CN103801110A - Preparation method of deoxynivalenol immunoaffinity column - Google Patents
Preparation method of deoxynivalenol immunoaffinity column Download PDFInfo
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- CN103801110A CN103801110A CN201210439205.0A CN201210439205A CN103801110A CN 103801110 A CN103801110 A CN 103801110A CN 201210439205 A CN201210439205 A CN 201210439205A CN 103801110 A CN103801110 A CN 103801110A
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Abstract
The invention discloses a preparation method of an immunoaffinity column based on deoxynivalenol monoclonal antibodies. An affinity adsorbent obtained through coupling of monoclonal antibodies obtained through cell fusion and cyanogen bromide activated agarose 4B is filled in a solid phase extraction tube to obtain the immunoaffinity column capable of specific adsorption of deoxynivalenol. The immunoaffinity column prepared by the invention can specifically combine with deoxynivalenol, has a maximal binding capacity of about 240ngDON, and reaches recovery rate of no less than 90% for three times of repeated usage. The invention can be used in the pretreatment method of detection of deoxynivalenol residues in cereals and cereal products.
Description
Technical field
The invention belongs to detection technique field, relate to particularly a kind of preparation method of immune affinity column of vomitoxin.
Background technology
Vomitoxin, claims again deoxynivalenol bacterium refining alcohol (Deoxynivalenol, DON), is that the one separated from the corn being polluted by Fusarium graminearum in 1970 can nauseant toxin, mainly by Fusarium graminearum (
fusarium graminearum) produce, belong to the mould refining compounds of group of single-ended spore Type B.More stable to heat, the general cooking and heating all can not destroy its toxicity, more responsive to alkaline environment, wash grain with sodium carbonate liquor, can remove the DON of 70% left and right.
After animal DON is poisoning, show as vomiting, anorexia, Body weight loss, miscarriage, weak son and stillborn foetus, the death rate increases.After mankind DON is poisoning, there will be nauseating, vomit, have a stomach upset, suffer from abdominal pain, suffer from diarrhoea, have a headache, a Halo, also may have the symptom of dry, general weakness, small number of patients there will be Blushing, fever.DON also has the harm such as very strong carcinogenic, teratogenesis, mutagenic toxicity, immunotoxicity, and food and agricultural organization of united state (FAO) and the World Health Organization (WHO) are defined as one of the most dangerous naturally-occurring food contaminant.1998, in the appraisal report of announcing in international cancer research institution, vomitoxin was listed in three class carcinogenic substances.
Many countries have formulated the limit standard of vomitoxin in cereal, and the U.S. formulates edible wheat vomitoxin Permissible limit≤2 mg/kg for the mankind, Permissible limit≤0.75 mg/kg in European Union's regulation flour and corn flour.The limitation of the deoxynivalenol in China GB 2715-2005 " grain sanitary standard " in regulation wheat, barley, corn and finished product of grain thereof is 1000 μ g/kg, and the limitation of the deoxynivalenol of GB 2761-2005 " mycotoxin limitation in food " regulation wheat and maize is 1000 μ g/kg.
The conventional detection of vomitoxin mostly at present is instrument detection method, as high performance liquid chromatography, LC-MS (LC-MS), gas chromatography mass spectrometry (GC-MS) etc.Instrumental method detection has highly sensitive, the advantage of high specificity, but the purity requirement to sample is higher, need to pass through complicated purification process, traditional Sample pretreatment technology, as liquid-liquid extraction, SPE, solid phase microextraction etc., complex steps, lack and selectively, be easily subject to that other compositions disturb, the degree of accuracy is low.And vomitoxin toxicity is violent, in pretreatment process, operating personnel are exposed in the environment of vomitoxin repeatedly, have increased the risk of experiment.Therefore set up Sample Pretreatment Technique efficiently and effectively and become problem demanding prompt solution in vomitoxin detection analysis.
Immune affinity chromatographic column (Immunoaffinity Chromatography, IAC) be a kind of novel Sample pretreatment technology, it is that immune response is combined with chromatogram analysis method, utilize high degree of specificity and the affinity of antigen-antibody combination, specific antigen or antibody are attached on chromatography carrier by suitable method, to effectively separate and the purifying immune substance of complementation separately.Extensive use in the analysis of antibody, hormone, polypeptide, virus and subcellular fraction compound at present.The present invention aims to provide a kind of preparation method of vomitoxin immune affinity column, as the pre-treating method of cereal and cereal products detection vomitoxin.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of vomitoxin immune affinity column, the affinity column of developing is to be filled in solid-phase extraction column after adopting sepharose 4B (CNBr-Sepharose 4B) coupling of vomitoxin monoclonal antibody and cyanogen bromide-activated, the testing sample that contains vomitoxin for purifying.
The preparation method of vomitoxin immune affinity column of the present invention, carries out according to following step:
(1) coupling: get vomitoxin monoclonal antibody after purifying and swelling after CNBr-Sepharose 4B gel be jointly positioned in coupling buffer, under room temperature, shake coupling;
(2) sealing: add the sealing buffer solution of its 5～10 times of volumes in coupling gel product, concussion reaction 2h under room temperature;
(3) washing: alternately wash 3 times with acetate buffer and the Tris-HCL buffer solution of gel volume after 5～10 times of sealings respectively, then wash 2 times with the PBS buffer solution of gel volume after 5～10 times of sealings, prepare immune affinity sorbent;
(4) dress post: the immune affinity sorbent obtaining is filled in solid-phase extraction column, adds natural subsidence after PBS buffer solution, obtain vomitoxin immune affinity column, 4 degree are preserved stand-by.
Described solid phase carrier can be cellulose, sephadex, acrylamide gel, Ago-Gel etc., is preferably Sepharose 4B.
The mass ratio of described vomitoxin monoclonal antibody and CNBr-Sepharose 4B dry powder is 1:80; Described coupling buffer is 0.1 mol/L NaHCO3 solution; The buffer solution that dissolves CNBr-Sepharose 4B dry powder is 1.0 mmol/L HCL.
Described sealing buffer solution is 0.1 mol/L Tris-HCL(pH 8.0), in order to seal the activation site of not coupling.
Described acetate buffer is the acetate buffer of 0.1 mol/L, pH4.0; The Tris-HCL buffer solution that described Tris-HCL buffer solution is 0.1 mol/L, pH8.0.
Vomitoxin immune affinity column prepared by the present invention can detect the residual pre-treating method of vomitoxin for cereal and cereal products.There is following distinguishing feature: the probability that 1) has reduced operating personnel and touch vomitoxin; 2) be combined with vomitoxin by specific antibody, primary purification can be removed most chaff interferences; 3) prepared immune affinity column maximum binding capacity is 240ng DON; 4) prepared immune affinity column is reused three times, and the rate of recovery is not less than 90%.
The specific embodiment
Further set forth the present invention below in conjunction with specific embodiment.These embodiment are only for the present invention is described, and are not used for limiting the scope of the invention.
The preparation of embodiment 1 vomitoxin immune affinity column
(1) coupling
Get the monoclonal antibody for vomitoxin of 10mg after caprylic acid-ammonium purifying dialysed overnight in 0.1 mol/L NaHCO3 solution, concentration is adjusted to 2mg/mL;
Take the dry glue 800mg of CNBr-Sepharose 4B, fully swelling in 10mL 1.0 mmol/L HCL; The 0.1 mol/L NaHCO3 solution washing amassing with 10 times of above wet colloids 3 times, the centrifugal 1min of 5000rpm;
To wet glue and antibody fully mixes, and under room temperature, stirs and spends the night;
With 50mL 0.1 mol/L NaHCO3 solution washing, collect cleaning solution, ultraviolet is identified, calculates coupling rate.The computing formula of coupling rate is:
The testing result of coupling rate shows, the coupling rate of vomitoxin monoclonal antibody and CNBr-Sepharose 4B is 92.45%.
(2) sealing
Jel product after above-mentioned coupling is proceeded to 0.1 mol/L Tris-HCL(pH 8.0) buffer solution, slowly stirs 2h, in order to seal the activation site of not coupling under room temperature.
(3) washing
Use respectively acetate buffer (0.1 mol/L, pH4.0) and the Tris-HCL buffer solution (0.1 mol/L, pH8.0) of gel volume after 5～10 times of sealings alternately to wash 3 times, then with the PBS buffer solution washing (0.1 mol/L, pH7.4) of gel volume after 5～10 times of sealings 2 times.
(4) dress post
The immune affinity sorbent obtaining is filled in solid-phase extraction column, adds natural subsidence after PBS buffer solution, obtain vomitoxin immune affinity column, 4 degree are preserved stand-by.
The mensuration of the maximum column capacity of immune affinity column: prepare three vomitoxin immune affinity columns, the standard solution that contains 50ng/mL vomitoxin with 10mL is respectively added on immune affinity column continuously, utilize under natural gravity and flow out, collect respectively efflux, every pipe 1mL, with indirect competition ELISA mensuration vomitoxin content.Found that in 1～4 pipe and only have a small amount of vomitoxin, the 5th pipe starts vomitoxin content to be increased gradually, does not absorb since the 7th pipe.The maximum column capacity of the immune affinity column therefore obtaining is about 240ng.As shown in Figure 1.
Determining of immune affinity column repeatability: prepare three vomitoxin immune affinity columns, repeat respectively loading, washing, wash-out, regenerative operation, repeat every other day test, then measure column capacity and the rate of recovery of immune affinity column with competitive ELISA.Result is as shown in table 1, and prepared immune affinity column is reused three times, column capacity 20% left and right that declines, and the rate of recovery is still higher than 90%.
The mensuration of table 1 vomitoxin immune affinity column reusability
| Loading number of times | 1 | 2 | 3 | 4 | 5 |
| Column capacity (ng) | 245±3 | 220±5 | 196±4 | 153±5 | 98±8 |
| The rate of recovery (%) | 98.7±1.8 | 93.2±2.6 | 90.5±5.7 | 82.1±6.4 | 67.9±6.4 |
Claims (6)
1. a preparation for vomitoxin immune affinity column, is characterized in that, is made up of the vomitoxin monoclonal antibody of vomitting of solid phase carrier and expection coupling, and concrete steps are:
(1) coupling: get vomitoxin monoclonal antibody after purifying and swelling after CNBr-Sepharose 4B gel be jointly positioned in coupling buffer, under room temperature, shake coupling;
(2) sealing: add the sealing buffer solution of its 5～10 times of volumes in coupling gel product, concussion reaction 2h under room temperature;
(3) washing: alternately wash 3 times with acetate buffer and the Tris-HCL buffer solution of gel volume after 5～10 times of sealings respectively, then wash 2 times with the PBS buffer solution of gel volume after 5～10 times of sealings, prepare immune affinity sorbent;
(4) dress post: the immune affinity sorbent obtaining is filled in solid-phase extraction column, adds natural subsidence after PBS buffer solution, obtain vomitoxin immune affinity column, 4 degree are preserved stand-by.
2. the preparation method of vomitoxin immune affinity column according to claim 1, is characterized in that described solid phase carrier is CNBr-Sepharose 4B.
3. the preparation method of vomitoxin immune affinity column according to claim 1, is characterized in that described vomitoxin monoclonal antibody and the mass ratio of CNBr-Sepharose 4B dry powder are 1:80; Described coupling buffer is 0.1 mol/L NaHCO3 solution; The buffer solution that dissolves CNBr-Sepharose 4B dry powder is 1.0 mmol/L HCL.
4. the preparation method of vomitoxin immune affinity column according to claim 1, is characterized in that described sealing buffer solution is 0.1 mol/L Tris-HCL(pH 8.0), to seal the activation site of not coupling.
5. the preparation method of vomitoxin immune affinity column according to claim 1, is characterized in that wherein the acetate buffer described in step (3) is the acetate buffer of 0.1 mol/L, pH4.0; The Tris-HCL buffer solution that described Tris-HCL buffer solution is 0.1 mol/L, pH8.0.
6. vomitoxin immune affinity column can be used for cereal and the residual pre-treating method of cereal products detection vomitoxin.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104383718A (en) * | 2014-12-04 | 2015-03-04 | 江西省农业科学院农产品质量安全与标准研究所 | Preparation method of kanamycin aptamer affinity column |
| CN104645666A (en) * | 2014-11-15 | 2015-05-27 | 于秋香 | Trichothecin purification column and purification method thereof |
| CN104707362A (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 affinity column for purifying 3-acetyl deoxynivalenol, aflatoxin, ochratoxin A and zearalenone |
| CN107262074A (en) * | 2017-01-23 | 2017-10-20 | 北京美正生物科技有限公司 | A kind of deoxynivalenol aptamers affinity column and its production and use |
| CN107807034A (en) * | 2017-10-31 | 2018-03-16 | 北京农业质量标准与检测技术研究中心 | A kind of vomitoxin aptamer affinity column and preparation method and application |
| CN110196321A (en) * | 2019-06-14 | 2019-09-03 | 中国计量科学研究院 | OLA and MQCA immune affinity column and the preparation method and application thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104645666A (en) * | 2014-11-15 | 2015-05-27 | 于秋香 | Trichothecin purification column and purification method thereof |
| CN104645666B (en) * | 2014-11-15 | 2016-09-28 | 于秋香 | A kind of nitric acid oxidation processing method of nano-graphite carbon black |
| CN104383718A (en) * | 2014-12-04 | 2015-03-04 | 江西省农业科学院农产品质量安全与标准研究所 | Preparation method of kanamycin aptamer affinity column |
| CN104383718B (en) * | 2014-12-04 | 2016-04-06 | 江西省农业科学院农产品质量安全与标准研究所 | The preparation method of kanamycins aptamers affinity column |
| CN104707362A (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 affinity column for purifying 3-acetyl deoxynivalenol, aflatoxin, ochratoxin A and zearalenone |
| CN107262074A (en) * | 2017-01-23 | 2017-10-20 | 北京美正生物科技有限公司 | A kind of deoxynivalenol aptamers affinity column and its production and use |
| CN107807034A (en) * | 2017-10-31 | 2018-03-16 | 北京农业质量标准与检测技术研究中心 | A kind of vomitoxin aptamer affinity column and preparation method and application |
| CN110196321A (en) * | 2019-06-14 | 2019-09-03 | 中国计量科学研究院 | OLA and MQCA immune affinity column and the preparation method and application thereof |
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Application publication date: 20140521 |