CN102183601A - Preparation method of metandienone polyclonal antibody immuno-affinity column and application thereof - Google Patents
Preparation method of metandienone polyclonal antibody immuno-affinity column and application thereof Download PDFInfo
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- CN102183601A CN102183601A CN2011100009356A CN201110000935A CN102183601A CN 102183601 A CN102183601 A CN 102183601A CN 2011100009356 A CN2011100009356 A CN 2011100009356A CN 201110000935 A CN201110000935 A CN 201110000935A CN 102183601 A CN102183601 A CN 102183601A
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Abstract
The invention relates to a preparation method of a metandienone polyclonal antibody immuno-affinity column and an application thereof, and belongs to the technical fields of immuno-affinity chromatography and veterinary drug residue detection. A preparation method and an application of a metandienone polyclonal antibody immuno-affinity column are disclosed. A immuno-affinity column with specific adsorption to metandienone is prepared by purifying a polyclonal antibody obtained from an immunized animal, coupling the antibody with CNBr-Sepharose4B to obtain an immuno-affinity adsorbent, and filling a solid-phase extraction tube with the adsorbent. The immuno-affinity column prepared by the invention can specifically bind to metandienone with a maximal binding capacity of 160 ng DMT; when an eluate of an 80% methanol-aqueous solution is used, the average recovery rate of standard addition is 97.87%; after repeated use for 3 times, the recovery rate is not less than 90%. The invention is applicable to the rapid detection of metandienone.
Description
Technical field
The invention belongs to immunoaffinity chromatography and detection of veterinary drugs in food technical field, relate to the preparation method and the application thereof of protobolin polyclonal antibody affinity column.
Background technology
(1-dehydro-17-methyltestosterone DMT), is a kind of protein assimilation male steroid hormone to protobolin.Because it has effects such as strong promotion albumen synthesizes, whets the appetite, improves food conversion ratio, and often illegally is used for the herding production field.After DMT is absorbed by the body, can disturb the hormonal balance of human body self, produce serious toxic and side effect.
At present, the detection method that DMT is commonly used mostly is the instrument detecting method, as high performance liquid chromatography (HPLC), LC-MS (LC-MS) and gas chromatography mass spectrometry (GC-MS) etc.These instrument detecting methods are had relatively high expectations to sample purity, need through certain pre-treatment, and traditional sample pre-treatments technology, as liquid-liquid extraction (LLE), Solid-Phase Extraction (SPE), solid phase micro extraction (SPME) etc., complex steps, lack selectivity, be subjected to the interference of other compositions, accuracy low easily.Therefore, setting up efficiently and effectively, the sample pre-treatments technology has become the matter of utmost importance that needs solution in the DMT check and analysis.
Immune affinity chromatographic (IAC) is a kind of new and effective sample pre-treatments technology.Its principle is to utilize specific reversible combination between antigen-antibody to realize extraction and purified treatment to the target substance in the complex sample.In recent years, the immune affinity chromatographic technology has become a main developing direction of detection of veterinary drugs in food in conjunction with conventional instrumental analysis.But have not yet to see the preparation of DMT polyclonal antibody affinity column and the report of use thereof.
Summary of the invention
The object of the present invention is to provide a kind of preparation and using method of DMT immune affinity column, the affinity column of being developed is that the sepharose 4B of DMT polyclonal antibody and cyanogen bromide-activated (CNBr-Sepharose 4B) coupling is filled in the solid phase extraction tube, is used for the testing sample that purifying contains DMT.
The preparation method of DMT polyclonal antibody affinity column of the present invention is prepared according to following step:
(1) coupling: get immune animal and purified DMT polyclonal antibody and the CNBr-Sepharose 4B wet gel after the swelling and place the coupling buffer coupling of at room temperature vibrating;
(2) sealing: the coupling gel product is changed in the sealing damping fluid of its 5-10 times volume over to oscillating reactions 2 h under the room temperature;
(3) washing: the acetate buffer and the Tris-HCl damping fluid of gel volume alternately wash 3 times after doubly sealing with 5-10 respectively earlier, and the PBS damping fluid of gel volume washs 2 times after doubly sealing with 5-10 then, prepares immune affinity sorbent;
(4) dress post: the immune affinity sorbent that obtains is packed in the solid phase extraction tube, and natural subsidence after the adding PBS damping fluid is made DMT polyclonal antibody affinity column, and is stand-by in 4 ℃ of preservations;
Wherein the mass ratio of DMT polyclonal antibody described in the step (1) and the dried glue of CNBr-Sepharose 4B is 1:100; Described coupling buffer is pH 8.3, contain the 0.1 mol/L NaHCO of 0.5 mol/L NaCl
3Coupling buffer; The solution of described swelling CNBr-Sepharose 4B is 1 mmol/L HCl solution;
Wherein the sealing damping fluid described in the step (2) is pH 8.0, contains the 0.1 mol/L Tris-HCl sealing damping fluid of 0.5 mol/L NaCl;
Wherein the acetate buffer described in the step (3) is pH 4.0, contains the 0.1 mol/L acetate buffer of 0.5 mol/L NaCl; Described Tris-HCl damping fluid is pH 8.0, contain the 0.1 mol/L Tris-HCl damping fluid of 0.5 mol/L NaCl; Described PBS damping fluid is the PBS damping fluid of pH 7.4,0.01 mol/L;
The application of protobolin polyclonal antibody immunoaffinity column of the present invention can be applied to the fast detecting of protobolin.
Beneficial effect of the present invention: the immune affinity column of (1) the present invention preparation can combine with the DMT specificity, and primary purification can be removed most chaff interferences, can obtain the higher DMT of purity.(2) Zhi Bei immune affinity column maximum binding capacity is about 160 ng DMT; When (3) eluent of Cai Yonging was the methanol-water solution of 80 %, average recovery of standard addition was 97.87 %; (4) immune affinity column of preparation is reused 3 times, and the recovery is not less than 90 %.
Description of drawings
Fig. 1 wherein. IAC post outside drawing of the present invention;
Fig. 2. IAC post elution requirement of the present invention selects to optimize figure;
Fig. 3. IAC post maximum binding capacity figure of the present invention;
Fig. 4. IAC post icELISA evaluation method canonical plotting of the present invention;
Fig. 5. IAC post HPLC evaluation method canonical plotting of the present invention.
Embodiment
The following examples of the present invention are only as the further specifying of content of the present invention, can not be as scope perhaps in the qualification of the present invention.The invention will be further described below by embodiment.
The preparation of embodiment 1 DMT polyclonal antibody immunoaffinity column
(1) coupling
The DMT polyclonal antibody of gained behind immune animal, the purifying is placed pH 8.3, contains the 0.1 mol/L NaHCO of 0.5 mol/L NaCl
3Dialysed overnight in the coupling buffer, and be diluted to behind 0.5 mg/mL standby;
Take by weighing the dried glue of CNBr-Sepharose 4B, abundant swelling obtains the wet glue of certain volume in the 1 mmol/L HCl solution of 5-10 mL.Wash 2 times centrifugal 1 min of 4000 r/min fast with the long-pending coupling buffer of wet colloid more than 10 times;
Wet glue after the washing is settled to 2 mL, adds the antibody-solutions of 0.5 mg/mL rapidly, the mass ratio that makes dried glue of CNBr-Sepharose 4B and antibody is 100:1, more than 2.5 h of oscillating reactions under the room temperature;
After coupling reaction finishes, remove the antibody of not coupling, collect cleansing solution, using formula C with the coupling buffer washing of coupling gel volume more than 10 times
Protein(mg/mL)=1.45 A
280 nm-0.74 A
260 nmCalculate the antibody amount of not coupling, the coupling rate that obtains is more than 90 %;
(2) sealing
With the coupling gel product of CNBr-Sepharose 4B and antibody change over to its 5-10 times volume pH 8.0, contain in the 0.1 mol/L Tris-HCl sealing damping fluid of 0.5 mol/L NaCl oscillating reactions 2 h sealing reactive group under the room temperature;
(3) washing
Successively alternately wash 3 times, then with the PBS washing of pH 7.4,0.01 mol/L of 5-10 times of coupling gel volume 2 times with the pH 4.0 of 5-10 times of coupling gel volume, the 0.1 mol/L acetate buffer that contains 0.5 mol/L NaCl, pH 8.0, the 0.1 mol/L Tris-HCl that contains 0.5 mol/L NaCl;
(4) dress post
With the affinity adsorbent that obtains be packed into its volume ratio be in the solid phase extraction tube of 4:1, make DMT polyclonal antibody affinity column, preserve stand-by in 4 ℃ of refrigerators.Prepared immune affinity column outside drawing is seen Figure of description 1.What show among the figure is the IAC post that has loaded above affinity adsorbent, and pillar is made up of two parts: shaft and sleeve pipe.Liquid wherein is PBS.
The condition optimizing method of test one, DMT polyclonal antibody immunoaffinity column of the present invention:
1, determining of embodiment 1 preparation-obtained DMT polyclonal antibody immunoaffinity column optimum washing engaging condition: prepare 5 IAC posts as stated above, the DMT solution of sample 100 ng on every post, after PBS fully washs, use 60 % respectively, 70 %, 80 %, 90 %, 100 % methanol-water eluant solutions, collecting eluent is spin-dried on 50 ℃ Rotary Evaporators, standard items content in the eluent is measured with the HPLC method in methyl alcohol redissolution back with 1 mL, calculate recovery rate, when methanol concentration during greater than 80 %, the recovery all more than 95 %, therefore selects the methanol-water solution of 80 less % of organic solvent concentration to be best elution requirement.Elution requirement selects optimization figure to see Figure of description 2;
2, the mensuration of embodiment 1 preparation-obtained DMT polyclonal antibody immunoaffinity column maximum binding capacity: prepare two IAC posts, use the DMT of 6 mL, 50 ng/mL (300 ng) to cross post respectively, the every mL of sample solution collects a pipe, measures DMT content in every component with indirect competitive ELISA (icELISA).Calculate according to following formula: the DMT total amount of maximum binding capacity (the DMT total amount that promptly the is adsorbed)=last sample total amount of DMT-wash.Found that in the 1-3 mL sample solution and have only a small amount of DMT then have greater than 30 ng during last sample the 4th mL to be washed, 5 mL begin not have absorption.Therefore, the maximum binding capacity of the coupling 1.0 mg IAC posts that resist is about 160 ng more.Binding capacity is determined shown in Figure of description 3;
3, the mensuration of embodiment 3 preparation-obtained DMT polyclonal antibody immunoaffinity column recovery of standard addition: go up 20 ng/mL, 40 ng/mL of sample 2.5 mL, the DMT of three kinds of variable concentrations of 60 ng/mL respectively, every kind of concentration do two groups parallel.Measure the recovery with HPLC after collecting cleansing solution.The results are shown in Table 1.Behind suitable eluent wash-out, the recovery all reaches more than 90 % after measured, and average recovery rate is 97.87 %;
The recovery of standard addition of table 1. IAC post
| Mark product concentration (ng/mL) | Mark product additions (ng) | The recovery (%) |
| 20 | 50 | 105.83 |
| 40 | 100 | 95.50 |
| 60 | 150 | 92.28 |
4, embodiment 4 preparation-obtained DMT polyclonal antibody immunoaffinity columns are reused determining of number of times: prepare two IAC posts, repeat sample, washing, wash-out, regenerative operation respectively, every 1-2 days revision tests, measure the column capacity and the recovery of IAC post more respectively with icELISA and HPLC.The result is as shown in table 2, and this post is reused 3 times, and this moment, column capacity descended about 25 %, and the recovery still is higher than 90 %.
The mensuration of the reusability of table 2. IAC post
| Last sample number of |
1 | 2 | 3 | 4 | 5 |
| Column capacity (ng) | 167.01 | 143.76 | 126.95 | 83.50 | 54.25 |
| The recovery (%) | 101.70 | 96.64 | 92.55 | 89.07 | 53.52 |
The method of evaluating performance of test two, DMT polyclonal antibody immunoaffinity column of the present invention:
(1) foundation of icELISA evaluation method
After bag is closed, the standard solution that adds earlier 50 μ L series concentration (0.001,0.01,0.1,1.0,2.5,5.0,10., 25,50,100,500 ng/mL), every kind of 2 of concentration are parallel, add one of 50 μ L optimum diluting multiples again and resist, the absorbance A of each concentration at 450 nm measured with enzyme mark detector in colour developing sealing back
iAnd the absorbance A of contrast when being 0 ng/mL (be concentration)
0, with A
i/ A
0Be ordinate, the logarithm value of concentration of standard solution is the horizontal ordinate mapping.After the match of Origin software, the range of linearity that obtains typical curve is 1-100 ng/mL.This method standard curve map is seen Figure of description 4;
(2) foundation of HPLC evaluation method
The chromatographic condition of HPLC: Shim-pack VP-ODS chromatographic column (250 * 4.6 mm); Moving phase: the V(acetonitrile): V(water)=80:20; The UV detecting device detects wavelength: λ=250 nm; Column temperature: 25 ℃; Sample size: 20 μ L; Flow velocity: 0.8 mL/min.
Get standard operation liquid (1 mg/mL), be mixed with series concentration (5,10,50,100,200,300 ng/mL), measure, carry out linear fit with each concentration and its corresponding peak area with HPLC.This typical curve linear relationship is better, and its equation of linear regression is: y=1443.3x-1207.2 (R
2=0.9999), the range of linearity is 5-300 ng/mL.This method standard curve map is seen Figure of description 5.
Being preferred embodiment of the present invention only in sum, is not to be used for limiting practical range of the present invention.Be that all equivalences of doing according to the content of the present patent application claim change and modification, all should be technology category of the present invention.
Claims (5)
1. the preparation method of DMT polyclonal antibody affinity column of the present invention is prepared according to following step:
(1) coupling: get immune animal and purified DMT polyclonal antibody and the CNBr-Sepharose 4B wet gel after the swelling and place the coupling buffer coupling of at room temperature vibrating;
(2) sealing: the coupling gel product is changed in the sealing damping fluid of its 5-10 times volume over to oscillating reactions 2 h under the room temperature;
(3) washing: the acetate buffer and the Tris-HCl damping fluid of gel volume alternately wash 3 times after doubly sealing with 5-10 respectively earlier, and the PBS damping fluid of gel volume washs 2 times after doubly sealing with 5-10 then, prepares immune affinity sorbent;
(4) dress post: the immune affinity sorbent that obtains is packed in the solid phase extraction tube, and natural subsidence after the adding PBS damping fluid is made DMT polyclonal antibody affinity column, and is stand-by in 4 ℃ of preservations.
2. a kind of protobolin polyclonal antibody affinity column according to claim 1 is characterized in that wherein the mass ratio of the DMT polyclonal antibody described in the step (1) and the dried glue of CNBr-Sepharose 4B is 1:100; Described coupling buffer is pH 8.3, contain the 0.1 mol/L NaHCO of 0.5 mol/L NaCl
3Coupling buffer; The solution of described swelling CNBr-Sepharose 4B is 1 mmol/L HCl solution.
3. a kind of protobolin polyclonal antibody affinity column according to claim 1 is characterized in that wherein the sealing damping fluid described in the step (2) is pH 8.0, contains the 0.1 mol/L Tris-HCl sealing damping fluid of 0.5 mol/L NaCl.
4. a kind of protobolin polyclonal antibody affinity column according to claim 1 is characterized in that wherein the acetate buffer described in the step (3) is pH 4.0, contains the 0.1 mol/L acetate buffer of 0.5 mol/L NaCl; Described Tris-HCl damping fluid is pH 8.0, contain the 0.1 mol/L Tris-HCl damping fluid of 0.5 mol/L NaCl; Described PBS damping fluid is the PBS damping fluid of pH 7.4,0.01 mol/L.
5. the application of protobolin polyclonal antibody affinity column can be applied to the fast detecting of protobolin.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102407098A (en) * | 2011-11-15 | 2012-04-11 | 南昌大学 | Preparation method of immunoaffinity chromatography medium and application in tetraodotoxin purification |
| 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 |
| CN105738628A (en) * | 2014-12-12 | 2016-07-06 | 上海复星长征医学科学有限公司 | Method of purifying goat-anti-human plasma apolipoprotein A-I monoclonal antibody |
| 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 |
| CN111138538A (en) * | 2020-02-25 | 2020-05-12 | 四川农业大学 | Goose pituitary prolactin protein immunoaffinity chromatography column, preparation method and purification method |
-
2011
- 2011-01-05 CN CN2011100009356A patent/CN102183601A/en active Pending
Non-Patent Citations (2)
| Title |
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| 张勋 等: "去氢甲睾酮抗体的制备与鉴定", 《细胞与分子免疫学杂志》 * |
| 赵思俊 等: "免疫亲和色谱-HPLC-FLD法测定动物肝脏中10种喹诺酮类药物残留", 《分析化学》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102407098A (en) * | 2011-11-15 | 2012-04-11 | 南昌大学 | Preparation method of immunoaffinity chromatography medium and application in tetraodotoxin purification |
| CN105738628A (en) * | 2014-12-12 | 2016-07-06 | 上海复星长征医学科学有限公司 | Method of purifying goat-anti-human plasma apolipoprotein A-I monoclonal antibody |
| 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 |
| 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 |
| CN111138538A (en) * | 2020-02-25 | 2020-05-12 | 四川农业大学 | Goose pituitary prolactin protein immunoaffinity chromatography column, preparation method and purification method |
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Application publication date: 20110914 |