CN101433825B - Method for extracting salinomycin compound from animal sample and special immune affinity sorbent thereof - Google Patents
Method for extracting salinomycin compound from animal sample and special immune affinity sorbent thereof Download PDFInfo
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Abstract
The invention discloses a method for extracting salinomycin compounds from an animal sample and a special immunoaffinity absorbent thereof. The immunoaffinity absorbent for extracting the salinomycin compounds from the animal sample consists of a solid phase carrier and a salinomycin monoclonal antibody which is coupled with the solid phase carrier; the salinomycin monoclonal antibody is an antibody obtained by taking a conjugate of salinomycin semiantigen and carrier protein as immunogen; and the salinomycin compounds are salinomycin or methylsalinomycin. The immunoaffinity absorbent and a chromatographic column use the high specific salinomycin monoclonal antibody, have high selectivity, ensure the reliability of detection effect, greatly simplify the pretreatment processes of samples at the same time, are particularly applicable to the pretreatment of a trace amount of salinomycin and methylsalinomycin in muscle and liver, and improve the analysis quality. A detection method of the invention can efficiently detect the content of the salinomycin and the methylsalinomycin, and remedy the disadvantages that the direct assay of the samples by a single immunoassay technology has less information amount, poor quantification accuracy, or low selectivity of physical and chemical methods and so on, so the immunoaffinity absorbent, the chromatographic column, a reagent kit and the methods for extracting and detecting the salinomycin compounds in the animal sample are suitable to be promoted and applied.
Description
Technical field
The present invention relates to a kind of method and special immune affinity sorbent thereof that in animal sample, extracts the salinomycin compounds.
Background technology
Along with development of life science, people have produced more and more keen interest to material in the biosome 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 approach.Immune affinity chromatographic (IAC, immunoaffinity chromatography) is a kind of analytical approach 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.
Salinomycin (Salinomycin) and NARASIN (Narasin) belong to polyethers microbiotic for animals, and most of gram-positive bacterias and part mould are played antibacterial action, and using separately has the good preventing effect to chicken coccidiasis.The use of salinomycin and NARASIN can not be excessive, otherwise it will accumulate in animal body in a large number, jeopardizes human health by food chain, and weight person can make institute's edible animal be poisoned to death.In Dec, 2002, China Ministry of Agriculture announced No. 235 civilian regulation salinomycin and NARASIN high residue amount in chicken muscle is 600 μ g/kg, and the high residue amount in skin, fat is 1200 μ g/kg, and the high residue amount in liver is 1800 μ g/kg.European Union no longer allowed salinomycin-sodium to add in feed from 2006.Therefore, detection salinomycin and the residual quantity of NARASIN in animal food are extremely important.
The method that detects salinomycin and NARASIN residual quantity mainly contains high efficiency liquid phase chromatographic analysis method (HPLC), liquid-matter online (LC-MS), liquid matter tandem mass spectrometry (LC-MS/MS) etc.; 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.
Summary of the invention
An object of the present invention is to provide a kind of immune affinity sorbent that is used for extracting the salinomycin compounds from animal sample.
The immune affinity sorbent that is used for extracting from animal sample the salinomycin compounds provided by the present invention is formed by solid phase carrier with the salinomycin monoclonal antibody of its coupling; Described salinomycin monoclonal antibody is that the conjugate with salinomycin haptens and carrier protein is the antibody that immunogene obtains; Described salinomycin compounds is salinomycin or NARASIN.
Wherein, described salinomycin haptens is by salinomycin and thionyl chloride condensation reaction are obtained the salinomycin acyl chlorides, more described salinomycin acyl chlorides and aminoacetic acid reaction is obtained; Described salinomycin monoclonal antibody is the salinomycin mouse monoclonal antibody.Described salinomycin monoclonal antibody can be the salinomycin mouse monoclonal antibody.
Described salinomycin monoclonal antibody specifically is to be produced by salinomycin monoclonal antibody hybridoma cell strain C-3-3CGMCCNo.2587 secretion.
Described salinomycin monoclonal antibody is that the conjugate with salinomycin haptens and carrier protein obtains as immunogene; Described salinomycin haptens obtains the salinomycin acyl chlorides with salinomycin and thionyl chloride by condensation reaction, again salinomycin acyl chlorides and aminoacetic acid reaction is obtained the salinomycin haptens.
Salinomycin 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 helps to make the synthetic salinomycin haptens of salinomycin reaction at the stronger polyclonal antibody of salinomycin antigentic specificity.Adopt carbodiimide method and carrier protein couplet to obtain immunogene the salinomycin haptens again.It is low or too high all unfavorable to immunity that haptens and the ratio that combines of carrier protein are crossed, and haptens is respectively 11:1,17:1,17:1 with the mol ratio that combines of OVA, RSA and KLH.
Described carrier protein can be common carrier albumen such as mouse haemocyanin, bovine serum albumin(BSA), rabbit anteserum albumen, human serum albumins, ovalbumin or hemocyanin;
Described solid phase carrier can be cellulose, sephadex, polyacrylamide gel, fritted glass, Ago-Gel, ultragel ACA22 etc., is preferably Sepharose 4B.
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 can comprise eluent, cleansing solution and preservation liquid in the described kit;
Described cleansing solution is to contain 12 hypophosphite monohydrate disodium hydrogens that potassium dihydrogen phosphate, final concentration that final concentration is 0.2-0.3g/L are 2.5-3.5g/L, potassium chloride that final concentration is 0.1-0.3g/L, sodium chloride that final concentration is 8.0-9.5g/L, 0.01-0.02M, the pH value phosphate buffer for 7.2-7.6; Described final concentration is the concentration of each material in described cleansing solution;
The described eluent solution that to be methyl alcohol mix with the 8-10:1 volume ratio with water;
Described preservation liquid is that to contain potassium dihydrogen phosphate, final concentration that final concentration is 0.2-0.3g/L be that the 12 hypophosphite monohydrate disodium hydrogens of 2.5-3.5g/L, the potassium chloride that final concentration is 0.1-0.3g/L, sodium chloride, the final concentration that final concentration is 8.0-9.5g/L are the NaN of 0.1-0.3g/L
3, 0.01-0.02M, pH value be the phosphate buffer of 7.2-7.6; Described final concentration is the concentration of each material in described preservation liquid;
Described cleansing solution is preferably that to contain 12 hypophosphite monohydrate disodium hydrogens that potassium dihydrogen phosphate, final concentration that final concentration is 0.27g/L are 2.9g/L, potassium chloride that final concentration is 0.2g/L, sodium chloride, 0.01M, pH value that final concentration is 8.8g/L be 7.4 phosphate buffer;
Described eluent is preferably the solution that methyl alcohol and water mix with the 9:1 volume ratio;
Described preservation liquid is preferably that to contain potassium dihydrogen phosphate, final concentration that final concentration is 0.27g/L be that the 12 hypophosphite monohydrate disodium hydrogens of 2.9g/L, the potassium chloride that final concentration is 0.2g/L, sodium chloride, the final concentration that final concentration is 8.8g/L are the NaN of 0.2g/L
3, 0.01M, pH value be 7.4 phosphate buffer.
Another object of the present invention provides a kind of method of extracting the salinomycin compounds in animal sample.
The method of extracting the salinomycin compounds in animal sample provided by the present invention may further comprise the steps:
1) pre-treatment of sample:
Get animal tissue's homogenate, add sulfate, add extract again and extract, carrying out centrifugal force then is the centrifugal of 3000g-4000g, gets supernatant as sample solution; Described extract is acetonitrile or methyl alcohol; Described centrifugal force is preferably 3500g;
2) sample solution that step 1) is obtained mixes with the PBS damping fluid, crosses above-mentioned immune affinity chromatographic column, carries out wash-out with above-mentioned arbitrary cleansing solution, water, above-mentioned arbitrary eluent successively then, collects and obtains salinomycin compounds solution; Described salinomycin compounds is salinomycin or NARASIN.
Wherein, described animal tissue sample comprises muscle, liver.
Last purpose of the present invention provides a kind of method that detects salinomycin kind compound content in the animal sample.
The method of salinomycin kind compound content in the detection animal sample provided by the present invention, be with the method for extracting the salinomycin compounds in the above-mentioned automatic matter sample from animal sample, extract obtain the salinomycin compounds after, the amount to described salinomycin compounds detects again.
Also belong to protection scope of the present invention by the monoclonal antibody of salinomycin monoclonal antibody hybridoma cell strain C-3-3CGMCC No.2587 secretion or the monoclonal hybridoma strain C-3-3CGMCC No.2587 of salinomycin.
Immune affinity sorbent of the present invention and the chromatographic column that is mounted with this adsorbent are fit to purification salinomycin and NARASIN from biological sample (as muscle, liver) based on immune response and chromatogram reaction, are convenient to retention analysis.In this immune affinity chromatographic column, the coupling rate of the monoclonal antibody of the monoclonal hybridoma strain C-3-3CGMCC No.2587 of salinomycin secretion and the Sepharose4B of cyanogen bromide-activated is 94%.Dynamically column capacity is 1800ng/mL (salinomycin), 2000ng/mL (NARASIN), and column capacity is about 25% of total column capacity after 20 times having used, and storage life is 1 year.
Immune affinity sorbent of the present invention and chromatographic column have been used the salinomycin monoclonal antibody of high specific, has high selectivity, guaranteed to detect the reliability of effect, also sample pretreatment process is simplified greatly simultaneously, be particularly useful for the pre-treatment of trace salt mycin and NARASIN in muscle and the liver, analyze quality and improve.The high selectivity of immune affinity sorbent makes the detectability of salinomycin analytical approach 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 reserve capability of component under the actual measurement sample condition.Also can provide qualitative information when extracting method of the present invention purifies component, method is simple to operate, good purification, and immune affinity chromatographic column can be reused, and can save a large amount of organic solvents, reduces analysis cost and environmental pollution.But the content of detection method efficient detection salinomycin of the present invention and NARASIN, remedied simple immunoassay directly measure sample information amount very little, the low deficiency that waits of quantitatively poor accuracy, or physico-chemical method selectivity.Therefore, immune affinity sorbent of the present invention, chromatographic column and kit, and the method for salinomycin compounds is suitable for applying in extraction and the detection animal sample.
Description of drawings
Fig. 1 is the chromatogram of salinomycin and NARASIN standard items 5 μ g/kg.
Fig. 2 is the liquid chromatogram that does not add the chicken muscle tissue of salinomycin and NARASIN standard items.
Fig. 3 is for adding the liquid chromatogram of 5 μ g/kg salinomycins and NARASIN sample chicken muscle tissue.
Fig. 4 is the chromatogram of salinomycin and NARASIN standard items 25 μ g/kg.
Fig. 5 is not for adding the chicken liver liquid chromatogram of salinomycin and NARASIN standard items.
Fig. 6 is for adding the chicken liver liquid chromatogram of 25 μ g/kg salinomycins and NARASIN standard items.
Embodiment
Employed experimental technique is conventional method if no special instructions among the following embodiment; Employed material reagent all can obtain from the purchase of market company like no specified otherwise.
The preparation of the immune chromatograph post of embodiment 1, purification salinomycin and NARASIN
Salinomycin is available from U.S. Sigma company, and its catalog number is 53003-10-4.
One, salinomycin MONOCLONAL ANTIBODIES SPECIFIC FOR
(1) salinomycin is haptenic synthetic:
Salinomycin and thionyl chloride are obtained the salinomycin acyl chlorides by condensation reaction, again salinomycin acyl chlorides and aminoacetic acid reaction are obtained the salinomycin haptens.
The synthetic concrete steps of haptens are:
(1) salinomycin and thionyl chloride mix with the mol ratio of 1:1, and reaction is 4 hours under room temperature (25 ℃) condition, obtains the salinomycin acyl chlorides;
(2) salinomycin acyl chlorides and aminoacetic acid mix with the volume ratio of 1:1, add pyridine and carry out catalysis, and stirring reaction under condition of ice bath obtains the salinomycin haptens.
(2) immunogenic synthetic:
Adopt carbodiimide method that salinomycin haptens and ovalbumin (OVA) coupling are obtained immunogene.
The concrete steps that immunogene is synthetic:
(1) get salinomycin haptens 5mg and be dissolved in 0.5ml N, in the dinethylformamide (DMF), to wherein adding 25mg carbodiimides (DEC), priming reaction 30min obtains I liquid under room temperature (25 ℃) condition again;
(2) get ovalbumin 20mg,, obtain II liquid with the dissolving of 2.7ml pH8.0PBS liquid;
(3) I liquid is added to II liquid after, room temperature (25 ℃) stirring reaction spends the night, and obtains immunogene.
Utilize ultra-violet absorption spectrum that haptens and carrier protein are identified, and press following formula calculations incorporated than (the haptens number that the per molecule carrier protein connects).
Haptens combines than being 11:1 with the mole of OVA in the immunogene that obtains.
It is 98.6% that synthetic immunogene adopts immunoelectrophoresis to measure its purity.
(3) animal immune: adopt the Balb/c mouse as immune animal, with salinomycin haptens and ovalbumin conjugate is immunogene, immunizing dose is 100 μ g/, Freund's complete adjuvant with immunogene and equivalent when head exempts from is mixed and made into emulsifying agent, the subcutaneous multi-point injection of nape portion, At intervals of two to three weeks are got the same dose immunogene and are added equivalent incomplete Freund mixing and emulsifying, and booster immunization once, four exempt from the pneumoretroperitoneum booster immunization once, extracting spleen cell after 3 days.
(4) Fusion of Cells: get immune BALB/c mouse splenocyte, merge with SP2/0 myeloma cell in 5:1 ratio (quantitative proportion).
Hybridoma cell cloneization: adopt indirect competitive ELISA to measure cell conditioned medium liquid, screen positive hole.Utilize limiting dilution assay that cloning is carried out in positive hole, up to the hybridoma cell strain that obtains the stably excreting monoclonal antibody, with this cell line called after salinomycin monoclonal antibody hybridoma cell strain C-3-3, this cell line is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on July 11st, 2008 and (is called for short CGMCC, address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCCNo.2587.
(5) cell cryopreservation and recovery: get the salinomycin monoclonal antibody hybridoma cell strain C-3-3CGMCC No.2587 that is in exponential phase and make 5 * 10 with cryopreserving liquid
6The cell suspension of individual/ml is sub-packed in frozen pipe, in the medium-term and long-term preservation of liquid nitrogen.Take out frozen pipe during recovery, put into 37 ℃ of water-bath middling speeds immediately and melt, behind the centrifugal removal cryopreserving liquid, move in the culture flask and cultivate.
(6) MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying
Increment cultivation: hybridoma CGMCC No.2587 is placed cell culture medium, under 37 ℃ of conditions, cultivate, with following sad-saturated ammonium sulfate method and DEME cellulose ion-exchange chromatography method carry out purifying with the nutrient solution that obtains, obtain monoclonal antibody ,-20 ℃ of preservations.
Described cell culture medium is to add calf serum and sodium bicarbonate in the RPMI-1640 nutrient culture media, making the final concentration of calf serum in cell culture medium is 20% (volumn concentration), and making the final concentration of sodium bicarbonate in cell culture medium is 0.2% (quality percentage composition); The pH of described cell culture medium is 7.4.
1, SAS saltouts: 1) 50% saturation degree is saltoutd: get above-mentioned cell culture fluid 5ml, the PBS that adds equivalent 0.01mol/L, pH7.4 (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) mixing, drip isopyknic saturated ammonium sulfate (pH7.4) solution (making the saturation degree of ammonium sulfate reach 50%) then gradually, the limit edged stirs, room temperature is placed 30min, and the centrifugal 30min of 3000g abandons supernatant and stays precipitation.2) 33% saturation degree is saltoutd: add 5ml0.01mol/L PBS respectively and (contain potassium dihydrogen phosphate 0.27g in the 1L solution in the precipitation that step 1) obtains, 12 hypophosphite monohydrate disodium hydrogen 2.86g, potassium chloride 0.2g, sodium chloride 8.8g) dissolution precipitation, add saturated ammonium sulfate solution again and reach 33% saturation degree, the limit edged stirs, and room temperature is placed 30min, abandons supernatant and stays precipitation.Repetitive operation 2 times.3) desalination: the PBS that gets 0.01mol/L, pH7.4 (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) dissolving step 2) precipitation that obtains, be loaded in the bag filter, be suspended from the PBS that fills 0.01mol/L, pH7.4 and (contain potassium dihydrogen phosphate 0.27g in the 1L solution, 12 hypophosphite monohydrate disodium hydrogen 2.86g, potassium chloride 0.2g, sodium chloride 8.8g) desalination in the beaker, be positioned over 4 ℃, change liquid every day 3-4 times, 1%BaCl
2Detection is in dislysate till the sulfate radical-free ion.4) dialysis finishes, and the centrifugal 5min of 3000g gets supernatant and puts-20 ℃ of refrigerators preservations, 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 800ml0.5mol/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 0.5mol/L NaOH solution-treated at last instead once, fully be washed to neutrality.2) balance: will (contain potassium dihydrogen phosphate 0.27g in the 1L solution through the PBS that the DE-52 cellulose that step 1) is handled is soaked in 0.01mol/L, pH7.4,12 hypophosphite monohydrate disodium hydrogen 2.86g, potassium chloride 0.2g, sodium chloride 8.8g) in, fully washing, remove redundant solution after leaving standstill, 2-3 time so repeatedly, pH reaches till 7.4 until supernatant.3) dress post: continuously add in chromatographic column with dropper the DE-52 cellulose after the balance, (contain potassium dihydrogen phosphate 0.27g in the 1L solution with eluent, 12 hypophosphite monohydrate disodium hydrogen 2.86g, potassium chloride 0.2g, sodium chloride 8.8g, pH7.4) continuous wash-out, fully stream is washed, till the pH value of effluent pH value and eluent is identical.4) application of sample: will (contain potassium dihydrogen phosphate 0.27g in the 1L solution with the phosphate buffer of 0.01mol/L, pH7.4 through the saltout antibody that obtains of step (1) SAS, 12 hypophosphite monohydrate disodium hydrogen 2.86g, potassium chloride 0.2g, sodium chloride 8.8g)) dilution, slowly add along the chromatography post jamb then, open the chromatographic column outlet, allow antibody diluent flow in the post bed, wash post jamb with phosphate buffer again.5) wash-out and collection: the adding eluent (contain potassium dihydrogen phosphate 0.27g in the 1L solution, 12 hypophosphite monohydrate disodium hydrogen 2.86g, potassium chloride 0.2g, sodium chloride 8.8g, pH7.4), every pipe 2ml collects, and detects the antibody elution situation while collecting with 20% sulfosalicylic acid.6) measure the 0 D value of antibody-solutions at 280nm and 260nm place with ultraviolet spectrophotometer, obtain the monoclonal antibody of the salinomycin monoclonal antibody hybridoma cell strain C-3-3CGMCC No.2587 secretion of purifying, the monoclonal antibody behind the purifying is preserved at-20 ℃ of refrigerators.
(7) monoclonal antibody is to the cross reaction experiment of NARASIN, horse Du mycin, coban
The indirect competitive ELISA method is measured the cross reaction of the salinomycin monoclonal antibody of hybridoma cell strain C-3-3CGMCC No.2587 generation to NARASIN, horse Du mycin, coban.The antibody mortifier adopts NARASIN, horse Du mycin, the coban of doubling dilution respectively; Concrete steps are: 1. with coating buffer antigen (salinomycin) is diluted to debita spissitudo, in the amount adding enzyme-linked reaction plate with every hole 100 μ L; 2. put into 37 ℃ of incubator 2h, outwell surplus liquid after the taking-up and wash plate three times with the washing lotion in 200 μ L/ holes, at interval each 3min pats dry afterwards; 3. the confining liquid that every hole adds 150 μ L is put into 37 ℃ of incubator 2h, directly pats dry after the taking-up; 4. the NARASIN, horse Du mycin, the every hole 50 μ L of coban that add doubling dilution; 5. add above-mentioned monoclonal antibody, every hole 50 μ L to every hole simultaneously by salinomycin monoclonal antibody hybridoma cell strain C-3-3CGMCC No.2587 secretion; 37 ℃ of incubation 30min; Wash plate 4 times with cleansing solution, dry; 6. every hole adds the ELIAS secondary antibody of 100 μ L, puts into 37 ℃ of incubator 30min, and the washing of taking-up back is the same, pats dry afterwards; 7. every hole adds tetramethyl benzidine (TMB) solution 100 μ L, puts into 37 ℃ of incubator 15min, takes out every hole, back and adds 50 μ L stop buffers; 8. use enzyme
Measure optical density value in the mark instrument.According to following formula, respectively according to IC
50(ng/mL) calculate cross reacting rate:
The salinomycin monoclonal antibody is respectively 80%, 1%, 0.1% to the cross reaction of NARASIN, horse Du mycin, coban.Show that monoclonal antibody of the present invention is to the high specificity of salinomycin and NARASIN.
Two, the preparation of immune chromatograph post (IAC)
(1) preparation of immune chromatograph post (IAC)
The preparation of matrix: get the Sepharose 4B dry freeze powder of 2 gram cyanogen bromide-activated, filling 1.0mmoll-
1The G of HCl
3Expand in the funnel.Use 200mL1.0mmoll
-1HCl fully washs, and whole process is no more than 15 minutes.
The preparation of antibody: with the NaHCO of 5ml0.1mol/L
3(NaCl that contains 0.5mol/L is pH8.4) with the monoclonal antibody dilution of the salinomycin monoclonal antibody hybridoma cell strain C-3-3CGMCC No.2587 of 35mg purifying secretion for solution.
Coupling reaction: the NaHCO that the colloidal sol that expands is used 0.1mol/L
3Solution (NaCl that contains 0.5mol/L, pH8.4) after the balance, changing the Sepharose 4B dry freeze powder of cyanogen bromide-activated in the said monoclonal antibody dilution and the mass ratio of antibody purification over to is 2g:35mg, mixes, 4 ℃ of following 150rpm stir 20-24hr.
Reactant liquor changes in the G3 funnel, use 100mL0.01M, the phosphate buffer of pH7.4 (contains potassium dihydrogen phosphate 0.27g in the 1L aqueous solution, 12 hypophosphite monohydrate disodium hydrogen 2.86g, potassium chloride 0.2g, sodium chloride 8.8g) washing, collect cleansing solution, ultraviolet is identified, will survey the ultraviolet absorptivity of its 260nm, 280nm after the cleansing solution dilution respectively.The formula that calculates the monoclonal antibody amount is:
Monoclonal antibody amount=(1.45 * OD
280nm-0.74 * OD
260nmThe volume of) * extension rate * solution
Calculating coupling rate computing formula is:
The coupling rate testing result of three repeated experiments shows that the coupling rate of the monoclonal antibody of salinomycin monoclonal antibody hybridoma cell strain C-3-3CGMCC No.2587 secretion and the Sepharose4B of cyanogen bromide-activated is 94%.
The sealing in activation site: with the gel after the above-mentioned coupling change over to 0.1mol/L Tris-HCl damping fluid (NaCl that contains 0.5mol/L, pH8.0) in, mix, 4 ℃ are slowly stirred 2hr down, to seal the activation site of not coupling.
Washing: (NaCl that contains 0.5mol/L, pH4.0) (NaCl that contains 0.5mol/L pH8.0) alternately washes 3 times to gel with 0.1mol/L Tris-HCl damping fluid with the 0.1mol/L acetate buffer of 5 times of volumes.PBS with 0.01mol/L, pH7.4 (contains potassium dihydrogen phosphate 0.27g in the 1L solution, 12 hypophosphite monohydrate disodium hydrogen 2.9g, potassium chloride 0.2g, sodium chloride 8.8g) after the balance, the phosphate buffer that the gel of draining changes 0.01mol/L, pH7.4 over to (contains potassium dihydrogen phosphate 0.27g, 12 hypophosphite monohydrate disodium hydrogen 2.9g, potassium chloride 0.2g in the 1L solution, sodium chloride 8.8g, NaN
30.2g) in, deposit under 4 ℃ standby.
Dress post: have the immunosorbent of the monoclonal antibody of salinomycin monoclonal antibody hybridoma cell strain C-3-3CGMCC No.2587 secretion to be transferred to coupling and contain G
3In the glass column of filter plate, make the immune chromatograph post (IAC post) that coupling has the monoclonal antibody of salinomycin monoclonal antibody hybridoma cell strain C-3-3CGMCC No.2587 secretion.
(2) the IAC column capacity determines
Employed PBS damping fluid is following composition if no special instructions in this experiment: contain potassium dihydrogen phosphate 0.27g in the 1L PBS solution, 12 hypophosphite monohydrate disodium hydrogen 2.9g, potassium chloride 0.2g, sodium chloride 8.8g.
The coupling of above-mentioned preparation there is the immune chromatograph post of the monoclonal antibody of salinomycin monoclonal antibody hybridoma cell strain C-3-3CGMCC No.2587 secretion, with the PBS balance of 10ml0.01mol/L, pH7.4.
To contain 5 μ gml
-1Salinomycin (SAL) and 5 μ gml
-1The PBS of NARASIN (NAR) is added to the IAC post continuously, and natural gravity flows out down.
After post reaches capacity (sample concentration is identical with application of sample liquid concentration in the effluent), successively, remove and disturb impurity in the extract with 10ml PBS, 10ml pure water washing IAC post.
Use 6ml methanol (volume ratio 90/10) with SAL, NAR wash-out at last, natural gravity flows out down, collects, and dries up, and gets 100 μ L after redissolving with 1mL methyl alcohol and advances the HPLC analysis.Wherein, HPLC chromatographic condition: C
18Reverse-phase chromatographic column (Supelcosil LC-18) (5 μ m, 250 * 4.6mm i.d.); Moving phase is that (940-30-30 v:v:v), adopts isocratic elution to methanol-water-acetate, and flow velocity is 1mL/min, temperature: 25 ℃ of room temperatures; Derivatization reagent is slowly to add 10mL sulfuric acid among the methyl alcohol 300mL, after mixing treats that temperature is reduced to room temperature, adds the 20g vanillic aldehyde, and mixing, flow velocity are 0.5mL/min, the derivatization temperature: 95 ℃; Detect wavelength: 520nm.
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.Its computing formula is:
Absolute column capacity (specific column capacity) is meant the maximum binding capacity of every milligram of sessile antibody to determinand.Its computing formula is:
3 repetitions are established in experiment, and the result takes the mean.The result shows that coupling has the dynamic column capacity and the absolute column capacity of immune chromatograph post of the monoclonal antibody of hybridoma cell strain salinomycin monoclonal antibody hybridoma cell strain C-3-3CGMCC No.2587 secretion to be respectively 1800ng/mL, 192ng/mL (SAL), 2000ng/mL, 213ng/mL (NAR).
Embodiment 2, contain the preparation of the kit of immune chromatograph post among the embodiment 1 and to the clean-up effect of salinomycin and NARASIN
1, purifies the preparation of the kit of salinomycin and NARASIN
This kit is by immune chromatograph post (IAC post), salinomycin, NARASIN standard solution, and cleansing solution, eluent is preserved liquid, and the sponge carriage is formed, and the sponge carriage is provided with hole and groove.Salinomycin, NARASIN standard solution are housed in the groove of sponge carriage, cleansing solution, 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 the immune chromatograph post of the monoclonal antibody of salinomycin monoclonal antibody hybridoma cell strain C-3-3CGMCC No.2587 secretion for the coupling of embodiment 1 preparation.
Described cleansing solution is that to contain 12 hypophosphite monohydrate disodium hydrogens that potassium dihydrogen phosphate, final concentration that final concentration is 0.27g/L are 2.9g/L, potassium chloride that final concentration is 0.2g/L, sodium chloride, 0.01M, pH value that final concentration is 8.8g/L be 7.4 phosphate buffer; Described final concentration is the concentration of each material in described cleansing solution;
The described eluent solution that to be methyl alcohol mix with the 9:1 volume ratio with water;
Described preservation liquid is that to contain potassium dihydrogen phosphate, final concentration that final concentration is 0.27g/L be that the 12 hypophosphite monohydrate disodium hydrogens of 2.9g/L, the potassium chloride that final concentration is 0.2g/L, sodium chloride, the final concentration that final concentration is 8.8g/L are the NaN of 0.2g/L
3, 0.01M, pH value be 7.4 phosphate buffer; Described final concentration is the concentration of each material in described preservation liquid;
To contain coupling has the kit of immune chromatograph post of the monoclonal antibody of salinomycin monoclonal antibody hybridoma cell strain C-3-3CGMCC No.2587 secretion to be placed on 4 ℃ respectively, and the term of validity is 12 months.
2, the extraction effect of salinomycin, NARASIN experiment
IAC extracts principle, with the monoclonal antibody and the inert base coupling of specific antibody salinomycin monoclonal antibody hybridoma cell strain C-3-3CGMCCNo.2587 secretion, and preparation immunosorbent, dress post.When the potpourri that contains salinomycin and NARASIN flows through the IAC post, sessile antibody is optionally in conjunction with salinomycin and NARASIN, 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, salinomycin is purified with NARASIN or separates.The IAC post is reusable after Regeneration Treatment.
The processing of test sample:
1) get the chicken muscle and the chicken liver tissue that do not contain salinomycin and NARASIN respectively and place refiner, homogenate is 3 minutes under 10000rpm.Place-20 ℃ of preservations standby.
2) do not add the sample preparation of salinomycin and NARASIN: take by weighing 5.0 chicken muscle and the chicken liver tissue samples that restrain homogenate respectively, place the 100ml polypropylene centrifuge tube respectively, in pipe, add the 10g anhydrous sodium sulfate more respectively, smash even with glass bar, add 20ml acetonitrile (muscle) or methyl alcohol (liver) after leaving standstill 5min, the whirling motion mixing vibrated 20 minutes, centrifugal 10 minutes of 3500g, get supernatant, repeat to extract once, merge supernatant, its heart bottle of packing into.50 ℃ of rotary evaporations go to the 10ml centrifuge tube to surplus 2-3ml with it, and with incorporating into behind the 2ml methanol wash heart bottle, centrifugal 10 minutes of 3500g adds to supernatant among the 20ml PBS, and mixing obtains sample solution.
Add the sample preparation of salinomycin and NARASIN: take by weighing 5.0 chicken muscle and the chicken liver tissue samples that restrain homogenate respectively, place the 100ml polypropylene centrifuge tube respectively, Xiang Guanzhong adds salinomycin and NARASIN standard items, make salinomycin and the NARASIN final concentration in chicken muscle homogenate be 5 μ g/kg, make salinomycin and the NARASIN final concentration in the chicken liver tissue be 25 μ g/kg, in pipe, add the 10g anhydrous sodium sulfate more respectively, smash even with glass bar, add 20ml acetonitrile (muscle) or methyl alcohol (liver) after leaving standstill 5min, the whirling motion mixing, vibrated 20 minutes, centrifugal 10 minutes of 3500g gets supernatant, repeats to extract once, merge supernatant, with its heart bottle of packing into.50 ℃ of rotary evaporations go to the 10ml centrifuge tube to surplus 2-3ml with it, and with incorporating into behind the 2ml methanol wash heart bottle, centrifugal 10 minutes of 3500g adds to supernatant among the 20ml PBS, and mixing obtains sample solution.
There is the immune chromatograph column equilibration of the monoclonal antibody of salinomycin monoclonal antibody hybridoma cell strain C-3-3CGMCC No.2587 secretion to arrive room temperature coupling, then above-mentioned sample solution is crossed post, with l0ml cleansing solution, the washing of 10ml pure water, purpose is in order to remove the impurity of non-specific adsorption.With 6ml eluent wash-out, at 50 ℃ of N
2Dry up under the air-flow, redissolve with 1mL methyl alcohol then that (the liver sample is with 1mL methanol (90/10, v/v) redissolve), crosses to get 100 μ L behind the organic filter membrane of 0.2 μ m and carry out HPLC and analyze, and measures the content of salinomycin and NARASIN.
Wherein, the chromatographic condition of muscle samples is: moving phase is that (940-30-30 v:v:v), adopts isocratic elution to methanol-water-acetate, and flow velocity is 1mL/min, temperature: room temperature; Derivatization reagent is slowly to add 10mL sulfuric acid among the methyl alcohol 300mL, after mixing treats that temperature is reduced to room temperature, adds the 20g vanillic aldehyde, and mixing, flow velocity are 0.5mL/min, the derivatization temperature: 95 ℃; Detect wavelength: 520nm.
The chromatographic condition of liver sample is: moving phase is that (935-50-15 v:v:v), adopts isocratic elution to methanol-water-acetate, and flow velocity is 0.8mL/min, temperature: room temperature; Derivatization reagent is slowly to add 10mL sulfuric acid among the methyl alcohol 500mL, after mixing treats that temperature is reduced to room temperature, adds the 20g vanillic aldehyde, and mixing, flow velocity are 0.7mL/min, the derivatization temperature: 95 ℃.
The chromatographic condition of standard items is: C
18Reverse-phase chromatographic column (Supelcosil LC-18) (5 μ m, 250 * 4.6mm i.d.); Moving phase is that (940-30-30 v:v:v), adopts isocratic elution to methanol-water-acetate, and flow velocity is 1mL/min, temperature: room temperature; Derivatization reagent is slowly to add 10mL sulfuric acid among the methyl alcohol 300mL, after mixing treats that temperature is reduced to room temperature, adds the 20g vanillic aldehyde, and mixing, flow velocity are 0.5mL/min, the derivatization temperature: 95 ℃; Detect wavelength: 520nm.The IAC post with the preservation liquid balance of 20ml be stored in 4 ℃ standby.The collection of illustrative plates of salinomycin 5 μ g/kg and NARASIN 5 μ g/kg standard items as shown in Figure 1; The collection of illustrative plates of salinomycin 25 μ g/kg and NARASIN 25 μ g/kg standard items as shown in Figure 4.
The result shows, carries out sample purification with IAC, and the interference medicament chromatographic peak can not separate fully, illustrates that the IAC non-specific adsorption of preparation is minimum.Wherein coupling have salinomycin monoclonal antibody hybridoma cell strain C-3-3CGMCC No.2587 secretion monoclonal antibody immune affinity chromatographic column for the clean-up effect of the chicken muscle that does not add salinomycin and NARASIN as shown in Figure 2, the clean-up effect of chicken muscle that adds salinomycin and NARASIN is shown in Figure 3; Coupling have salinomycin monoclonal antibody hybridoma cell strain C-3-3CGMCC No.2587 secretion monoclonal antibody immune affinity chromatographic column for the clean-up effect of the chicken liver that does not add salinomycin and NARASIN as shown in Figure 5, shown in Figure 6 for the clean-up effect of the chicken liver that adds salinomycin and NARASIN.
Salinomycin and NARASIN described in embodiment 3, the usefulness embodiment 2 in the kit test sample
1, sample pre-treatments:
1) get chicken muscle respectively and the chicken liver tissue places refiner, homogenate is 3 minutes under 10000rpm.Place-20 ℃ of preservations standby.
2) take by weighing 5.0 chicken muscle and the chicken liver tissue samples that restrain homogenate respectively, place the 100ml polypropylene centrifuge tube respectively, in pipe, add the 10g anhydrous sodium sulfate more respectively, smash even with glass bar, add 20ml acetonitrile (muscle) or methyl alcohol (liver) after leaving standstill 5min, the whirling motion mixing vibrated 20 minutes, centrifugal 10 minutes of 3500g, get supernatant, repeat to extract once, merge supernatant, its heart bottle of packing into.50 ℃ of rotary evaporations go to the 10ml centrifuge tube to surplus 2-3ml with it, and with incorporating into behind the 2ml methanol wash heart bottle, centrifugal 10 minutes of 3500g adds to supernatant among the 20ml PBS, and mixing obtains sample solution.
2, the extraction of salinomycin and NARASIN
There is the immune chromatograph column equilibration of the monoclonal antibody of salinomycin monoclonal antibody hybridoma cell strain C-3-3CGMCC No.2587 secretion to arrive room temperature coupling, then above-mentioned sample solution is crossed post, with 10ml cleansing solution, the washing of 10ml pure water, purpose is in order to remove the impurity of non-specific adsorption.With 6ml eluent wash-out, at 50 ℃ of N
2Dry up under the air-flow, (the liver sample is crossed the organic filter membrane of 0.2 μ m with 1mL methanol (90/10, v/v) redissolve), obtains salinomycin and NARASIN extract, gets 100 μ L and carries out subsequent analysis with the redissolution of 1mL methyl alcohol then.
3, HPLC analyzes, and measures the content of salinomycin and NARASIN.
Claims (14)
1. immune affinity sorbent that is used for extracting the salinomycin compounds from animal sample, it is formed by solid phase carrier with the salinomycin monoclonal antibody of its coupling; Described salinomycin monoclonal antibody is to be produced by salinomycin monoclonal antibody hybridoma cell strain C-3-3CGMCC No.2587 secretion.
2. be the immune affinity chromatographic column of filler with the described immune affinity sorbent of claim 1.
3. the kit that contains the described immune affinity sorbent of claim 1.
4. according to the described kit of claim 3, it is characterized in that: also comprise eluent, cleansing solution and preservation liquid in the described kit;
Described cleansing solution is to contain 12 hypophosphite monohydrate disodium hydrogens that potassium dihydrogen phosphate, final concentration that final concentration is 0.2-0.3g/L are 2.5-3.5g/L, potassium chloride that final concentration is 0.1-0.3g/L, sodium chloride that final concentration is 8.0-9.5g/L, 0.01-0.02M, the pH value phosphate buffer for 7.2-7.6; Described final concentration is the concentration of each material in described cleansing solution;
Described eluent be methyl alcohol with water with 8-10: the solution that 1 volume ratio is mixed;
Described preservation liquid is that to contain potassium dihydrogen phosphate, final concentration that final concentration is 0.2-0.3g/L be that the 12 hypophosphite monohydrate disodium hydrogens of 2.5-3.5g/L, the potassium chloride that final concentration is 0.1-0.3g/L, sodium chloride, the final concentration that final concentration is 8.0-9.5g/L are the NaN of 0.1-0.3g/L
3, 0.01-0.02M, pH value be the phosphate buffer of 7.2-7.6; Described final concentration is the concentration of each material in described preservation liquid.
5. according to the described kit of claim 4, it is characterized in that: described cleansing solution is that to contain 12 hypophosphite monohydrate disodium hydrogens that potassium dihydrogen phosphate, final concentration that final concentration is 0.27g/L are 2.9g/L, potassium chloride that final concentration is 0.2g/L, sodium chloride, 0.01M, pH value that final concentration is 8.8g/L be 7.4 phosphate buffer;
The described eluent solution that to be methyl alcohol mix with 9: 1 volume ratios with water;
Described preservation liquid is that to contain potassium dihydrogen phosphate, final concentration that final concentration is 0.27g/L be that the 12 hypophosphite monohydrate disodium hydrogens of 2.9g/L, the potassium chloride that final concentration is 0.2g/L, sodium chloride, the final concentration that final concentration is 8.8g/L are the NaN of 0.2g/L
3, 0.01M, pH value be 7.4 phosphate buffer.
6. the kit that contains the described immune affinity chromatographic column of claim 2.
7. according to the described kit of claim 6, it is characterized in that: also comprise eluent, cleansing solution and preservation liquid in the described kit;
Described cleansing solution is to contain 12 hypophosphite monohydrate disodium hydrogens that potassium dihydrogen phosphate, final concentration that final concentration is 0.2-0.3g/L are 2.5-3.5g/L, potassium chloride that final concentration is 0.1-0.3g/L, sodium chloride that final concentration is 8.0-9.5g/L, 0.01-0.02M, the pH value phosphate buffer for 7.2-7.6; Described final concentration is the concentration of each material in described cleansing solution;
Described eluent be methyl alcohol with water with 8-10: the solution that 1 volume ratio is mixed;
Described preservation liquid is that to contain potassium dihydrogen phosphate, final concentration that final concentration is 0.2-0.3g/L be that the 12 hypophosphite monohydrate disodium hydrogens of 2.5-3.5g/L, the potassium chloride that final concentration is 0.1-0.3g/L, sodium chloride, the final concentration that final concentration is 8.0-9.5g/L are the NaN of 0.1-0.3g/L
3, 0.01-0.02M, pH value be the phosphate buffer of 7.2-7.6; Described final concentration is the concentration of each material in described preservation liquid.
8. according to the described kit of claim 7, it is characterized in that: described cleansing solution is that to contain 12 hypophosphite monohydrate disodium hydrogens that potassium dihydrogen phosphate, final concentration that final concentration is 0.27g/L are 2.9g/L, potassium chloride that final concentration is 0.2g/L, sodium chloride, 0.01M, pH value that final concentration is 8.8g/L be 7.4 phosphate buffer;
The described eluent solution that to be methyl alcohol mix with 9: 1 volume ratios with water;
Described preservation liquid is that to contain potassium dihydrogen phosphate, final concentration that final concentration is 0.27g/L be that the 12 hypophosphite monohydrate disodium hydrogens of 2.9g/L, the potassium chloride that final concentration is 0.2g/L, sodium chloride, the final concentration that final concentration is 8.8g/L are the NaN of 0.2g/L
3, 0.01M, pH value be 7.4 phosphate buffer.
9. method of extracting the salinomycin compounds in animal sample may further comprise the steps:
1) pre-treatment of sample:
Get animal tissue's homogenate, add sulfate, add extract again and extract, carrying out centrifugal force then is the centrifugal of 3000g-4000g, gets supernatant as sample solution; Described extract is acetonitrile or methyl alcohol;
2) sample solution that step 1) is obtained mixes with the PBS damping fluid, cross the described immune affinity chromatographic column of claim 2, carry out wash-out with the eluent described in the cleansing solution described in the claim 7, water, the claim 7 successively then, collect and obtain salinomycin compounds solution; Described salinomycin compounds is salinomycin or NARASIN.
10. method according to claim 9 is characterized in that: in the step 1), described centrifugal force is 3500g.
11. according to claim 9 or 10 described methods, it is characterized in that: described animal tissue sample is muscle, liver.
12. a method that detects salinomycin kind compound content in the animal sample, be with method described in the claim 9 from animal sample, extract obtain the salinomycin compounds after, the amount to described salinomycin compounds detects again.
13. monoclonal antibody by salinomycin monoclonal antibody hybridoma cell strain C-3-3CGMCC No.2587 secretion.
14. salinomycin monoclonal antibody hybridoma cell strain C-3-3CGMCC No.2587.
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