CN1830545A - 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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- CN1830545A CN1830545A CN 200610007239 CN200610007239A CN1830545A CN 1830545 A CN1830545 A CN 1830545A CN 200610007239 CN200610007239 CN 200610007239 CN 200610007239 A CN200610007239 A CN 200610007239A CN 1830545 A CN1830545 A CN 1830545A
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Abstract
A method for purifying alpha-zearalol and/or beta-zearalol and/or zearalone and/or alpha-zearalenol by the special immunoaffinity chromatographic column carrying immunoaffinity adsorbent is disclosed. Said adsorbent is composed of solid carrier and its coupled alpha-zearalol monoclonal antibody. It features that the chromatography is used to measure the contents of alpha-zearalol and its metabolites, having high correctness.
Description
Technical field
The present invention relates to a kind of method and special immune affinity chromatographic column thereof that purifies α-Yu Michimeichun and metabolin thereof, particularly a kind of method and special immune affinity chromatographic column thereof that purifies α-Yu Michimeichun and/or β-ZER and/or zearelone and/or α-zearalenol.
Background technology
Along with development of life science, people have produced more and more keen interest to material in the organism and variation thereof, and the analysis of biological specimen just becomes the necessary means of exploring and finding the life secret.Because the substance in biological sample complexity, testing concentration is lower, and most of sampling amount seldom, and this just has higher requirement to the selectivity and the sensitivity of analytical method.Immune affinity chromatographic (IAC, immunoaffinity chromatography) is applied to the residue of veterinary drug analysis, and its high selectivity and high-affinity make purification process simplify undoubtedly.With GC, the HPLC coupling can make immunological technique and physics and chemistry technology obtain complementation aspect selectivity, separating power, speed and the sensitivity, avoided immunoassay (as ELISA, RIA) direct many deficiencies of working sample.At present, this method is widely used in the analysis of antibody, hormone, polypeptide, enzyme, recombinant protein, acceptor virus and micromolecular compound.
α-Yu Michimeichun, β-ZER, zearelone and α-zearalenol belong to thunder carboxylic acid lactone non-steroid anabolic hormone, α-Yu Michimeichun Ceng Zuowei growth promoter is applied to ruminant, have the promotion protein synthesis, β-ZER, zearelone are the metabolites of α-Yu Michimeichun.α-zearalenol is the metabolite of zearalenone.α-Yu Michimeichun, β-ZER, zearelone and α-zearalenol have weak estrogen action, its residual meeting in animal tissue influence human health, the lighter causes the human body disorder, weight person influences the normal development of secondary sex characters, externally condition is induced down, and this class material may be carcinogenic.Simultaneously, after ZER is discharged outside the animal body, also can cause secondary pollution and environmental pollution through drinking-water and food, European Union in 1998 clearly forbids hormone medicine is applied to Livestock Production.China forbids in 2002 that also it is used for all food animals, must not detect in all edible tissues.
The method that detects α-Yu Michimeichun, β-ZER, zearelone and α-zearalenol at present mainly contains high performance liquid chromatography (HPLC), gas-mass spectrometry method (GC-MS), liquid-matter coupling method (LC-MS) and ELISA (ELISA) etc.The pre-treatment of these methods utilizes liquid-liquid to distribute more, conventional SPE column purification with separate, all exist shortcomings such as processing procedure is loaded down with trivial details, clean-up effect is poor, the organic solvent waste is many, required time is grown to some extent.The affine technology of immunity is the new technology that is applied at analysis field the nineties, but with the α-Yu Michimeichun (α-Zearalanol in the immune affinity column while decontamination substrate, ZER), (α-ZOL) do not appear in the newspapers does not more have commercial IAC post and sells for β-ZER (TAL), zearelone (ZEN) and α-zearalenol.
Summary of the invention
The purpose of this invention is to provide a kind of method and special immune affinity sorbent thereof that purifies α-Yu Michimeichun and/or β-ZER and/or zearelone and/or α-zearalenol.
The immune affinity sorbent of purification α-Yu Michimeichun provided by the present invention and/or β-ZER and/or zearelone and/or α-zearalenol by solid phase carrier and with its coupling β-ZER, zearelone and α-zearalenol all are made up of the α-Yu Michimeichun monoclonal antibody of cross reaction; It is described that β-ZER, zearelone and α-zearalenol are all had the α-Yu Michimeichun monoclonal antibody of cross reaction is that conjugate with α-Yu Michimeichun haptens and carrier protein is that immunogene obtains; Described α-Yu Michimeichun haptens is that α-Yu Michimeichun and succinyl oxide are reacted the ZER-7-hemisuccinic acid ester that obtains, and is the α-Yu Michimeichun haptens.
Described carrier protein can be common carrier albumen such as bovine serum albumin(BSA) or ovalbumin.
α-Yu Michimeichun is a small-molecule substance, has only immunoreactivity, does not have immunogenicity, can not bring out body and produce immune response, must with the macromolecular carrier albumen coupling after just have immunogenicity.The present invention forms haptens with the reaction of α-Yu Michimeichun and succinyl oxide, the feature structure of having given prominence to the α-Yu Michimeichun haptenic group like this, and then adopt mixed anhydride method and carrier protein couplet to obtain immunogene the α-Yu Michimeichun.It is low or too high all unfavorable to immunity that α-Yu Michimeichun haptens and the ratio that combines of carrier protein are crossed, and the α-Yu Michimeichun haptens was respectively 8: 1 and 10: 1 with the mol ratio that combines of ovalbumin (OVA), bovine serum albumin (BSA).
Described α-Yu Michimeichun monoclonal antibody is preferably the α-Yu Michimeichun mouse monoclonal antibody.
Described α-Yu Michimeichun mouse monoclonal antibody is preferably the monoclonal antibody that α-Yu Michimeichun, β-ZER, zearelone and α-zearalenol is all had the monoclonal hybridoma strain A-1-1 CGMCCNo.1603 secretion of cross reaction.
The described monoclonal hybridoma strain A-1-1 CGMCC No.1603 that α-Yu Michimeichun, β-ZER, zearelone and α-zearalenol are all had a cross reaction has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on February 9th, 2006.
Described solid phase carrier can be cellulose, sephadex, polyacrylamide gel, cellular glass, Ago-Gel, ultragel ACA22 etc., is preferably Sepharose 4B.
Described immune affinity sorbent can be loaded into and make immune affinity chromatographic column in the post, and this immune affinity chromatographic column also belongs to protection scope of the present invention.
The kit that contains above-mentioned immune affinity sorbent or immune affinity chromatographic column also belongs to protection scope of the present invention.
Also comprise eluent in the described kit, described eluent can be methyl alcohol.
Also comprise cleaning solution in the described kit, preserve liquid; Described cleaning solution is pH7.4, and the phosphate buffer of 0.0lmol/L, described 0.01mol/L phosphate buffer are to contain potassium dihydrogen phosphate 0.27g among the 1L, 12 hypophosphite monohydrate disodium hydrogen 2.86g, potassium chloride 0.2g, the solution of sodium chloride 8.8g; Described preservation liquid is to contain potassium dihydrogen phosphate 0.27g in the 1L solution, 12 hypophosphite monohydrate disodium hydrogen 2.86g, potassium chloride 0.2g, sodium chloride 8.8g, NaN
30.2g, pH7.4.
This immune affinity sorbent is based on immune response and chromatogram reaction, be fit to purification α-Yu Michimeichun and/or β-ZER and/or zearelone and/or α-zearalenol from biological sample (as muscle, liver, lung, kidney, blood plasma), be convenient to retention analysis.In this immune affinity sorbent, to α-Yu Michimeichun, β-ZER, the coupling rate that zearelone and α-zearalenol all have the Sepharose 4B of the monoclonal antibody of monoclonal hybridoma strain A-1-1CGMCC No.1603 secretion of cross reaction and cyanogen bromide-activated is 96.7 ± 1.5%, this monoclonal antibody is to β-ZER, or zearelone and and α-zearalenol very high cross reaction is all arranged, α-Yu Michimeichun, β-ZER, the dynamic column capacity of zearelone and α-zearalenol is respectively 2640,2840,2731,2736ng/mL, absolute column capacity is respectively 377,405,390,391ng/mg, column capacity is about 43% of total column capacity after 15 times having used, and storage life is 1 year.
The method of purification α-Yu Michimeichun provided by the present invention and/or β-ZER and/or zearelone and/or α-zearalenol may further comprise the steps:
1) pre-treatment of sample:
Animal tissue's sample is added 5mL methyl alcohol with the amount of 5g, the whirling motion mixing, centrifugal 10 minutes of 3500g collects supernatant, place-20 ℃ 1 hour, cross 0.25 μ M miillpore filter and obtain sample solution.
2) sample solution that step 1) is obtained is crossed immune affinity chromatographic column, with the cleaning solution washing, uses above-mentioned eluent wash-out again, the α-Yu Michimeichun that is purified and/or β-ZER and/or zearelone and/or α-zearalenol solution then.
Immune affinity sorbent of the present invention is simplified sample pretreatment process greatly, be particularly useful for the pre-treatment of micro-α-Yu Michimeichun, β-ZER, zearelone and α-zearalenol in muscle, liver and the blood plasma, analyze quality and improve.The high selectivity of immune affinity sorbent makes the detectability of α-Yu Michimeichun and/or β-ZER and/or zearelone and/or α-zearalenol analytical method will depend primarily on sampling amount, and this is that simple physics and chemistry means are unapproachable; Immune affinity sorbent of the present invention has very strong reservation and concentrating capacity to component to be measured, as long as the application of sample amount is no more than column capacity, immune affinity sorbent is subjected to the influence of sample volume or concentration of component hardly to the reserve capability of component under the actual measurement sample condition.When purifying component, method of the present invention also can provide qualitative information.Method water operation of the present invention, simple to operate, good purification, immune affinity chromatographic column can be reused, and can save a large amount of organic solvents, reduces analysis cost and environmental pollution.Method of purification of the present invention is in conjunction with the content of chromatography efficient detection α-Yu Michimeichun and/or β-ZER and/or zearelone and/or α-zearalenol, remedied simple immunoassay directly measure sample information amount very little, quantitatively accurately poor, or the low deficiency that waits of physico-chemical method selectivity, embodied immunological technique and conventional physics and chemistry technology complementarity in analysis mechanisms.
Description of drawings
Fig. 1 is for adding α-Yu Michimeichun, β-ZER, zearelone and α-zearalenol ox muscle samples clean-up effect makings chromatogram
Fig. 2 is the former preparation flow figure of α-Yu Michimeichun monoclonal antibody immunity
The specific embodiment
Experimental technique among the following embodiment if no special instructions, is conventional method.
The preparation of the immune chromatograph post of embodiment 1, purification α-Yu Michimeichun and/or β-ZER and/or zearelone and/or α-zearalenol
1, the preparation of α-Yu Michimeichun mouse monoclonal antibody
Haptenic synthetic: as α-Yu Michimeichun and succinyl oxide reaction to be obtained ZER-7-hemisuccinic acid ester, be the α-Yu Michimeichun haptens.Concrete steps are as described below:
1) get α-Yu Michimeichun 520mg, succinyl oxide 480mg is dissolved in the 20ml pyridine, adds hot reflux 4 hours under nitrogen environment.
2) the product reduction vaporization of step 1) is dissolved in residue in the ethyl acetate, uses the 0.5mol/L sulfuric acid scrubbing then 3 times, and aqueous phase discarded obtains organic facies.
3) step 2) organic facies that obtains keeps water with saturated sodium carbonate solution extraction 3 times, again with ethyl acetate washing 3 times.
4) water is transferred pH to 4.0 with the HCl of 4mol/L, obtains ZER-7-hemisuccinic acid ester precipitation, uses ethyl acetate extraction 3 times, combining extraction liquid, and ZER-7-hemisuccinic acid ester promptly is dissolved in the organic facies.Organic facies is used anhydrous sodium sulfate drying with distilled water washing 2 times, and nitrogen dries up, and is dissolved in the chloroform, adds benzinum again, obtains ZER-7-hemisuccinic acid ester precipitation and is the α-Yu Michimeichun haptens.
The synthetic reaction equation of haptens is:
Immunogenic preparation: adopt mixed anhydride method,, make BSA-ZER or OVA-ZER conjugate, i.e. immunogene with α-Yu Michimeichun haptens and protein carrier bovine serum albumin (BSA) or ovalbumin (OVA) coupling.
Concrete steps (the immunogen preparing flow chart is as shown in Figure 2) as described below:
1) get ZER-7-hemisuccinic acid ester 300mg, tri-n-butylamine 132mg is dissolved in the 20ml diox, and solution is cooled to 10 ℃, adds isobutyl chlorocarbonate 100mg while stirring, and solution is cooled to 4 ℃, stirs 45min.
2) under 4 ℃ of conditions, get BSA 650mg, add 2.5ml lmol/L NaOH, add 130ml diox and water in 1: 1 ratio liquid mixture prepared, finally obtain BSA solution, the reaction liquid that obtains in the step 1) is added in this BSA solution, 4 ℃ are stirred adding lml 1mol/L NaOH after 1 hour, 4 ℃ were stirred distill water dialysis 12 hours 4 hours.
3) with lmol/L hydrochloric acid with step 2) solution that obtains transfers pH to 2.0, promptly produces the Z7BSA precipitation, and precipitation is placed 100ml distilled water, transfers pH to 11.0 with 1M NaOH, the precipitation dissolving.(4 ℃, 300ml), with 1M hydrochloric acid accent pH to 2.0, Z7BSA is precipitated out once more, collects, and repeats the acetone precipitation process again 3 times to add acetone.
4) the Z7BSA precipitation that step 3) is obtained is dissolved in the 300ml distilled water, transfers pH to 11.0 with 1mol/L NaOH.The centrifugal 10min of 2000g abandons precipitation, and supernatant is to 4 ℃ of distilled water dialysis 48h, changes one time dislysate in per 6～8 hours.After the dialysis, add the 0.0l% thimerosal, bottle packing ,-20 ℃ of preservations.
Animal immune: adopting the BALA/C mouse is immunogene as immune animal, with the conjugate BSA-ZER or the OVA-ZER conjugate of above-mentioned α-Yu Michimeichun haptens and protein carrier, immunizing dose is 100 μ g/, add isopyknic complete Freund's adjuvant emulsification, carry out first immunisation.After two weeks, get same amount immunizing antigen and add incomplete Freund's adjuvant, the immunity second time is carried out in emulsification.At interval two weeks, carry out immunity for the third time, method is with immunity for the second time.Every two weeks, carry out the 4th immunity again, method is with immunity for the second time.Merge preceding 3 days again to the BALB/C mice booster immunization once, lumbar injection 100 μ g antigens do not add adjuvant.Pluck the eyeball bloodletting before the fusion, obtain positive serum, then mouse is drawn the neck dislocation to put to death, get spleen.
Fusion of Cells: splenocyte carries out Fusion of Cells in 5: 1 ratios and SP2/0 myeloma cell.
Hybridoma cell cloneization: adopt limiting dilution assay screening hybridoma, up to what obtain complete homogeneity α-Yu Michimeichun, β-ZER, zearelone and α-zearalenol all had cross reactivity, monoclonal antibody and stable monoclonal hybridoma strain-α-Yu Michimeichun, β-ZER, zearelone and α-zearalenol are all the had monoclonal hybridoma strain A-1-1 CGMCCNo.1603 of cross reaction.The monoclonal hybridoma strain A-1-1 CGMCC No.1603 that α-Yu Michimeichun, β-ZER, zearelone and α-zearalenol is all had cross reaction has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on February 9th, 2006.
The preservation of monoclonal hybridoma: at liquid nitrogen-20 ℃ preservation down, 37 ℃ of water-bath quick-thawings during use.
Monoclonal antibody raised growth and purification: adopt in the body and induce method, the BALB/c mouse abdominal cavity is injected the sterilization paraffin oil, and pneumoretroperitoneum injections in 7-14 days all have the monoclonal hybridoma strain A-1-1 CGMCC No.1603 5 * 10 of cross reaction to α-Yu Michimeichun, β-ZER, zearelone and α-zearalenol
5-10
6Individual/as only, to gather ascites after 7-10 days.
2, Purification of Monoclonal Antibodies:
Adopt sad-saturated ammonium sulfate method monoclonal antibody purification.Its concrete steps are as follows:
Get the said method lumbar injection α-Yu Michimeichun, β-ZER, zearelone and α-zearalenol are all had the ascites 5mL that the monoclonal hybridoma strain A-1-1 CGMCC No.1603 of cross reaction obtains, the NaAc-HAc buffer solution 10mL that adds 0.06mol/L, pH4.0, stir, transfer pH to 7.2 with 1mol/L NaOH solution.It is sad at room temperature to add 165 μ L while stirring, stirs 30min.At 4 ℃ of centrifugal 30min of 6000g, get supernatant, transfer pH to 7.2 with 1mol/L NaOH solution.Then under 4 ℃ of conditions, add 15mL saturated ammonium sulfate solution while stirring, the final mass percentage composition that makes ammonium sulfate is 50%, after stirring 30min,, abandon supernatant at 4 ℃ of centrifugal 30min of 6000g, the PBS that precipitation is suspended in 5.5mL 0.01M pH7.2 (contains potassium dihydrogen phosphate 0.27g in the 1L solution, 12 hypophosphite monohydrate disodium hydrogen 2.86g, potassium chloride 0.2g, sodium chloride 8.8g) in.To precipitate suspension and add 4.5mL saturated ammonium sulfate solution while stirring, this operates under 4 ℃ of conditions and finishes.This moment, the ammonium sulfate final concentration was 45%, and behind the stirring 30min, 4 ℃ of centrifugal 30min of 6000g abandon supernatant, and precipitation is suspended among the 1mL 0.01M pH7.2PBS.To precipitate suspension and pack in the bag filter,, change liquid once in 4～6 hours, change liquid 2～3 times with the 0.01MpH7.2PBS dialysis.Measure the OD value of IgG solution with ultraviolet specrophotometer at 280nm and 260nm place, obtain α-Yu Michimeichun, β-ZER, zearelone and α-zearalenol are all had the monoclonal antibody of the monoclonal hybridoma strain A-1-1 CGMCC No.1603 secretion of cross reaction, the IgG behind the purifying preserves at-20 ℃ of refrigerators.
3, the preparation of immune chromatograph post (IAC)
The preparation of matrix: get the Sepharose 4B dry freeze powder of cyanogen bromide-activated, filling 1.0mmol 1
-1The G of HCl
3Expand in the funnel.
The preparation of IgG antibody: with the NaHCO of 0.1mol/L
3Solution all has the monoclonal antibody dilution of the monoclonal hybridoma strain A-1-1 CGMCC No.1603 secretion of cross reaction with purifying to α-Yu Michimeichun, β-ZER, zearelone and α-zearalenol, and transferring the pH value of solution is 8.4.
Coupling reaction: the NaHCO that the colloidal sol that expands is used 0.1mol/L
3Behind the solution equilibria, change above-mentioned NaHCO over to
3Solution dilution α-Yu Michimeichun, β-ZER, zearelone and α-zearalenol are all had in the monoclonal anti liquid solution of monoclonal hybridoma strain A-1-1 CGMCC No.1603 secretion of cross reaction, mix, 4 ℃ were slowly stirred 20-24 hour down.40mLPBS (contains potassium dihydrogen phosphate 0.27g in the 1L solution, 12 hypophosphite monohydrate disodium hydrogen 2.86g, potassium chloride 0.2g, sodium chloride 29.3g) flush away coupling antibody and survey its coupling rate not, the testing result of coupling rate shows, the coupling rate that α-Yu Michimeichun, β-ZER, zearelone and α-zearalenol is all had the Sepharose 4B of the monoclonal antibody of monoclonal hybridoma strain A-1-1 CGMCC No.1603 secretion of cross reaction and cyanogen bromide-activated is 96.7 ± 1.5%.
The sealing in activation site: the gel after the above-mentioned coupling is changed in the Tris-HCl buffer solution that fills 0.1mol/L, pH8.0, mix, 4 ℃ are slowly stirred 2hr down, to seal the activation site of not coupling.
Washing: gel alternately washes 3 times with 0.1mol/L, pH4.0 acetate buffer and 0.1mol/L, the pH8.0Tris-HCl buffer solution of 5 times of volumes.After PBS (containing potassium dihydrogen phosphate 0.27g in the 1L solution, 12 hypophosphite monohydrate disodium hydrogen 2.86g, potassium chloride 0.2g, the sodium chloride 8.8g) balance with 0.01mol/L, pH7.4, the gel of draining changes the 0.1%NaN that contains of 0.01mol/L, pH7.4 over to
3Phosphate buffer (contains potassium dihydrogen phosphate 0.27g, 12 hypophosphite monohydrate disodium hydrogen 2.86g, potassium chloride 0.2g, sodium chloride 8.8g, NaN in the 1L solution
31g), deposit under 4 ℃ standby.
The dress post: the immunosorbent of the monoclonal antibody that the monoclonal hybridoma strain A-1-1 CGMCC No.1603 that has pair α-Yu Michimeichun, β-ZER, zearelone and α-zearalenol to have cross reaction coupling secretes is transferred to and contains G
3In the chromatographic column of filter plate, the immune chromatograph post (IAC post) of the monoclonal antibody that the monoclonal hybridoma strain A-1-1 CGMCC No.1603 that making coupling has pair α-Yu Michimeichun, β-ZER, zearelone and α-zearalenol all to have cross reaction secretes.
5, the IAC column capacity determines
The coupling of step 4 preparation there is pair α-Yu Michimeichun, β-ZER, zearelone and α-zearalenol all have the immune chromatograph post of monoclonal antibody of the monoclonal hybridoma strain A-1-1 CGMCC No.1603 secretion of cross reaction, use 10ml 0.01mol/L respectively, the PBS of pH7.4 (contains potassium dihydrogen phosphate 0.27g in the 1L solution, 12 hypophosphite monohydrate disodium hydrogen 2.86g, potassium chloride 0.2g, sodium chloride 8.8g) washes post, IAC post gently teetertotters, drive the bubble in the post away, again with 10ml 0.01mol/L, the PBS of pH7.4 (contains potassium dihydrogen phosphate 0.27g in the 1L solution, 12 hypophosphite monohydrate disodium hydrogen 2.86g, potassium chloride 0.2g, sodium chloride 8.8g) balance.To contain 100ngml respectively
-1The PBS-methanol solution of α-Yu Michimeichun, β-ZER, zearelone and α-zearalenol (contains potassium dihydrogen phosphate 0.27g in the 1L solution, 12 hypophosphite monohydrate disodium hydrogen 2.86g, potassium chloride 0.2g, sodium chloride 8.8g, 200mL methyl alcohol) be added to the IAC post continuously, natural gravity flows out down.After post reaches capacity (it is identical with application of sample liquid concentration with α-zearalenol to flow out α-Yu Michimeichun in the liquid, β-ZER, zearelone), (contain potassium dihydrogen phosphate 0.27g in the 1L solution with 10ml PBS, 12 hypophosphite monohydrate disodium hydrogen 2.86g, potassium chloride 0.2g, sodium chloride 8.8g), 15ml water washing IAC post, use the methanol aqueous solution washing of 4ml 30% (volumn concentration) again, remove and disturb impurity in the extract.Use 4ml methyl alcohol with α-Yu Michimeichun, β-ZER, zearelone and α-zearalenol wash-out at last, natural gravity flows out down, collect, dry up, two-trimethyl silane trifluoroacetamide (BSTFA) reagent that adds 0.05ml, whirling motion, 60 ℃ of derivatization 15min carry out GC/MS and measure.Calculate dynamic column capacity and absolute column capacity.Dynamically column capacity (dynamic column capacity) is meant the obtained the maximum absorption of every milliliter of immunosorbent (or bed volume) to determinand.Absolute column capacity (specific column capacity) is meant the maximum binding capacity of every milligram of sessile antibody to determinand.The result shows that coupling has pair α-Yu Michimeichun, β-ZER, zearelone and α-zearalenol all have the α-Yu Michimeichun of immune chromatograph post of monoclonal antibody of the monoclonal hybridoma strain A-1-1 CGMCCNo.1603 secretion of cross reaction, β-ZER, the dynamic column capacity of zearelone and α-zearalenol is respectively 2640,2840,2731,2736ng/mL, this immune chromatograph post is to α-Yu Michimeichun, β-ZER, the absolute column capacity of zearelone and α-zearalenol is respectively 377,405,390,391ng/mg.
1, purifies the preparation of the kit of α-Yu Michimeichun and/or β-ZER and/or zearelone and/or α-zearalenol
Mainly by box body, immune chromatograph post (IAC post), standard α-Yu Michimeichun reagent, cleaning solution, eluent is preserved liquid, and the sponge carriage is formed, and the sponge carriage is provided with hole and groove.Standard α-Yu Michimeichun reagent is housed in the groove of sponge carriage, cleaning solution, eluent, the reagent bottle of preservation liquid is equipped with the IAC post in the hole of sponge carriage.Wherein the immune chromatograph post all has the immune chromatograph post of monoclonal antibody of the monoclonal hybridoma strain A-1-1 CGMCCNo.1603 secretion of cross reaction for the coupling of embodiment 1 preparation has pair α-Yu Michimeichun, β-ZER, zearelone and α-zearalenol.
Cleaning solution is that (0.01M, pH7.4), compound method is phosphate buffer: contain potassium dihydrogen phosphate 0.27g in the 1L solution, 12 hypophosphite monohydrate disodium hydrogen 2.86g, potassium chloride 0.2g, sodium chloride 8.8g.
Eluent is chromatographic grade methyl alcohol.
Preserving liquid is to contain potassium dihydrogen phosphate 0.27g in the 1L solution, 12 hypophosphite monohydrate disodium hydrogen 2.86g, potassium chloride 0.2g, sodium chloride 8.8g, NaN
30.2g, pH7.4.
It is 12 months that the kit of above-mentioned immune chromatograph post is placed on 4 ℃ of terms of validity respectively.
2, the clean-up effect of α-Yu Michimeichun, β-ZER, zearelone and α-zearalenol experiment
IAC extracts principle, the monoclonal antibody and the inert base coupling that specific antibody are all had the monoclonal hybridoma strain A-1-1 CGMCC No.1603 secretion of cross reaction to α-Yu Michimeichun, β-ZER, zearelone and α-zearalenol, the preparation immunosorbent, the dress post.When containing α-Yu Michimeichun or/and β-ZER or/and zearelone or/and the mixture of α-zearalenol when flowing through the IAC post, sessile antibody is optionally in conjunction with α-Yu Michimeichun and/or β-ZER and/or zearelone and/or α-zearalenol, the sample impurity that other is not identified then flows out the IAC post in the clear, after washing, with the antigen-antibody complex wash-out that dissociates, α-Yu Michimeichun and/or β-ZER and/or zearelone and/or α-zearalenol are purified or separate.The IAC post is reusable after regeneration is handled.
The processing of test sample: get each 5g of ox muscle samples (taking from the ox of not contacted α-Yu Michimeichun class material) in the 50ml plastic centrifuge tube, each sample adds α-Yu Michimeichun, β-ZER, zearelone and α-zearalenol standard items respectively by 2.5 μ g/kg concentration, add 5mL methyl alcohol more respectively, whirling motion 1min, the centrifugal 10min of 3500g, supernatant is changed in another clean centrifuge tube, repeat to extract, merge supernatant.Put into-20 ℃ of refrigerator-freezer 1hr, cross 0.25 μ M miillpore filter.Got the cleaning solution mixing in coating solution 5mL and the 20mL mentioned reagent box, standby.
There are pair α-Yu Michimeichun, β-ZER, zearelone and α-zearalenol all to have the immune chromatograph column equilibration of monoclonal antibody of monoclonal hybridoma strain A-1-1 CGMCC No.1603 secretion of cross reaction the coupling of above-mentioned preparation to room temperature, wash with the cleaning solution in the 10ml mentioned reagent box, then above-mentioned sample solution is crossed post, use the 15ml water washing, and then the washing of the methanol aqueous solution of adding 4ml30% (volumn concentration), purpose is in order to remove the impurity of non-specific adsorption.With 4ml eluent wash-out, at 50 ℃ of N
2Dry up under the air-flow, add 50 μ l derivatization reagent BSTFA then, whirling motion, 60 ℃ of derivatizations 15 minutes, carrying out GC/MS then and analyze, measure the content of α-Yu Michimeichun, is contrast with α-Yu Michimeichun, β-ZER, zearelone and α-zearalenol standard items.The IAC post is kept in 4 ℃ of refrigerators standby with the preservation liquid balance of 20ml.Measurement result shows, carries out sample purification with IAC, and the interference medicament chromatographic peak can not separate fully, illustrates that the IAC non-specific adsorption of preparation is minimum.Wherein coupling have pair α-Yu Michimeichun, β-ZER, zearelone and α-zearalenol all have cross reaction monoclonal hybridoma strain A-1-1 CGMCC No.1603 secretion monoclonal antibody the immune chromatograph post for the clean-up effect in the ox muscle samples of α-Yu Michimeichun, β-ZER, zearelone and the α-zearalenol standard items that add 2.5 μ g/kg concentration as shown in Figure 1,1 is zearelone among the figure, 2 is α-Yu Michimeichun, 3 is β-ZER, and 4 is α-zearalenol; A is for adding α-Yu Michimeichun, β-ZER, zearelone and α-zearalenol beef sample (each 2.5ng/g), and B is α-Yu Michimeichun, β-ZER, zearelone and α-zearalenol standard items (each 2.0ng/g).
Claims (10)
1. an immune affinity sorbent that purifies α-Yu Michimeichun and/or β-ZER and/or zearelone and/or α-zearalenol is formed by solid phase carrier with the α-Yu Michimeichun monoclonal antibody of its coupling; Described α-Yu Michimeichun monoclonal antibody is that the conjugate with α-Yu Michimeichun haptens and carrier protein is that immunogene obtains; Described α-Yu Michimeichun haptens is that α-Yu Michimeichun and succinyl oxide are reacted the ZER-7-hemisuccinic acid ester that obtains, and is the α-Yu Michimeichun haptens.
2, adsorbent according to claim 1 is characterized in that: described solid phase carrier is cellulose, sephadex, polyacrylamide gel, cellular glass, Ago-Gel or ultragel ACA22, is preferably Sepharose 4B; Described α-Yu Michimeichun monoclonal antibody is the α-Yu Michimeichun mouse monoclonal antibody.
3, adsorbent according to claim 2 is characterized in that: the monoclonal antibody that described α-Yu Michimeichun mouse monoclonal antibody is secreted for the monoclonal hybridoma strain A-1-1 CGMCC No.1603 that α-Yu Michimeichun, β-ZER, zearelone and α-zearalenol is all had cross reaction.
4, adsorbent according to claim 1 is characterized in that: described carrier protein is bovine serum albumin(BSA) or ovalbumin.
5, be mounted with the immune affinity chromatographic column of arbitrary described immune affinity sorbent among the claim 1-4.
6, contain the kit of arbitrary described immune affinity sorbent among the claim 1-4 or contain the kit of the described immune affinity chromatographic column of claim 5.
7, according to the described kit of claim 6, it is characterized in that: comprise also in the described kit that eluent, described eluent are methyl alcohol.
8, according to the described kit of claim 7, it is characterized in that: also comprise cleaning solution in the described kit, preserve liquid; Described cleaning solution is pH7.4, and the phosphate buffer of 0.01mol/L, described 0.01mol/L phosphate buffer are to contain potassium dihydrogen phosphate 0.27g among the 1L, 12 hypophosphite monohydrate disodium hydrogen 2.86g, potassium chloride 0.2g, the solution of sodium chloride 8.8g; Described preservation liquid is to contain potassium dihydrogen phosphate 0.27g in the 1L solution, 12 hypophosphite monohydrate disodium hydrogen 2.86g, potassium chloride 0.2g, sodium chloride 8.8g, NaN
30.2g, pH7.4.
9, a kind of method that purifies α-Yu Michimeichun and/or β-ZER and/or zearelone and/or α-zearalenol may further comprise the steps:
1) pre-treatment of sample:
Animal tissue's sample is added 5mL methyl alcohol with the amount of 5g, the whirling motion mixing, centrifugal 10 minutes of 3500g collects supernatant, place-20 ℃ 1 hour, cross 0.25 μ M miillpore filter and obtain sample solution;
2) sample solution that step 1) is obtained is crossed the described immune affinity chromatographic column of claim 5, then with the described cleaning solution washing of claim 8, use the described eluent wash-out of claim 7 again, the α-Yu Michimeichun that is purified and/or β-ZER and/or zearelone and/or α-zearalenol solution.
10, method according to claim 9 is characterized in that: described animal tissue sample comprises muscle, liver, lung, kidney and blood plasma.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100420946C (en) * | 2006-02-15 | 2008-09-24 | 中国农业大学 | Method for purifying alpha-zeranol and special immunity affinity chromatographic column therefor |
| 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 |
| CN106645697A (en) * | 2016-11-09 | 2017-05-10 | 百奥森（江苏）食品安全科技有限公司 | A kit for detecting zeranol in foods |
| 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 |
| CN113960314A (en) * | 2021-10-15 | 2022-01-21 | 北京勤邦生物技术有限公司 | Zearalanol immunoaffinity column and preparation method and application thereof |
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2006
- 2006-02-15 CN CNB2006100072397A patent/CN100406115C/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100420946C (en) * | 2006-02-15 | 2008-09-24 | 中国农业大学 | Method for purifying alpha-zeranol and special immunity affinity chromatographic column therefor |
| 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 |
| CN106645697A (en) * | 2016-11-09 | 2017-05-10 | 百奥森（江苏）食品安全科技有限公司 | A kit for detecting zeranol in foods |
| 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 |
| CN113960314A (en) * | 2021-10-15 | 2022-01-21 | 北京勤邦生物技术有限公司 | Zearalanol immunoaffinity column and preparation method and application thereof |
| 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 |
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