CN100402522C - Method for purifying halofuginone and its special immune affinity chromatographic column - Google Patents
Method for purifying halofuginone and its special immune affinity chromatographic column Download PDFInfo
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- CN100402522C CN100402522C CNB2006100035491A CN200610003549A CN100402522C CN 100402522 C CN100402522 C CN 100402522C CN B2006100035491 A CNB2006100035491 A CN B2006100035491A CN 200610003549 A CN200610003549 A CN 200610003549A CN 100402522 C CN100402522 C CN 100402522C
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Abstract
The present invention discloses a method for purifying halofuginone and immune affinity chromatographic columns thereof. An adsorbing agent carried by the immune affinity chromatographic columns for purifying halofuginone is composed of a solid-phase carrier and a polyclonal halofuginone antibody or a monoclonal halofuginone antibody coupled with the solid-phase carrier; the polyclonal halofuginone antibody or the monoclonal halofuginone antibody is obtained by using a conjugate of halofuginone semiantigens and carrier protein as an immunogen; the halofuginone semiantigen is a succinic acid derivative of halofuginone obtained by dissolving hydrobromic acid halofuginone in N,-N, dimethyl formamide and then adding trimethylsilane iminazole. The purifying method of the present invention can combine a chromatographic method to effectively detect the content of the halofuginone. Thus, the disadvantages of less information quantity and poor quantitative accuracy of a method for directly detecting samples only by an immunoassay technology or low selectivity by a physicochemical method, etc. are compensated. The complementarity of the immunological technique and the conventional physicochemical technology in an analysis mechanism is embodied.
Description
Technical field
The present invention relates to a kind of method and special immune affinity chromatographic column thereof of purifying halofuginone.
Background technology
Along with development of life science, people have produced more and more keen interest to intravital material of biology 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 procedure.Immune affinity chromatographic (IAC, immunoaffinity chromatography) is a kind of analytical procedure 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.
Halofuginone hydrobromide (Halofuginone) is a kind of broad-spectrum anti-parasite medicine, to tender, the murder by poisoning of chicken, heap type, displacement, huge, the Bu Shi Eimeria is all effective, and sporozoite, first-generation schizont and the s-generation schizont of coccidia all had tangible inhibitory or killing effect.Also effective to most of gram-positive microorganisms, and do not have cross resistance.In aviculture, halofuginone hydrobromide is widely used for preventing and is treated the coccidiosis of poultry as a kind of fodder additives.Like this with regard to the potential danger that enters in the people's food chain.Many countries have set up the method for detecting residue of halofuginone hydrobromide and have made residual regulation of limiting the quantity of.In the U.S., the national residue detection that halofuginone hydrobromide has been put into FSIS in the works, the high residue that is defined in halofuginone hydrobromide in the chicken gizzard detects and is limited to 0.16mgkg
-1, the off-drug period is 4 days.China's regulation halofuginone hydrobromide maximum residue limit(MRL) in chicken gizzard, muscle is respectively 0.13mgkg
-1And 0.1mgkg
-1, the off-drug period is five days.
The method that detects halofuginone hydrobromide at present mainly contains vapor-phase chromatography (GC-UV), high performance liquid chromatography (HPLC-UV), the online LC-MS-MS of liquid-matter and euzymelinked immunosorbent assay (ELISA) ELISA etc.The pre-treatment of these methods utilizes liquid-liquid partition, conventional SPE column purification with separate, all exist shortcomings such as treating processes is loaded down with trivial details, decontamination effect improving 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 does not appear in the newspapers with the halofuginone hydrobromide in the immune affinity column decontamination substrate, does not more have commercial IAC post and sells.
Summary of the invention
The method and the special immune affinity sorbent thereof that the purpose of this invention is to provide a kind of purifying halofuginone.
The immune affinity sorbent of purifying halofuginone provided by the present invention is formed by solid phase carrier with its link coupled halofuginone hydrobromide polyclonal antibody or monoclonal antibody; Described halofuginone hydrobromide polyclonal antibody or halofuginone hydrobromide monoclonal antibody are that the conjugate with halofuginone hydrobromide haptens and carrier proteins is that immunogen obtains; Described halofuginone hydrobromide haptens is that Hydrogen bromide halofuginone hydrobromide (HAL) is dissolved in N,-N in the dimethyl formamide (DMF), adds N-TMS imidazoles (TMSI again, N-Trimethysiylimidazole) react, the succinic acid derivative that obtains halofuginone hydrobromide is the halofuginone hydrobromide haptens.
Halofuginone hydrobromide is a small-molecule substance, has only immunoreactivity, does not have immunogenicity, can not bring out body and produce immunne response, must with the macromolecular carrier albumen coupling after just have immunogenicity.The present invention uses N with Hydrogen bromide halofuginone hydrobromide (HAL);-N; dimethyl formamide (DMF) acidylate; pick out a spacerarm that contains 4 carbon and formed haptens; given prominence to the feature structure of halofuginone hydrobromide haptenic group like this, helped to prepare at the stronger polyclonal antibody of halofuginone hydrobromide antigen-specific.Again halofuginone hydrobromide is adopted N-hydroxy succinic acid imines method (active fat method) and carrier protein couplet to obtain immunogen.Haptens is crossed the low or too high generation that all is unfavorable for specific antibody with the ratio that combines of carrier proteins, and haptens was respectively 5: 1 and 9.5: 1 with the mol ratio that combines of ovalbumin (OVA), bovine serum albumin (BSA).
Described solid phase carrier has Mierocrystalline cellulose, dextrane gel, polyacrylamide gel, sintered glass, sepharose, ultragel ACA22 etc., is preferably Sepharose 4B.
Described halofuginone hydrobromide antibody can be mouse source, Ma Yuan, Yang Yuan, rabbit source or cavy source antibody, and described halofuginone hydrobromide monoclonal antibody is preferably the halofuginone hydrobromide mouse monoclonal antibody, and described halofuginone hydrobromide polyclonal antibody is preferably the halofuginone hydrobromide rabbit polyclonal antibody.
Described halofuginone hydrobromide mouse monoclonal antibody is preferably the monoclonal hybridoma strain A-2--2 CGMCC No.1608 excretory monoclonal antibody of halofuginone hydrobromide.
The monoclonal hybridoma strain A-2--2CGMCC No.1608 of described halofuginone hydrobromide 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 proteins is common carrier albumen such as bovine serum albumin 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 test kit that contains above-mentioned immune affinity sorbent or immune affinity chromatographic column also belongs to protection scope of the present invention.
Also comprise elutriant in the described test kit, described elutriant is chromatographic grade methyl alcohol.
Also comprise washings in the described test kit and preserve liquid; Described washings is 0.01mol/L, and the phosphate buffered saline buffer of pH7.4 is for containing potassium primary phosphate 0.27g, 12 hypophosphite monohydrate disodium hydrogen 2.86g, Repone K 0.2g, sodium-chlor 8.8g in the 1L solution; Described preservation liquid is to contain potassium primary phosphate 0.27g in the 1L solution, 12 hypophosphite monohydrate disodium hydrogen 2.86g, Repone K 0.2g, sodium-chlor 8.8g, NaN
30.2g, pH7.4.
This immunity is affine attached dose and chromatographic column are fit to purifying halofuginone from biological sample (as muscle, liver, lung, kidney blood plasma) based on immune response and chromatogram reaction, are convenient to retention analysis.In attached dose of affine absorption of this immunity and chromatographic column, the coupling rate of the monoclonal hybridoma strain A-2--2 CGMCC No.1608 excretory monoclonal antibody of rabbit polyclonal antibody IgG and hybridoma cell strain halofuginone hydrobromide and the Sepharose 4B of cyanogen bromide-activated is respectively 96.7 ± 1.5%, 97.3 ± 1.5%.Dynamically column capacity is 1500-6000ng, and column capacity is about 51% of former total column capacity after using 20 times.
The method of purifying halofuginone provided by the present invention may further comprise the steps:
1) pre-treatment of sample:
Adding 5ml concentration in 2g animal tissues sample is 0.5mgl
-1Trypsinase is 10% NaCO with the quality percentage composition
3The solution adjust pH is 7-8, and enzymolysis 3h in 40 ℃ of water-baths takes out and puts to room temperature, and the quality percentage composition that adds 2ml is 10% NaCO
3Solution, mixing adds the 10ml ethyl acetate again, whirling motion 40s leaves standstill 3min at mixture of ice and water, centrifugal two minutes of 2000g, get supernatant liquor, add the 0.125mol/L ammonium acetate solution of 5ml, whirling motion 1min leaves standstill 3min at mixture of ice and water, 2000g is centrifugal two minutes immediately, collect normal hexane that lower layer of water is added to 5ml jolting 20s gently, remove ethyl acetate and normal hexane, the water that obtains is a sample solution;
2) sample solution that step 1) is obtained is crossed immune affinity chromatographic column, with the washings washing, uses above-mentioned elutriant wash-out more then, obtains the halofuginone hydrobromide solution of purifying.
Immune affinity sorbent of the present invention and chromatographic column have highly selective, and sample pretreatment process is simplified greatly, are particularly useful for the pre-treatment of micro-HAL in muscle, liver and the blood plasma, analyze quality and improve.The highly selective of immune affinity sorbent makes the detectability of halofuginone hydrobromide analytical procedure will depend primarily on sampling amount, and this is that simple physics and chemistry means are unapproachable; Immune affinity sorbent of the present invention and chromatographic column have 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 save power 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.Purifying method of the present invention is in conjunction with the content of chromatography efficient detection halofuginone hydrobromide, 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 high performance liquid chromatography design sketch of Hydrogen bromide halofuginone hydrobromide in the fish muscle of immune affinity chromatographic column purification
Embodiment
Experimental technique among the following embodiment if no special instructions, is ordinary method.
The preparation of the immune chromatograph post of embodiment 1, purifying halofuginone
1, the preparation of halofuginone hydrobromide rabbit polyclonal antibody
Halofuginone hydrobromide is haptenic synthetic: (HAL) is dissolved in N with the Hydrogen bromide halofuginone hydrobromide, and-N is in the dimethyl formamide (DMF), add N-TMS imidazoles (TMSI, N-Trimethysiylimidazole), the white precipitate of generation is the halofuginone hydrobromide succinic acid derivative, is the halofuginone hydrobromide haptens.
Immunogenic preparation: adopt N-hydroxy succinic acid imines to live (active fat method),, make immunogen BSA-HAL or OVA-HAL with halofuginone hydrobromide haptens and protein carrier bovine serum albumin (BSA) or ovalbumin (OVA) coupling.
Animal immune: adopt new zealand white rabbit as immune animal, immunizing dose is 1mgml
-1BSA-HAL conjugate or 1mgml
-1The OVA-HAL 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.Each immunity is 3-4 week at interval, has immunity 5 times altogether, and last immunity does not add the direct intramuscular injection of adjuvant, and blood sampling detects behind the last immune 7-10d, and behind the mensuration serum titer, serum is collected in the carotid artery bloodletting.
2, the preparation of halofuginone hydrobromide mouse monoclonal antibody
Animal immune: adopting the BALA/C mouse as immune animal, is immunogen with the conjugate BSA-HAL or the OVA-HAL of above-mentioned halofuginone hydrobromide haptens and protein carrier, and immunizing dose is 50 μ g/.The first immunisation emulsification of immunogen and equal-volume complete Freund's adjuvant.After one month, get same amount immunizing antigen and add incomplete Freund's adjuvant emulsification, carry out booster immunization.Carried out booster immunization once more with method after one month, two exempt from blood sampling in back 10 days, measure antibody titer, extracting spleen cell.
Cytogamy: splenocyte carries out cytogamy in 5: 1 ratio and SP2/0 myeloma cell.
Hybridoma cell cloneization: adopt limiting dilution assay screening hybridoma, up to the monoclonal hybridoma strain A-2--2CGMCC No.1608 of monoclonal antibody that obtains complete homogeneity and stable halofuginone hydrobromide.The monoclonal hybridoma strain A-2--2CGMCC No.1608 of halofuginone hydrobromide 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 mass production and purification: adopt in the body and induce method, the BALB/c mouse abdominal cavity is injected the sterilization paraffin oil, monoclonal hybridoma strain A-2--2CGMCC No.16085 * 10 of 7 days pneumoretroperitoneum injection halofuginone hydrobromides
5-10
6Individual/as only, to gather ascites after 7-10 days.
The preservation of monoclonal antibody: preserve 37 ℃ of water-bath quick-thawings during use at-20 ℃ down.
3, the purifying of IgG:
Adopt saturated sulfuric acid amine salt method (SAS) and DEME cellulose ion-exchange chromatography method purifying antiserum(antisera) or ascites.Its concrete steps are as follows:
(1) SAS saltouts: 1) 50% saturation ratio is saltoutd: get the rabbit anti-serum of above-mentioned preparation or the mouse ascites fluid 5ml that obtains of monoclonal hybridoma strain A-2--2 CGMCC No.1608 of injection halofuginone hydrobromide, the PBS that adds equivalent 0.01mol/L, pH7.4 (contains potassium primary phosphate 0.27g in the 1L solution, 12 hypophosphite monohydrate disodium hydrogen 2.86g, Repone K 0.2g, sodium-chlor 8.8g) mixing, dropwise add isopyknic saturated ammonium sulphate (pH7.4) solution then, the limit edged stirs, room temperature is placed 30min, the centrifugal 30min of 3000g, abandoning supernatant.2) 33% saturation ratio is saltoutd: step 1) gained precipitation is dissolved in 5ml 0.01mol/L PBS, adds saturated ammonium sulphate solution again and reach 33% saturation ratio, the limit edged stirs, and room temperature is placed 30min, abandoning supernatant.This step repetitive operation 2 times.3) desalination: with step 2) precipitation that obtains is dissolved among the PBS of 0.01mol/L, pH7.4 and (contains potassium primary phosphate 0.27g in the 1L solution, 12 hypophosphite monohydrate disodium hydrogen 2.86g, Repone K 0.2g, sodium-chlor 8.8g), be loaded on and inhale in the bag, be suspended from the PBS that fills 0.01mol/L, pH7.4 and (contain potassium primary phosphate 0.27g in the 1L solution, 12 hypophosphite monohydrate disodium hydrogen 2.86g, Repone K 0.2g, sodium-chlor 8.8g) dialysis desalting in the beaker, be positioned over 4 ℃, change liquid 3-4 every day, with 1%BaCl
2Detection is in dialyzate till the sulfate radical-free ion.4) dialysis finishes, and the centrifugal 5min of 3000g gets supernatant liquor and carries out the DEME-cellulose ion-exchange chromatography.
(2) DEME-cellulose ion-exchange chromatography: 1) the cellulosic processing of DE-52: take by weighing 2g DE-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, B suction filtration behind the 1h, then extremely neutral with the redistilled water thorough washing.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.2) balance: will (contain potassium primary phosphate 0.27g in the 1L solution through the PBS that the DE-52 Mierocrystalline cellulose that step 1) is handled is soaked in 0.01mol/L, pH7.4,12 hypophosphite monohydrate disodium hydrogen 2.86g, Repone K 0.2g, sodium-chlor 8.8g) in, thorough washing, remove redundant solution after leaving standstill, 2-3 time so repeatedly, pH reaches till 7.4 until supernatant liquor.3) dress post: continuously add in chromatography column with dropper the DE-52 Mierocrystalline cellulose after the balance, with the elutriant (PBS of 0.01mol/L, pH7.4, contain potassium primary phosphate 0.27g in the 1L solution, 12 hypophosphite monohydrate disodium hydrogen 2.86g, Repone K 0.2g, sodium-chlor 8.8g) continuous wash-out, fully stream is washed, till the pH value of effluent liquid pH value and elutriant is identical.4) application of sample: the SAS gained antibody of saltouing (is contained potassium primary phosphate 0.27g with the phosphate buffered saline buffer of 0.01mol/L, pH7.4 in the 1L solution, 12 hypophosphite monohydrate disodium hydrogen 2.86g, Repone K 0.2g, sodium-chlor 8.8g) dilution, slowly add along the chromatography post jamb then, open the chromatography column outlet, allow the IgG diluent flow in the post bed, wash post jamb with phosphate buffered saline buffer again.5) wash-out and collection: add the PBS of elutriant 0.01mol/L, pH7.4, contain potassium primary phosphate 0.27g in the 1L solution, 12 hypophosphite monohydrate disodium hydrogen 2.86g, Repone K 0.2g, sodium-chlor 8.8g), every pipe 2ml collects, and detects IgG wash-out situation while collecting with 20% sulphosalicylic acid.6) measure the OD value of IgG solution with ultraviolet spectrophotometer, show the halofuginone hydrobromide rabbit polyclonal antibody that obtains purifying and the monoclonal hybridoma strain A-2--2 CGMCC No.1608 excretory monoclonal antibody of halofuginone hydrobromide at 280nm and 260nm place.IgG behind the purifying preserves at-20 ℃ of refrigerators.
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 l
-1The G of HCl
3Expand in the funnel.
The preparation of IgG antibody: with the NaHCO of 0.01mol/L
3Solution is with the monoclonal hybridoma strain A-2--2 CGMCC No.1608 excretory monoclonal antibody dilution of the rabbit polyclonal antibody IgG or the halofuginone hydrobromide of purifying, and transferring the pH value of solution is 8.4.
Linked reaction: with the NaHCO of expansible colloidal sol with 0.01mol/L
3Behind the solution equilibria, change in the above-mentioned antibody-solutions, mix, 4 ℃ are slowly stirred 20-24hr down.The detected result of coupling rate shows that the coupling rate of the monoclonal hybridoma strain A-2--2 CGMCC No.1608 excretory monoclonal antibody of rabbit polyclonal antibody IgG and halofuginone hydrobromide and the Sepharose 4B of cyanogen bromide-activated is respectively 96.7 ± 1.5%, 97.3 ± 1.5%.
The sealing in activation site: the gel after the above-mentioned coupling is changed in the Tris-HCl damping fluid that fills 0.1mol/L, pH8.0, mix, 4 ℃ are slowly stirred 2hr down, to seal not link coupled activation site.
Washing: gel alternately washes 3 times with 0.1mol/L, pH4.0 acetate buffer and 0.1mol/L, the pH8.0Tris-HCl damping fluid of 5 times of volumes.After PBS (containing potassium primary phosphate 0.27g in the 1L solution, 12 hypophosphite monohydrate disodium hydrogen 2.86g, Repone K 0.2g, the sodium-chlor 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 buffered saline buffer (contains potassium primary phosphate 0.27g, 12 hypophosphite monohydrate disodium hydrogen 2.86g, Repone K 0.2g, sodium-chlor 8.8g, NaN in the 1L solution
31g), deposit under 4 ℃ standby.
Dress post: have the immunosorbent of the monoclonal hybridoma strain A-2--2CGMCC No.1608 excretory monoclonal antibody of halofuginone hydrobromide rabbit polyclonal antibody IgG or halofuginone hydrobromide to be transferred to coupling and contain G
3In the glass column of filter plate, make the immune affinity chromatographic column (IAC post) that coupling has the monoclonal hybridoma strain A-2--2CGMCC No.1608 excretory monoclonal antibody of rabbit polyclonal antibody or halofuginone hydrobromide.
5, the IAC column capacity determines
Immune affinity chromatographic column with step 4 preparation, PBS with 10ml 0.01mol/L, pH7.4 (contains potassium primary phosphate 0.27g in the 1L solution, 12 hypophosphite monohydrate disodium hydrogen 2.86g, Repone K 0.2g, sodium-chlor 8.8g) wash post, IAC post gently teetertotters, drive the bubble in the post away, the PBS with 10ml 0.01mol/L, pH7.4 (contains potassium primary phosphate 0.27g, 12 hypophosphite monohydrate disodium hydrogen 2.86g in the 1L solution again, Repone K 0.2g, sodium-chlor 8.8g) balance.To contain 100ngml
-1The PBS solution of Hydrogen bromide halofuginone hydrobromide (HAL) (containing potassium primary phosphate 0.27g in the 1L solution, 12 hypophosphite monohydrate disodium hydrogen 2.86g, Repone K 0.2g, sodium-chlor 8.8g) is added to the IAC post continuously, and natural gravity flows out down.After post reaches capacity (concentration of HAL is identical with application of sample liquid concentration in the effluent liquid),, remove interference impurity with 20ml PBS (containing potassium primary phosphate 0.27g in the 1L solution, 12 hypophosphite monohydrate disodium hydrogen 2.86g, Repone K 0.2g, sodium-chlor 8.8g) washing IAC post.Use 3ml methyl alcohol with the HAL wash-out at last, natural gravity flows out down, collects, and dries up, and to 1ml, crosses 0.25 millipore filtration with the moving phase constant volume, carries out HPLC and measures, and calculates 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 column volume) to determinand.Absolute column capacity (specific columncapacity) is meant the maximum binding capacity of every milligram of sessile antibody to determinand.The result shows that coupling has the dynamic column capacity and the absolute column capacity of the immune affinity chromatographic column of rabbit polyclonal antibody IgG to be respectively 1800ng/mL and 400ng/mg, and coupling has the dynamic column capacity of immune affinity chromatographic column and the absolute column capacity of the monoclonal hybridoma strain A-2--2CGMCC No.1608 excretory monoclonal antibody of halofuginone hydrobromide to be respectively 2000ng/mL and 600ng/mg.
Embodiment 2, coupling have rabbit polyclonal antibody IgG or mouse monoclonal antibody immune affinity chromatographic column test kit preparation and to the decontamination effect improving of halofuginone hydrobromide
1, the preparation of the test kit of purifying halofuginone
Mainly by box body, immune affinity chromatographic column (IAC post), the HAL standardized solution, diluent, washings, elutriant is preserved liquid, and the sponge carriage is formed, and the sponge carriage is provided with hole and groove.The HAL standardized solution is housed in the groove of sponge carriage, diluent, washings, elutriant, the reagent bottle of preservation liquid is equipped with the IAC post in the hole of sponge carriage.Wherein the immune chromatograph post has the immune affinity chromatographic column of the monoclonal hybridoma strain A-2--2 CGMCC No.1608 excretory monoclonal antibody of rabbit polyclonal antibody IgG or halofuginone hydrobromide for the coupling of embodiment 1 preparation.
Wherein, washings is that (0.01M pH7.4), consists of phosphate buffered saline buffer: contain potassium primary phosphate 0.27g in the 1L solution, 12 hypophosphite monohydrate disodium hydrogen 2.86g, Repone K 0.2g, sodium-chlor 8.8g;
Elutriant is chromatographic grade methyl alcohol;
Preserving liquid is to contain potassium primary phosphate 0.27g in the 1L solution, 12 hypophosphite monohydrate disodium hydrogen 2.86g, Repone K 0.2g, sodium-chlor 8.8g, NaN
30.2g, pH7.4.
2, the extraction effect of halofuginone hydrobromide experiment
IAC extracts principle, with the monoclonal hybridoma strain A-2--2CGMCC No.1608 excretory monoclonal antibody and the inert base coupling of specific antibody halofuginone hydrobromide rabbit polyclonal antibody IgG or halofuginone hydrobromide, and preparation immunosorbent, dress post.When the mixture that contains halofuginone hydrobromide flows through the IAC post, fixed antibody is optionally in conjunction with halofuginone hydrobromide, and 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, halofuginone hydrobromide is purified or separates.The IAC post is reusable after manipulation of regeneration.
The processing of test sample: animal tissues's sample: get fish muscle respectively, the liver of pig, lung, muscle, the heart, each tissue sample 2g of kidney are in the 50ml plastic centrifuge tube, this concentration by 50 μ g/kg of every increment is added the halofuginone hydrobromide standard substance, adds trypsin 5ml, 0.5mgl
-1), with the quality percentage composition 10% NaCO
3The solution adjust pH is between 7-8.Enzymolysis 3h in 40 ℃ of water-baths.Taking-up is put to room temperature, and the quality percentage composition that adds 2ml is 10%NaCO
3Solution, mixing adds the 10ml ethyl acetate again, and whirling motion 40s leaves standstill 3min at mixture of ice and water, and 2000g is centrifugal two minutes immediately, and supernatant liquor is changed in another clean centrifuge tube.Extract once the combined ethyl acetate layer again with the 10ml ethyl acetate.Add the 0.125M ammonium acetate solution of 5ml, whirling motion 1min leaves standstill 3min at mixture of ice and water, and 2000g is centrifugal two minutes immediately, collects water layer, and the 0.125M ammonium acetate solution that adds 5ml again extracts once again.Combining water layer, the normal hexane of adding 5ml is jolting 20s gently, and taking off layer water is sample solution.
Respectively coupling there is the immune affinity chromatographic column of the monoclonal hybridoma strain A-2--2CGMCC No.1608 excretory monoclonal antibody of rabbit polyclonal antibody IgG or halofuginone hydrobromide to equilibrate to room temperature, respectively with the flushing of the washings in the test kit of 10ml, then above-mentioned sample solution is crossed post, with the washing of 3ml washings, remove the impurity of non-specific adsorption.With 3ml elutriant wash-out, then at 40 ℃ of N
2Evaporate to dryness under the air-flow, moving phase (the 250ml acetonitrile that adds 1ml, 150ml 0.25mol/L ammonium acetate buffer (transferring pH=4.3 with glacial acetic acid) is settled to 1000ml with pure water then, 0.45 μ m membrane filtration), whirling motion, cross 0.25 μ m millipore filtration, under HPLC-UV, measure the content of halofuginone hydrobromide, wherein, standard substance are 50 μ g/kg Hydrogen bromide halofuginone hydrobromide standard substance, dilute with diluent (containing 9.64g amine acetate and 15ml Glacial acetic acid in the 1L solution).The IAC post with the preservation liquid balance of 20ml be stored in 4 ℃ standby.The result shows, carry out sample purification with IAC, interference medicament chromatographic peak not, can separate fully, the IAC non-specific adsorption that preparation is described is minimum, wherein coupling have halofuginone hydrobromide monoclonal hybridoma strain A-2--2 CGMCC No.1608 excretory monoclonal antibody immune affinity chromatographic column for the decontamination effect improving of fish muscle as shown in Figure 1, A is standardized solution color atlas (50ng/mL) among Fig. 1; B is blank fish muscle (not adding the fish muscle samples of a halofuginone hydrobromide) color atlas; C is for adding sample chromatogram figure (50ng/g).
Claims (10)
1. the immune affinity sorbent of a purifying halofuginone is made up of monoclonal hybridoma strain A-2-2CGMCC No.1608 excretory monoclonal antibody by solid phase carrier sepharose Sepharose 4B with its link coupled.
2. be mounted with the immune affinity chromatographic column of the described immune affinity sorbent of claim 1.
3. the test kit that contains the described immune affinity sorbent of claim 1.
4. according to the described test kit of claim 3, it is characterized in that: comprise also in the described test kit that elutriant, described elutriant are methyl alcohol.
5. according to the described test kit of claim 4, it is characterized in that: also comprise washings in the described test kit and preserve liquid; Described washings is pH7.4, and the phosphate buffered saline buffer of 0.01mol/L, described 0.01mol/L phosphate buffered saline buffer are to contain potassium primary phosphate 0.27g in the 1L solution, 12 hypophosphite monohydrate disodium hydrogen 2.86g, Repone K 0.2g, sodium-chlor 8.8g; Described preservation liquid is to contain potassium primary phosphate 0.27g in 11 solution, 12 hypophosphite monohydrate disodium hydrogen 2.86g, Repone K 0.2g, sodium-chlor 8.8g, NaN
30.2g, pH7.4.
6. the test kit that contains the described immune affinity chromatographic column of claim 2.
7. according to the described test kit of claim 6, it is characterized in that: comprise also in the described test kit that elutriant, described elutriant are methyl alcohol.
8. according to the described test kit of claim 7, it is characterized in that: also comprise washings in the described test kit and preserve liquid; Described washings is pH7.4, and the phosphate buffered saline buffer of 0.01mol/L, described 0.01mol/L phosphate buffered saline buffer are to contain potassium primary phosphate 0.27g in the 1L solution, 12 hypophosphite monohydrate disodium hydrogen 2.86g, Repone K 0.2g, sodium-chlor 8.8g; Described preservation liquid is to contain potassium primary phosphate 0.27g in the 1L solution, 12 hypophosphite monohydrate disodium hydrogen 2.86g, Repone K 0.2g, sodium-chlor 8.8g, NaN
30.2g, pH7.4.
9. the method for a purifying halofuginone may further comprise the steps:
1) pre-treatment of sample:
It is 0.5mgl that animal tissues's sample is added 5ml concentration according to the amount of 2g
-1Trypsinase is 10% NaCO with the quality percentage composition
3The solution adjust pH is 7-8, and enzymolysis 3h in 40 ℃ of water-baths takes out and puts to room temperature, and the quality percentage composition that adds 2ml is 10% NaCO
3Solution, mixing adds the 10ml ethyl acetate again, whirling motion 40s, leave standstill 3min at mixture of ice and water, centrifugal two minutes of 2000g takes out supernatant liquor, the 0.125mol/L ammonium acetate solution that adds 5ml, whirling motion 1min leaves standstill 3min at mixture of ice and water, and 2000g is centrifugal two minutes immediately, collect normal hexane that lower layer of water is added to 5ml jolting 20s gently, taking off layer water is sample solution;
2) sample solution that step 1) is obtained is crossed the described immune affinity chromatographic column of claim 2, with the described washings washing of claim 8, uses the described elutriant wash-out of claim 7 again, the halofuginone hydrobromide solution that is purified then.
10. method according to claim 9 is characterized in that: described animal tissues sample comprises muscle, liver, lung, kidney and blood plasma.
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| CN102539587B (en) * | 2012-01-04 | 2013-11-06 | 河南科技大学 | Preparation method of halofuginone molecularly-imprinted solid-phase extraction small column and application |
| CN102580693B (en) * | 2012-01-04 | 2013-08-07 | 河南科技大学 | Preparation method and application of phloroglucinol molecularly imprinted polymers in plants |
Citations (4)
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|---|---|---|---|---|
| WO2001042787A2 (en) * | 1999-12-08 | 2001-06-14 | Syngenta Participations Ag | Immunoassay for neonicotinyl insecticides |
| CN1588068A (en) * | 2004-09-07 | 2005-03-02 | 上海大学 | Detecting method for aflatoxins |
| CN1651428A (en) * | 2004-02-06 | 2005-08-10 | 上海因诺生化科技有限公司 | Preparation method of hydrobromic acid antifebrile dichroanone |
| CN1696698A (en) * | 2005-05-19 | 2005-11-16 | 中国农业大学 | Affinity chromatography-enzyme immunoassay method for PAT protein, and dedicated kit |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001042787A2 (en) * | 1999-12-08 | 2001-06-14 | Syngenta Participations Ag | Immunoassay for neonicotinyl insecticides |
| CN1651428A (en) * | 2004-02-06 | 2005-08-10 | 上海因诺生化科技有限公司 | Preparation method of hydrobromic acid antifebrile dichroanone |
| CN1588068A (en) * | 2004-09-07 | 2005-03-02 | 上海大学 | Detecting method for aflatoxins |
| CN1696698A (en) * | 2005-05-19 | 2005-11-16 | 中国农业大学 | Affinity chromatography-enzyme immunoassay method for PAT protein, and dedicated kit |
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| Title |
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| Determination of halofuginone in eggs byliquidchromatography/tandem mass spectrometry aftercleanupwith immunoaffinity chromatography. L.Mortier et al.Rapid Communications in Mass Spectrometry,,No.18. 2004 |
| Determination of halofuginone in eggs byliquidchromatography/tandem mass spectrometry aftercleanupwith immunoaffinity chromatography. L.Mortier et al.Rapid Communications in Mass Spectrometry,,No.18. 2004 * |
| 氢溴酸常山酮对鲟鱼的急性毒性及其残留检测方法的研究. 侯亚莉.中国农业大学硕士学位论文. 2005 |
| 氢溴酸常山酮对鲟鱼的急性毒性及其残留检测方法的研究. 侯亚莉.中国农业大学硕士学位论文. 2005 * |
| 鸡组织中常山酮残留的ELISA检测方法和HPLC检测方法研究. 吴宁鹏.中国农业大学硕士学位论文. 2004 |
| 鸡组织中常山酮残留的ELISA检测方法和HPLC检测方法研究. 吴宁鹏.中国农业大学硕士学位论文. 2004 * |
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