CN103665160B - The method of purifying water soluble nano-sized iron oxide mouse IgG class monoclonal antibody conjugate - Google Patents
The method of purifying water soluble nano-sized iron oxide mouse IgG class monoclonal antibody conjugate Download PDFInfo
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Abstract
The invention discloses the method for applicable mass-producing efficiently purifying water soluble oxidized Fe nanometer particles and IgG class monoclonal antibody conjugates, belong to biological technical field.Complicated for current ferric oxide nano particles antibody coupling matter purifying process; the rate of recovery is low; be difficult to large-scale production; the residual carboxyl of the present invention by adopting single-ended amination polyoxyethylene glycol to close ferric oxide nano particles and antibody coupling matter; reduce the surperficial zeta-potential of coupled product; adjustment solution ph to 4.5 ~ 5.0, reduce coupled product net charge content in the solution further, realize common supercentrifugal process mass-producing efficiently purifying ferric oxide nano particles and antibody coupling matter.This invention simplifies the experimental implementation flow process of ferric oxide nano particles and antibody coupling matter, reduce the requirement to separating device, be applicable to purify oxidized Fe nanometer particles IgG class monoclonal antibody conjugates in enormous quantities, yield is more than 90%, and the light characteristic of coupled product and biological activity have no considerable change.
Description
Technical field
The invention belongs to biological technical field, relate to the novel method of the modification of nano material biology and conjugate purification.
Background technology
Since application cell integration technology in 1975 prepares monoclonal antibody first, obtain with the Basic-crosses knurl technology of natural hybridization technology and developed rapidly.A large amount of monoclonal antibodies is successfully obtained, and is widely used in immunology, biological chemistry, pharmacology, cytobiology, microbiology and the every field such as clinical.Prepare at present the hybridoma cell strain of mouse, rat and people, wherein most widely usedly remain mouse hybridoma cell strain.Therefore, mouse IgG class monoclonal antibody naturally becomes the main and monoclonal antibody be most widely used of a class.This antibody-like not only has important scientific research value, and has huge commercial value.But mouse IgG class monoclonal antibody does not have the character such as optical, electrical of similar nano material, therefore often needs some materials in conjunction with other in actual applications, as fluorescent substance, organic dye, magneticsubstance etc.
After last century, the sixties manually produced magnetic nanoparticle, the aspects such as the synthesis of magnetic Nano material and application obtain and develop rapidly.Magnetic nano-particle is used widely in every field in recent years, as: bioseparation, drug transport, crosses heat cure, fixing, the immunoassay of enzyme, tissue repair and nuclear magnetic resonance etc.But, magnetic nano-particle has higher surface energy, solution mutually in there is strong gathering tendency, magnetic nano-particle is very limited in application aspect, particularly in biological and medicine and other fields, require that magnetic nano-particle has good stability, biocompatibility and biodegradability.By surface and copolymerized and surface modification, the surface energy of nanoparticle can be reduced, obtain solubility or the good nanoparticle of dispersibility thus.The magnetic compound particles formed after treatment both had magnetic, had surface active groups again, can further with the different kinds of molecules couplings such as medicine, antibody, protein, enzyme, cell and DNA, external magnetic field target organs, tissue or tumour can be utilized.The kind of magnetic nano-particle has a lot, compares and common are Ni, Fe, Co and alloy thereof, ferric oxide, ferrite, wherein with ferric oxide (γ-Fe
2o
3, Fe
3o
4) being most widely used of magneticsubstance.In numerous magnetic Nano biomaterial, Fe
3o
4nanoparticle (ironoxidenanoparticles, IOs) (particle diameter is less than 10nm), owing to having superior superparamagnetic characteristic, non-immunogenic, hypotoxicity and good biocompatibility, illustrates unique advantage at many biomedical aspects.But magnetic nano-particle does not have specificity and the selectivity of antibody molecule, antibody molecule in its surperficial coupling is therefore often needed to form magnetic nano-particle monoclonal antibody conjugates.Like this, this conjugate just has specificity and the selectivity of the special physico-chemical properties such as the magnetic of magnetic nano-particle and antibody molecule simultaneously.
As a kind of novel biomarker, ferric oxide nano particles is due to its special physico-chemical property, after being combined with organic dye or fluorescence dye, in cell marking, active somatic cell imaging, living animal body, the field such as fluorescent microscopic imaging and fluorescently-labeled immunology quick diagnosis technology has more significant advantage.Wherein the water soluble oxidized Fe nanometer particles on carboxyl function group surface is most widely used, and adopts the amphiphilic polymers of many carboxyls and hydrophobic chain to be the water soluble oxidized Fe nanometer particles most popular method that oil insoluble oxidation Fe nanometer particles is converted into carboxyl surface.Carboxylated ferric oxide nano particles mouse IgG class monoclonal antibody coupling common method is the active ester method of EDC/NHSS mediation.In ferric oxide nano particles and mouse IgG class monoclonal anti physique coupling process, mouse IgG class monoclonal antibody free in ferric oxide nano particles mouse IgG class monoclonal antibody conjugates and solution is carried out high efficiency separation, is the prerequisite ensureing ferric oxide nano particles mouse IgG class monoclonal antibody conjugates service efficiency.At present, ferric oxide nano particles mouse IgG class labeling of monoclonal antibodies Probe Purification method mainly comprises following several.The first, Ultracentrifugation Method.Because water soluble oxidized Fe nanometer particles nano particle diameter is little, specific surface area is large, nano material and mouse IgG class monoclonal antibody conjugates surface are containing a large amount of carboxyls, therefore adopt conventional centrifugal (lower than 30,000g centrifugal force), ferric oxide nano particles mouse IgG class monoclonal antibody conjugates organic efficiency generally (being less than 50%) on the low side, to ferric oxide nano particles mouse IgG class monoclonal antibody conjugates organic efficiency is increased to more than 90%, centrifugal speed generally need be greater than 50,000g.The common purification process of the second is gel chromatography.The method utilizes ferric oxide nano particles mouse IgG class monoclonal antibody conjugates and the molecular weight difference (showing as the difference of optics and aquation particle diameter) of mark mouse IgG class monoclonal antibody, utilizes exclusion chromatography principle to be separated.The third conventional separation method is ultra-filtration and separation method.This method utilize the ultra-filtration membrane of super filter tube to dam a kind of method that mark mouse IgG class monoclonal antibody and ferric oxide nano particles mouse IgG class monoclonal antibody conjugates carry out being separated by molecular weight difference.Also has the method for purifying and separating based on sepharose and agarose-polyacrylamide hybrid gel electrophoresis etc. simultaneously.In a word, although utilize above purification process can obtain the ferric oxide nano particles mouse IgG class monoclonal antibody conjugates of better quality, but still there is operational condition harshness, flow process is complicated, and the defects such as yield is low, are difficult to accomplish scale production.
Summary of the invention
Water soluble oxidized Fe nanometer particles is a kind of good Nanoparticle labeling material, this material by with antibody, Streptavidin, albumin A, Protein G and part or acceptor molecule coupling, the numerous areas such as nuclear magnetic resonance (MRI) contrast medium, cellular segregation, bacteria distribution, drug targeting transport, targeted therapy and immune quick diagnosis can be widely used in.But traditional ferric oxide nano particles monoclonal antibody conjugates purification process exists operational condition harshness, flow process is complicated, and yield is low and be difficult to defects such as accomplishing scale production.
The object of this invention is to provide a kind of easy and simple to handle, separation efficiency is high, the novel method of purify oxidized Fe nanometer particles mouse IgG class monoclonal antibody conjugates in enormous quantities, specifically comprise the following steps:
The method of purify oxidized Fe nanometer particles and IgG class monoclonal antibody conjugates, comprise the steps: that the ferric oxide nano particles of water-soluble carboxyl modified activates by (1), add IgG class monoclonal antibody solution, behind adjustment pH value of solution to 7.0 ~ 9.0, linked reaction; (2), after linked reaction terminates, single-ended amination polyoxyethylene glycol (HO-PEG-NH in solution, is added
3) close the carboxyl of ferric oxide nano particles remained on surface in coupled product, reacting solution pH value is adjusted to slightly acidic; (3) high speed centrifugation, abandons supernatant liquor, gets precipitation.
After step (3), also have precipitation with containing 25% glycerine, 0.01%NaN
30.05mol/LpH7.0 ~ 7.5 phosphate buffered saline buffer dissolving step.
Water soluble oxidized Fe nanometer particles used is the ferric oxide nano particles of nucleocapsid structure, shell is made up of parents' polymkeric substance, outside is a large amount of hydrophilic carboxyl surfaces, and internal layer is that long chain hydrophobic group is by being wrapped in shell inside with the hydrophobic interaction of trioctylphosphine oxide by oil insoluble oxidation Fe nanometer particles.
In described step (1), activation is for be dissolved in pH5.0 ~ 6.0 by the ferric oxide nano particles of water-soluble carboxyl modified, in 0.05mol/L borate buffer solution, add 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine and N-hydroxy thiosuccinimide respectively, 37 DEG C are reacted 2 hours, activated water-soluble ferric oxide nano particles carboxyl.
The mol ratio of described 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine and N-hydroxy thiosuccinimide and ferric oxide nano particles is 100 ~ 200:1, is preferably 150:1; The mol ratio of described IgG class monoclonal antibody and ferric oxide nano particles is 1 ~ 10:1;
After linked reaction terminates, in solution, add single-ended amination polyoxyethylene glycol (HO-PEG-NH
3) to single-ended amination polyoxyethylene glycol final concentration be 1 ~ 2%, fully mix 15 ~ 30 minutes;
Reacting solution pH value is adjusted to slightly acidic for pH value is adjusted to 4.5 ~ 5.0 in (2) by step, is preferably 5.0;
SuperMag described in step (3)
tMmagneticstrength is 100T/m or high speed centrifugation centrifugal force is 28,000 ~ 30,000g.
Described IgG class monoclonal antibody is aspergillus flavus resisting toxin B1 monoclonal antibody, anti-ochratoxin monoclonal antibody, anti-zearalenone monoclonal antibody, anti-enrofloxacin monoclonal antibody, anti-Clenbuterol hydrochloride monoclonal antibody, anti-salbutamol monoclonal antibody, anti-malachite green monoclonal antibody, anti-ractopamine monoclonal antibody, anti-salmonella monoclonal antibody, anti-Shigellae monoclonal antibody or anti-campylobacter jejuni monoclonal antibody.
Principle is shown in Fig. 1.
The ferric oxide nano particles preparation method of described water-soluble carboxyl modified is:
Under room temperature, get 0.1nmolFeO (OH), 1.5mL oleic acid and 18.75mL1-octadecylene Homogeneous phase mixing, by mixing solutions heated and stirred to 320 DEG C under nitrogen pressure.When question response solution becomes amber transparent by black muddiness, the carboxylate salt of iron is formed.When solution becomes black transparent, Z 250 (Fe
3o
4) nanocrystal solution formation.To above-mentioned Z 250 (Fe
3o
4) add acetone soln iron oxide precipitation nanoparticle in nanocrystal solution, be then further purified with the chloroform/acetone solution that volume ratio is 1:10, until form iron oxide nanocrystals powder.Get 1g polymaleic anhydride octadecyl, 1.2g2-(2-amino ethoxy) ethanol and a certain amount of Fe respectively
3o
4crystal powder (Fe
3o
4the mol ratio of crystal powder/polymkeric substance is 1:10) be scattered in 1mL chloroformic solution, then add 1mL phosphate buffered saline buffer (PBS, 10 μMs), remove completely in 40 DEG C of rotary evaporations to chloroform, finally obtain water-soluble carboxylated Fe
3o
4nanoparticle.
The ferric oxide nano particles of water-soluble carboxyl modified is the ferric oxide nano particles of nucleocapsid structure, shell is made up of parents' polymkeric substance, outside is a large amount of hydrophilic carboxyl surfaces, internal layer is long chain hydrophobic group, by oil insoluble oxidation Fe nanometer particles being wrapped in shell inside with the hydrophobic interaction of trioctylphosphine oxide.Ferric oxide nano particles is dissolved in pH6.0, in 0.05mol/L borate buffer solution, add 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine and N-hydroxy thiosuccinimide that mol ratio is 100 ~ 200:1,37 DEG C are reacted 2 hours, ferric oxide nano particles surface carboxyl groups are converted into the active ester of stable under acidic conditions.Add mouse IgG class monoclonal antibody solution, wherein mouse IgG class monoclonal antibody and ferric oxide nano particles mol ratio are 10:1, with 1MNaOH solution adjustment pH value of solution to 8.0 ~ 9.0.Under the condition of pH8.0 ~ 9.0, amino in mouse IgG class monoclonal antibody amino acid residue is easy to protonated, and the carboxyl of the active esterification in ferric oxide nano particles surface issues unboiled water solution at weak basic condition, form stable amido linkage with protonated amino coupling, thus mouse IgG class monoclonal antibody is coupled to ferric oxide nano particles surface.After ferric oxide nano particles and monoclonal antibody coupling, due to sterically hindered reason, ferric oxide nano particles surface still can remain a large amount of not coupled carboxyl, therefore ferric oxide nano particles monoclonal antibody conjugates still keeps larger zeta-potential, therefore adopts common centrifugal method ferric oxide nano particles monoclonal antibody conjugates effectively cannot be separated with non-conjugated monoclonal antibodies.In order to reduce the zeta-potential on ferric oxide nano particles monoclonal antibody conjugates surface, the present invention is by single-ended amination polyoxyethylene glycol (HO-PEG-NH
3) amino and the free carboxy generation ion-exchange of ferric oxide nano particles surface, make ferric oxide nano particles monoclonal antibody conjugates remain carboxyl and be converted to hydroxyl, improve ferric oxide nano particles monoclonal antibody conjugates iso-electric point, simultaneously further by the single-ended amination polyoxyethylene glycol of high density (HO-PEG-NH
3) (2% ~ 5%) destroy the hydration layer of mouse IgG class monoclonal anti surface, when solution ph is adjusted to 5.0, ferric oxide nano particles monoclonal antibody conjugates is under common high speed centrifugation (28,000g ~ 30,000g) can realize effectively being separated with conjugated monoclonal antibodies non-in solution, wherein the rate of recovery of ferric oxide nano particles and monoclonal antibody conjugates is greater than 90%.
In order to verify single-ended amination polyoxyethylene glycol (HO-PEG-NH
3) whether effectively can reduce the zeta-potential of carboxylated ferric oxide nano particles, we take particle diameter as the carboxylated water soluble oxidized Fe nanometer particles of 8nm is raw material, with single-ended amination polyoxyethylene glycol (HO-PEG-NH
3) be encapsulant, adopt EDC method to close ferric oxide nano particles surface carboxyl groups.Then adopt 0.1MHCl or NaOH to adjust carboxylated ferric oxide nano particles (concentration is 0.1 μM) pH respectively to 2,3,4,5,6 and 7, add the 0.1MHCl of isodose or NaOH solution in the hydroxylation ferric oxide nano particles solution of same concentrations simultaneously.The ferric oxide nano particles of the different pH value of above two classes is analyzed through Malvern surface potential particle instrument, the results are shown in Table 1.As known from Table 1, under same pH, single-ended amination polyoxyethylene glycol (HO-PEG-NH
3) the hydroxylation ferric oxide nano particles surface potential modified is significantly lower than hydroxylation ferric oxide nano particles, when solution ph is down between 4 ~ 5, hydroxylation ferric oxide nano particles zeta-potential declines obviously.
Under table 1 condition of different pH, the surface potential of carboxylated and hydroxylation ferric oxide nano particles
| pH 2 | pH 3 | pH 4 | pH 5 | pH 6 | pH 7 | |
| Carboxylated ferric oxide nano particles | -7.26 | -10.91 | -15.11 | -30.29 | -53.51 | -60.23 |
| Hydroxylation ferric oxide nano particles | 8.99 | -2.35 | -5.58 | -27.71 | -48.38 | -52.01 |
Technical solution of the present invention is adopted to have following beneficial effect:
1, the inventive method is by adding single-ended amination polyoxyethylene glycol (HO-PEG-NH
3) close ferric oxide nano particles surface not by the carboxyl reacted, carboxyl is made to change carboxyl into, reduce the zeta-potential on water soluble oxidized Fe nanometer particles mouse IgG class monoclonal antibody conjugates surface, destroy the hydration layer of mouse IgG class monoclonal anti surface simultaneously, be conducive to water soluble oxidized Fe nanometer particles mouse IgG class monoclonal antibody and separate out from reaction soln.
2, the inventive method is by acid adjustment, changes reacting solution pH value to 5.0, water soluble oxidized Fe nanometer particles monoclonal antibody conjugates is easily separated out in the solution.
3, the technology of the present invention is by adding single-ended amination polyoxyethylene glycol (HO-PEG-NH
3) and acid adjustment, water soluble oxidized Fe nanometer particles monoclonal antibody conjugates is made to be more prone to separate out from reaction soln, adopt common centrifugal method 28,000g ~ 30, water soluble oxidized Fe nanometer particles monoclonal antibody conjugates and non-conjugated monoclonal antibodies just can be carried out high efficiency separation (separation efficiency reaches more than 90%) by 000g.Compared with traditional water soluble oxidized Fe nanometer particles monoclonal antibody conjugates purification process, have simple to operate, equipment requirements low (common laboratory all can reach), purification efficiency high (more than 90%) and can accomplishing scale production.
Accompanying drawing explanation
Fig. 1 the inventive method principle schematic.
Embodiment
In order to make the present invention clearly understand, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
The collocation method of phosphate buffered saline buffer (PBS, 0.05M, pH7.4): NaCl40g, Na
2hPO
413.5g, KH
2pO
41.0g, KCl1.0g are dissolved in 1L ultrapure water.With 0.1MNaOH adjust pH to 8.0 ~ 9.0.
The compound method of borate buffer solution (0.05M, pH6.0): get 1.0g boric acid and be dissolved in 1L ultrapure water.Adjust pH to 6.0.
Mouse IgG class monoclonal antibody involved in embodiment: aspergillus flavus resisting toxin B1 monoclonal antibody, anti-ochratoxin monoclonal antibody, anti-zearalenone monoclonal antibody, anti-enrofloxacin monoclonal antibody, anti-Clenbuterol hydrochloride monoclonal antibody, anti-salbutamol monoclonal antibody, anti-malachite green monoclonal antibody, anti-ractopamine monoclonal antibody, anti-salmonella monoclonal antibody, anti-Shigellae monoclonal antibody, or anti-campylobacter jejuni monoclonal antibody provides by Wuxi Zhongde Bore Bioisystech Co., Ltd.
It is the water-soluble carboxyl ferric oxide nano particles of shell that embodiment 1 is synthesized with amphiphilic polymer
Under room temperature, get 0.1nmolFeO (OH), 1.5mL oleic acid and 18.75mL1-octadecylene Homogeneous phase mixing, by mixing solutions heated and stirred to 320 DEG C under nitrogen pressure.When question response solution becomes amber transparent by black muddiness, the carboxylate salt of iron is formed.When solution becomes black transparent, Z 250 (Fe
3o
4) nanocrystal solution formation.To above-mentioned Z 250 (Fe
3o
4) add acetone soln iron oxide precipitation nanoparticle in nanocrystal solution, be then further purified with the chloroform/acetone solution that volume ratio is 1:10, until form iron oxide nanocrystals powder.Get 1g polymaleic anhydride octadecyl, 1.2g2-(2-amino ethoxy) ethanol and a certain amount of Fe respectively
3o
4crystal powder (Fe
3o
4the mol ratio of crystal powder/polymkeric substance is 1:10) be scattered in 1mL chloroformic solution, then add 1mL phosphate buffered saline buffer (PBS, 10 μMs), remove completely in 40 DEG C of rotary evaporations to chloroform, finally obtain water-soluble carboxylated Fe
3o
4nanoparticle.
It is 8.0 ± 0.5nm that water-soluble carboxyl ferric oxide nano particles (0.5nmol/L) after synthesis measures its median size through transmission electron microscope (JEOL2100F).
It is 8nm water-soluble carboxyl ferric oxide nano particles and anti-enrofloxacin monoclonal antibody coupling and purifying process that embodiment 2 experimental techniques prepare particle diameter
The particle diameter getting 5mL embodiment 1 gained is that the carboxylated water soluble oxidized Fe nanometer particles of 8nm (concentration is 5mg/mL) mixes with the 0.05mol/L borate buffer solution of equal-volume pH6.0; Adding respectively with ferric oxide nano particles mol ratio is 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine and the N-hydroxy thiosuccinimide of 150:1,37 DEG C of reactions 2 hours; Adding with ferric oxide nano particles mol ratio is the anti-enrofloxacin monoclonal antibody solution of 10:1, behind 1MNaOH solution adjustment pH value of solution to 8.0 ~ 9.0, and room temperature reaction 3 hours; Adding final concentration in the most backward solution is 1% single-ended amination polyoxyethylene glycol (HO-PEG-NH
3), adjust pH to 5.0 with 1MHCl solution further.18,000rpm(about 29,000g) 4 DEG C of centrifugal 30min, abandon supernatant, precipitation with containing 25% glycerine, 0.01%NaN
30.05mol/L phosphate buffered saline buffer (pH7.0 ~ 7.5) dissolve and namely obtain not containing the anti-enrofloxacin monoclonal antibody conjugate of water soluble oxidized Fe nanometer particles of free anti-enrofloxacin monoclonal antibody.Experimental result, the water soluble oxidized Fe nanometer particles anti-enrofloxacin monoclonal antibody conjugate of technical solution of the present invention synthesis is through 1% single-ended amination polyoxyethylene glycol (HO-PEG-NH
3) after process, the rate of recovery of water soluble oxidized Fe nanometer particles anti-enrofloxacin monoclonal antibody conjugate centrifugal purification is 91.1%.
It is 8nm water-soluble carboxyl ferric oxide nano particles and the coupling of anti-Clenbuterol hydrochloride monoclonal antibody and purifying process that embodiment 3 experimental techniques prepare particle diameter
The particle diameter getting 5mL embodiment 1 gained is that the carboxylated water soluble oxidized Fe nanometer particles of 8nm (concentration is 5mg/mL) mixes with the 0.05mol/L borate buffer solution of equal-volume pH6.0; Adding respectively with ferric oxide nano particles mol ratio is 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine and the N-hydroxy thiosuccinimide of 150:1,37 DEG C of reactions 2 hours; Adding with ferric oxide nano particles mol ratio is the anti-Clenbuterol hydrochloride monoclonal antibody solution of 10:1, behind 1MNaOH solution adjustment pH value of solution to 8.0 ~ 9.0, and room temperature reaction 3 hours; Adding final concentration in the most backward solution is 1% single-ended amination polyoxyethylene glycol (HO-PEG-NH
3), adjust pH to 5.0 with 1MHCl solution further.18,000rpm(about 29,000g) 4 DEG C of centrifugal 30min, abandon supernatant, precipitation with containing 25% glycerine, 0.01%NaN
30.05mol/L phosphate buffered saline buffer (pH7.0 ~ 7.5) dissolve and namely obtain not containing the anti-Clenbuterol hydrochloride monoclonal antibody conjugates of water soluble oxidized Fe nanometer particles of free anti-Clenbuterol hydrochloride monoclonal antibody.Experimental result, the water soluble oxidized Fe nanometer particles anti-Clenbuterol hydrochloride monoclonal antibody conjugates of technical solution of the present invention synthesis is through 1% single-ended amination polyoxyethylene glycol (HO-PEG-NH
3) after process, the rate of recovery of water soluble oxidized Fe nanometer particles anti-Clenbuterol hydrochloride monoclonal antibody conjugates centrifugal purification is 91.7%.
It is 8nm water-soluble carboxyl ferric oxide nano particles and the coupling of anti-salbutamol monoclonal antibody and purifying process that embodiment 4 experimental techniques prepare particle diameter
The particle diameter getting 5mL embodiment 1 gained is that the carboxylated water soluble oxidized Fe nanometer particles of 8nm (concentration is 5mg/mL) mixes with the 0.05mol/L borate buffer solution of equal-volume pH6.0; Adding respectively with ferric oxide nano particles mol ratio is 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine and the N-hydroxy thiosuccinimide of 150:1,37 DEG C of reactions 2 hours; Adding with ferric oxide nano particles mol ratio is the anti-salbutamol monoclonal antibody solution of 10:1, behind 1MNaOH solution adjustment pH value of solution to 8.0 ~ 9.0, and room temperature reaction 3 hours; Adding final concentration in the most backward solution is 1% single-ended amination polyoxyethylene glycol (HO-PEG-NH
3), adjust pH to 5.0 with 1MHCl solution further.18,000rpm(about 29,000g) 4 DEG C of centrifugal 30min, abandon supernatant, precipitation with containing 25% glycerine, 0.01%NaN
30.05mol/L phosphate buffered saline buffer (pH7.0 ~ 7.5) dissolve and namely obtain not containing the anti-salbutamol monoclonal antibody conjugates of water soluble oxidized Fe nanometer particles of free anti-salbutamol monoclonal antibody.Experimental result, the water soluble oxidized Fe nanometer particles anti-salbutamol monoclonal antibody conjugates of technical solution of the present invention synthesis is through 1% single-ended amination polyoxyethylene glycol (HO-PEG-NH
3) after process, the rate of recovery of water soluble oxidized Fe nanometer particles anti-salbutamol monoclonal antibody conjugates centrifugal purification is 90.9%.
It is 8nm water-soluble carboxyl ferric oxide nano particles and the coupling of anti-malachite green monoclonal antibody and purifying process that embodiment 5 experimental techniques prepare particle diameter
The particle diameter getting 5mL embodiment 1 gained is that the carboxylated water soluble oxidized Fe nanometer particles of 8nm (concentration is 5mg/mL) mixes with the 0.05mol/L borate buffer solution of equal-volume pH6.0; Adding respectively with ferric oxide nano particles mol ratio is 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine and the N-hydroxy thiosuccinimide of 150:1,37 DEG C of reactions 2 hours; Adding with ferric oxide nano particles mol ratio is the anti-malachite green monoclonal antibody solution of 10:1, behind 1MNaOH solution adjustment pH value of solution to 8.0 ~ 9.0, and room temperature reaction 3 hours; Adding final concentration in the most backward solution is 1% single-ended amination polyoxyethylene glycol (HO-PEG-NH
3), adjust pH to 5.0 with 1MHCl solution further.18,000rpm(about 29,000g) 4 DEG C of centrifugal 30min, abandon supernatant, precipitation with containing 25% glycerine, 0.01%NaN
30.05mol/L phosphate buffered saline buffer (pH7.0 ~ 7.5) dissolve and namely obtain not containing the anti-malachite green monoclonal antibody conjugates of water soluble oxidized Fe nanometer particles of free anti-malachite green monoclonal antibody.Experimental result, the water soluble oxidized Fe nanometer particles anti-malachite green monoclonal antibody conjugates of technical solution of the present invention synthesis is through 1% single-ended amination polyoxyethylene glycol (HO-PEG-NH
3) after process, the rate of recovery of water soluble oxidized Fe nanometer particles anti-malachite green monoclonal antibody conjugates centrifugal purification is 92.5%.
It is 8nm water-soluble carboxyl ferric oxide nano particles and anti-ractopamine monoclonal antibody coupling and purifying process that embodiment 6 experimental techniques prepare particle diameter
The particle diameter getting 5mL embodiment 1 gained is that the carboxylated water soluble oxidized Fe nanometer particles of 8nm (concentration is 5mg/mL) mixes with the 0.05mol/L borate buffer solution of equal-volume pH6.0; Adding respectively with ferric oxide nano particles mol ratio is 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine and the N-hydroxy thiosuccinimide of 150:1,37 DEG C of reactions 2 hours; Adding with ferric oxide nano particles mol ratio is the anti-ractopamine monoclonal antibody solution of 10:1, behind 1MNaOH solution adjustment pH value of solution to 8.0 ~ 9.0, and room temperature reaction 3 hours; Adding final concentration in the most backward solution is 1% single-ended amination polyoxyethylene glycol (HO-PEG-NH
3), adjust pH to 5.0 with 1MHCl solution further.18,000rpm(about 29,000g) 4 DEG C of centrifugal 30min, abandon supernatant, precipitation with containing 25% glycerine, 0.01%NaN
30.05mol/L phosphate buffered saline buffer (pH7.0 ~ 7.5) dissolve and namely obtain not containing the anti-ractopamine monoclonal antibody conjugate of water soluble oxidized Fe nanometer particles of free anti-ractopamine monoclonal antibody.Experimental result, the water soluble oxidized Fe nanometer particles anti-ractopamine monoclonal antibody conjugate of technical solution of the present invention synthesis is through 1% single-ended amination polyoxyethylene glycol (HO-PEG-NH
3) after process, the rate of recovery of water soluble oxidized Fe nanometer particles anti-ractopamine monoclonal antibody conjugate centrifugal purification is 92.9%.
It is 8nm water-soluble carboxyl ferric oxide nano particles and the coupling of anti-ochratoxin monoclonal antibody and purifying process that embodiment 7 experimental techniques prepare particle diameter
The particle diameter getting 5mL embodiment 1 gained is that the carboxylated water soluble oxidized Fe nanometer particles of 8nm (concentration is 5mg/mL) mixes with the 0.05mol/L borate buffer solution of equal-volume pH6.0; Adding respectively with ferric oxide nano particles mol ratio is 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine and the N-hydroxy thiosuccinimide of 150:1,37 DEG C of reactions 2 hours; Adding with ferric oxide nano particles mol ratio is the anti-ochratoxin monoclonal antibody solution of 10:1, behind 1MNaOH solution adjustment pH value of solution to 8.0 ~ 9.0, and room temperature reaction 3 hours; Adding final concentration in the most backward solution is 1% single-ended amination polyoxyethylene glycol (HO-PEG-NH
3), adjust pH to 5.0 with 1MHCl solution further.18,000rpm(about 29,000g) 4 DEG C of centrifugal 30min, abandon supernatant, precipitation with containing 25% glycerine, 0.01%NaN
30.05mol/L phosphate buffered saline buffer (pH7.0 ~ 7.5) dissolve and namely obtain not containing the anti-ochratoxin monoclonal antibody conjugates of water soluble oxidized Fe nanometer particles of free anti-ochratoxin monoclonal antibody.Experimental result, the water soluble oxidized Fe nanometer particles anti-ochratoxin monoclonal antibody conjugates of technical solution of the present invention synthesis is through 1% single-ended amination polyoxyethylene glycol (HO-PEG-NH
3) after process, the rate of recovery of water soluble oxidized Fe nanometer particles anti-ochratoxin monoclonal antibody conjugates centrifugal purification is 90.6%.
It is 8nm water-soluble carboxyl ferric oxide nano particles and the coupling of anti-zearalenone monoclonal antibody and purifying process that embodiment 8 experimental techniques prepare particle diameter
The particle diameter getting 5mL embodiment 1 gained is that the carboxylated water soluble oxidized Fe nanometer particles of 8nm (concentration is 5mg/mL) mixes with the 0.05mol/L borate buffer solution of equal-volume pH6.0; Adding respectively with ferric oxide nano particles mol ratio is 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine and the N-hydroxy thiosuccinimide of 150:1,37 DEG C of reactions 2 hours; Adding with ferric oxide nano particles mol ratio is the anti-zearalenone monoclonal antibody solution of 10:1, behind 1MNaOH solution adjustment pH value of solution to 8.0 ~ 9.0, and room temperature reaction 3 hours; Adding final concentration in the most backward solution is 1% single-ended amination polyoxyethylene glycol (HO-PEG-NH
3), adjust pH to 5.0 with 1MHCl solution further.18,000rpm(about 29,000g) 4 DEG C of centrifugal 30min, abandon supernatant, precipitation with containing 25% glycerine, 0.01%NaN
30.05mol/L phosphate buffered saline buffer (pH7.0 ~ 7.5) dissolve and namely obtain not containing the anti-zearalenone monoclonal antibody conjugates of water soluble oxidized Fe nanometer particles of free anti-zearalenone monoclonal antibody.Experimental result, the water soluble oxidized Fe nanometer particles anti-zearalenone monoclonal antibody conjugates of technical solution of the present invention synthesis is through 1% single-ended amination polyoxyethylene glycol (HO-PEG-NH
3) after process, the rate of recovery of water soluble oxidized Fe nanometer particles anti-zearalenone monoclonal antibody conjugates centrifugal purification is 91.5%.
It is the water-soluble carboxyl ferric oxide nano particles of 8nm and aspergillus flavus resisting toxin B1 monoclonal antibody coupling and purifying process that embodiment 9 experimental techniques prepare particle diameter
The particle diameter getting 5mL embodiment 1 gained is that the carboxylated water soluble oxidized Fe nanometer particles of 8nm (concentration is 5mg/mL) mixes with the 0.05mol/L borate buffer solution of equal-volume pH6.0; Adding respectively with ferric oxide nano particles mol ratio is 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine and the N-hydroxy thiosuccinimide of 150:1,37 DEG C of reactions 2 hours; Adding with ferric oxide nano particles mol ratio is the aspergillus flavus resisting toxin M1 monoclonal antibody solution of 10:1, behind 1MNaOH solution adjustment pH value of solution to 8.0 ~ 9.0, and room temperature reaction 3 hours; Adding final concentration in the most backward solution is 1% single-ended amination polyoxyethylene glycol (HO-PEG-NH
3), adjust pH to 5.0 with 1MHCl solution further.18,000rpm(about 29,000g) 4 DEG C of centrifugal 30min, abandon supernatant, precipitation with containing 25% glycerine, 0.01%NaN
30.05mol/L phosphate buffered saline buffer (pH7.0 ~ 7.5) dissolve and namely obtain not containing the water soluble oxidized Fe nanometer particles aspergillus flavus resisting toxin B1 monoclonal antibody conjugates of free aspergillus flavus resisting toxin B1 monoclonal antibody.Experimental result, the water soluble oxidized Fe nanometer particles aspergillus flavus resisting toxin B1 monoclonal antibody conjugates of technical solution of the present invention synthesis is through 1% single-ended amination polyoxyethylene glycol (HO-PEG-NH
3) after process, the rate of recovery of water soluble oxidized Fe nanometer particles aspergillus flavus resisting toxin B1 monoclonal antibody conjugates centrifugal purification is 92.7%.
It is the water-soluble carboxyl ferric oxide nano particles of 8nm and the coupling of anti-salmonella monoclonal antibody and purifying process that embodiment 10 experimental techniques prepare particle diameter
The particle diameter getting 5mL embodiment 1 gained is that the carboxylated water soluble oxidized Fe nanometer particles of 8nm (concentration is 5mg/mL) mixes with the 0.05mol/L borate buffer solution of equal-volume pH6.0; Adding respectively with ferric oxide nano particles mol ratio is 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine and the N-hydroxy thiosuccinimide of 150:1,37 DEG C of reactions 2 hours; Adding with ferric oxide nano particles mol ratio is the anti-salmonella monoclonal antibody solution of 10:1, behind 1MNaOH solution adjustment pH value of solution to 8.0 ~ 9.0, and room temperature reaction 3 hours; Adding final concentration in the most backward solution is 1% single-ended amination polyoxyethylene glycol (HO-PEG-NH
3), adjust pH to 5.0 with 1MHCl solution further.18,000rpm(about 29,000g) 4 DEG C of centrifugal 30min, abandon supernatant, precipitation with containing 25% glycerine, 0.01%NaN
30.05mol/L phosphate buffered saline buffer (pH7.0 ~ 7.5) dissolve and namely obtain not containing the water soluble oxidized Fe nanometer particles anti-salmonella monoclonal antibody conjugates of free anti-salmonella monoclonal antibody.Experimental result, the water soluble oxidized Fe nanometer particles anti-salmonella monoclonal antibody conjugates of technical solution of the present invention synthesis is through 1% single-ended amination polyoxyethylene glycol (HO-PEG-NH
3) after process, the rate of recovery of water soluble oxidized Fe nanometer particles anti-salmonella monoclonal antibody conjugates centrifugal purification is 93.1%.
It is 8nm water-soluble carboxyl ferric oxide nano particles and the coupling of anti-Shigellae monoclonal antibody and purifying process that embodiment 11 experimental techniques prepare particle diameter
The particle diameter getting 5mL embodiment 1 gained is that the carboxylated water soluble oxidized Fe nanometer particles of 8nm (concentration is 5mg/mL) mixes with the 0.05mol/L borate buffer solution of equal-volume pH6.0; Adding respectively with ferric oxide nano particles mol ratio is 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine and the N-hydroxy thiosuccinimide of 150:1,37 DEG C of reactions 2 hours; Adding with ferric oxide nano particles mol ratio is the anti-Shigellae monoclonal antibody solution of 10:1, behind 1MNaOH solution adjustment pH value of solution to 8.0 ~ 9.0, and room temperature reaction 3 hours; Adding final concentration in the most backward solution is 1% single-ended amination polyoxyethylene glycol (HO-PEG-NH
3), adjust pH to 5.0 with 1MHCl solution further.18,000rpm(about 29,000g) 4 DEG C of centrifugal 30min, abandon supernatant, precipitation with containing 25% glycerine, 0.01%NaN
30.05mol/L phosphate buffered saline buffer (pH7.0 ~ 7.5) dissolve and namely obtain not containing the anti-Shigellae monoclonal antibody conjugates of water soluble oxidized Fe nanometer particles of free anti-Shigellae monoclonal antibody.Experimental result, the water soluble oxidized Fe nanometer particles anti-Shigellae monoclonal antibody conjugates of technical solution of the present invention synthesis is through 1% single-ended amination polyoxyethylene glycol (HO-PEG-NH
3) after process, the rate of recovery of water soluble oxidized Fe nanometer particles anti-Shigellae monoclonal antibody conjugates centrifugal purification is 90.8%.
It is 8nm water-soluble carboxyl ferric oxide nano particles and the coupling of anti-campylobacter jejuni monoclonal antibody and purifying process that embodiment 12 experimental techniques prepare particle diameter
The particle diameter getting 5mL embodiment 1 gained is that the carboxylated water soluble oxidized Fe nanometer particles of 8nm (concentration is 5mg/mL) mixes with the 0.05mol/L borate buffer solution of equal-volume pH6.0; Adding respectively with ferric oxide nano particles mol ratio is 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine and the N-hydroxy thiosuccinimide of 150:1,37 DEG C of reactions 2 hours; Adding with ferric oxide nano particles mol ratio is the anti-campylobacter jejuni monoclonal antibody solution of 10:1, behind 1MNaOH solution adjustment pH value of solution to 8.0 ~ 9.0, and room temperature reaction 3 hours; Adding final concentration in the most backward solution is 1% single-ended amination polyoxyethylene glycol (HO-PEG-NH
3), adjust pH to 5.0 with 1MHCl solution further.18,000rpm(about 29,000g) 4 DEG C of centrifugal 30min, abandon supernatant, precipitation with containing 25% glycerine, 0.01%NaN
30.05mol/L phosphate buffered saline buffer (pH7.0 ~ 7.5) dissolve and namely obtain not containing the anti-campylobacter jejuni monoclonal antibody conjugates of water soluble oxidized Fe nanometer particles of free anti-campylobacter jejuni monoclonal antibody.Experimental result, the water soluble oxidized Fe nanometer particles anti-campylobacter jejuni monoclonal antibody conjugates of technical solution of the present invention synthesis is through 1% single-ended amination polyoxyethylene glycol (HO-PEG-NH
3) after process, the rate of recovery of water soluble oxidized Fe nanometer particles anti-campylobacter jejuni monoclonal antibody conjugates centrifugal purification is 93.0%.
Claims (9)
1. the method for a purifying water soluble nano-sized iron oxide mouse IgG class monoclonal antibody conjugate, it is characterized in that comprising the following steps: the ferric oxide nano particles of water-soluble carboxyl modified activates by (1), add IgG class monoclonal antibody solution, behind adjustment pH value of solution to 7.0 ~ 9.0, linked reaction; (2), after linked reaction terminates, add the carboxyl that ferric oxide nano particles remained on surface in coupled product closed by single-ended amination polyoxyethylene glycol in solution, reacting solution pH value is adjusted to slightly acidic; (3) be 100T/mSuperMag by magneticstrength
tMseparator or high speed centrifugation, abandon supernatant liquor, gets precipitation;
In described step (1), activation is for be dissolved in pH5.0 ~ 6.0 by the ferric oxide nano particles of water-soluble carboxyl modified, in 0.05mol/L borate buffer solution, add 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine and N-hydroxy thiosuccinimide respectively, 37 DEG C are reacted 2 hours, activated water-soluble ferric oxide nano particles carboxyl;
Reacting solution pH value is adjusted to slightly acidic for pH value is adjusted to 4.5 ~ 5.0 in (2) by step.
2. method according to claim 1, after it is characterized in that step (3), also has precipitation with containing 25% glycerine, 0.01%NaN
30.05mol/LpH7.0 ~ 7.5 phosphate buffered saline buffer dissolving step.
3. method according to claim 1, it is characterized in that the ferric oxide nano particles of water-soluble carboxyl modified is the ferric oxide nano particles of nucleocapsid structure, shell is made up of parents' polymkeric substance, outside is a large amount of hydrophilic carboxyl surfaces, and internal layer is that long chain hydrophobic group is by being wrapped in shell inside with the hydrophobic interaction of trioctylphosphine oxide by oil insoluble oxidation Fe nanometer particles.
4. method according to claim 1, is characterized in that the mol ratio of described 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine and N-hydroxy thiosuccinimide and ferric oxide nano particles is 100 ~ 200:1; The mol ratio of described IgG class monoclonal antibody and ferric oxide nano particles is 1 ~ 10:1.
5. method according to claim 4, is characterized in that the mol ratio of described 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine and N-hydroxy thiosuccinimide and ferric oxide nano particles is 150:1; The mol ratio of described IgG class monoclonal antibody and ferric oxide nano particles is 1 ~ 10:1.
6. method according to claim 1, after it is characterized in that linked reaction terminates, adding single-ended amination polyoxyethylene glycol to single-ended amination polyoxyethylene glycol final concentration is in the solution 1% ~ 2%, fully mixes 15 ~ 30 minutes.
7. method according to claim 1, is characterized in that, in step (2), reacting solution pH value is adjusted to slightly acidic for pH value is adjusted to 5.0.
8. method according to claim 1, its feature is being that the high speed centrifugation centrifugal force described in step (3) is 28,000 ~ 30,000g.
9. method according to claim 1, is characterized in that described IgG class monoclonal antibody is aspergillus flavus resisting toxin B1 monoclonal antibody, anti-ochratoxin monoclonal antibody, anti-zearalenone monoclonal antibody, anti-enrofloxacin monoclonal antibody, anti-Clenbuterol hydrochloride monoclonal antibody, anti-salbutamol monoclonal antibody, anti-malachite green monoclonal antibody, anti-ractopamine monoclonal antibody, anti-salmonella monoclonal antibody, anti-Shigellae monoclonal antibody or anti-campylobacter jejuni monoclonal antibody.
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