CN100408163C - Method of purifying abamectin kind medicine and its immune affinity chromatographic column - Google Patents
Method of purifying abamectin kind medicine and its immune affinity chromatographic column Download PDFInfo
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- CN100408163C CN100408163C CNB2006100072607A CN200610007260A CN100408163C CN 100408163 C CN100408163 C CN 100408163C CN B2006100072607 A CNB2006100072607 A CN B2006100072607A CN 200610007260 A CN200610007260 A CN 200610007260A CN 100408163 C CN100408163 C CN 100408163C
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Abstract
The present invention discloses a method for purifying avermectin medicines and an immune affinity chromatography column thereof. The immune affinity chromatography column for purifying avermectin loads with immune affinity adsorbents which comprise solid phase carriers and avermectin polyclonal antibodies or monoclonal antibodies coupled with the solid phase carriers, wherein the avermectin polyclonal antibodies or the avermectin monoclonal antibodies are obtained by using couplers of avermectin haptens and carrier proteins as immunogens. The avermectin semiantigens are avermectin succinic acid derivants obtained by esterifying avermectin-4'-OH and succinic anhydride. The avermectin content is effectively detected with a purification method combined with a chromatography, and the present invention overcomes the defects of fewer information contents, low quantitative accuracy, low selectivity of a rationalizing method and the like in the direct measurement of a sample by a simple immunoassay technique, and embodies the complementary performance of an immunological technique and a conventional rationalizing technique in a analysis mechanism.
Description
Technical field
The present invention relates to a kind of purifying abamectin kind medicine (Aermectins, AVMs), comprise AVM (Aermectin, AVM), doractin (Doramectin, DOR), dust is than rhzomorph (Eprinomectin, EP) and ivermectin (Ivermectin, method IVM) and special immune affinity chromatographic column thereof.
Background technology
Along with development of life science, people have produced more and more keen interest to material in the organism and variation thereof, and the analysis of biological specimen just becomes the necessary means of exploring and finding the life secret.Because the substance in biological sample complexity, testing concentration is lower, and most of sampling amount seldom, and this just has higher requirement to the selectivity and the sensitivity of analytical method.Immune affinity chromatographic (IAC, immunoaffinity chromatography) is a kind of analytical method that immune response is combined with chromatogram analysis method.Its high selectivity and high-affinity make analytic process simplify undoubtedly.In residue of veterinary drug is analyzed, the simplest and the most effective application mode of IAC is (as HPLC as the physical and chemical determination technology, GC) sample purification means, this method for combined use can make immunological technique and physics and chemistry technology obtain complementation aspect selectivity, separating power, speed and the sensitivity, and avoided immunoassay (as ELISA, RIA) direct many deficiencies of working sample.At present, this method is widely used in the analysis of antibody, hormone, polypeptide, enzyme, recombinant protein, acceptor virus and micromolecular compound.
AVMs comprises AVM (Avermectin, AVM), ivermectin (Ivermectin, IVM), doractin (Doramectin, DOR), the general Reno rhzomorph of dust (dust is than rhzomorph, according to general rhzomorph, Eprinomectin, EP), moxidectin (Moxidectin, MOX) and plug draw rhzomorph (Selamectin, medicine such as SEL), wherein, the apokoinou construction of AVMs as shown in Figure 2.
AVM (Avermectin, AVM), doractin, dust belongs to macrolides broad-spectrum anti-parasite Tri-Biocin than rhzomorph and ivermectin, the multiple nematode such as the Colombia nodular worm that the pest-resistant spectrum of this class medicine are comprised cattle and sheep, radiation nodular worm and larva thereof, trichostrongyle, the gentle genus of blood nematode, Parafilaria bovicola, all kinds of roundworms of equus, secondary roundworm, pinworm, the blood nematode that softens, the tailfiber larva of a tapeworm or the cercaria of a schistosome of coiling, the esophageal orifice nematode of pig, the lung filaria, the trichina and the larva of a tapeworm or the cercaria of a schistosome of dividing a word with a hyphen at the end of a line thereof, the packing larva of a tapeworm or the cercaria of a schistosome, kidney worm (Eustrongylus gigas), the roundworm of dog, pinworm, heartworm, vermin of hookworm and various animals such as acarid, tick, lice, the fly class and the fly class larva of a tapeworm or the cercaria of a schistosome, the pest-resistant spectrum of this class medicines such as tsutsugamushi mite of itching day comprises multiple nematode such as the Colombia nodular worm of cattle and sheep, radiation nodular worm and larva thereof, trichostrongyle, the gentle genus of blood nematode, Parafilaria bovicola, all kinds of roundworms of equus, secondary roundworm, pinworm, the blood nematode that softens, the tailfiber larva of a tapeworm or the cercaria of a schistosome of coiling, the esophageal orifice nematode of pig, the lung filaria, the trichina and the larva of a tapeworm or the cercaria of a schistosome of dividing a word with a hyphen at the end of a line thereof, the packing larva of a tapeworm or the cercaria of a schistosome, kidney worm (Eustrongylus gigas), the roundworm of dog, pinworm, heartworm, vermin of hookworm and various animals such as acarid, tick, lice, the fly class and the fly class larva of a tapeworm or the cercaria of a schistosome, tsutsugamushi mite etc. itches day.Many countries have set up the method for detecting residue of Avermectins medicine and have made residual regulation of limiting the quantity of.AVMs is as fat-soluble compound, in animal body the residual effect time longer, thereby WHO classifies it as height and poisons compound, the residue detection work in its animal-derived food causes the very big concern of various countries, and formulates MRL.
Detect AVM at present, doractin, dust mainly contains high performance liquid chromatography-fluorescence (HPLC-FLD), liquid-matter online (LC-MS-MS) and ELISA (ELISA) etc. than the method for rhzomorph and ivermectin.The pre-treatment of these methods utilizes liquid-liquid to distribute, conventional SPE column purification with separate, all exist shortcomings such as processing procedure is loaded down with trivial details, clean-up effect is poor, the organic solvent waste is many, required time is grown to some extent.The affine technology of immunity is the new technology that is applied at analysis field the nineties, but with the AVM in the immune affinity column decontamination substrate, doractin, dust does not appear in the newspapers than rhzomorph and ivermectin, does not more have commercial IAC post sale.
Summary of the invention
The purpose of this invention is to provide a kind of purifying abamectin and/or ivermectin and/or doractin and/or dust method and special immune affinity chromatographic column thereof than rhzomorph.
Purifying abamectin provided by the present invention and/or doractin and/or dust are formed by solid phase carrier with the AVM polyclonal antibody or the monoclonal antibody of its coupling than the immune affinity sorbent of rhzomorph and/or ivermectin; Described AVM polyclonal antibody or AVM monoclonal antibody are that the conjugate with AVM haptens and carrier protein is that immunogene obtains; Described AVM haptens is that C4 " connect on the position succinyl oxide obtain the 4 "-hydroxyl-succinyl oxide AVM (4 " o-succinoyl AVM) at AVM is the AVM haptens.
AVM is a small-molecule substance, has only immunoreactivity, does not have immunogenicity, can not bring out body and produce immune response, must with the macromolecular carrier albumen coupling after just have immunogenicity.The present invention obtains immunogene with carrier protein couplet again with the AVM succinic anhydride acylation.It is low or too high all unfavorable to immunity that haptens and the ratio that combines of carrier protein are crossed, and haptens was respectively 31.6: 1 and 23: 1 with the mol ratio that combines of ovalbumin (OVA), bovine serum albumin (BSA).
Described solid phase carrier can be cellulose, sephadex, polyacrylamide gel, cellular glass, Ago-Gel, ultragel ACA22 etc., is preferably Sepharose 4B.
Described AVM polyclonal antibody can be mouse source, Ma Yuan, Yang Yuan, rabbit source or cavy source antibody, described AVM monoclonal antibody is preferably the AVM mouse monoclonal antibody, and described AVM polyclonal antibody is preferred AVM rabbit polyclonal antibody.
Described AVM mouse monoclonal antibody is preferably the monoclonal hybridoma strain A-1-4CGMCC No.1606 that AVM, ivermectin, doractin and Ai Bi rhzomorph is all had cross reaction.
The described monoclonal hybridoma strain A-1-4CGMCC No.1606 that AVM, ivermectin, doractin and Ai Bi rhzomorph are all had a cross reaction has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on February 9th, 2006.
Described carrier protein can be common carrier albumen such as bovine serum albumin(BSA) or ovalbumin.
Described immune affinity sorbent can be loaded into and make immune affinity chromatographic column in the post, and this immune affinity chromatographic column also belongs to protection scope of the present invention.
The kit that contains above-mentioned immune affinity sorbent or immune affinity chromatographic column also belongs to protection scope of the present invention.
Also comprise eluent in the described kit, described eluent can be methyl alcohol.
Also comprise cleaning solution I, cleaning solution II, cleaning solution II I in the described kit and preserve liquid; Described preservation liquid can be 0.01M, and the phosphate buffer of pH 7.4, described 0.01M, the phosphate buffer of pH 7.4 can be and contain 0.2g KH among the 1L
2PO
4, 0.2g KCl, 2.9g Na
2HPO
4.12H
2O, 8.8g NaCl, 0.02gNaN
3Solution; Described cleaning solution I can be the methyl alcohol that contains volumn concentration 20% among the 1L, 0.2gKH
2PO
4, 0.2g KCl, 2.9g Na
2HPO
4.12H
2O and 29.3g NaCl, the solution of pH 7.4; Described cleaning solution II is that volumn concentration is 20% methanol solution; It is 50% methanol solution that described cleaning solution II I can be volumn concentration.
This immune affinity sorbent and the chromatographic column that contains this immune affinity sorbent are based on immune response and chromatogram reaction, be fit to purifying abamectin from biological sample (as muscle, liver, lung, kidney blood plasma), doractin, dust is convenient to retention analysis than rhzomorph and ivermectin.In this immune affinity sorbent, the coupling rate of the Sepharose 4B of AVM mouse monoclonal antibody and AVM rabbit polyclonal antibody and cyanogen bromide-activated is respectively 99.7%, 98.1%, coupling has the immune affinity chromatographic column of AVM mouse monoclonal antibody to AVM, ivermectin, dust is respectively 3884ng/mL, 3896ng/mL, 3612ng/mL, 3897ng/mL than rhzomorph and the dynamic column capacity of doractin, and absolute column capacity is respectively 545ng/mg IgG, 549ng/mg IgG, 506ng/mg IgG, 542ng/mgIgG; Dynamic column capacity is 1300-1500ng/mL after having used 15 times.
Purification AVM provided by the present invention and/or ivermectin and/or doractin and/or dust may further comprise the steps than the method for rhzomorph:
1) pre-treatment of sample:
Get animal tissue's homogenate and add 15mL methyl alcohol by the amount of 5 ± 0.01g, abundant mixing, middling speed vibration 30min on oscillator then, centrifugal 10 minutes of 3000rpm obtains supernatant and is sample solution;
2) getting the 3ml sample solution mixes with the above-mentioned preservation liquid of 20mL, cross above-mentioned immune affinity chromatographic column, then successively with 20mL cleaning solution I, 15mL cleaning solution II and 3mL cleaning solution II I washing, use 4mL eluent wash-out then, collect eluent, obtain the AVM of purifying and/or ivermectin and/or doractin and/or dust than rhzomorph solution.
Immune affinity chromatographic column of the present invention has high selectivity, and sample pretreatment process is simplified greatly, is particularly useful for the pre-treatment of micro-Avermectins medicine in muscle, liver and the blood plasma, analyzes quality and improves.The high selectivity of immune affinity sorbent makes AVM, ivermectin, and dust will depend primarily on sampling amount than the detectability of rhzomorph and doractin analytical method, and this is that simple physics and chemistry means are unapproachable; Immune affinity chromatographic column of the present invention has very strong reservation and concentrating capacity to component to be measured, as long as the application of sample amount is no more than column capacity, immune affinity sorbent is subjected to the influence of sample volume or concentration of component hardly to the reserve capability of component under the actual measurement sample condition.When purifying component, method of the present invention also can provide qualitative information.Method water operation of the present invention, simple to operate, good purification, immune affinity chromatographic column can be reused, and can save a large amount of organic solvents, reduces analysis cost and environmental pollution.Purification method of the present invention is in conjunction with chromatography efficient detection AVM, ivermectin, dust is than the content of rhzomorph and doractin, remedied simple immunoassay directly measure sample information amount very little, quantitatively accurately poor, or the low deficiency that waits of physico-chemical method selectivity, embodied immunological technique and conventional physics and chemistry technology complementarity in analysis mechanisms.
Description of drawings
Fig. 1 is the standard items of AVMs, blank musculature and the liquid chromatogram that adds sample
Fig. 2 is the apokoinou construction figure of AVMs
The specific embodiment
Experimental technique among the following embodiment if no special instructions, is conventional method.
1, the sero-fast preparation of AVM rabbit polyclonal
Haptenic synthetic three steps of branch of AVM:
(1) 5-o-t-BuMe
2Si-AVM-4 " OH's is synthetic: the 1.0g AVM is placed three mouthfuls of heart bottles of 25mL, 6mL N, N-dimethylformamide (DMF) dissolving adds the 0.47g imidazoles, mixes.Under the mechanical agitation, with the 0.52g t-BuMe of 2mL DMF dilution
2SiCl (tert-butyldimethylchlorosilane) dropwise adds, 30 ℃ of reaction 2h.Adding 100mL ethyl acetate (EtoAc) in reactant liquor mixes.Mixed liquor 50mL water washing 3 times.Separate the EtoAc layer, MgSO
4Drying, decompression concentrate and obtain little yellow dope.Should little yellow dope CH
2Cl
2Dissolving, the silica gel column chromatography separated product obtains 5-o-t-BuMe after the drying
2Si-AVM-4 " OH.
(2) 5-o-t-BuM
2" o-succinoyl's is synthetic: get 0.50g 5-o-t-BuM for Si-AVM-4
2" OH places three mouthfuls of heart bottles of 50mL to Si-R-4, adds 12mLCH
2Cl
2Make its dissolving, add 0.28g 4-dimethylamino naphthyridine (DMAP), 0.45g triethylamine and 0.92g succinyl oxide successively, refluxed in the water-bath 2.5 hours, solution becomes brown, black (irrelevant with reaction raw materials) by colourless.Evaporated under reduced pressure CH
2Cl
2After, add the 100mL ether, go in the 250mL separatory funnel after removing by filter insoluble matter, ether layer is used the 100mL water washing 2 times again with 100mL 3.6% (quality percentage composition) HCl washing 2 times.MgSO
4Drying concentrates, and gets faint yellow dope, uses CH
2Cl
2After the dissolving, with thin-layer chromatography (TLC) product is separated that (each composition volume ratio of solvent is CH
2Cl
2: oxolane (THF): CH
3OH=95: 5: 5).San Tiao district band appears in TLC, and middle maximum district's band is scraped, and uses CH
2Cl
2And CH
3OH (volume ratio is 1: 1) drip washing, decompression concentrates, and vacuum drying 24h (lucifuge) obtains 5-o-t-BuM
2Si-AVM-4 " o-succinoyl.
(3) with step 2) product that obtains sloughs protecting group 5-o-t-BuM
2Si-AVM obtains 4, and " o-succinoyl-AVM, the succinic acid derivative of this AVM is the AVM haptens.
Immunogenic preparation: this test adopts N-hydroxy-succinamide ester method (NHS) with AVM haptens and carrier protein bovine serum albumin(BSA) (BSA) or ovalbumin (OVA) coupling, obtains the BSA-AVM conjugate or the OVA-AVM conjugate is immunizing antigen.
The concrete steps that immunogene is synthesized are as follows:
1) gets 12mg AVM haptens and place the 5mL round-bottomed flask, add 0.4mL dimethyl formamide (DMF) and make the dissolving of AVM haptens, add 2.7mg N-hydroxy-succinamide ester and 4.7mg 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC.HCl) again, after the mixing, room temperature (18-20 ℃) lucifuge stirred 10 hours.
2) stir down, the reactant liquor that in 1h step 1) is obtained dropwise is added to the borate buffer solution (0.2M, pH 9.4) that 6mL contains 30mg BSA or OVA and 0.6ml DMF.It is muddy that solution begins to show slightly, and has small amount of precipitate to occur at last, continues to stir 1 hour, goes to 4 ℃ again and stirred 10-12 hour down, carries out following dialysis purifying immediately.
3) with step 2) reactant liquor that obtains changes bag filter over to (by molecular weight 10,000Da) under 4 ℃ of stirrings, dialysis 2d changes PBS therebetween three times.With the centrifugal 5min of dialysate 1000rpm, remove precipitation, the supernatant that obtains is immunogene BSA-AVM conjugate or OVA-AVM conjugate.
Animal immune: adopt new zealand white rabbit as immune animal, immunizing dose is 1mgml
-1BSA-AVM conjugate or 1mgml
-1The OVA-AVM conjugate.The New Zealand rabbit is raised immunoprophylaxis after several weeks, and first immunisation is with the emulsification of 1ml complete Freund's adjuvant, and booster immunization is with the emulsification of 1ml incomplete Freund's adjuvant.In each immunity interval 3-4 week, immunity is 5 times altogether, does not add the direct intramuscular injection of adjuvant for the last time, and blood sampling detects behind the last immune 7-10d, and after mensuration serum titer and the cross reaction, serum is collected in the arteria carotis bloodletting.
2, the preparation of AVM mouse monoclonal antibody
Animal immune: adopting the BALA/C mouse is immunogene as immune animal, with the conjugate BSA-AVM conjugate or the OVA-AVM conjugate of above-mentioned AVM haptens and protein carrier, immunizing dose is that 50 μ g/ (volume is 0.1ml) adds isopyknic complete Freund's adjuvant emulsification, carries out first immunisation.After one month, get same amount immunizing antigen and add incomplete Freund's adjuvant, booster immunization is carried out in emulsification, carries out booster immunization once more with method after one month, and two exempt from blood sampling in back 10 days, after mensuration antibody titer and the cross reaction, and extracting spleen cell.
Fusion of Cells: splenocyte carries out Fusion of Cells in 5: 1 ratios and SP2/0 myeloma cell.
Hybridoma cell cloneization: adopt limiting dilution assay screening hybridoma, up to monoclonal antibody that obtains complete homogeneity and stable monoclonal hybridoma strain A-1-4CGMCC No.1606, this monoclonal hybridoma strain all has cross reaction to AVM, ivermectin, doractin and Ai Bi rhzomorph.The monoclonal hybridoma strain A-1-4CGMCCNo.1606 that AVM, ivermectin, doractin and Ai Bi rhzomorph is all had cross reaction has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on February 9th, 2006.
Monoclonal antibody raised growth and purification: adopt in the body and induce method, the BALB/c mouse abdominal cavity is injected the sterilization paraffin oil, and pneumoretroperitoneum injections in 7-14 days all have monoclonal hybridoma strain A-1-4CGMCC No.16065 * 10 of cross reaction to AVM, ivermectin, doractin and Ai Bi rhzomorph
5-10
6Individual/as only, to gather ascites after 7-10 days.
The preservation of monoclonal antibody: at liquid nitrogen-20 ℃ preservation down, 37 ℃ of water-bath quick-thawings during use.
3, the purifying of IgG:
Adopt saturated sulfuric acid amine salt method (SAS) and DEME cellulose ion-exchange chromatography method purifying antiserum or ascites, at first IgG is slightly carried and concentrates, with DEAE52 cellulose anion exchange method IgG is further purified again with the saturated ammonium sulfate method.Its concrete steps are as follows:
Slightly carry: get 3mL AVM polyclonal antibody serum or AVM, ivermectin, doractin and Ai Bi rhzomorph are all had the monoclonal hybridoma strain A-1-4CGMCC No.1606 ascites and the 3mLPBS (0.01M of cross reaction, pH 7.0), mixing, draw saturated ammonium sulfate solution 6mL, the limit slowly drips the limit and stirs in adding serum or the ascites solution, makes the saturation degree of ammonium sulfate reach 50%.Behind 4 ℃ of placement 60min, the centrifugal 15min of 3000g.The gained precipitation is dissolved among the 3mL PBS (0.01M, pH 7.0) again, slowly drips saturated ammonium sulfate solution 1.6mL, make the ammonium sulfate saturation degree reach 35%.Behind 4 ℃ of placement 20min, with the centrifugal 15min of 3000g.Precipitation is to precipitate once in 35% the ammonium sulfate with said method in saturation degree again.
Desalination: precipitation is dissolved among (0.0175M, pH 6.7) PBS the bag filter of packing into, and bag filter is placed the PBS (0.0175M of 1000mL, pH 6.7) stir dialysis 24 hours down in 4 ℃ in the solution, the displacement buffer solution is 3 times in this process, the IgG solution of slightly being carried.
Purifying: take by weighing 2g DE-52 (DEAE-52) cellulose powder, place the beaker that fills double distilled water, fully stir, remove redundant solution after leaving standstill, add 0.5mol/L NaOH solution then, stir, Buchner funnel suction filtration behind the 1h then fully washs to neutrality with redistilled water.Handle with same method with 0.5mol/L HCl solution again.Use the 0.5mol/LNaOH solution-treated at last instead once, fully be washed to neutrality, and in the chromatographic column of then DEAE-52 that handles being packed into (2.0 * 20cm), in 0.0175M, balance among the PBS of pH 6.7.The IgG solution that the gained of saltouing is slightly carried splashes into chromatographic column, treat that whole samples enter post after, close the post end opening, and on post, covering 3-5cm 0.0175M, the PBS wash-out of pH 6.7 connects the wash-out bottle, the control flow velocity is 1.0mL/min, collects protein component with UV-detector.The eluent that will contain antibody protein precipitates with 50% saturated ammonium sulfate, after the desalination of gained precipitation, obtain ivermectin, dust are all had the AVM rabbit polyclonal antibody of cross reaction or AVM, ivermectin, doractin and Ai Bi rhzomorph are all had the monoclonal antibody of the monoclonal hybridoma strain A-1-4CGMCC No.1606 secretion of cross reaction than rhzomorph and doractin, with the antibody packing and in-20 ℃ of preservations.
4, the preparation of immune chromatograph post (IAC)
The preparation of matrix: get the Sepharose 4B dry freeze powder of cyanogen bromide-activated, filling 1.0mmol 1
-1The G of HCl
3Expand in the funnel.
Coupling reaction: with gel 0.1mol/L NaHCO
3Behind the solution equilibria, (be dissolved in 5mL 0.1mol/L NaHCO with the above-mentioned antibody purification of 20mg
3Solution) mix, stir 20h at 4 ℃.
Reactant liquor changes in the G3 funnel, uses 100mL 0.01M, and the phosphate buffer of pH 7.4 (contains potassium dihydrogen phosphate 0.27g in the 1L solution, 12 hypophosphite monohydrate disodium hydrogen 2.86g, potassium chloride 0.2g, sodium chloride 8.8g) washing, collect cleaning solution, ultraviolet is identified, calculates the coupling rate.Coupling rate computing formula is:
The testing result of coupling rate shows, rabbit polyclonal antibody and AVM, ivermectin, doractin and Ai Bi rhzomorph are all had the monoclonal antibody of monoclonal hybridoma strain A-1-4CGMCC No.1606 secretion of cross reaction and the coupling rate of the Sepharose 4B of cyanogen bromide-activated is respectively 99.7 ± 1.5%, 98.1 ± 1.6%.
The sealing in activation site: the gel after the above-mentioned coupling is changed in the Tris-HCl buffer solution that fills 0.1mol/L, pH8.0, mix, 4 ℃ are slowly stirred 2hr down, to seal the activation site of not coupling.
Washing: gel alternately washes 3 times with 0.1mol/L, pH4.0 acetate buffer and 0.1mol/L, the pH8.0Tris-HCl buffer solution of 5 times of volumes.After PBS (containing potassium dihydrogen phosphate 0.27g in the 1L solution, 12 hypophosphite monohydrate disodium hydrogen 2.86g, potassium chloride 0.2g, the sodium chloride 8.8g) balance with 0.01mol/L, pH7.4, the gel of draining changes the 0.1%NaN that contains of 0.01mol/L, pH7.4 over to
3Phosphate buffer (contains potassium dihydrogen phosphate 0.27g, 12 hypophosphite monohydrate disodium hydrogen 2.86g, potassium chloride 0.2g, sodium chloride 8.8g, NaN in the 1L solution
31g), deposit under 4 ℃ standby.
The dress post: the immunosorbent of monoclonal antibody that coupling is had the AVM rabbit polyclonal antibody or AVM, ivermectin, doractin and Ai Bi rhzomorph are all had a monoclonal hybridoma strain A-1-4CGMCC No.1606 secretion of cross reaction is transferred to and contains G
3In the glass column of filter plate, make the immune chromatograph post (IAC post) of monoclonal antibody that coupling has rabbit polyclonal antibody or AVM, ivermectin, doractin and Ai Bi rhzomorph all had the monoclonal hybridoma strain A-1-4CGMCC No.1606 secretion of cross reaction.
5, the IAC column capacity determines
The coupling of step 4 preparation there is rabbit polyclonal antibody or AVM, ivermectin, doractin and Ai Bi rhzomorph all had the immune chromatograph post of monoclonal antibody of the monoclonal hybridoma strain A-1-4CGMCC No.1606 secretion of cross reaction, wash post with preservation liquid respectively, balance.The IAC post that teetertotters is gently driven the bubble in the post away.2mL is contained 2000ngml
-1AVM (AVM), 2000ngml
-1Ivermectin (IVM), 2000ngml
-1Dust is than rhzomorph (EP) and 2000ngml
-1The preservation liquid of the hybrid standard product of 4 kinds of medicines of doractin is added to respectively on the immune affinity chromatographic column continuously, and natural gravity flows out down.(sample concentration is identical with application of sample liquid concentration in the outflow liquid) successively uses 20mL cleaning solution I (methyl alcohol that contains volumn concentration 20% among the 1L, 0.2g KH after post reaches capacity
2PO
4, 0.2g KCl, 2.9g Na
2HPO
4.12H
2O and 29.3g NaCl, pH 7.4), 15mL cleaning solution II (volumn concentration is 20% methanol solution) and 3mL cleaning solution II I (volumn concentration is 50% methanol solution) washing immune affinity chromatographic column are removed interference impurity.Use 4ml eluent (methyl alcohol) with the Avermectins medicine wash-out at last, natural gravity flows out down, collects, and dries up, and carries out HPLC and measures.Calculate dynamic column capacity and absolute column capacity.Dynamically column capacity (dynamic column capacity) is meant the obtained the maximum absorption of every milliliter of immunosorbent (or bed volume) to determinand.Absolute column capacity (specificcolumn capacity) is meant the maximum binding capacity of every milligram of sessile antibody to determinand.The result shows that the dynamic column capacity and the absolute column capacity of immune chromatograph post of monoclonal antibody that coupling has pair AVM, ivermectin, doractin and Ai Bi rhzomorph all to have the monoclonal hybridoma strain A-1-4CGMCC No.1606 secretion of cross reaction is respectively 3884ng/mL, 545ng/mg IgG (AVM); 3896ng/mL, 549ng/mg IgG (ivermectin); 3612ng/mL, 506ng/mg IgG (dust is than rhzomorph); 3897ng/mL, 542ng/mgIgG (doractin).
1, the preparation of the kit of AVM
This kit is mainly by box body, immune chromatograph post (IAC post), AVM standard liquid, the ivermectin standard liquid, doractin standard liquid, dust be than rhzomorph standard liquid, cleaning solution I, cleaning solution II, cleaning solution II I, eluent is preserved liquid, the sponge carriage is formed, and the sponge carriage is provided with hole and groove.The AVM standard liquid is housed in the groove of sponge carriage, the ivermectin standard liquid, the doractin standard liquid, dust is than rhzomorph standard liquid, wash liquid I, cleaning solution II, cleaning solution II I, eluent, the reagent bottle of preservation liquid is equipped with the IAC post in the hole of sponge carriage.Wherein the immune chromatograph post has rabbit polyclonal antibody for the coupling of embodiment 1 preparation or AVM, ivermectin, doractin and Ai Bi rhzomorph is all had the immune chromatograph post of monoclonal antibody of the monoclonal hybridoma strain A-1-4CGMCCNo.1606 secretion of cross reaction.
Eluent is a methyl alcohol.
Preservation liquid is 0.01M, and the phosphate buffer of pH 7.4 promptly contains 0.2gKH among the 1L
2PO
4, 0.2g KCl, 2.9gNa
2HPO
4.12H
2O, 8.8gNaCl, 0.02g NaN
3Solution;
Cleaning solution I is the methyl alcohol that contains volumn concentration 20% among the 1L, 0.2gKH
2PO
4, 0.2gKCl, 2.9gNa
2HPO
4.12H
2O, and 29.3gNaCl, the solution of pH 7.4;
Cleaning solution II is that volumn concentration is 20% methanol solution;
Cleaning solution II I is that volumn concentration is 50% methanol solution.
The kit of immune chromatograph post of monoclonal antibody that will contain coupling has rabbit polyclonal antibody or AVM, ivermectin, doractin and Ai Bi rhzomorph are all had a monoclonal hybridoma strain A-1-4CGMCC No.1606 secretion of cross reaction is placed on 4 ℃ respectively.
2, the purifying effect of Avermectins medicine experiment
IAC extracts principle, with specific antibody AVM rabbit polyclonal antibody or AVM, ivermectin, doractin and Ai Bi rhzomorph are all had the monoclonal antibody and the inert base coupling of the monoclonal hybridoma strain A-1-4CGMCC No.1606 secretion of cross reaction, the preparation immunosorbent, the dress post.When containing AVM and/or ivermectin and/or doractin and/or dust mixture and flow through the IAC post than rhzomorph, sessile antibody optionally compares rhzomorph in conjunction with AVM and/or ivermectin and/or doractin and/or dust, the sample impurity that other is not identified then flows out the IAC post in the clear, after washing, with the antigen-antibody complex wash-out that dissociates, AVM and/or ivermectin and/or doractin and/or dust are purified than rhzomorph.The IAC post is reusable after regeneration is handled.
The processing of test sample: animal tissue's sample: get pig muscle respectively, the liver of ox, lung, muscle, the heart, each tissue sample homogenate 5.0 ± 0.01g of kidney, in the 50ml plastic centrifuge tube, each sample adds AVM respectively by 10ng/g concentration, ivermectin, doractin and Ai Bi rhzomorph standard items, after leaving standstill 15min, add methyl alcohol 15mL, abundant mixing, 30min vibrates on oscillator, the centrifugal 10min of 3000rpm, get supernatant 3mL, mix with the preservation liquid of 20mL, as sample solution.
The immune affinity chromatographic column of monoclonal antibody that respectively coupling is had rabbit polyclonal antibody IgG or AVM, ivermectin, doractin and Ai Bi rhzomorph are all had a monoclonal hybridoma strain A-1-4CGMCC No.1606 secretion of cross reaction equilibrates to room temperature, then above-mentioned sample solution is crossed post, natural gravity flows out down, successively with 20mL cleaning solution I, 15mL cleaning solution II and 3mL cleaning solution II I washing immune affinity chromatographic column, use 4mL eluent wash-out at last, collect eluent, nitrogen dries up.
Add 100 μ L derivatization reagent A liquid (is that 3: 4 1-methylimidazole and acetonitrile formed by volume ratio), whirling motion 0.5 minute, add 100 μ L derivatization reagent B liquid (is that 2: 5 acetic anhydride and acetonitrile formed by volume ratio) again, whirling motion 0.5min, airtight, in 96 ℃ of derivative reactions 100 minutes, be settled to 2.5mL with acetonitrile, cross 0.45 μ m syringe filter membrane, advance HPLC and analyze (chromatographic condition: C
18Reverse-phase chromatographic column, Inertsil ODS-3 (4.6mm * 250mm, particle diameter 5 μ m); Flowing is 97% methanol solution mutually for volumn concentration; Flow velocity is 1mL/min; Sample size is 20 μ L; Excitation wavelength is 365nm, emission wavelength 475nm).The IAC post is kept in 4 ℃ of refrigerators standby with the preservation liquid balance of 20ml.The result shows, carry out sample purification with IAC, interference medicament chromatographic peak not, can separate fully, illustrate that the IAC non-specific adsorption of the present invention's preparation is minimum, wherein the AVM in the pig muscle, ivermectin, doractin and Ai Bi rhzomorph clean-up effect as shown in Figure 1, among Fig. 1, A is the standard solution that contains AVM, ivermectin, doractin and each 10ng/mL of Ai Bi rhzomorph; B is for adding 10ng/g AVM, 10ng/g ivermectin, 10ng/g doractin and the 10ng/g dust pig muscle concentration of specimens than rhzomorph standard items; C is not for adding the pig muscle tissue of AVM, ivermectin, doractin and Ai Bi rhzomorph; Among Fig. 1,1 be dust than rhzomorph B1a, 2 is AVM B1a, 3 is doractin, 4 is ivermectin B1a.
Claims (12)
1. purifying abamectin and/or ivermectin and/or doractin and/or dust are formed by solid phase carrier with the AVM monoclonal antibody of its coupling than the immune affinity sorbent of rhzomorph; Described AVM monoclonal antibody is the monoclonal antibody of monoclonal hybridoma strain A-1-4 CGMCC No.1606 secretion.
2. adsorbent according to claim 1 is characterized in that: described solid phase carrier is cellulose, sephadex, polyacrylamide gel, cellular glass, Ago-Gel or ultragel ACA22.
3. adsorbent according to claim 2 is characterized in that: described solid phase carrier is Sepharose 4B.
4. the immune affinity chromatographic column that is filler with the arbitrary described immune affinity sorbent of claim 1-3.
5. the kit that contains the arbitrary described immune affinity sorbent of claim 1-3.
6. according to the described kit of claim 5, it is characterized in that: comprise also in the described kit that eluent, described eluent are methyl alcohol.
7. according to the described kit of claim 6, it is characterized in that: also comprise cleaning solution I, cleaning solution II, cleaning solution II I in the described kit and preserve liquid; Described preservation liquid is 0.01M, and the phosphate buffer of pH7.4, described 0.01M, the phosphate buffer of pH7.4 are to contain 0.2g KH among the 1L
2PO
4, 0.2g KCl, 2.9gNa
2HPO
4.12H
2O, 8.8gNaCl, 0.02g NaN
3Solution; Described cleaning solution I is the methyl alcohol that contains volumn concentration 20% among the 1L, 0.2gKH
2PO
4, 0.2gKCl, 2.9gNa
2HPO
4.12H
2O and 29.3gNaCl, the solution of pH 7.4; Described cleaning solution II is that volumn concentration is 20% methanol solution; Described cleaning solution II I is that volumn concentration is 50% methanol solution.
8. the kit that contains the described immune affinity chromatographic column of claim 4.
9. described according to Claim 8 kit is characterized in that: comprise also in the described kit that eluent, described eluent are methyl alcohol.
10. according to the described kit of claim 9, it is characterized in that: also comprise cleaning solution I, cleaning solution II, cleaning solution II I in the described kit and preserve liquid; Described preservation liquid is 0.01M, and the phosphate buffer of pH7.4, described 0.01M, the phosphate buffer of pH7.4 are to contain 0.2g KH among the 1L
2PO
4, 0.2g KCl, 2.9gNa
2HPO
4.12H
2O, 8.8gNaCl, 0.02g NaN
3Solution; Described cleaning solution I is the methyl alcohol that contains volumn concentration 20% among the 1L, 0.2gKH
2PO
4, 0.2gKCl, 2.9gNa
2HPO
4.12H
2O and 29.3gNaCl, the solution of pH7.4; Described cleaning solution II is that volumn concentration is 20% methanol solution; Described cleaning solution II I is that volumn concentration is 50% methanol solution.
11. purifying abamectin and/or ivermectin and/or doractin and/or dust may further comprise the steps than the method for rhzomorph:
1) pre-treatment of sample:
Get animal tissue's homogenate and add 15mL methyl alcohol by the amount of 5 ± 0.01g, abundant mixing, middling speed vibration 30min on oscillator then, centrifugal 10 minutes of 3000rpm obtains supernatant and is sample solution;
2) getting the preservation liquid described in the claim 7 of 3ml sample solution and 20mL mixes, cross the described immune affinity chromatographic column of claim 4, then successively with the cleaning solution II I washing described in cleaning solution I, the cleaning solution II described in the 15mL claim 7 and the 3mL claim 7 described in the 20mL claim 7, use the eluent wash-out described in the 4mL claim 6 then, collect eluent, obtain the AVM of purifying and/or doractin and/or ivermectin and/or dust than rhzomorph solution.
12. method according to claim 11 is characterized in that: described animal tissue sample comprises muscle, liver, lung, the heart, kidney and blood plasma.
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| CN102258987B (en) * | 2011-04-11 | 2013-03-13 | 江苏大学 | Preparation and use of dehydro methyltestosterone immunoaffinity column with chitosan (CTS) as vector |
| CN107082784B (en) * | 2017-04-10 | 2019-08-09 | 中国农业大学 | A kind of polyclonal antibody and its preparation method and application for detecting moxidectin |
| CN109180760B (en) * | 2018-08-30 | 2021-09-17 | 华中农业大学 | Ivermectin derivative, monoclonal antibody of anti-avermectin drug and application of monoclonal antibody |
| CN109541216B (en) * | 2018-10-26 | 2022-10-21 | 北京勤邦生物技术有限公司 | Enzyme linked immunosorbent assay kit for detecting ivermectin and abamectin and application thereof |
| CN112326815B (en) * | 2020-10-16 | 2023-06-06 | 丽珠集团新北江制药股份有限公司 | Method for detecting various antibiotic residues in livestock tissues by liquid chromatography-mass spectrometry |
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