CN103170309B - Ractopamine antibody immunoaffinity chromatographic column and application thereof - Google Patents
Ractopamine antibody immunoaffinity chromatographic column and application thereof Download PDFInfo
- Publication number
- CN103170309B CN103170309B CN201310077763.1A CN201310077763A CN103170309B CN 103170309 B CN103170309 B CN 103170309B CN 201310077763 A CN201310077763 A CN 201310077763A CN 103170309 B CN103170309 B CN 103170309B
- Authority
- CN
- China
- Prior art keywords
- ractopamine
- antibody
- kit
- chromatographic column
- immune affinity
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a ractopamine antibody immunoaffinity chromatographic column and an application thereof. The filler of the ractopamine antibody immunoaffinity chromatographic column provided by the invention is formed by cross-linking of ractopamine hapten and solid phase carrier, wherein the structural formula of the ractopamine hapten is as shown in formula I; the solid phase carrier is agarose gel, cellulose, dextrangel, polyacrylamide gel, porous glass or polyacrylamide-agarose gel. Compared with the antibody purifying technologies including the existing salting-out technology, the conventional chromatographic technology and the like, the ractopamine antibody immunoaffinity chromatographic column has the main advantages as follows: firstly, the hapten ractopamine can be combined with the solid-phase carrier by introducing amino group to the ractopamine; and secondly, the specific affinity of the antigen and the antibody is utilized to effectively remove non-specific antibody and other hybrid proteins, so that the purifying efficiency of the ractopamine antibody is improved, and the operation is simple. Formula I is shown in the specification.
Description
Technical field
The present invention relates to a kind of Anti-ractopamine antibody immune affinity chromatographic column and application thereof.
Background technology
Ractopamine is a kind of medical material, has physiological effect widely, the cardiotonic drug of available treatment congestive heart failure disease, and be usually used in the treatment of bronchial astehma, bronchial spasm and obstetrics' disease.When its consumption is increased to 5-10 times of quantity, can increase muscle growth, reduce lipopexia, be good nutrient distribution agent and growth promoter.U.S. FDA, approval in 2000, can, for Animal nutrition again ingredients, be widely used for animal husbandry and aquaculture.Can improve the daily gain of animal simultaneously, improve efficiency of feed utilization, improve the protein content of animal.Once but its in animal body residual enters human body through food chain, can produce significant damage to eater, larger to patient's harm such as heart disease, diabetes, hypertension, hyperthyroidism, glaucoma, hypertrophy of the prostates especially, even dead, as clenbuterol hydrochloride (Ractopamine be in " clenbuterol hydrochloride medicine " a kind of) once caused hundreds of people's poisoning in Shanghai; In Taiwan, contain clenbuterol hydrochloride due to the pork from imported from America, almost provoke a political controversial issue.China forbids the medicine promoting animal growth agent such as beta-2-agonists to be used at present.But for a long time, various because illegally using the poisoning that beta-2-agonists causes to happen occasionally.For hitting unsanctioned use, the health and safety of Protection of consumer, in the urgent need to sound relevant detection method.
At present, the conventional method of detection of veterinary drugs in food has the physico-chemical analysis methods such as gas-chromatography, high performance liquid chromatography and gas chromatography mass spectrometry.Although these method high specificities, highly sensitive, sample pre-treatments complex operation step, cost is higher, and the screening that is not also suitable for batch samples detects.Immunochemical analyses in view of the advantage in uniqueness aspect the qualitative, quantitative of antigen-antibody and easy and simple to handle fast, cost is low, sensitivity is higher, analyzing samples amount is large advantage made up the deficiency of physico-chemical analysis, in the residue detection of Ractopamine, play a part more and more important, and the composition of antiserum or ascites is very complicated, some foreign proteins may affect reacting of antibody and antigen, so the purifying of Anti-ractopamine antibody just seems particularly important.
The chromatography that current antibody purification process is mainly salting out method, organic solvent precipitation method, various principles and affinity purification (normally Protein A or Protein G) etc., these all can not obtain very pure specific antibody, even affinity purification, also can only obtain the mixture of specific antibody and non-specific antibody, and both can not be isolated to specific antibody.Therefore study a kind of effectively purification process of separation and purification specific antibody, there is higher learning value and application prospect.
Summary of the invention
An object of the present invention is to provide a kind of Anti-ractopamine antibody immune affinity sorbent.
The immune affinity sorbent of Anti-ractopamine antibody provided by the present invention is by Ractopamine haptens and solid phase carrier is crosslinked forms; The haptenic structure of described Ractopamine is suc as formula shown in I; Described solid phase carrier is Ago-Gel, cellulose, sephadex, polyacrylamide gel, cellular glass or ultragel ACA22.
Formula I
In the present invention, described solid phase carrier is specially Sepharose4B.More concrete, in one embodiment of the invention, described Sepharose4B WeiGE company product, article No. is 17-5437-01.
Described Ractopamine haptens specifically can be prepared according to the method comprising the steps: the ratio that is 1:1:5 according to mol ratio by Ractopamine, N-(4-brombutyl) phthalimide, hydrazine hydrate is reacted, and obtains described Ractopamine haptens (amino activation).
In one embodiment of the invention, the preparation method of described immune affinity sorbent comprises the steps: the Sepharose4B solid phase carrier of described Ractopamine haptens and cyanogen bromide-activated crosslinked, and seal processing, obtain described immune affinity sorbent.Described crosslinked being specially (taking 0.84g sodium acid carbonate by the Sepharose4B of described Ractopamine haptens and described cyanogen bromide-activated at the sodium bicarbonate solution of 0.1M pH8.0, be dissolved in 100ml deionized water) middle room temperature (20-25 ℃) reaction 4h, the proportioning of the Sepharose4B of described Ractopamine haptens, described cyanogen bromide-activated and the sodium bicarbonate solution of described 0.1M pH8.0 (take 0.84g sodium acid carbonate, be dissolved in 100ml deionized water) specifically can be 4mg:1ml:2ml; The monoethanolamine aqueous solution (measure 5.98ml monoethanolamine, be dissolved in 80ml deionized water, regulate pH to 8.0 with 0.1M HCl) the sealing 2h under room temperature (20-25 ℃) being specially with 1MpH8.0 is processed in described sealing.In said method, after described being cross-linked, also comprise that sodium bicarbonate solution (take 0.84g sodium acid carbonate, be dissolved in 100ml deionized water) the washing filler with 0.1M pH8.0 is removed the haptenic step of unconjugated described Ractopamine.
A further object of the present invention is to provide a kind of immune affinity chromatographic column of Anti-ractopamine antibody.
The immune affinity chromatographic column of Anti-ractopamine antibody provided by the present invention, take described immune affinity sorbent as filler.
In one embodiment of the invention, the preparation method of described immune affinity chromatographic column comprises the steps: to pack described immune affinity sorbent into chromatographic column, with acetate buffer solution (0.1M pH4.0, contain 0.5M NaCl) and Tris-HCl buffer solution (0.1M pH8.0, contain 0.5M NaCl) alternately wash filler three times (complete once alternately washing for once), obtain described immune affinity chromatographic column.
Described acetate buffer solution (0.1M pH4.0 contains 0.5M NaCl) composed as follows: sodium acetate 8.2g/L, acetic acid 1.04ml/L, NaCl29.22g/L, surplus is water, pH4.0.
Described Tris-HCl buffer solution (0.1M pH8.0 contains 0.5M NaCl) composed as follows: Tris12.114g/L, NaCl29.22g/L, surplus is water, pH8.0.
Kit containing described immune affinity sorbent or described immune affinity chromatographic column also belongs to protection scope of the present invention.
Described kit also can comprise eluent; Described eluent is the 0.1M glycine solution of pH3.0.
Described kit also can comprise cleaning solution; Described cleaning solution is the 0.01M phosphate buffer that contains 0.14M NaCl of pH7.2.The solvent of described 0.01M phosphate buffer is water, and solute and concentration thereof are as follows: NaCl8.5g/L, KCl0.02g/L, Na
2hPO412H
2o2.9g/L, NaH
2pO42H
2o0.593g/L.
Described kit also can comprise neutralizer; Described neutralizer is 1M pH8.8Tris-HCl buffer solution.Described 1MpH8.8Tris-HCl buffer solution composed as follows: Tris121.14g/L, surplus is water, pH8.8(adjusts pH with 0.1M HCl).Concrete compound method is as follows: take 121.14g Tris, adding distil water 800ml, adjusts pH to 8.8 with 0.1M HCl, and adding distil water is to 1000ml.
The method of preparing described kit also belongs to protection scope of the present invention.
The preparation method of described kit specifically can comprise the steps: described immune affinity sorbent or described immune affinity chromatographic column, packs separately: described eluent, described cleaning solution and described neutralizer with at least one difference in following.
Described immune affinity sorbent, or described immune affinity chromatographic column, or the application of described kit in Anti-ractopamine antibody purifying also belongs to protection scope of the present invention.
Also object of the present invention is to provide a kind of method of purifying Anti-ractopamine antibody.
The method of purifying Anti-ractopamine antibody provided by the present invention, be specially and utilize described immune affinity chromatographic column, or described kit purifying Anti-ractopamine antibody, can comprise the steps: sample to be purified to join described immune affinity chromatographic column, with described cleaning solution, wash to ultraviolet specrophotometer mensuration OD
280below value to 0.01, recycle described eluent and carry out wash-out, then, with described neutralizer neutralization, the proportioning of described eluent and described neutralizer is 3000 μ L:360 μ L, obtains the Anti-ractopamine antibody after purifying.
Above-mentioned all described Anti-ractopamine antibodies all can be Ractopamine monoclonal antibody or Ractopamine polyclonal antibody.In the present invention, described Anti-ractopamine antibody is Ractopamine monoclonal antibody, be specially by this hybridoma cell strain of hybridoma cell strain RAC CGMCC No.3776(and be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, the preservation center numbering of registering on the books: CGMCC No.3776, this hybridoma cell strain on August 3rd, 2010 application patent in disclosed, number of patent application is 201010244260.5, and application publication number is CN101915845A) monoclonal antibody that produces.
With existingly saltout, the purification technique of the antibody such as traditional chromatography compares, major advantage of the present invention is:
(1) utilize Ractopamine is introduced to amino processing, can make haptens Ractopamine be combined with solid phase carrier;
(2) utilize the specificity of antigen and antibody affine, effectively removed non-specific antibody and other foreign proteins, improve the purification efficiency of Anti-ractopamine antibody, and simple to operate.
Accompanying drawing explanation
Fig. 1 is the haptenic positive ion mass spectrum figure of Ractopamine.
Fig. 2 is the haptenic hydrogen nuclear magnetic resonance spectrogram of Ractopamine.
Fig. 3 is Anti-ractopamine antibody purifying collection of illustrative plates.
Fig. 4 is antibody titer measurement result before and after Anti-ractopamine antibody purifying.
Fig. 5 is SDS-PAGE testing result before and after Anti-ractopamine antibody purifying.Wherein, swimming lane M is molecular weight of albumen standard; Swimming lane 1 is antibody (sample-ascites to be purified) before purifying; Swimming lane 2 is antibody after purifying (affinity purification eluent); Swimming lane 3 is worn liquid for affinity purification stream.
The specific embodiment
The experimental technique using in following embodiment if no special instructions, is conventional method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
Ractopamine: Sigma company, article No. 34198.
DMF: Chemical Reagent Co., Ltd., Sinopharm Group, article No. 81007718.
N-(4-brombutyl) phthalimide: Sigma company, article No. 100919.
Triethylamine: Beijing chemical reagents corporation, article No. 80134392.
Ethyl acetate: Chemical Reagent Co., Ltd., Sinopharm Group, article No. 10009418.
Sodium chloride: Chemical Reagent Co., Ltd., Sinopharm Group, article No. 10019318.
Methyl alcohol: Chemical Reagent Co., Ltd., Sinopharm Group, article No. 10014118.
Carrene: Chemical Reagent Co., Ltd., Sinopharm Group, article No. 80047318.
Hydrazine hydrate: Aladdin reagent (Shanghai) Co., Ltd., article No. 87006570.
Sodium acid carbonate: Chemical Reagent Co., Ltd., Sinopharm Group, article No. 10018992.
Anhydrous sodium acetate: Chemical Reagent Co., Ltd., Sinopharm Group, article No. 10018818.
Glacial acetic acid: Chemical Reagent Co., Ltd., Sinopharm Group, article No. 10000218.
Tris:Amresco company, article No. 0497.
Concentrated hydrochloric acid: Chemical Reagent Co., Ltd., Sinopharm Group, article No. 10011094.
The Sepharose4B filler of cyanogen bromide-activated: purchased from GE company, article No. 17-5437-01.
Monoethanolamine: Sigma company, article No. 398136.
Glycine: Chemical Reagent Co., Ltd., Sinopharm Group, article No. 62011516.
Potassium chloride: Chemical Reagent Co., Ltd., Sinopharm Group, article No. 10016318.
Na
2hPO
412H
2o: Chemical Reagent Co., Ltd., Sinopharm Group, article No. 100203190.
NaH
2pO
42H
2o: Chemical Reagent Co., Ltd., Sinopharm Group, article No. 20040717.
Proclin-300:Sigma company, article No. 48912-U.
Triton x-100:Sigma company, article No. T9284.
HRP mark sheep anti mouse two is anti-: Jackson ImmunoResearch company, article No. 111-035-003.
3,3', 5,5'-tetramethyl benzidine (TMB): Sigma company, article No. ST056501.
Marker: Beijing Quanshijin Biotechnology Co., Ltd, article No. DR101.
Acrylamide: Amresco company, article No. 0341.
The two acrylamides of N-N '-methene: Amresco company, article No. 0172.
TEMED (N-N-N`-N`-tetramethylethylenediamine): Amresco company, article No. 0761.
Beta-mercaptoethanol: Amresco company, article No. M131.
Dodecyl sodium sulfate:, article No. 30166627.
Bromophenol blue: Amresco company, article No. 0449.
Glycerine: Chemical Reagent Co., Ltd., Sinopharm Group, article No. 10010618.
Ammonium persulfate: Sigma company, A3678.
Coomassie brilliant blue R_250: Sigma company, article No. 27816.
Methyl alcohol: Chemical Reagent Co., Ltd., Sinopharm Group, article No. 10014118.
Glacial acetic acid: Chemical Reagent Co., Ltd., Sinopharm Group, article No. 10000218.
The concentrated sulfuric acid: Chemical Reagent Co., Ltd., Sinopharm Group, article No. 73108460.
Casein: Sigma company, article No. C6554.
Sucrose: Chemical Reagent Co., Ltd., Sinopharm Group, article No. 10021418.
Calf serum: Zhengzhou Yikang bioengineering Co., Ltd.
The preparation of embodiment 1, Anti-ractopamine antibody immune affinity chromatographic column
One, the haptenic preparation of Ractopamine and Structural Identification
1, the haptenic preparation of Ractopamine
Ractopamine is dissolved in to N, in dinethylformamide (DMF), in single port bottle, stir, add N-(4-brombutyl) phthalimide, Ractopamine and N-(4-brombutyl) phthalimide mol ratio 1:1, add again triethylamine (as catalyst), 100-110 ℃ of stirring and refluxing 8-16h, thin-layer chromatography monitoring to reacting completely (the band number on thin-layer chromatography with react that band number is different before, and after stable, react completely), add a small amount of shrend reaction of going out, ethyl acetate (ETOAC), saturated sodium-chloride washing organic phase, dry, be spin-dried for, (fixing is silica gel to thin-layer chromatography purified product mutually, mobile phase is methyl alcohol: carrene=3:50(volume ratio), the Rf value R of product
f=0.3), product after purifying is dissolved in ethanol, add hydrazine hydrate (in whole reaction, the mol ratio of described Ractopamine, described N-(4-brombutyl) phthalimide, described hydrazine hydrate is 1:1:5) By Hydrolysis At Room Temperature 24h, reaction finishes rear convection drying, be spin-dried for, obtain Ractopamine haptens.
2, the haptenic Structural Identification of Ractopamine
(1) Mass Spectrometric Identification
Mass spectrum condition: electron source: ESI source, dry gas temperature: 350 ℃, sprayer pressure: 15.00psi, dry gas flow velocity: 5.00l/min, taper hole voltage :-34.7--6.0Volt, quality of scanning scope: 100-800m/z, voltage multiplie voltage: 1696Volt, dynode voltage: 7.0Volt.
Positive ion mass spectrum figure as shown in Figure 1, MS(ESIsource, positive): 372.3(M+1), prove that the haptenic molecular weight of Ractopamine is 372.3.
(2) nuclear-magnetism is identified
The haptenic hydrogen nuclear magnetic resonance spectrogram of Ractopamine result is as shown in Figure 2: H
1nMR (300MHz, deuterated methanol) δ: 8.16 (dd, 1H), 7.59 (dd, 1H), 7.02～6.95 (m, 3H), 6.69-6.54 (m, 3H), 4.37 (dt, 1H) 2.80-2.71 (m, 3H), 2.48-2.31 (m, 6H), 1.70 (dd, 2H), 1.52 (dd, 2H), 1.19 (s, 2H), 1.01～0.80 (m3H).
Owing to making solvent with deuterated methanol, the hydroxyl effect in it and molecule, so should have 2H(5.0(dd, the 2H of phenylol near 5.0)); Should there be amino 2H and the 1H of hydroxyl (3H) in 2.0 places.
By above-mentioned evaluation, obtain the haptenic structural formula of described Ractopamine suc as formula shown in I.
Formula I
Two, the preparation of immune affinity chromatographic column
The sodium bicarbonate solution of 0.1M pH8.0: take 0.84g sodium acid carbonate, adding distil water is to 100ml.
Sodium-acetate buffer (0.1M pH4.0 contains 0.5M NaCl): take sodium acetate 8.2g, 1.04ml acetic acid, 29.22g NaCl, adding distil water is settled to 1000ml.
Tris-HCl buffer solution (0.1M pH8.0 contains 0.5M NaCl): take 12.114g Tris, 29.22g NaCl, adding distil water 800ml, adjusts pH to 8.0 with 0.1M HCl, and adding distil water is settled to 1000ml.
Get Ractopamine haptens prepared by 4mg step 1, with the Sepharose4B(of 1ml cyanogen bromide-activated purchased from GE company, article No. 17-5437-01) filler, in the sodium bicarbonate solution of 2ml0.1M pH8.0, fully mix room temperature reaction 4h; This sodium bicarbonate buffer liquid washing filler of 10ml of take is removed after the Ractopamine haptens of combination, with monoethanolamine (sigma company, the article No. 398136) aqueous solution of 2ml1M pH8.0, seals filler 2h under room temperature; Then filler is packed in 8ml chromatography pillar, use respectively 5ml acetate buffer solution (0.1M pH4.0, contain 0.5M NaCl) and Tris-HCl buffer solution (0.1M pH8.0, contain 0.5M NaCl) alternately wash filler three times (complete once alternately washing for once), obtain stand-by Anti-ractopamine antibody immune affinity chromatographic column.
Preparation and the application thereof of embodiment 2, Anti-ractopamine antibody immune affinity chromatographic column kit
One, the preparation of Anti-ractopamine antibody immune affinity chromatographic column kit
Anti-ractopamine antibody immune affinity chromatographic column kit, Anti-ractopamine antibody immune affinity chromatographic column, cleaning solution, eluent and the neutralizer of by embodiment 1, being prepared gained form.
The 0.01M phosphate buffer that contains 0.14M NaCl of cleaning solution: pH7.2, described 0.01M phosphate buffer: NaCl8.5g, KCl0.02g, Na
2hPO412H
2o2.9g, NaH
2pO42H
2o0.593g, adding distil water is to 1000mL.
0.1M glycine-HCl aqueous solution of eluent: pH3.0, takes 7.507g glycine, and adding distil water 800ml adjusts pH to 8.0 with 0.1M HCl, and adding distil water is to 1000ml.
Neutralizer: 1M pH8.8Tris-HCl buffer solution, take 121.14g Tris, adding distil water 800ml, adjusts pH to 8.8 with 0.1MHCl, and adding distil water is to 1000ml.
Two, utilize the kit purifying Anti-ractopamine antibody of step 1
1, purifying
Sample to be purified: Balb/c mouse peritoneal is only injected to sterilizing paraffin oil 0.4mL/, within 7 days, this hybridoma cell strain of pneumoretroperitoneum injection hybridoma cell strain RAC CGMCC No.3776(is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, the preservation center numbering of registering on the books: CGMCC No.3776, this hybridoma cell strain on August 3rd, 2010 application patent in disclosed, number of patent application is 201010244260.5, application publication number is CN101915845A), 5 * 10
5individual/only, after 7 days, to gather ascites as sample to be purified.
After sample to be purified is diluted with the cleaning solution in step 1, getting 2ml sample liquid adds in the Anti-ractopamine antibody immune affinity chromatographic column in step 1 kit, efflux packs in this post, seal again flow export, at room temperature react 30min, with the cleaning solution in step 1, clean chromatographic column, to OD
280value is collected foreign protein stream lower than 0.01(and is worn liquid, the reference substance detecting as purification effect in following step 2); Eluent elution chromatography post with in step 1, makes to be attached to the antibody dissociation on chromatographic column, to OD
280value is lower than 0.01, during collection, in collecting pipe, add in advance the neutralizer (effluent volume of collecting is 3000 μ l, and the proportioning of eluent and neutralizer is 3000 μ l:360 μ l) in 360 μ l step 1, to regulate the pH of component, antibody after acquisition purifying.
Attention: utilizing Anti-ractopamine antibody immune affinity chromatographic column to carry out in Sample Purification on Single process, the temperature of chromatographic column and each solution all needs to be returned to room temperature, otherwise the speed of antigen-antibody combination can be slack-off; In addition, note can not containing solid impurity (as multigelation is removed cotton-shaped lipid for twice, with the membrane filtration of 0.45 μ m) in ascites to be purified, otherwise can block pillar, affect pillar service life.
Anti-ractopamine antibody purifying collection of illustrative plates as shown in Figure 3, as can be seen from the figure, in step 1 under the cleaning of cleaning solution, foreign protein, non-specific antibody and unconjugated specific antibody can flow out with cleaning solution (peak A), and while being changed in step 1 eluent wash-out, the specific antibody being attached on chromatographic column can be dissociated (peak B).
2, purification effect analysis
(1) indirect elisa method is measured purifying front and back antibody titer
Coated buffer solution: the sodium carbonate buffer of 0.05mol/L pH9.6, fill a prescription as Na
2hCO
31.59g, NaHCO
32.93g, adding distil water is to 1000mL.
The formula of 0.01M phosphate buffer is: NaCl8.5g, KCl0.02g, Na
2hPO412H
2o2.9g, NaH
2pO42H
2o0.593g, adding distil water is to 1000mL.
Confining liquid: Na
2hPO
412H
2o5.80g, NaH
2pO
42H
2o0.593g, NaCl8.0g, casein 2.50g, sucrose 50.00g, calf serum 50ml, ProClin-300300 μ l, adding distil water is to 1000mL, pH7.4.
Antibody diluent: NaCl16g, KCl0.04g, Na
2hPO
412H
2o2.9g, NaH
2pO
42H2O0.593g, proclin-300300 μ l, triton x-100500 μ l, adding distil water is to 1000mL.
Adopt indirect elisa method to measure purifying front and back antibody titer, operating procedure is specific as follows:
1) coated: in 96 hole ELISA Plates, to add the Ractopamine haptens solution (with coated buffer solution dilution) of preparing in 2 μ g/mL embodiment 1 step 1 of 100 μ L, the not contrast of envelope antigen is set simultaneously, 4 ℃ of coated spending the night, with PBS buffer solution washing 3 times.
2) sealing: add the confining liquid in 150 μ L/ holes, hatch 1h at 37 ℃, abandon confining liquid, wash 3 times, pat dry.Be placed in 4 ℃ of Refrigerator stores standby.
3) add testing sample: draw testing sample (antibody before purifying or after purifying, regulate concentration to after 0.1mg/ml with antibody diluent carry out gradient dilution (1 *, 10 *, 20 *, 40 *, 80 *, 100 *, 200 *, 400 *, 800 *)) 100 μ l, add in corresponding ELISA Plate, hatch 30min for 37 ℃, wash plate 4 times, pat dry.Each concentration gradient is done 3 parallel laboratory tests.
The contrast (negative control hole) that replaces detected sample with PBS is set simultaneously.
4) add ELIAS secondary antibody: get HRP mark sheep anti mouse two anti-(Jackson ImmunoResearch company, article No. 111-035-003), 1:5000 doubly dilutes after (diluting with antibody diluent) by volume, 100 μ l/ holes, hatch 30min for 37 ℃, wash 4 times, pat dry.
5) colour developing: 20 * TMB is diluted to 1 * TMB, adds by 100 μ l/ holes, 37 ℃ of colour developing 15-30min.
6) stop: add stop buffer (2M H
2sO
4) 50 μ l/ holes.
7) reading: measure each hole light absorption value with 450nm, 630nm dual wavelength, be greater than 2.1 with the ratio (P/N) with negative control hole (replacing contrasting of testing sample with PBS) OD value and be limited, as the critical point that is judged as antibody titer.
ELISA result decision method: the antibody maximum dilution multiple with P/N>2.1 represents.
The light absorption value testing result of antibody as shown in Figure 4, calculates according to light absorption value before and after purifying, the tiring as 1:3000 of antibody (sample to be purified) before purifying, the tiring as 1:12000 of the antibody after purifying.Anti-ractopamine antibody after purifying, under identical initial concentration, it is tired compared with improving approximately 4 times before purifying.
(2) SDS-PAGE analyzes purifying front and back antibody purity
With antibody (eluent) after antibody before purifying (sample-ascites to be purified), purifying, and the foreign protein stream of collecting in step 1 is worn liquid, carry out SDS-PAGE analysis, every kind of sample is 10 μ g Tot Prots at the applied sample amount of swimming lane separately, thereby detects antibody purity before and after purifying.
SDS-PAGE testing result as shown in Figure 5, as can be seen from the figure, most of Anti-ractopamine antibody (heavy chain size is about 50KD, and light chain size is about the object band of 25KD) is all in eluent, and the stream amount of antibody in liquid of wearing greatly reduces, after purifying, the purity of antibody is very high.
Claims (18)
1. an immune affinity sorbent for Anti-ractopamine antibody, is cross-linked and is formed by Ractopamine haptens and solid phase carrier; The haptenic structure of described Ractopamine is suc as formula shown in I:
Described solid phase carrier is Ago-Gel, cellulose, sephadex, polyacrylamide gel, cellular glass or ultragel ACA22;
Described Anti-ractopamine antibody is the monoclonal antibody being produced by hybridoma cell strain RAC CGMCC No.3776.
2. immune affinity sorbent according to claim 1, it is characterized in that: described Ractopamine haptens is prepared according to the method comprising the steps: the ratio that is 1:1:5 according to mol ratio by Ractopamine, N-(4-brombutyl) phthalimide, hydrazine hydrate is reacted, and obtains described Ractopamine haptens.
3. an immune affinity chromatographic column for Anti-ractopamine antibody, is characterized in that: the immune affinity sorbent that described immune affinity chromatographic column be take described in claim 1 or 2 is filler.
4. the kit that contains immune affinity sorbent described in claim 1 or 2.
5. kit according to claim 4, is characterized in that: described kit also comprises eluent; Described eluent is the 0.1M glycine solution of pH3.0.
6. kit according to claim 4, is characterized in that: described kit also comprises cleaning solution; Described cleaning solution is the 0.01M phosphate buffer that contains 0.14M NaCl of pH7.2.
7. kit according to claim 4, is characterized in that: described kit also comprises neutralizer; Described neutralizer is 1M pH8.8Tris-HCl buffer solution.
8. the kit that contains immune affinity chromatographic column described in claim 3.
9. kit according to claim 8, is characterized in that: described kit also comprises eluent; Described eluent is the 0.1M glycine solution of pH3.0.
10. kit according to claim 8, is characterized in that: described kit also comprises cleaning solution; Described cleaning solution is the 0.01M phosphate buffer that contains 0.14M NaCl of pH7.2.
11. kits according to claim 8, is characterized in that: described kit also comprises neutralizer; Described neutralizer is 1M pH8.8Tris-HCl buffer solution.
12. prepare the method for arbitrary described kit in claim 5-7, comprise the steps: described immune affinity sorbent, pack separately: described eluent, described cleaning solution and described neutralizer with at least one difference in following.
13. prepare the method for arbitrary described kit in claim 9-11, comprise the steps: described immune affinity chromatographic column, pack separately: described eluent, described cleaning solution and described neutralizer with at least one difference in following.
The application of immune affinity sorbent in Anti-ractopamine antibody purifying described in 14. claims 1 or 2;
Described Anti-ractopamine antibody is the monoclonal antibody being produced by hybridoma cell strain RAC CGMCC No.3776.
The application of immune affinity chromatographic column in Anti-ractopamine antibody purifying described in 15. claims 3;
Described Anti-ractopamine antibody is the monoclonal antibody being produced by hybridoma cell strain RAC CGMCC No.3776.
The application of arbitrary described kit in Anti-ractopamine antibody purifying in 16. claim 4-11;
Described Anti-ractopamine antibody is the monoclonal antibody being produced by hybridoma cell strain RAC CGMCC No.3776.
17. utilize the method for immune affinity chromatographic column purifying Anti-ractopamine antibody described in claim 3, comprise the steps: sample to be purified to join described immune affinity chromatographic column, with cleaning solution, wash, recycling eluent carries out wash-out, then with neutralizer neutralization, obtain the Anti-ractopamine antibody after purifying;
Described cleaning solution is the 0.01M phosphate buffer that contains 0.14M NaCl of pH7.2; Described eluent is the 0.1M glycine solution of pH3.0; Described neutralizer is 1M pH8.8Tris-HCl buffer solution;
Described Anti-ractopamine antibody is the monoclonal antibody being produced by hybridoma cell strain RAC CGMCC No.3776.
18. utilize the method for arbitrary described kit purifying Anti-ractopamine antibody in claim 8-11, comprise the steps: sample to be purified to join described immune affinity chromatographic column, with cleaning solution, wash, recycling eluent carries out wash-out, then with neutralizer neutralization, obtain the Anti-ractopamine antibody after purifying;
Described cleaning solution is the 0.01M phosphate buffer that contains 0.14M NaCl of pH7.2; Described eluent is the 0.1M glycine solution of pH3.0; Described neutralizer is 1M pH8.8Tris-HCl buffer solution;
Described Anti-ractopamine antibody is the monoclonal antibody being produced by hybridoma cell strain RAC CGMCC No.3776.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310077763.1A CN103170309B (en) | 2013-03-12 | 2013-03-12 | Ractopamine antibody immunoaffinity chromatographic column and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310077763.1A CN103170309B (en) | 2013-03-12 | 2013-03-12 | Ractopamine antibody immunoaffinity chromatographic column and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103170309A CN103170309A (en) | 2013-06-26 |
| CN103170309B true CN103170309B (en) | 2014-10-29 |
Family
ID=48630756
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310077763.1A Active CN103170309B (en) | 2013-03-12 | 2013-03-12 | Ractopamine antibody immunoaffinity chromatographic column and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103170309B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN105435758A (en) * | 2015-12-02 | 2016-03-30 | 江苏农牧科技职业学院 | Immunoaffinity chromatographic column adopting astragaloside IV as ligand and application of immunoaffinity chromatographic column |
| CN108490082A (en) * | 2017-11-02 | 2018-09-04 | 苏州大学 | A kind of Ractopamine specific immunity affinity chromatography chromatography glue and its preparation method and application |
| CN108865978A (en) * | 2018-07-25 | 2018-11-23 | 辽宁润基生物科技有限公司 | A method of separation and purifying excretion body |
| CN110950960B (en) * | 2019-11-26 | 2021-05-14 | 中国农业大学 | Preparation method of small molecule compound antibody based on high-throughput sequencing and hybrid hybridoma technology |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4361509A (en) * | 1981-12-14 | 1982-11-30 | Scripps Clinic And Research Foundation | Ultrapurification of factor VIII using monoclonal antibodies |
| 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 |
| CN101993488B (en) * | 2009-08-27 | 2014-05-21 | 深圳市三方圆生物科技有限公司 | Ractopamine immunogen, coating antigen and use thereof in colloid-gold test paper |
-
2013
- 2013-03-12 CN CN201310077763.1A patent/CN103170309B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN103170309A (en) | 2013-06-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103170309B (en) | Ractopamine antibody immunoaffinity chromatographic column and application thereof | |
| WO2014005298A1 (en) | Enzyme-linked immunosorbent assay kit for detecting dinitolmide and use thereof | |
| JPH04230855A (en) | Reagent and method for detecting polychlorinated biphenyl | |
| CN102967709B (en) | Detect enzyme linked immunological kit and the application thereof of zearalenone medicine | |
| CN112877296B (en) | Anti-phenacetin monoclonal antibody hybridoma cell strain AD and preparation method and application thereof | |
| CN103215230B (en) | Hybridoma cell strain AFM1B7, monoclonal antibody thereof and aflatoxin M1 flow lag immunization time-resolved fluorescence quick test kit | |
| CN106556592B (en) | The chemical luminescence reagent kit and preparation method thereof of quantitative detection people TK1 | |
| CN104569404A (en) | Method for detecting olaquindox by directly-competing TRFIA (Time Resolved Fluorescence Immunoassay) | |
| CN105242047A (en) | Aflatoxin B1 graphene oxide immunochromatographic test strip and application thereof | |
| CN106674138B (en) | A kind of novel haptens of Tebuconazole and its synthetic method and application | |
| CN103613563A (en) | Preparation method of benzothiostrobin hapten, artificial antigen and specific antibody and application thereof | |
| CN109180519B (en) | Olaquindox metabolite antigen, antibody, enzyme-linked immunosorbent assay kit and detection method | |
| CN105588940A (en) | Preparation of time-resolved fluorescence immunoassay kit for plasticizer detection | |
| CN101921730B (en) | Monoclonal antibody of ractopamine and preparation method and application thereof | |
| Zhang et al. | Fluorescence polarization immunoassay based on fragmentary hapten for rapid and sensitive screening of polymyxins in human serum | |
| CN109239368A (en) | Measure the method and kit of progesterone | |
| CN105758846A (en) | Chemiluminescence enzyme-linked immunosorbent assay reagent kit for detecting clenbuterol | |
| CN110724192B (en) | Hybridoma cell strain secreting serpentine monoclonal antibody and application thereof | |
| CN101995460B (en) | Ractopamine residual time resolution immunoassay kit and detection method thereof | |
| CN110003300B (en) | Derivative of 17-hydroxysteroid, detection reagent and preparation method | |
| CN111763658A (en) | Hybridoma cell strain secreting anti-dinitrotolamine monoclonal antibody and application thereof | |
| CN105435758A (en) | Immunoaffinity chromatographic column adopting astragaloside IV as ligand and application of immunoaffinity chromatographic column | |
| CN102295698B (en) | Cyclosporine A immunogen, cyclosporine A specific antibody, detection reagent, and detection kit | |
| CN114280289A (en) | Magnetic particle chemiluminescence detection kit and detection method thereof | |
| CN109061150B (en) | Time-resolved fluorescence immunochromatographic test strip for detecting metalaxyl and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |