beta-zearalanol, zearalenone and alpha-zearalenol.
The monoclonal hybridoma cell strain A-1-1CGMCC No.1603 which has cross reaction on alpha-zearalol, beta-zearalol, zearalenone and alpha-zearalenol is preserved in the 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 which can be methanol.
The kit also comprises a washing solution and a preservation solution; the washing solution is a phosphate buffer solution with the pH value of 7.4 and the concentration of 0.01mol/L, and the 0.01mol/L phosphate buffer solution is a 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 preserving fluidThe solution 1L 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 immunoaffinity adsorbent is based on immune reaction and chromatographic reaction, and is suitable for purifying alpha-zearalol and/or beta-zearalol and/or zearalenone and/or alpha-zearalenol from biological samples (such as muscle, liver, lung, kidney, plasma), and is convenient for residue analysis. In the immunoaffinity adsorbent, the coupling rate of monoclonal antibody secreted by monoclonal hybridoma cell strain A-1-1CGMCC No.1603 and cyanogen bromide activated Sepharose 4B which have cross reaction on alpha-corn gibberellin, beta-corn gibberellin, corn gibberellin and alpha-corn gibberellin is 96.7 +/-1.5 percent, the monoclonal antibody has high cross reaction to beta-corn gibberellin or corn gibberellin and alpha-corn gibberellin enol, the capacities of dynamic columns of the alpha-corn gibberellin, the beta-corn gibberellin, the corn gibberellin and the alpha-corn gibberellin enol are 2640, 2840, 2731 and 2736ng/mL respectively, the absolute column capacities are 377, 405, 390 and 391ng/mg respectively, after 15 times of use, the column capacity is about 43 percent of the total column capacity, and the storage life is 1 year.
The method for purifying alpha-zearalanol and/or beta-zearalanol and/or zearalenone and/or alpha-zearalenol provided by the invention comprises the following steps:
1) pretreatment of a sample:
5mL of methanol is added into an animal tissue sample according to the amount of 5g, the mixture is vortexed and mixed evenly, 3500g of the mixture is centrifuged for 10 minutes, the supernatant is collected and placed at the temperature of minus 20 ℃ for 1 hour, and the mixture is filtered through a 0.25 mu M microporous filter membrane to obtain a sample solution.
2) Passing the sample solution obtained in the step 1) through an immunoaffinity chromatographic column, washing by using a washing solution, and eluting by using the eluent to obtain a purified alpha-zearalanol and/or beta-zearalanol and/or zearalenone and/or alpha-zearalenol solution.
The immunoaffinity adsorbent of the invention greatly simplifies the pretreatment process of samples, is especially suitable for the pretreatment of trace alpha-zearalanol, beta-zearalanol, zearalenone and alpha-zearalenol in muscle, liver and plasma, and improves the analysis quality. The high selectivity of the immunoaffinity adsorbent ensures that the detection limit of an alpha-zearalanol and/or beta-zearalanol and/or zearalenone and/or alpha-zearalenol 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 content of alpha-zearalanol and/or beta-zearalanol and/or zearalenone and/or alpha-zearalenol, makes up the defects of too little 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 gas chromatogram of the purification effect of bovine muscle samples supplemented with alpha-zearalanol, beta-zearalanol, zearalenone and alpha-zearalenol
FIG. 2 is a flow chart of the preparation of the immunogen of the monoclonal antibody to alpha-zearalanol
Detailed Description
The experimental procedures in the following examples are conventional unless otherwise specified.
EXAMPLE 1 preparation of an immunochromatographic column for purifying alpha-zearalanol and/or beta-zearalanol and/or zearalenone and/or alpha-zearalenol
1. Preparation of alpha-zearalanol mouse monoclonal antibody
Synthesis of hapten: reacting the alpha-corn gibberellin with succinic anhydride to obtain ZER-7-hemisuccinate enzyme, namely the alpha-corn gibberellin hapten. The method comprises the following specific steps:
1) 520mg of alpha-zearalanol and 480mg of succinic anhydride are dissolved in 20ml of pyridine and heated under reflux for 4 hours under a nitrogen atmosphere.
2) The reaction product of step 1) was evaporated under reduced pressure, the residue was dissolved in ethyl acetate, and then washed with 0.5mol/L sulfuric acid 3 times, and the aqueous phase was discarded to obtain an organic phase.
3) Extracting the organic phase obtained in the step 2) with a saturated sodium carbonate solution for 3 times, retaining the water phase, and washing with ethyl acetate for 3 times.
4) The pH of the aqueous phase was adjusted to 4.0 with 4mol/L HCl to obtain a precipitate of ZER-7-hemisuccinate, which was extracted 3 times with ethyl acetate, and the combined extracts were dissolved in the organic phase. Washing the organic phase with distilled water for 2 times, drying with anhydrous sodium sulfate, drying with nitrogen, dissolving in chloroform, adding petroleum ether to obtain ZER-7-hemisuccinate precipitate as alpha-zearalanol hapten.
The reaction formula for hapten synthesis is:
preparation of immunogen: the alpha-corn gibberellin hapten is coupled with protein carrier Bovine Serum Albumin (BSA) or Ovalbumin (OVA) by adopting a mixed anhydride method to prepare a BSA-ZER or OVA-ZER conjugate, namely immunogen.
The specific steps are as follows (the immunogen preparation flow chart is shown in figure 2):
1) 300mg of ZER-7-hemisuccinate and 132mg of tri-n-butylamine are dissolved in 20ml of dioxane, the solution is cooled to 10 ℃, 100mg of isobutyl chloroformate is added while stirring, the solution is cooled to 4 ℃, and stirring is carried out for 45 min.
2) Adding 650mg of BSA (bovine serum albumin) into 2.5ml of 1mol/L sodium hydroxide and adding 130ml of a mixed solution of dioxane and water in a ratio of 1: 1 at 4 ℃ to finally obtain a BSA solution, adding the reaction liquid obtained in the step 1) into the BSA solution, stirring at 4 ℃ for 1 hour, adding 1ml of 1mol/L sodium hydroxide, stirring at 4 ℃ for 4 hours, and dialyzing with distilled water for 12 hours.
3) The solution obtained in step 2) was adjusted to pH 2.0 with 1mol/L hydrochloric acid to give a precipitate of Z7BSA, and the precipitate was dissolved in 100ml of distilled water and adjusted to pH 11.0 with 1M sodium hydroxide. Acetone (4 ℃, 300ml) was added, the pH was adjusted to 2.0 with 1M hydrochloric acid, Z7BSA was again precipitated, collected and the acetone precipitation process was repeated 3 times.
4) The precipitate of Z7BSA obtained in step 3) is dissolved in 300ml of distilled water and adjusted to pH 11.0 with 1mol/L sodium hydroxide. Centrifuging for 10min at 2000g, removing the precipitate, dialyzing the supernatant with distilled water at 4 ℃ for 48h, and replacing the dialysate every 6-8 h. After dialysis, 0.01% thimerosal was added, and the mixture was stored at-20 ℃ in small vials.
Animal immunization: BALA/C mice are used as immune animals, a conjugate BSA-ZER or OVA-ZER conjugate of the alpha-zearalanol hapten and a protein carrier is used as an immunogen, the immune dose is 100 mu g/mouse, and an equal 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: the hybridoma cells are screened by adopting a limiting dilution method until completely homogeneous monoclonal hybridoma cell strains A-1-1CGMCC No.1603 which have cross reaction on alpha-zearalanol, beta-zearalanol, zearalenone and alpha-zearalenol are obtained. A monoclonal hybridoma cell strain A-1-1CGMCC No.1603 which has cross reaction on alpha-zearalol, beta-zearalol, zearalenone and alpha-zearalenol is preserved in the China general microbiological culture Collection center (CGMCC for short) at 2006, 2 and 9 months.
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, injecting monoclonal hybridoma cell strain A-1-1CGMCC No. 16035 × 10 with cross reaction to alpha-zearalanol, beta-zearalanol, zearalenone and alpha-zearalenol in abdominal cavity after 7-14 days5-106Ascites were collected 7-10 days later.
2. 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-1-1CGMCC No.1603 which has cross reaction on alpha-zearalanol, beta-zearalanol, zearalenone and alpha-zearalenol by the method is added with 10mL of NaAc-HAc buffer solution with the concentration of 0.06mol/L and the pH value of 4.0 and stirred uniformly, and the pH value is adjusted to 7.2 by using 1mol/L of 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 0.01MpH7.2PBS, and changing the solution once after 4-6 hours and 2-3 times. Measuring OD values of IgG solution at 280nm and 260nm with ultraviolet spectrophotometer to obtain monoclonal antibody secreted by monoclonal hybridoma cell strain A-1-1CGMCC No.1603 with cross reaction to alpha-corn gibberellin, beta-corn gibberellin, corn gibberellin and alpha-corn gibberellin enol, and storing the purified IgG in refrigerator at-20 deg.c.
3. Preparation of immunochromatographic columns (IAC)
Preparation of a matrix: taking cyanogen bromide activated Sepharose 4B dry powder, and placing in a container 1.0mmol 1-1G of HCl3The funnel is expanded.
Preparation of antibody IgG: with 0.1mol/L NaHCO3The solution is diluted by monoclonal antibody secreted by the purified monoclonal hybridoma cell strain A-1-1CGMCC No.1603 which has cross reaction to alpha-zearalanol, beta-zearalanol, zearalenone and alpha-zearalenol, and the pH value of the solution is adjusted to 8.4.
Coupling reaction: the expanded sol was treated with 0.1mol/L NaHCO3After equilibration of the solution, transfer to NaHCO as described above3Monoclonal hybridoma cell strain A-1-1CGMCC No.1603 secreted by solution diluted monoclonal hybridoma cell strain with cross reaction on alpha-zearalanol, beta-zearalanol, zearalenone and alpha-zearalenolThe cloned antibody solution is mixed and stirred slowly at 4 ℃ for 20-24 hours. 40ml of LPBS (1L of solution contains 0.27g of potassium dihydrogen phosphate, 2.86g of 12-hydrated disodium hydrogen phosphate, 0.2g of potassium chloride and 29.3g of sodium chloride) is washed to remove unconjugated antibody and the conjugation rate is measured, and the detection result of the conjugation rate shows that the conjugation rate of the monoclonal antibody secreted by the monoclonal hybridoma cell strain A-1-1CGMCC No.1603, which has cross reaction on alpha-zearalol, beta-zearalol, zearalenone and alpha-zearalenol, and the Sepharose 4B activated by cyanogen bromide is 96.7 +/-1.5%.
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: the immunoadsorbent coupled with monoclonal antibody secreted by monoclonal hybridoma cell strain A-1-1CGMCC No.1603 with cross reaction to alpha-zearalanol, beta-zearalanol, zearalenone and alpha-zearalenol is transferred into a medium containing G3An immunochromatographic column (IAC column) coupled with monoclonal antibodies secreted by monoclonal hybridoma cell strains A-1-1CGMCC No.1603, which have cross reaction on alpha-zearalanol, beta-zearalanol, zearalenone and alpha-zearalenol, is prepared in a chromatographic column of a filter plate.
5. Determination of IAC column Capacity
Coupling the mixture prepared in step 4 with para-alpha-zearalanol, beta-zearalanol, zearalenone andan immunochromatographic column of monoclonal antibodies secreted by monoclonal hybridoma cell strains A-1-1CGMCC No.1603 with all alpha-zearalenol having cross reaction is washed by 10ml of PBS (0.27 g of potassium dihydrogen phosphate, 2.86g of 12-hydrated disodium hydrogen phosphate, 0.2g of potassium chloride and 8.8g of sodium chloride) with 0.01mol/L and pH7.4 (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), gently shaken up and down to remove air bubbles in the column, and then balanced by 10ml of PBS (0.01 mol/L and pH7.4 (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). Respectively contain 100 ng/ml-1A PBS-methanol solution of α -zearalol, β -zearalol, zearalenone and β 1-zearalenol (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, 200mL of methanol) was continuously applied to an IAC column and allowed to flow out under natural gravity. When the column was saturated (the concentrations of β 2-zearalanol, β 0-zearalanol, zearalenone and β 4-zearalenol in the effluent were the same as those of the sample addition solution), 10ml of PBS (1L of which 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 15ml of water were used to wash the IAC column, and 4ml of 30% (volume percent) aqueous methanol was used to wash the column, thereby removing interfering impurities from the extract. And finally, eluting the alpha-zearalanol, the beta 3-zearalanol, the zearalenone and the alpha-zearalenol by using 4ml of methanol, flowing out under natural gravity, collecting, drying, adding 0.05ml of bis-trimethylsilane trifluoroacetamide (BSTFA) reagent, performing vortex movement, derivatizing for 15min at 60 ℃, and performing GC/MS (gas chromatography/Mass Spectrometry) 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 capacities of the dynamic columns of the alpha-zearalanol, the beta-zearalanol, the zearalenone and the alpha-zearalenol of the immunochromatographic column coupled with the monoclonal antibody secreted by the monoclonal hybridoma cell strain A-1-1CGMCC No.1603 which has cross reaction on the alpha-zearalanol, the beta-zearalanol, the zearalenone and the alpha-zearalenol are 2640, 2840, 2731 and 2736 respectivelyng/mL, the absolute column capacities of the immunochromatographic column to alpha-zearalanol, beta-zearalanol, zearalenone and alpha-zearalenol are 377, 405, 390 and 391ng/mg respectively.
Example 2 preparation of a kit for immunochromatography column to which murine monoclonal antibody is coupled and its purification Effect on alpha-zearalol
1. Preparation of kit for purifying alpha-zearalanol and/or beta-zearalanol and/or zearalenone and/or alpha-zearalenol
The kit mainly comprises a kit body, an immunochromatographic column (IAC column), a standard alpha-zearalanol reagent, a washing solution, an eluent, a preservation solution and a sponge bracket, wherein holes and grooves are arranged on the sponge bracket. A reagent bottle filled with a standard alpha-zearalanol reagent, a washing solution, an eluent and a 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 prepared in example 1 and coupled with monoclonal antibodies secreted by monoclonal hybridoma cell strains A-1-1CGMCCNo.1603 which have cross reaction on alpha-zearalanol, beta-zearalanol, zearalenone and alpha-zearalenol.
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.
The eluent was methanol of chromatographic grade.
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 immunochromatography column kit is respectively placed at 4 ℃ for 12 months.
2. Purification effect experiment of alpha-zearalanol, beta-zearalanol, zearalenone and alpha-zearalenol
The IAC extraction principle is that monoclonal antibody secreted by monoclonal hybridoma cell strain A-1-1CGMCC No.1603 with specific antibody having cross reaction to alpha-corn gibberellin, beta-corn gibberellin, corn gibberellin and alpha-corn gibberellin alcohol is coupled with inert substrate to prepare immunoadsorbent, which is packed in column. When the mixture containing alpha-corn gibberellin or/and beta-corn gibberellin or/and alpha-corn gibberellin enol flows through the IAC column, the immobilized antibody selectively binds to the alpha-corn gibberellin and/or beta-corn gibberellin and/or alpha-corn gibberellin enol, other unidentified sample impurities flow out of the IAC column without being hindered, and after washing, the antigen-antibody complex is dissociated and eluted, and the alpha-corn gibberellin and/or beta-corn gibberellin and/or alpha-corn gibberellin enol are purified or separated. The IAC column can be reused after regeneration treatment.
Treatment of the detection sample: taking 5g of each cattle muscle sample (taken from cattle not contacted with alpha-zearalanol substances) into a 50mL plastic centrifuge tube, respectively adding alpha-zearalanol, beta-zearalanol, zearalenone and alpha-zearalenol standard products into each sample according to the concentration of 2.5 mu g/kg, respectively adding 5mL of methanol, whirling for 1min, centrifuging for 10min at 3500g, transferring the supernatant into another clean centrifuge tube, repeatedly extracting, and combining the supernatants. Placing in a freezer at-20 deg.C for 1hr, and filtering with 0.25 μ M microporous membrane. 5mL of the membrane solution is uniformly mixed with 20mL of the washing solution in the kit for later use.
The immunochromatography column coupled with the monoclonal antibody secreted by the monoclonal hybridoma cell line A-1-1CGMCC No.1603 which has cross reaction to alpha-zearalanol, beta-zearalanol, zearalenone and alpha-zearalenol and is prepared is balanced to room temperature, is washed by 10ml of washing liquid in the kit, then the sample solution passes through the column, is washed by 15ml of water, and then is washed by adding 4ml of 30% (volume percentage content) methanol water solution, so as to remove non-specifically adsorbed impurities. Eluting with 4ml of eluent, drying at 50 ℃ under N2 airflow, adding 50 μ l of derivatization reagent BSTFA, whirling, derivatizing at 60 ℃ for 15 minutes, performing GC/MS analysis, and determining the content of alpha-zearalanol by using standard substances of alpha-zearalanol, beta-zearalanol, zearalenone and alpha-zearalenol as controls. 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 indicates that the prepared IAC has extremely low nonspecific adsorption. The purification effect of an immunochromatographic column coupled with a monoclonal antibody secreted by a monoclonal hybridoma cell strain A-1-1CGMCC No.1603 which has cross reaction on alpha-zearalanol, beta-zearalanol, zearalenone and alpha-zearalenol on a bovine muscle sample added with a standard product of alpha-zearalanol, beta-zearalanol, zearalenone and alpha-zearalenol at the concentration of 2.5 mu g/kg is shown in figure 1, wherein 1 is zearalenone, 2 is alpha-zearalanol, 3 is beta-zearalenol, and 4 is alpha-zearalenol; a is beef sample added with alpha-corn gibberellin, beta-corn gibberellin, corn gibberellin and alpha-corn gibberellin enol (2.5 ng/g each), and B is alpha-corn gibberellin, beta-corn gibberellin, corn gibberellin and alpha-corn gibberellin enol standard (2.0 ng/g each).
Claims (2)
1. A method for purifying α -zearalanol and/or β -zearalanol and/or zearalenone and/or α -zearalenol comprising the steps of:
1) pretreatment of a sample:
adding 5g of 5mL of methanol into an animal tissue sample, mixing uniformly by vortexing, centrifuging 3500g for 10 minutes, collecting supernatant, placing at-20 ℃ for 1 hour, and filtering through a 0.25 mu M microporous filter membrane to obtain a sample solution;
2) washing the immunoaffinity chromatography column loaded with the immunoaffinity adsorbent with 10ml of washing solution, dissolving the sample obtained in step 1)Passing through immunoaffinity chromatography column, washing with 15ml water, adding 4ml methanol water solution with 30% volume percent content, eluting with 4ml eluent, and eluting at 50 deg.C with N2Drying under air flow to obtain purified alpha-zearalanol and/or beta-zearalanol and/or zearalenone and/or alpha-zearalenol solution; wherein,
the immunoaffinity adsorbent consists of a solid phase carrier and an alpha-zearalanol 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-1-1CGMCC No. 1603;
the washing solution is a phosphate buffer solution with the pH value of 7.4 and the concentration of 0.01mol/L, and the 0.01mol/L 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 eluent is methanol.
2. The method of claim 1, wherein: the animal tissue samples include muscle, liver, lung, kidney and plasma.
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| CN104707583A (en) * | 2015-01-05 | 2015-06-17 | 重庆出入境检验检疫局检验检疫技术中心 | Composite immunization affinity column for purifying beta-zeranol and chloramphenicol, preparation method and application thereof |
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| CN107344966A (en) * | 2017-07-05 | 2017-11-14 | 河南省农业科学院 | A kind of method that ZER monoclonal antibody is prepared based on analogue cross reactivity |
| CN108956822A (en) * | 2018-08-22 | 2018-12-07 | 中国农业科学院饲料研究所 | The evaluation method of detoxification efficiency in a kind of zearalenone detoxifying agent body |
| CN113960314B (en) * | 2021-10-15 | 2023-07-11 | 北京勤邦科技股份有限公司 | Corn gibberellic alcohol immunoaffinity column and preparation method and application thereof |
-
2006
- 2006-02-15 CN CNB2006100072397A patent/CN100406115C/en not_active Expired - Fee Related
Non-Patent Citations (8)
| Title |
|---|
| 快速检测动物组织中玉米赤霉醇残留的方法. 顾鸣等.中国卫生检验杂志，第13卷第06期. 2003 |
| 快速检测动物组织中玉米赤霉醇残留的方法. 顾鸣等.中国卫生检验杂志，第13卷第06期. 2003 * |
| 牛尿中玉米赤霉醇残留酶联免疫检测方法的研究. 王鹤佳等.中国兽医杂志，第40卷第11期. 2004 |
| 牛尿中玉米赤霉醇残留酶联免疫检测方法的研究. 王鹤佳等.中国兽医杂志，第40卷第11期. 2004 * |
| 玉米赤霉醇单克隆抗体的制备及鉴定. 王鹤佳等.中国兽医杂志，第40卷第10期. 2004 |
| 玉米赤霉醇单克隆抗体的制备及鉴定. 王鹤佳等.中国兽医杂志，第40卷第10期. 2004 * |
| 玉米赤霉醇及其残留检测. 王鹤佳等.中国兽药杂志，第37卷第05期. 2003 |
| 玉米赤霉醇及其残留检测. 王鹤佳等.中国兽药杂志，第37卷第05期. 2003 * |
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