CN103100237A - Mini-type efficient ractopamine immunoaffinity purification enriching column preparation and application - Google Patents
Mini-type efficient ractopamine immunoaffinity purification enriching column preparation and application Download PDFInfo
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- CN103100237A CN103100237A CN2012105893140A CN201210589314A CN103100237A CN 103100237 A CN103100237 A CN 103100237A CN 2012105893140 A CN2012105893140 A CN 2012105893140A CN 201210589314 A CN201210589314 A CN 201210589314A CN 103100237 A CN103100237 A CN 103100237A
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Abstract
The invention provides an efficient and accurate enrichment type ractopamine immunoaffinity purification enriching column as well as a preparation method and an application thereof. The immunoaffinity purification enriching column provided by the invention comprises an active agarose filler coupled with a ractopamine specific antibody, and a plastic column in which the affinitive filler is loaed. The ractopamine specific antibody coupled with the affinitive filler is obtained by extracting through using an immunoaffinity method; and the immunoaffinity purification enriching column is obtained by loading the immunoaffinity filler in the special plastic column. The immunoaffinity purification enriching column has small loaded filler volume and strong ractopamine enrichment capacity, is used by combining a chromatography with a colloidal gold test strip, and can be used for efficiently and accurately detecting the content of ractopamine.
Description
Technical field
The present invention relates to a kind of bio-separation, purification enrichment device, especially a kind of preparation method and application thereof of micro high efficiency Ractopamine immune affinity chromatographic column.
Background technology
Ractopamine all belongs to beta-stimulants, can improve lean meat percentage, promote growth of animal.Because of its economic benefit obvious, interests order about the raiser or breeding enterprise uses in a large number, cause in livestock products Ractopamine residual in a large number, after edible these class livestock products of people, can cause muscular tremor, palpitaition, neuroticism, headache, the poisoning symptom such as dizzy, nauseating, particularly larger to blood pressure, cardiac harm, long-term use may cause chromosome aberration, brings out malignant tumour.Ractopamine is in many countries, particularly European Union and China, and being under an embargo is used as the domestic animal growth accelerator.So it is very important that Rct opamine residue in the food of animal origin is strictly detected.
Because substance in biological sample is complicated, testing concentration is low, and most of sampling amounts are few, and China's clear forbids that Ractopamine and derivative thereof use in the feeding process of animal.This just has higher requirement to sensitivity, the degree of accuracy of analytical method.Adopt immune affinity chromatographic column to extract Ractopamine in sample, then prove conclusively with high performance liquid chromatography (HPLC-UV), gas-matter on-line method (GC-UV), the quick ELISA test strip method of collaurum the trend that is inevitable.But, antibody poor specificity, antibody and filler coupling that the technology of existing extraction Ractopamine immune affinity chromatographic column exists unstable, the non-affine method of the rear filler of activation to purify are unstable, cause the antibody that is coupled on filler to come off, fill column volume (the dress column volume is more than 1 milliliter) bigger than normal, cause when extracting Ractopamine that elution volume is large, bioaccumulation efficiency is low, lowered the degree of accuracy and the sensitivity of conclusive evidence.
Summary of the invention
The purpose of this invention is to provide immunoaffinity purification enriching column of a kind of efficient, accurate purification enrichment Ractopamine and its production and use.
The technical scheme that Bian of the present invention gets:
The purpose of this invention is to provide immunoaffinity purification enriching column preparation method and the application thereof of a kind of efficient, specific isolation purifying, enrichment Ractopamine (Sal).
For reaching above-mentioned purpose, the present invention adopts specific reaction and dissociable characteristic Ag+Ab (solid phase) ó Ag-Ab (solid phase) principle of antigen-antibody for 2 times, and is specific as follows:
At first utilize this principle that the Ractopamine acetonyl ester is coupled to and make Sal-Agarose affinity column on activated agarose (Agarose), be used for extracting the Ractopamine specific polyclonal antibody.
Recycle this principle and will extract the anti-Ractopamine specific antibody (Anti-Sal) of gained and be coupled to and make Anti-Sal-Agarose immune affinity column on Agarose, for separating of the Ractopamine in purifying, enrichment sample to be checked.
According to above-mentioned mechanism, the present invention adopts following concrete technical step:
A kind of preparation of Ractopamine immunoaffinity purification enriching column is characterized in that:
1) will be through the Ractopamine specific polyclonal antibody of immunoaffinity purification and the agarose mixing of periodic acid activation, room temperature reaction is 30 minutes on shaking table, then add sodium borohydride reduction at 4 ℃ of refrigerators after standing 30 minutes, clean unconjugated antibody with distilled water, clean filler with phosphate buffer, 10 times of packing volumes/time, wash 4 times, add equal-volume glycerine after the drip-dry phosphate buffer, mixing obtains the affine filler of Ractopamine immunity;
2) filter membrane is loaded onto in the lower end in special plastic column, the affine filler of Ractopamine immunity is packed in post, and 25 μ L/ posts, the filler upper strata covers filter membrane, namely gets described Ractopamine immunoaffinity purification enriching column.
Described Ractopamine immunoaffinity purification enriching column, comprise coupling have immunoaffinity purification the Ractopamine specific polyclonal antibody the agarose filler and load the tailormadepiston post of this filler.
the Ractopamine specific polyclonal antibody of described immunoaffinity purification is to obtain derivative Ractopamine acetyl bromide ester solution with Ractopamine and bromoacetyl chloride reaction, with reverse this solution of high-efficient liquid phase chromatogram HPLC purifying, obtain the Ractopamine acetyl bromide ester after purifying, with Ractopamine acetyl bromide ester and the carrier protein couplet after purifying, obtain this conjugate as the immunogen immune animal, obtain antiserum, the immune affinity column of using with the Ractopamine acetyl bromide ester after this purifying and activated sepharose 4B reaction preparation antibody purification simultaneously, extract antiserum with this one step of affinity column and obtain the Ractopamine specific polyclonal antibody.
Described Ractopamine acetyl bromide ester preparation method obtains derivative Ractopamine acetyl bromide ester solution with the bromoacetyl chloride reaction with the Ractopamine standard items under triethylamine catalysis.This Ractopamine bromoacetyl chloride ester solution is gone out with respect to behind Ractopamine standard items peaks to move with the reverse column chromatography separating purification of preparative high performance liquid chromatography instrument, and the single main peak component with Ractopamine uv absorption spectra feature is collected the Ractopamine acetyl bromide ester that this main peak component obtains purifying.
Described Ractopamine acetyl bromide ester and carrier protein couplet prepare immunogene, with common carrier albumen such as lauryl sodium sulfate SDS denatured bovine serum protein BSA or ovalbumin OVA, be the reaction of NHS-iodoacetic acid with iodoacetic acid-hydroxysuccinimide eater again, with dithiothreitol (DTT) DTT reduction, obtain BSA or the OVA of sulfhydrylation again; After removing unnecessary dithiothreitol (DTT) by sephadex G 25 Sephadex desalinations, Ractopamine acetyl bromide ester is mixed with BSA or the OVA of sulfhydrylation, transfer pH=8.5, shake reaction 60 minutes in room temperature, what obtain after G25 Sephadex desalination is immunogene.
the described immune affinity column of using with the Ractopamine acetyl bromide ester after purifying and activated agarose reaction preparation antibody purification, that activated agarose and lysine reaction are obtained the amino agarose of spending, again with after amidized agarose and the reaction of NHS-iodoacetic acid, reduce with DTT again, obtain the agarose of sulfhydrylation, agarose and the quick mixing of Ractopamine acetyl bromide ester with sulfhydrylation, room temperature reaction 60 minutes, obtain chemical covalent bond complex Ractopamine acetyl bromide-agarose filler, filler is filled post, wash away unnecessary DTT, the immune affinity column that the Ractopamine specific antibody that obtains purifying is used.
The technology of described Ractopamine immunoaffinity purification enriching column and the coupling of Ractopamine colloidal gold strip.
Advantage of the present invention:
1) antibody of this affinity column coupling used is for what purify through immune affine one-step method, and the antibody specificity is high; Adopt high density, large capacity specific antibody to be coupled to the agarose filler of activation, make affinity column antibody of the present invention and filler in conjunction with stable, column capacity, the rate of recovery obviously are better than current similar product, reach despumation and disturb, improve detection accuracy and reliability.
2) this affinity column loads the microminiaturization of filler, and filler dress column volume only needs 25 μ L/ posts, and elution volume is dwindled, and improves very significantly the sensitivity that detects.
3) to reprint the plastic column of filler be special in the present invention, can reach flexible grasp loading volume, improve the effect of column efficiency.
Description of drawings
Fig. 1 is the Ractopamine chromatogram through the preparative high performance liquid chromatography separation and purification, and main compositions derived therefrom is collected the peak.
Fig. 2 is the IC50 figure of Ractopamine polyclonal antibody indirect competitive ELISA.
Fig. 3 is Ractopamine immunoaffinity purification enriching column and Ractopamine colloidal gold strip coupling design sketch, and 1 in figure, 2 is with the negative urine of colloidal gold strip test Ractopamine pig; 2,3 is 0.01ng/mL for adding the Ractopamine standard items to make concentration with the colloidal gold strip detection in the negative urine of Ractopamine; 5,6 to add the Ractopamine standard items to make concentration in the negative urine of Ractopamine be the eluent of 0.01ng/mL after through the enrichment of Ractopamine immunoaffinity purification enriching column in order to detect with colloidal gold strip.
The specific embodiment
It is in order further to understand better the present invention that following embodiment is provided, and never content of the present invention and protection domain is consisted of any restriction.
The preparation of embodiment 1 Ractopamine specific polyclonal antibody.
1.1 the preparation of Ractopamine acetyl bromide fat:
1.1.1 get in dimethyl fumarate (DMF) solution that the Ractopamine of 50 mg is dissolved in 500 μ L, fully dissolved freezing 10 minutes, then add 25 μ L triethylamines to shake up after shaking up freezing 2 minutes with the bromoacetyl chloride of 40 μ L, 60 ℃ of reactions 30 minutes; 50% methyl alcohol that adds 500 μ L.
1.1.2 centrifugal, get supernatant oppositely column chromatography separating purification of preparative high performance liquid chromatography instrument (C-18 HPLC).Collect respectively with respect to the peak that moves behind Ractopamine standard items peaks, and the key component (about 20 mg) of Ractopamine ultraviolet light (UV) absorption spectrum feature, freeze-drying are arranged.
1.1.3 the derivative after the collection freeze-drying redissolves with 1 mL methyl alcohol immediately, with 0.1 mol/L Na
2CO
3Transfer PH〉8.
1.2 Ractopamine immunogene, the preparation of pairing detectable antigens
1.2.1 Ractopamine immunogene
Get 250 mg BSA, add 5 mL 0.1 mol/L Na
2CO
3, 0.5 mL 10% the SDS mixing after be cooled to room temperature after boiling sex change in 5 minutes, add the NHS-iodoacetic acid active ester that 100 mg now make (iodoacetic acid-NHS), 4 ℃ of lucifuges were reacted 30 minutes, desalination immediately, collect 10 mL, then add the dithiothreitol (DTT) (DTT) of 50 mg to keep pH〉8.5 times room temperature lucifuge reactions 60 minutes, then add 50 mg DTT to boil reduction in 5 minutes.Be cooled to room temperature, BSA after reduction removes the Ractopamine acetyl bromide ester 400 μ L through the HPLC purifying that add above-mentioned 1.1.3 after unnecessary DTT through desalting column G25 Sephadex, with 0.1 mol/L Na
2CO
3Transfer pH=8.5, the reaction of room temperature lucifuge generates Ractopamine acetyl bromide-BSA covalent bond complex after 60 minutes, preserves stand-by through G25 Sephadex desalination, packing.
1.2.2 pairing detectable antigens
Get 10 mg ovalbumin OVA, be dissolved in the 0.1 mol/L Na that 400 μ L contain 0.1 mol/L EDTA
2CO
3Solution fully after the dissolving, boils sex change in 5 minutes after adding the SDS mixing of 40 μ L 10%, then adds the DTT of 6 mg to boil reduction in 10 minutes.Be cooled to room temperature, the OVA after reduction adds above-mentioned Ractopamine acetyl bromide ester 200 μ L through the HPLC purifying after desalting column G25 Sephadex removes unnecessary DTT, with 0.1 mol/L Na
2CO
3Transfer pH=8.5, the reaction of room temperature lucifuge generated Ractopamine acetyl bromide-OV covalent bond complex after 60 minutes, and what obtain after G25 Sephadex desalination is the pairing detectable antigens.
1.3 Ractopamine polyclonal antiserum preparation
Be immune animal with new zealand white rabbit, vaccine concentration is 1 mg/mL, first immunisation adds 0.5 mL PBS with 0.5 mL vaccine and adds 1 mL Freund's complete adjuvant mixing multi-point injection, booster immunization adds 0.75 mLPBS with 0.25 mL vaccine and adds 1 mL Freunds incomplete adjuvant mixing and divide 2 injections, after first immunisation, at interval of 2 all booster immunizations once, gather 0.5 mL serum in 35-40 days and detect titre, detect with the coated indirect ELISA of making serum of pairing detectable antigens, serum titer carries out a large amount of serum preparations after reaching 1:6400.
1.4 extract the affinity column preparation that antibody is used
Be dissolved in 10 mL 0.1 mol/L Na 1.4.1 take lysine (Lysine) 2 g
2CO
3In, join the Agarose mixing that 20 mL activate, 60 ℃ were reacted 5 minutes, shake up and again react 10 minutes, turn 4 ℃ standing 20 minutes, add sodium borohydride 100 mg after cooling, the shaking table shaken over night, inferior daily 5 times of packing volumes distillation washing obtains Lysine-Agarose 20 mL.Drain moisture standby.
1.4.2 20 mL Lysine-Agarose are added the 2 saturated Na of mL
2CO
3, PH〉and 8, dropwise add 15 mL acetone on oscillator.
1.4.3 add 100 mg activation iodoacetic acid (now with the current), PH〉shake lucifuge reaction 30 minutes 8.5 time
1.4.4 drain the Lysine-Agarose through the iodoacetic acid activation, the distillation washing with 10 times of Lysine-Agarose volumes is discharged to small beaker to filler, adds with 0.1 mol/L Na
2CO
3DTT solution (200 mg/10 mL) 10 mL of preparation, moment is mixing both, room temperature lucifuge reaction 60 minutes.
1.4.5 with 5 times of packing volume washings, remove remaining DTT, obtain sulfhydrylation filler (SH Agarose)
1.4.6 the Ractopamine acetyl bromide ester 400 μ l through the HPLC purifying of 1.1.3 are splashed in filler, add 0.1 mol/L Na
2CO
35 mL, the reaction of room temperature lucifuge is spent the night, and the dress post after fully cleaning with PBSt, then is neutralized to PH=7 with PBS.
000ne-a:N often improves significantly one step of inspection and extracts the i.e. 1.5 anti-Ractopamine specific antibody preparations of antiserum
The immune serum of 150 mL is flow through the made affinity column of step 1.4.6.After using successively 1 mol/L sodium chloride purged of impurities of the PBSt of 10 times of packing volumes, 10 times of packing volumes, the anti-Anti-ractopamine antibody of the specificity on post is with 2% acetic acid wash-out.The antibody of wash-out removes acetic acid with the bag filter dialysis, and freeze-drying is standby.
Detect through indirect ELISA and indirect competitive ELISA, result shows that the made polyclonal antibody of the present invention tires very highly, and specificity is very high.
Agarose 12 mL of sodium periodate activation change in pillar 2.1 learn from else's experience, and wash with 400 mL distillations, and drip-dry is after 0.1 mol/L Na of 2 times of volumes of Agarose
2CO
3, drip-dry is standby;
2.2 with 1200 mg through the D-tag of immunoaffinity purification specific polyclonal antibody with 0.1 mol/L Na
2CO
3Dissolving makes concentration reach 20 mg/mL, fully centrifuging and taking supernatant after the dissolving;
2.3 supernatant adds in the affinity column of step 2.1, collects efflux and again crosses post, 3-5 time repeatedly;
2.4 with 80 mL distilled water, unconjugated antibody is come out, is collected together;
2.5 be sodium borohydride solution 4.8 mL of 10 mg/mL with the distilled water compound concentration, add in pillar, the vibration mixing, 4 ℃ were reacted 30 minutes;
2.6 the PBS with 10 times of packing volumes washes filler, drip-dry PBS moves into filler in clean test tube, adds the glycerine that equates with packing volume, namely gets Ractopamine immunoaffinity purification enrichment filler.
2.7 filter membrane is loaded onto in the lower end in the tailormadepiston post, Ractopamine immunoaffinity purification enrichment filler is loaded in post, and 25 μ l/ posts, then the filler upper strata covers filter membrane, namely gets described Ractopamine immunoaffinity purification enriching column.
2.8 this immunoaffinity purification enriching column preservation condition is :-20 ℃.
3.1 with known blank (being defined as after testing the Ractopamine feminine gender) urine 2 mL.Adding the Ractopamine standard items to make concentration is 2.5 μ g/mL, 5000 rev/mins centrifugal 10 minutes, get supernatant stand-by.
3.2 take out Ractopamine immunoaffinity purification enriching column of the present invention from-20 ℃ of refrigerators, equilibrium at room temperature 20 minutes, drip-dry store buffer liquid, crossing with 1 mL PBST 3% acetic acid aqueous solution 500 μ l that post cleans filler, 0.05% polysorbas20 and 1 mg/mL BSA successively crosses post and cleans, cross in post with PBS again and filler to neutral PH=7.0 ± 0.5, drip-dry PBS, stand-by;
3.2 sample 2 mL that handle well are crossed post, connect efflux and repeated post 1 time.
Clean 3.3 cross post with 2 mL PBST, then cross post with 1.5 mL PBS and clean.
3.4 with the aqueous solution 100 μ L wash-outs that contain 80% methyl alcohol 3% acetic acid, collect all eluents; With containing 1% acetic acid aqueous solution 300 μ L wash-outs, collect all eluents again.Whole elution process is collected 400 μ L eluents altogether.
3.5 detect the content of Ractopamine in eluent with high performance liquid chromatography.
The capacity of testing result explanation Ractopamine immunoaffinity purification enriching column reaches 10000 ng/mL, and so high capacity has good enrichment effect to Ractopamine.
Embodiment 4 Ractopamine immunoaffinity purification enriching column determination of recovery rates
4.1 with known blank (being defined as after testing the Ractopamine feminine gender) urine 5 mL.Adding the Ractopamine standard items to make concentration is 100 ng/mL, 5000 rev/mins centrifugal 10 minutes, get supernatant stand-by.
4.2 take out Ractopamine immunoaffinity purification enriching column from-20 ℃ of refrigerators, equilibrium at room temperature 20 minutes, drip-dry store buffer liquid, crossing post with the 3% acetic acid aqueous solution 500 μ L that cross post cleaning, 0.05% polysorbas20 and 1 mg/mL BSA with 1 mL PBST successively cleans, cross in post with PBS again and filler to neutral PH=7.0 ± 0.5, drip-dry PBS, stand-by;
4.3 sample 5 mL that handle well are crossed the affinity purification enriching column, connect efflux and repeated post 1 time.
Clean 4.4 cross post with 1 mL PBST, then cross post with 1 mL PBS and clean.
4.5 first use eluent wash-out 100 uL of 80% methyl alcohol+3% acetic acid+distilled water, then wash 300 uL with 3% acetic acid.Total amount is 400 uL.
4.6 with high performance liquid chromatography detect added standard items but do not cross the sample of post and eluent in the content of Ractopamine.
The affine filler recovery rate of measurement result explanation is greater than 70%.
Embodiment 5 Ractopamine immunoaffinity purification enriching columns and the coupling of Ractopamine colloidal gold strip
5.1 get negative each 2 colloidal gold strips of 25 μ L points of urine of Ractopamine, determine that it is negative urine (as 1 in Fig. 3,2).
5.2 get this feminine gender urine 100 mL, adding the Ractopamine standard items to make concentration is 0.01 ng/mL, respectively gets 2 colloidal gold strips of 25 μ L points, test strips still is shown as feminine gender (as 3 in Fig. 3,4) as a result.
5.3 take out Ractopamine immunoaffinity purification enriching column from-20 ℃ of refrigerators, equilibrium at room temperature 20 minutes, drip-dry store buffer liquid, 3% acetic acid aqueous solution 200 μ L with 1 mL PBST, 0.05% polysorbas20 and 1mg/mL BSA cross the post cleaning successively, cross in post with PBS again and filler to neutral PH=7.0 ± 0.5, drip-dry PBS, stand-by;
5.4 sample 100 mL that handle well are crossed the affinity purification enriching column, connect efflux and repeated post 1 time.
Clean 5.5 cross post with 1 mL PBST, then cross post with 1 mL PBS and clean.
5.6 with the 3% acetic acid aqueous solution 150 μ L wash-outs that contain 0.05% polysorbas20 and 1 mg/mL BSA, collect eluent.
5.7 transfer eluent PH=7.0 ± 0.5. with saturated trishydroxymethylaminomethane Tris
5.8 respectively get 2 colloidal gold strips of eluent 25 μ L points after neutralization, test strips is shown as the positive (as 5 in Fig. 3,6) as a result.Explanation can be reduced to the detection lower limit 0.01ng/mL. and compare with independent detection lower limit 5 ng/mL with the colloidal gold strip direct-detection through the enrichment of affinity purification enriching column, and detection sensitivity has improved 500 times.
Claims (9)
1. the preparation of a micro high efficiency Ractopamine immunoaffinity purification enriching column is characterized in that:
1) will be through the Ractopamine specific polyclonal antibody of immunoaffinity purification and the agarose mixing of periodic acid activation, room temperature reaction is 30 minutes on shaking table, then add sodium borohydride reduction at 4 ℃ of refrigerators after standing 30 minutes, clean unconjugated antibody with distilled water, clean filler with phosphate buffer, 10 times of packing volumes/time, wash 4 times, add equal-volume glycerine after the drip-dry phosphate buffer, mixing obtains the affine filler of Ractopamine immunity;
2) filter membrane is loaded onto in the lower end in special plastic column, the affine filler of Ractopamine immunity is packed in post, and 25 μ L/ posts, the filler upper strata covers filter membrane, namely gets described Ractopamine immunoaffinity purification enriching column.
2. the Ractopamine immunoaffinity purification enriching column of method according to claim 1 preparation, comprise coupling have immunoaffinity purification the Ractopamine specific polyclonal antibody the agarose filler and load the tailormadepiston post of this filler.
3. the preparation of Ractopamine immunoaffinity purification enriching column according to claim 1, it is characterized in that: the Ractopamine specific polyclonal antibody of described immunoaffinity purification is to obtain derivative Ractopamine acetyl bromide ester solution with Ractopamine and bromoacetyl chloride reaction, obtain Ractopamine acetyl bromide ester after purifying with reverse this solution of high-efficient liquid phase chromatogram HPLC purifying, with Ractopamine acetyl bromide ester and the carrier protein couplet after purifying, the conjugate that obtains is as the immunogen immune animal, obtain antiserum, the immune affinity column of using with the Ractopamine acetyl bromide ester after this purifying and activated agarose reaction preparation antibody purification simultaneously, extract antiserum with this one step of affinity column and obtain the Ractopamine specific polyclonal antibody.
4. the preparation of Ractopamine immunoaffinity purification enriching column according to claim 3 is characterized in that: described Ractopamine acetyl bromide ester is that the Ractopamine standard items are obtained derivative Ractopamine acetyl bromide ester solution with the bromoacetyl chloride reaction under triethylamine catalysis.
5. this Ractopamine bromoacetyl chloride ester solution is gone out with respect to behind Ractopamine standard items peaks to move with the reverse column chromatography separating purification of preparative high performance liquid chromatography instrument, and the single main peak component with Ractopamine uv absorption spectra feature is collected the Ractopamine acetyl bromide ester that this main peak component obtains purifying.
6. the preparation of Ractopamine immunoaffinity purification enriching column according to claim 3 is characterized in that: described Ractopamine acetyl bromide ester is that the Ractopamine standard items are obtained derivative Ractopamine acetyl bromide ester solution with the bromoacetyl chloride reaction under triethylamine catalysis.
7. this Ractopamine bromoacetyl chloride ester solution is gone out with respect to behind Ractopamine standard items peaks to move with the reverse column chromatography separating purification of preparative high performance liquid chromatography instrument, and the single main peak component with Ractopamine uv absorption spectra feature is collected the Ractopamine acetyl bromide ester that this main peak component obtains purifying.
8. the preparation of Ractopamine immunoaffinity purification enriching column according to claim 3, it is characterized in that: the described immune affinity column of using with the Ractopamine acetyl bromide ester after purifying and activated agarose reaction preparation antibody purification, that activated agarose and lysine reaction are obtained amidized agarose, again with after amidized agarose and the reaction of NHS-iodoacetic acid, reduce with DTT again, obtain the agarose of sulfhydrylation, agarose and the quick mixing of Ractopamine acetyl bromide ester with sulfhydrylation, room temperature reaction 60 minutes, obtain chemical covalent bond complex Ractopamine acetyl bromide-agarose filler, filler is filled post, wash away unnecessary DTT, the immune affinity column that the Ractopamine specific antibody that obtains purifying is used.
9. Ractopamine immunoaffinity purification enriching column according to claim 1 and 2 and the coupling of Ractopamine colloidal gold strip.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104784973A (en) * | 2015-04-24 | 2015-07-22 | 重庆出入境检验检疫局检验检疫技术中心 | Compound immunoaffinity column for purifying phenobarbital and ractopamine as well as preparation method and application of compound immunoaffinity column |
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| CN1916591A (en) * | 2006-08-08 | 2007-02-21 | 中国农业科学院农业质量标准与检测技术研究所 | Ractopamine column, preparation method and usage |
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|---|---|---|---|---|
| IL89479A (en) * | 1988-03-07 | 1994-08-26 | Lilly Co Eli | Compositions comprising ractopamine and growth hormone |
| CN1687783A (en) * | 2005-05-24 | 2005-10-26 | 中国农业大学 | Affinity chromatography-enzyme linked immunity test method for ractopamine and dedicated kit |
| CN201051101Y (en) * | 2006-04-03 | 2008-04-23 | 河南省动物免疫学重点实验室 | Quick testing paper for ractopamine residue |
| CN1916591A (en) * | 2006-08-08 | 2007-02-21 | 中国农业科学院农业质量标准与检测技术研究所 | Ractopamine column, preparation method and usage |
| CN201181297Y (en) * | 2008-01-30 | 2009-01-14 | 北京望尔生物技术有限公司 | Colloidal gold test paper card for detecting Ractopamine medicine residue |
| CN102553297A (en) * | 2011-12-31 | 2012-07-11 | 南宁市蓝光生物技术有限公司 | Preparation and application of miniature high-efficiency clenbuterol immuno-affinity chromatography column |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103100237B (en) | 2015-12-02 |
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