A kind of Ractopamine immuno magnetic cell separation enrichment kit and application thereofTechnical field
The present invention relates to a kind of Ractopamine immunomagnetic beads kit and the method for separating and concentrating to Ractopamine in sample thereof.Belong to field of immunology.
Background technology
Ractopamine (Ractopamine, RAC), chemical name is 1-(4-hydroxy phenyl)-2 [1-methyl-3-(4-hydroxy phenyl)-the third is amino]-ethylate hydrochlorate, be beta-receptor excitant with Clenbuterol (clenbuterol hydrochloride), be commonly used to treatment bronchitis and asthma clinically.Research in recent years shows, RAC has control Animal nutrition metabolic pathway, strengthens the effects such as feed conversion rate, is therefore used as novel growth-promoting additive.But have residual in the body of the animals such as pig due to Ractopamine and human body can be entered by biologic chain mode, showing toxicity when accumulation exceedes a certain amount of, occur the symptoms such as tachycardia, arrhythmia cordis and myalgia.In recent years, along with government is to the enhancing of " clenbuterol hydrochloride " control and monitoring, RAC is applied in pig aquaculture as " clenbuterol hydrochloride " substitute by illegal retailer.RAC is defined as forbidden drug by the Ministry of Agriculture of China No. 176 bulletins in 2002.Correspondingly, for Ractopamine detection technique researched and proposed higher requirement.Simply, fast, accurate, sensitive Ractopamine detection technique is also more and more subject to the extensive concern of food enterprise, testing agency.
Rct opamine residue analysis mainly adopts the methods such as immunoassay, gas chromatography-mass spectrography (GC-MS), HPLC MS (HPLC-MS), Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).Sample pre-treatments due to above-mentioned detection method all needs to use purification plant, comprises C18 post, graphitized carbon solid-phase extraction column, florisil silica solid-phase extraction column, silica gel SPE solid-phase extraction column etc.Above-mentioned solid phase extraction column is expensive, be about 500-2000 unit/, and purifying step is loaded down with trivial details, needs multiple organic solvent.Ractopamine immuno magnetic cell separation technology is owing to including Fe3o4the finishing functional group of nanometer magnetic bead can be bonded Ractopamine immunomagnetic beads with Ractopamine monoclonal antibody specific, by externally-applied magnetic field utilize the specific immune response of antibody from mixed solution enrichment be separated Ractopamine, the purifying carrier of Ractopamine immunomagnetic beads is dispersed, monoclonal antibody specific and the join probability detecting target material can be improved, thus the recovery of Ractopamine in raising sample, add the accuracy of Ractopamine detection method.Associative key between the magnetic bead of immunomagnetic beads and antibody is chemical bond key, and combination degree is comparatively firm; Immune affinity column is the special SPE pillar utilizing the specific binding characteristics of antigen and antibody.The covalent bond that antibody and carrier exist is the physical bond power such as hydrophobic forces and ion exchange power, its do not have in conjunction with dynamics the chemical bond key of immunomagnetic beads in conjunction with great efforts.And the carrier of immune affinity column is solid phase, antibody can be low compared with immunomagnetic beads with the join probability detecting target material.
Summary of the invention
Technical matters to be solved by this invention is to provide a kind of Ractopamine immuno magnetic cell separation enrichment kit, when adopting this kit to carry out the separation and concentration of Ractopamine in sample, there is higher specificity, the recovery and accuracy, simplify sample pre-treatments step, avoid in sample pretreatment process, using multiple and a large amount of organic solvents.
For achieving the above object, the invention provides a kind of Ractopamine immuno magnetic cell separation enrichment kit, it comprises the magnetic bead of coupling ractopamine monoclonal antibody, redissolution liquid and magnet, the magnetic bead of described coupling ractopamine monoclonal antibody is that 1:500 ~ 800 mix and are dissolved in coupling in 2-(N-morpholino) ethyl sulfonic acid monohydrate and are prepared from by ractopamine monoclonal antibody and the mass ratio of magnetic bead, described ractopamine monoclonal antibody is that the conjugate obtained by Ractopamine haptens and bovine serum albumin(BSA) prepares as immunogen immune Balb/c mouse.
Described Ractopamine haptens is oxidized through Pyridinium dichromate by Ractopamine, obtain ketone group Ractopamine intermediate, again intermediate and para hydroxybenzene hydrazine are carried out condensation reaction, the spacerarm treating carboxyl is introduced at phenyl ring ortho position, obtain para hydroxybenzene hydrazine Ractopamine haptens, yield is 88%, and its structural formula is such as formula shown in I.
Described redissolution liquid contains 0.2% bovine serum albumin(BSA), 0.04% Tween-20 and 0.02%NaN3phosphate buffer or the TRIS buffer of pH7.4, described percentage composition is mass percentage, and the magnetic bead of described coupling ractopamine monoclonal antibody is preserved in redissolution liquid.
Described magnet produces externally-applied magnetic field, makes the magnetic bead of coupling ractopamine monoclonal antibody be gathered on magnet, reaches the object being separated magnetic bead.
Present invention also offers the method for Ractopamine in separation and concentration sample, comprise the following steps:
1) separation and concentration of Ractopamine in sample is carried out with kit;
2) will the magnetic bead methanol-eluted fractions of the coupling ractopamine monoclonal antibody of Ractopamine being enriched with, reclaiming methanol solution, for detecting analysis.
Beneficial effect of the present invention is as follows:
1) kit of the present invention has higher specificity and accuracy, catching concentration and can reach 20ng/mg (every milligram of immunomagnetic beads can catch 20ng Ractopamine), sample TIANZHU XINGNAO Capsul >=85% Ractopamine;
2) magnetic bead of the coupling ractopamine monoclonal antibody in kit of the present invention and liquid come through the easy quick separating of magneticaction, detachment process is simple, the operating process that centrifugal and filtration etc. are loaded down with trivial details can be saved, save time, when removing externally-applied magnetic field, memory that magnetic bead is nonmagnetic, can be dispersed, do not occur clustering phenomena;
3) magnetic bead has certain mechanical degrees and chemical stability, the degraded of certain density acid-base solution and microorganism can be tolerated, magnetisable material in its structure is not easily oxidized, and this physicochemical property of magnetic particle stablize feature, and its magnetic is not easily declined;
4) rinse of magnetic bead and wash-out use deionized water and methyl alcohol respectively, and the magnetic bead of the coupling ractopamine monoclonal antibody of 0.1mL only needs 1mL methanol-eluted fractions, and the amount of agents useful for same is less, the comparatively environmental protection of the rinse of magnetic bead and elution process.
Accompanying drawing explanation
Fig. 1 is Ractopamine hapten synthesis reaction equation.
Fig. 2 is Ractopamine haptens hydrogen nuclear magnetic resonance spectrogram.
Embodiment
Embodiment 1: the preparation of the concrete component of kit
1. Ractopamine hapten synthesis
Ractopamine haptens is oxidized through Pyridinium dichromate by Ractopamine, then carry out condensation reaction with para hydroxybenzene hydrazine, introduces band carboxyl spacerarm, obtain para hydroxybenzene hydrazine Ractopamine haptens at phenyl ring ortho position.
Get 0.1g Ractopamine 1.0g to add in acetonitrile and dissolve, then add Pyridinium dichromate 0.78g, stir, add 500 μ L acetic acid again, stir 10h in 80 DEG C, after stopping reaction, acetonitrile is removed with Rotary Evaporators, add water and extraction into ethyl acetate again, after layering, remove aqueous phase, organic phase anhydrous sodium sulfate drying evaporate to dryness, upper silicagel column, the volume ratio of mobile phase chloroform and methyl alcohol is chloroform: methyl alcohol=10:1, carries out wash-out separation, obtains intermediate 0.93g; Get intermediate 0.93g and add ethanol dissolving, add para hydroxybenzene hydrazine 0.72g, at 70 DEG C, stir 6h, after stopping reaction, by solvent evaporate to dryness, obtain yellow solid, add ethyl acetate wash crystallization again, obtain para hydroxybenzene hydrazine Ractopamine haptens 0.87g, yield is 88%.Synthetic reaction formula as shown in Figure 1.Get above-mentioned haptens product to measure through nucleus magnetic hydrogen spectrum, as shown in Figure 2, the position of chemical shift δ=11ppm is carboxyl hydrogen, and the position of chemical shift δ=7.85 is the hydrogen of phenyl ring in phenylhydrazine, and the existence of these characteristic peaks proves coupling success, and haptens structure is correct.Nuclear-magnetism qualification result:1hNMR (CDCl3, 300MHZ) and δ: 11.0 (s, 1H, COOH), 6.84-7.85 (d, 12H, ArH), 5.35 (s, 1H, OH), 2.79 (m, 1H ,-CH-), 2.55 (t, 2H, CH2), 2.60 (t, 2H ,-CH2-), 2.0 (s, 1H, NH), 1.7 (m, 2H,-CH2-), 1.12 (s, 3H ,-CH3).
2. immunogenic preparation
Get 13mg para hydroxybenzene hydrazine Ractopamine haptens, be dissolved in 1mLN, in dinethylformamide (DMF); Get after 30mg ethylene dichloride (EDC) and N-hydroxy-succinamide (NHS) 0.2mL water fully dissolves, add in haptens solution, stirred at ambient temperature 24h, reactant liquor A can be obtained; Take bovine serum albumin(BSA) (BSA) 50mL, making it fully to be dissolved in 4mLpH value is in the phosphate buffer of 7.2, is dropwise slowly added drop-wise in BSA solution by reactant liquor A, and in stirred at ambient temperature 24h; Dialyse 3 days in 4 DEG C with the phosphate buffer of 0.01mol/L, change 3 dislysates every day, to remove unreacted small-molecule substance, obtain Ractopamine immunogene; Packing, saves backup in-20 DEG C.
3. the preparation of ractopamine monoclonal antibody
1) animal immune: by the above-mentioned immunogene (RAC-BSA) prepared by 100 μ g/, mix with physiological saline solution immunogene and Freund's complete adjuvant equal-volume, the female mouse of neck dorsal sc injection immunity Balb/c in 6 ~ 8 week age, within after initial immunity the 7th, 14,28 day, mix with immunogene and incomplete Freund's adjuvant equal-volume, each supplementary immunization once, with immune complex 100 μ g/ only merge first 3 days, supplementary immunization is once more not add Freund's adjuvant.
2) Fusion of Cells: carry out according to a conventional method, the splenocyte getting immune mouse mixes with the murine myeloma cell being in exponential phase (SP2/0), then the fusion agent (PEG4000) slowly adding preheating in 45 seconds merges, suspend evenly with HAT nutrient culture media, add appropriate feeder cells again, be incubated at 96 well culture plates, in 37 DEG C, 5%CO2cultivate in incubator, within 5 days, partly change liquid with HT nutrient culture media afterwards, when 9 days, entirely change liquid.
3) screening of hybridoma: after Fusion of Cells, when cell grows to 1/4 of culture hole area, adopts a point step screening method screening hybridoma.Primary election adopts indirect ELISA method, with envelope antigen (wrapping by concentration and positive serum dilutability by its best of square formation method conventional titration in advance) coated elisa plate, add measured hole culture supernatant, hatch, Ractopamine standard solution 50 μ L is added after cleaning, add cell conditioned medium liquid 50 μ L and sheep anti-mouse igg-HRP50 μ L again, in 37 DEG C of reaction 30min, wash plate, add substrate solution nitrite ion 100 μ L again, lucifuge reaction 15min at 25 DEG C, then add stop buffer 50 μ L, measure OD450nmvalue drops to less than 50% of control wells, is judged to the positive, and detecting through 2 ~ 3 times is all positive hole, carries out subcloning immediately with limiting dilution assay.
4) monoclonal antibody preparation: 2 ~ 3 subclones are built the hybridoma after strain and expands cultivation, collects supernatant indirect ELISA mensuration and tires, frozen; And get 8 ~ 10 week age Balb/c mouse peritoneal injecting fluid paraffin 0.5mL/, lumbar injection hybridoma 1 ~ 2 × 10 after 7 ~ 10 days6/ only, extract mouse ascites after 7 ~ 10 days, centrifuging and taking supernatant, mensuration is tired, and frozen for subsequent use.
5. the preparation of the magnetic bead of coupling ractopamine monoclonal antibody
1) magnetic bead activation
Surface has the magnetic bead of-COOH group (to be purchased from DYNAL, particle diameter is 2.8 μm), its content is 0.1eq/g ~ 0.3eq/g, get 100 μ L magnetic beads, with containing pH5.0,0.05% the concentration of Tween-20 be that 2-(N-morpholine) ethyl sulfonic acid monohydrate (MES) 100mL of 25mmol/L washes twice, remove supernatant after Magneto separate; Before magnetic bead activation, prepare EDC and the NHS solution of 50mmol/L respectively with the above-mentioned MES solution of 4 DEG C of storages; Respectively to each 50 μ L of EDC and NHS solution adding new configuration in the centrifuge tube that magnetic bead is housed, vortex mixes, room temperature activation 30min; Centrifuge tube is placed on Magneto separate frame and carries out Magneto separate 4min, remove supernatant, then add wherein 100 μ L, pH5.0,25mmol/L MES cleaning 2 ~ 3 times after can obtain the magnetic bead that there is activated carboxylic on surface.Described percentage composition is mass percentage.
2) preparation of the magnetic bead of coupling ractopamine monoclonal antibody
10 μ g ractopamine monoclonal antibody are dissolved in the MES of 60 μ L, pH5.0,25mmol/L, add the magnetic bead of 5mg activation wherein, or 5 μ g ractopamine monoclonal antibody are dissolved in the MES of 60 μ L, pH5.0,25mmol/L, add the magnetic bead of 4mg activation wherein, and regulate cumulative volume to 100 μ L with above-mentioned concentration MES solution, gently mix magnetic bead and ractopamine monoclonal antibody; Coupling 30min or 4 DEG C of coupling 2h under room temperature condition, can utilize vortex instrument to make magnetic bead keep mixing state during this period; Centrifuge tube is placed on Magneto separate frame and carries out Magneto separate 5min, removes supernatant; In order to the unreacted-COOH of cancellation, 100 μ L can be added, the trishydroxymethylaminomethane (TRIS) of pH7.2 ~ pH7.6 reacts 15min or 100 μ L, pH8.0, phosphate buffer that ethanolamine concentration is 50mmol/L closes magnetic bead; The magnetic bead closed with the phosphate buffer cleaning of 100 μ L, 0.2%BSA, 0.1% Tween-20 3 ~ 5 times, redissolves magnetic bead in the BSA, 0.08% Tween-20, the 0.02%NaN that contain 0.4%5phosphate buffer in, in 2 DEG C ~ 8 DEG C preservations.Described percentage composition is mass percentage.
Embodiment 2: the establishment of kit
Set up and detect Ractopamine magnetic immuno magnetic cell separation enrichment kit, make it contain following component:
The magnetic bead of coupling ractopamine monoclonal antibody
Redissolution liquid
Magnet
Being joined by the magnetic bead of coupling ractopamine monoclonal antibody and redissolve in liquid, is 10mg/mL to final concentration.
Embodiment 3: kit is to the method for separating and concentrating of Ractopamine in sample
1) get the magnetic bead being dissolved with coupling ractopamine monoclonal antibody and redissolve liquid 0.1mL in 10mL centrifuge tube, with 5mL deionized water rinse magnetic bead 1-2 time, centrifuge tube is left standstill 2-3min on magnet, guarantees that magnetic bead is all adsorbed on magnet, be separated magnetic bead and washing lotion with magnet at every turn;
2) joined by urine sample 5mL in the centrifuge tube of the 10mL that the good coupling ractopamine monoclonal antibody magnetic bead of rinse is housed, mixing, reacts 20min under room temperature; Or by animal tissue, Feed Sample with after homogenizer homogeneous, take 5.0 ± 0.05g sample in sample bottle, add 1.5 ± 0.05g sodium chloride respectively, 20mL50% methanol solution, with vortex instrument vortex 5min, the centrifugal 5min of 3000r/min under room temperature, or after leaving standstill, get 10mL supernatant liquid filtering replacement centrifugal process, draw 5mL centrifuged supernatant/filtrate, add 5mL deionized water, mixing, draws centrifuged supernatant/filtrate and the mixed liquor 5mL of deionized water and the magnetic bead of coupling ractopamine monoclonal antibody and mixes, at room temperature react 20min;
3) after having reacted, be separated magnetic bead with magnet, with 5mL washed with de-ionized water magnetic bead, clean twice;
4) 1mL methyl alcohol is joined be equipped with through step 3) in the 10mL centrifuge tube of cleaned magnetic bead, mixing, leaves standstill
1min, is separated magnetic bead with magnet, reclaims methanol solution, for detecting analysis.
Embodiment 4: the mensuration of kit quality
1. the quantity of the catch of kit and the recovery
Kit quantity of the catch is defined as: the magnetic bead of every milligram of coupling ractopamine monoclonal antibody can catch the maximum of Ractopamine.Prepare Ractopamine concentration and be respectively 10ng/mL, 15ng/mL, 20ng/mL, 25ng/mL, 30ng/mL sample solution, every part of pig urine, ox urinates, sheep urinates, feed, pork, pork liver, beef, meat samples solution is 1mL, getting ractopamine monoclonal antibody immunomagnetic beads 0.1mL is added in above-mentioned sample respectively, separation and the enrichment of Ractopamine in sample is carried out with kit, and detect crossing sample with the magnetic bead process of coupling ractopamine monoclonal antibody by Ractopamine enzyme linked immunological (ELISA) detection kit, magnetic capture amount and the sample TIANZHU XINGNAO Capsul of coupling ractopamine monoclonal antibody are as shown in table 1:
The magnetic capture amount of table 1 coupling ractopamine monoclonal antibody and sample TIANZHU XINGNAO Capsul result
As can be seen from Table 1, in sample, Ractopamine concentration is 10ng/mL, during 15ng/mL and 20ng/mL, through Ractopamine immuno magnetic cell separation enrichment kit to after Ractopamine separation and concentration in sample, detect through ELISA detection method again, show that Ractopamine TIANZHU XINGNAO Capsul scope is 89.0%-100.0%, when in sample, Ractopamine concentration is 25ng/mL and 30ng/mL, show that Ractopamine TIANZHU XINGNAO Capsul scope is 61.0%-78.8%, show that Ractopamine immuno magnetic cell separation enrichment kit is 20ng/mL to the quantity of the catch of Ractopamine in sample.
2. specificity
Using Ractopamine as standard, if the cross reacting rate of Ractopamine is 100%, the medicine for kit cross reaction Journal of Sex Research is and Ractopamine structure or intimate competition medicine: salbutamol, clenbuterol, phenolethanolamine A, bromine chlorine Boot sieve, bromine Boot sieve, Terbutaline, methylol Ractopamine, Cimaterol, Tulobuterol, horse spray special sieve, match Boot sieve, Ke Lunpante, Zilpaterol, spraying special sieve, gram logical sequence third sieve, Mabuterol, Clorprenaline.Adding above-mentioned 18 kinds of medicine (comprising Ractopamine) concentration respectively in pig urine, ox urine, sheep urine, feed, pork, pork liver, beef, meat samples is 20ng/mL, operate by kit step, after separation and concentration is carried out to Ractopamine in sample, utilize ELISA detection method to detect sample, testing result lists in table 2:
Table 2 kit specific test
As can be seen from Table 2, kit has higher specificity to Ractopamine, to Ractopamine structure or the equal no cross reaction of intimate competition medicine.