CN103933947B - For blood purification material removing rheumatoid factor and preparation method thereof - Google Patents
For blood purification material removing rheumatoid factor and preparation method thereof Download PDFInfo
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Abstract
The invention provides a kind of blood purification material for removing rheumatoid factor and preparation method, belonging to field of biomedicine technology.This blood purification material forms by solid phase carrier with by the aglucon that chemical coupling is fixed on solid phase carrier; Wherein, solid phase carrier is polysaccharide natural macromolecular material, and aglucon is 1-amido-3-(2-(4-pyridine radicals)-ehtylmercapto)-2-propyl alcohol, the ligand density be fixed on solid phase carrier by chemical coupling is the dry solid phase carrier of 1.4 ~ 2.8mmol/g.This blood purification material can rheumatoid factor in selective absorption blood, limited to the non-specific adsorption of other plasma component such as human serum albumins, IgG, and preparation cost is low, physicochemical properties are stablized, can be used as the removing of adsorption stuffing for rheumatoid factor in rheumatoid disease human blood of apparatus for purifying blood.
Description
Technical field
The present invention relates to a kind of for blood purification material removing rheumatoid factor and its preparation method and application, belong to field of biomedicine technology.
Background technology
Rheumatoid factor (Rheumatoid factors, RF) is a kind of IgM type antitypy IgG antibody, has been proved with the emergence and development of a series of rheumatoid disease closely related, as rheumatoid arthritis, systemic loupus erythematosus etc.For these diseases, the concentration being reduced rheumatoid factor in patient blood by the mode of external removing obviously can improve symptom, alleviation and therapeutic action (B.C.McLeod are played to disease, Introduction to the Third Special Issue:Clinical Applications of TherapeuticApheresis.J Clin Apheresis, 2000,15 (1-2): 1-5), earn widespread respect in clinical for removing the blood purification technology of rheumatoid factor.
Blood purification technology is the methods for the treatment of that a kind of manual intervention regulates blood related component.Since last century, the eighties obtained development and application, it develops into a kind of important clinical treatment means gradually.The blood of patient is by outside lead body, after purifier in defeated ex vivo, purifier is for removing metabolic waste, morbid substance, the unnecessary moisture content etc. in blood in this process, unbalance to correct with conditioning engine body environment, thus reaches the object of disease therapy.
The adsorbent being applied to blood purification is generally made up of two parts: as adsorbent matrix polymer carrier and utilize chemical crosslinking to be fixed on the ligand molecule of carrier surface.Adsorbent matrix generally adopts the porous material of good hydrophilic property.Good adsorbent matrix also needs to possess the features such as physicochemical properties are stable, non-specific adsorption is little and blood compatibility is good.There is the macromolecular material of number of different types to can be used as the carrier matrix of blood-purifying adsorbing agent at present, comprise polysaccharide natural polymer, as agarose, cellulose, glucan, shitosan etc.; Synthesized polymer material, as polyacrylamide, polyimides, polyvinyl alcohol etc.; Inorganic material, as silica, Bio-Glas etc.Ligand molecule is by being covalently bonded on carrier material, and its structures and characteristics determines the action effect of adsorbent.Adsorbent at present for antibody-based molecules Adsorption mainly adopts two kinds of functional ligand: bion and micromolecular compound type.
Protein A adsorbent is a kind of typical bion blood-purifying adsorbing agent, and it adopts albumin A as adsorption function base.Albumin A is a kind of aureus cell wall-held protein, and full name staphylococcal protein A (Staphylococcal Protein A, SPA) is single chain polypeptide, molecular weight 4.2kDa.It has reversible affinity interaction to the mankind and other mammiferous panimmunity globulin, particularly has stronger binding ability to the Fc district of IgG.Albumin A to the combination of rheumatoid factor mainly based on the ability of its wide spectrum binding domain-immunoglobulin.At present, albumin A has been used to the removing of autoantibody and immune complex in clinical middle autoimmune disease patient's body as a kind of typical affinity ligand.Prosorba and Immunosorba(Fresenius, Germany) be two kinds of protein A adsorbent be most widely used at present, they are respectively using silica gel and Ago-Gel as carrier matrix, wherein Prosorba has obtained the certification of food and drug administration (FDA), is used for the treatment of ITP and rheumatoid arthritis.Domestic also have like product to come out (CN1367181A).Adopt albumin A as the adsorbent of aglucon for this class, its advantage is to utilize affinity interaction natural between biomolecule, antagonist quasi-molecule has very high Selective recognition ability, the scavenging action to a large class autoantibody can be realized, therefore can be used for the treatment of the disease that a series of autoantibody is relevant broad spectrum activity.But its shortcoming is that the binding ability of albumin A to IgM type antibody will be significantly smaller than the binding ability to IgG type antibody.Albumin A mainly acts on the Fc fragment of IgG, but the Fc fragment of IgM concentrates on molecular center position, and degree of exposure is lower, and associated proteins A's is sterically hindered larger.Therefore, albumin A will, lower than the removal effect to other IgG type autoantibodies, want to obtain comparatively ideal result for the treatment of the removal effect of rheumatoid factor, needs very high treatment intensity, and this will cause the loss of a large amount of normal IgG.In addition, as having bioactive protein molecule, need to obtain from staphylococcus aureus or genetic engineering bacterium, its production cost is very high.The market price of current Prosorba adsorption column is about 1000 Euros, and the Immunosorba adsorption column market price of a pair changeable use is about 10000 Euros.Meanwhile, protein ligand is unstable in immobilization and preservation process, easy in inactivation.Although can regenerate, reuse, also there is the hidden danger being difficult to the problem of on-line cleaning sterilizing and ligand molecule and coming off.These shortcomings limit the Clinical practice of protein A adsorbent in rheumatoid disease treatment above.
Except albumin A, report is also had to adopt hot polymerization IgG as the affine function base of absorption rheumatoid factor.The application of hot polymerization IgG mainly has the biologically active in conjunction with sex change IgG based on IgM type rheumatoid factor, and the hot polymerization IgG therefore prepared by the mode of artificial thermal denaturation is immobilized after host surface, also has the ability of selective removal rheumatoid factor.But due to high aglucon production cost and the potential safety hazard that may exist, the adsorbent of this type is detected in laboratory research, not yet realizes industrial applications.
Compared with large biological molecule, the micromolecular compound of chemical synthesis all has significant advantage on production cost and physical and chemical stability, so a lot of research is also conceived to screen and design the Small molecular aglucon that can be applied to blood purification.
Hydrophobic amino acid reports and apply maximum Small molecular groups for antibody absorption at present.Japan Asahi medical company develops two kinds respectively with the sorbent material that tryptophan and phenylalanine are aglucon, and trade name is respectively Immusorba TR (IM-TR) and Immusorba PH (IM-PH).These two kinds of adsorbents are all using polyvinyl alcohol microparticles as carrier matrix, and tryptophan or phenylalanine molecule are coupled at stromal surface by amino group.The active force feature of these two kinds of adsorbents, mainly based on aromatic rings and carboxylic group, therefore infers that the interaction of they and antibody molecule combines hydrophobic effect and electrostatic interaction.Adsorption experiment shows that IM-TR and IM-PH does not have considerable non-specific adsorption to albumin in blood plasma and fibrinogen, but to autoantibodies such as Anti-DNA antibody, anti-AchR antibody and Anti-C1q antibodies, there is general removal effect, be used to the treatment of the autoimmune diseases such as Guillain Barre syndrome, systemic loupus erythematosus, myasthenia gravis in Japan.A kind of histidine that utilizes is provided as the sorbent material of adsorption group in Chinese patent literature CN1666784A.In addition, the report (Chinese patent literature CN1476908A) adopting polyaminoacid to be used in this field as adsorption group is also had.In the application study of adsorbent, also having a small amount of research to confirm also there is Adsorption effect to a certain degree to rheumatoid factor in this kind of adsorbent based on hydrophobic amino acid.Such as, someone reports and adopts phenylalanine as ligand cou in polyvinylalcohol microsphere bulb matrix, the Adsorption (king is superfine, Chinese biomedical engineering journal, 2009,28(4) for rheumatoid factor: 561-566).
In addition, inventor place team also develops the Adsorption of two kinds of Small molecular aglucons for autoantibody in autoimmune disease patient's body, comprise benzoic acid (Chinese patent literature CN101185880A) and 4-mercaptoethyl pyridine (J.Ren, et al., J Biomed Mater Res Part A, 2011:98A:589 – 595).For the Adsorption of rheumatoid factor, although current existing Small molecular aglucon adsorbent all shows binding ability to a certain degree under laboratory evaluation condition, in the experimental evaluation of further simulating clinical treatment, effect is often unsatisfactory.Main cause is, the adsorbent based on Small molecular aglucon being representative with aromatic amino acid and 4-mercaptoethyl pyridine is all wide spectrum antibody adsorbents of molecular characterization exploitation for IgG type antibody and design, and IgM type antibody has significant difference with IgG type antibody on molecular scale and design feature: 1. the molecular weight of IgM is six times of IgG; Although 2. both have the identical conserved sequence of part, still there is larger difference in molecular characterization.Therefore the adsorbent for IgG design is improper to the rheumatoid factor based on IgM.For 4-mercaptoethyl pyridine, although we find that this molecule all has adsorption capacity in various degree to Multiple Antibodies component in human blood, the binding ability of it and non-IgG type antibody (as IgM, IgA and IgE) and compound thereof is far away from the adsorption effect to IgG.If for the adsorbing therapy of IgM type rheumatoid factor, by in the face of the problem similar with albumin A, namely rheumatoid factor adsorption capacity is more weak, if improve the clearance of rheumatoid factor by strengthening treatment intensity, the loss of a large amount of normal IgG will be caused, the risk bringing immunologic function to weaken (J.Ren, et al., J Biomed Mater Res Part A, 2011:98A:589 – 595).
In essence, 4-mercaptoethyl pyridine is as the special adsorption function base for the exploitation of IgG type antibody, and the action site of it and IgG mainly concentrates on the Fc fragment of IgG, and wherein hydrophobic effect and π-pi-conjugated has played important function.Ability to function Fc fragment being wrapped in intramolecular IgM, 4-mercaptoethyl pyridine is difficult to effectively embody.Therefore the adsorptive selectivity to IgM antibody-like will be improved, realizing removing with regard to needing the special molecular characterization for IgM to carry out brand-new design and the structure optimization of ligand molecule the selective absorption of rheumatoid factor, making it on space structure and active force, be more suitable for IgM.
Summary of the invention
The object of the present invention is to provide a kind of for blood purification material removing rheumatoid factor and preparation method thereof.Rheumatoid factor in this blood purification material reply human blood has adsorption capacity selective and higher preferably, and has stable physicochemical properties and the production cost of relative moderate.
The present inventor has carried out ligand molecule design for the architectural feature of IgM, by the screening of computer molecular docking Binding experiment, finally determine a brand-new long-chain polyfunctional compound: 1-amido-3-(2-(4-pyridine radicals)-ehtylmercapto)-2-propyl alcohol (1-amino-3-(2-(pyridin-4-yl) ethylthio) propan-2-ol) is as the selective binding aglucon of IgM.1-amido-3-(2-(4-pyridine radicals)-ehtylmercapto)-2-propyl alcohol molecule compared with the aglucon used in prior art (benzoic acid, aromatic amino acid, 4-mercaptoethyl pyridine), the basis of hydrophobic heterocyclic group introduces multiple electron rich group (hydroxyl, amido).Molecular docking result shows, and the binding ability of itself and IgM type antibody will be significantly higher than IgG type antibody; And action site analysis shows, these electron rich groups are more prone to the Fab fragment in conjunction with IgM, have more effective binding sites at the outer surface of IgM.Because IgM molecular size is about 6 times of IgG, multiple Fab fragment is exposed to molecular surface, and the combination of strengthening ligand molecule and Fab significantly will strengthen the ability to function of aglucon and IgM and rheumatoid factor.Meanwhile, IgM has larger surface area, and compared with absorption IgG molecule, realizing effectively adsorbing required ligand density to IgM and rheumatoid factor can be lower.By conservative control coupling group density, the differentiation of adsorbent to IgM and IgG just can be realized.Based on the factor of above two aspects, by optimizing the immobilized reaction condition of aglucon further on the basis of preferred aglucon, determine the sorbing material synthetic schemes that is applicable to rheumatoid factor in selective clearing human blood.
Above-mentioned ligand molecule 1-amido-3-(2-(4-pyridine radicals)-ehtylmercapto) structural formula of-2-propyl alcohol is as follows:
Specifically, blood purification material for removing rheumatoid factor of the present invention forms by solid phase carrier with by aglucon two parts that chemical coupling is fixed on solid phase carrier, solid phase carrier is cross-linked polysaccharides, described aglucon is 1-amido-3-(2-(4-pyridine radicals)-ehtylmercapto)-2-propyl alcohol, the ligand density be fixed on solid phase carrier by chemical coupling is the dry solid phase carrier of 1.4 ~ 2.8mmol/g.
Ligand density affects the adsorption effect of blood purification material to rheumatoid factor: ligand density improves, and the rheumatoid factor binding capacity of unit volume blood purification material improves thereupon; But density height to a certain extent after, simultaneously also can bring more non-specific adsorption, especially to the combination of IgG.The present inventor finds after deliberation, and suitable ligand density is the dry solid phase carrier of 1.4 ~ 2.8mmol/g.Wherein, moisture free solid phase carrier after described dry solid phase carrier refers to drying process.
Solid phase carrier for blood purification material should have good hydrophily and itself should not produce absorption to protein, should have good blood compatibility simultaneously, can not cause the bad reactions such as clotting mechanism activation.As such solid support material, such as, can enumerate: polysaccharide natural polymer, as agar, agarose, cellulose, glucan, shitosan etc.; Synthesized polymer material, as the polyacrylamide, polyimides, polyvinyl alcohol etc. that are substituted or are unsubstituted; Inorganic material, as silica etc.Consider that polysaccharide natural polymer has good biocompatibility and the successful Application at blood purification Material Field thereof, the present invention selects cross-linked polysaccharides class material as carrier, more preferably agarose wherein.
Described solid phase carrier preferably adopts the form of porous microsphere.In addition, solid phase carrier needs enough permeabilities and metastable space structure, its glue hole through molecular weight preferably 150,000 to 5,000,000Da.Because the molecular weight of IgM antibody-like and immune complex thereof is often 900, more than 000Da in the molecules of interest that blood purification material will adsorb.
Method for the preparation of above-mentioned blood purification material of the present invention comprise the steps: first to introduce on solid phase carrier can with aglucon 1-amido-3-(2-(4-pyridine radicals)-ehtylmercapto) active group that reacts of amine groups on-2-propyl alcohol, then by chemical coupling reaction by 1-amido-3-(2-(4-pyridine radicals)-ehtylmercapto)-2-propyl alcohol is covalently bound to surface of solid phase carriers.
The immobilization of ligand molecule on solid phase carrier be rely on the amine groups on ligand molecule and solid phase carrier can reactive group covalently bound by chemical reaction.The group that can react with the amido on above-mentioned ligand molecule comprises benzimidazole carbamate base, sulfuryl chlorio, carboxyl, epoxy radicals, haloalkyl, succinimido, trifluoroethyl sulfonic acid mono methoxy etc.If solid phase carrier containing the active group that can react with amido, does not then need to adopt some activating reagents to make it produce amido reactive group.This kind of activating reagent often has double-functional group, such as carbonyl dimidazoles, epoxychloropropane, epoxy bromopropane, dichloro (bromine) propyl alcohol, dibromobutane, diglycidyl ether, divinylsulfone, cyanogen bromide etc.In addition, the standard process for fixation of any amine groups can also be used, those skilled in the art can see pertinent texts or handbook, such as Immobilized Affinity Ligand Techniques(Hermanson etc., Academic Press, 1992) and Bioconjugate Techniques(Greg T.Hermanson, Academic Press, Inc, 1996).For the solid phase carrier with hydroxyl, as cross-linked polysaccharides class carriers such as agarose, cellulose, glucans, the hydroxyl that can pass through the activated carrier surfaces such as carbonyl dimidazoles, epoxychloropropane, epoxy bromopropane, cyanogen bromide generates active group, recycles the group coupling ligand molecule that these can react with amido.Because cyanogen bromide is extremely toxic substance, comparatively large to human body and environmental hazard during activated carrier, therefore avoid using as far as possible.The present invention preferably uses carbonyl dimidazoles, epoxychloropropane, epoxy bromopropane as activating reagent, more preferably easily controls the carbonyl dimidazoles of support-activated degree as activating reagent.
When using carbonyl dimidazoles to prepare above-mentioned blood purification material as activating reagent, the active group that solid phase carrier is introduced is benzimidazole carbamate active group, specifically comprises the steps:
(1) activated solid support
With carbonyl dimidazoles activation, its addition is that 0.9 ~ 1.5g/10mL wets solid phase carrier; Reaction is carried out in acetone, 15 ~ 30 DEG C of reaction 1 ~ 2h, and product acetone washs; By the activation degree regulating carbonyl dimidazoles can control carrier, and then obtain the blood purification material with suitable ligand density (the dry solid phase carrier of 1.4 ~ 2.8mmol/g).Wherein, described wet solid phase carrier refers to the solid phase carrier removing non-binding state water through vacuum filtration, and wet solid phase carrier to be activated and the volume ratio of acetone are preferably 1:1 ~ 1:2.
(2) coupling aglucon
By 1-amido-3-(2-(4-pyridine radicals)-ehtylmercapto)-2-propyl alcohol is covalently bound to surface of solid phase carriers, reaction condition is: the 1-amido-3-(2-(4-pyridine radicals adding 5-10 times of volume in the suspension of the solid phase carrier activated containing equal-volume carbonyl dimidazoles and acetone)-ehtylmercapto)-2-propyl alcohol, 20-35 DEG C of reaction 2-3h, then cleans by acetone, 0.1M HCl solution, 0.1M NaOH solution, deionized water respectively several times repeatedly.
By this step, can by ligand molecule 1-amido-3-(2-(4-pyridine radicals)-ehtylmercapto) amido in-2-propyl alcohol is covalently bound on the active group that generated at carrier surface by carbonyl dimidazoles, thus realizes the chemical coupling of ligand molecule and carrier.
(3) post processing
Use the NaOH solution of pH10 ~ 13, to the coupling that step (2) obtains, the solid phase carrier of aglucon carries out post processing, the active group being derived from carbonyl dimidazoles that hydrolysis solid phase carrier does not react with aglucon.
The active group (i.e. benzimidazole carbamate active group) being derived from carbonyl dimidazoles do not reacted with aglucon also may be there is in the solid phase carrier that step (2) obtains.These active groups are the latencies producing non-specific adsorption, therefore it may be necessary above-mentioned post processing and it hydrolysis are removed.
Application of the present invention refers to that above-mentioned blood purification material is for the preparation of the application of removing in the apparatus for purifying blood of rheumatoid factor in rheumatoid disease human blood, specifically, this material adsorption stuffing of can be used as apparatus for purifying blood is for the removing of rheumatoid factor in rheumatoid disease human blood and other IgM type autoantibody and CIC ELISA.
The invention has the beneficial effects as follows that this blood purification materials'use micromolecular compound is as aglucon, has good stability relative to protein ligand, does not exist by the hidden danger of proteolytic degradation.Meanwhile, the ligand structure adopted is simple, synthesis is convenient, and its production cost is more much lower than protein.And blood purification material of the present invention can tolerate the harsh treatment conditions such as strong acid, highly basic, organic solvent, heating, absorption property also can not change by it affects.In addition, relative to the antibody sorbing material based on micromolecular compound aglucon that other adopt at present, blood purification material of the present invention can show higher IgM adsorptive selectivity, limited to the non-specific adsorption of other plasma component such as human serum albumins, IgG, the loss of IgG antibody-like at utmost can be reduced while removing rheumatoid factor.
Detailed description of the invention
The present invention is described in detail below in conjunction with embodiment; but the following examples are only the present invention's preferably embodiment; protection scope of the present invention is not limited thereto; anyly be familiar with those skilled in the art in the technical scope that the present invention discloses; be equal to according to technical scheme of the present invention and inventive concept thereof and replace or change, all should be encompassed within protection scope of the present invention.
One, ligand molecule 1-amido-3-(2-(4-pyridine radicals)-ehtylmercapto) synthesis of-2-propyl alcohol
Obtain through ammoniacal liquor open loop after there is substitution reaction by 4-mercaptoethyl pyridine (CAS:2127-05-1) and epoxychloropropane (CAS:106-89-8), concrete grammar is as follows:
Add 4-mercaptoethyl pyridine (5g) and the epoxychloropropane (3.33g) of equimolar amounts in 25ml dimethyl sulfoxide (DMSO), mix, then in reaction system, add 1ml triethylamine, 25 DEG C are reacted 6 hours.After question response terminates, under agitation reactant liquor is added dropwise in 100ml ammonia spirit (10%), continues reaction 12 hours.Reaction terminates latter standing 1 hour, collects organic phase, repeatedly clean with 0.1M sodium hydroxide solution after solution phase-splitting, adopts silicagel column separated product.Product purity is 95% by analysis, and the rate of recovery is 65%.
Two, the preparation of blood purification material
Comparative example 1: preparation 1-amido-3-(2-(4-pyridine radicals)-ehtylmercapto)-2-propyl alcohol coupling density is the Sepharose blood purification material (through carbonyl dimidazoles activation) of the dry glue of 0.75mmol/g
1) get abundant sedimentation (spending the night) volume is the cross-linked agarose gel SepharoseCL-6B(glue hole of 10mL is afterwards 10 through molecular weight, 000 ~ 4,000,000Da), wash to include water except removing photoresist several times with 200mL anhydrous propanone, then gel is dispersed in isopyknic acetone, and ensures that whole system is anhydrous.Take 0.5g carbonyl dimidazoles (CDI) and mix (CDI consumption be 0.5g/10mL wet glue) with above-mentioned gel suspension, gained mixture is stirred 1h in 15 DEG C of outstanding oars.After reaction terminates, gel 200mL anhydrous propanone cleans several times, obtains the cross-linked agarose gel with benzimidazole carbamate active group.
2) gel that 10mL CDI activates is dispersed in isopyknic acetone, in gel rubber system, press acetone suspension 5 times of volumes add 1-amido-3-(2-(4-pyridine radicals)-ehtylmercapto)-2-propyl alcohol, stirs 2h by gained mixture in 20 DEG C of outstanding oars.After reaction terminates, wash several times by 100mL acetone, 50mL0.1M HCl solution, 50mL0.1M NaOH solution, 100mL deionized water respectively, obtain the Ago-Gel of coupling aglucon.
3) cake that wet by the gel cleaned up joins in the NaOH solution of 50mL pH12, is placed in 30 DEG C of shaking tables with the rotating speed of 130rmp concussion reaction 10h with active group remaining on closed gel.Reactant mixture gets filter cake after suction filtration, and successively with 50mL deionized water, 50mL1M NaCl solution, the washing of 50mL deionized water, its ligand cou density of elementary analysis (nitrogen content) is the dry glue of 0.75mmol/g.
Comparative example 2: preparation 1-amido-3-(2-(4-pyridine radicals)-ehtylmercapto)-2-propyl alcohol coupling density is the Sepharose blood purification material (through carbonyl dimidazoles activation) of the dry glue of 1.12mmol/g
Concrete steps are with embodiment 1, and difference is: CDI consumption increases to 0.8g/10mL and to wet glue, and priming reaction carries out 1h at 30 DEG C; During coupling aglucon, 1-amido-3-(2-(4-pyridine radicals)-ehtylmercapto)-2-propyl alcohol consumption is 10 times of volumes, reacts and carries out 2h at 35 DEG C; The NaOH solution of pH13 is adopted during hydrolysed residual imidazoles activated group.Its ligand cou density of elementary analysis is the dry glue of 1.12mmol/g.
Embodiment 1: preparation 1-amido-3-(2-(4-pyridine radicals)-ehtylmercapto)-2-propyl alcohol coupling density is the Sepharose blood purification material (through carbonyl dimidazoles activation) of the dry glue of 1.4mmol/g
Concrete steps are with embodiment 1, and difference is: CDI consumption increases to 0.9g/10mL and to wet glue, and priming reaction carries out 2h at 20 DEG C; During coupling aglucon, react and carry out 2h at 25 DEG C; During hydrolysed residual imidazoles activated group, adopt the NaOH solution of pH10.Its ligand cou density of elementary analysis is the dry glue of 1.4mmol/g.
Embodiment 2: preparation 1-amido-3-(2-(4-pyridine radicals)-ehtylmercapto)-2-propyl alcohol coupling density is the Sepharose blood purification material (through carbonyl dimidazoles activation) of the dry glue of 2.03mmol/g
Concrete steps are with embodiment 1, and difference is: CDI consumption increases to 1.1g/10mL and to wet glue, and priming reaction carries out 1h at 25 DEG C; During coupling aglucon, 1-amido-3-(2-(4-pyridine radicals)-ehtylmercapto)-2-propyl alcohol consumption is 10 times of volumes, reacts and carries out 3h at 25 DEG C; During hydrolysed residual imidazoles activated group, adopt the NaOH solution of pH10.Its ligand cou density of elementary analysis is the dry glue of 2.03mmol/g.
Embodiment 3: preparation 1-amido-3-(2-(4-pyridine radicals)-ehtylmercapto)-2-propyl alcohol coupling density is the Sepharose blood purification material (through carbonyl dimidazoles activation) of the dry glue of 2.61mmol/g
Concrete steps are with embodiment 1, and difference is: CDI consumption increases to 1.2g/10mL and to wet glue, and priming reaction carries out 1h at 25 DEG C; During coupling aglucon, 1-amido-3-(2-(4-pyridine radicals)-ehtylmercapto)-2-propyl alcohol consumption is 10 times of volumes, reacts and carries out 3h at 25 DEG C; During hydrolysed residual imidazoles activated group, adopt the NaOH solution of pH13.Its ligand cou density of elementary analysis is the dry glue of 2.61mmol/g.
Embodiment 4: preparation 1-amido-3-(2-(4-pyridine radicals)-ehtylmercapto)-2-propyl alcohol coupling density is the Sepharose blood purification material (through carbonyl dimidazoles activation) of the dry glue of 2.82mmol/g
Concrete steps are with embodiment 1, and difference is: CDI consumption increases to 1.5g/10mL and to wet glue, and priming reaction carries out 1h at 25 DEG C; During coupling aglucon, 1-amido-3-(2-(4-pyridine radicals)-ehtylmercapto)-2-propyl alcohol consumption is 10 times of volumes, reacts and carries out 3h at 25 DEG C; During hydrolysed residual imidazoles activated group, adopt the NaOH solution of pH10.Its ligand cou density of elementary analysis is the dry glue of 2.82mmol/g.
Comparative example 3: preparation 1-amido-3-(2-(4-pyridine radicals)-ehtylmercapto)-2-propyl alcohol coupling density is the Sepharose blood purification material (through carbonyl dimidazoles activation) of the dry glue of 3.06mmol/g
Concrete steps are with embodiment 1, and difference is: CDI consumption increases to 1.6g/10mL and to wet glue, and priming reaction carries out 2h at 30 DEG C; During coupling aglucon, 1-amido-3-(2-(4-pyridine radicals)-ehtylmercapto)-2-propyl alcohol consumption is 10 times of volumes, reacts and carries out 3h at 25 DEG C; During hydrolysed residual imidazoles activated group, adopt the NaOH solution of pH10.Its ligand cou density of elementary analysis is the dry glue of 3.06mmol/g.
Comparative example 4: preparation 1-amido-3-(2-(4-pyridine radicals)-ehtylmercapto)-2-propyl alcohol coupling density is the Sepharose blood purification material (through carbonyl dimidazoles activation) of the dry glue of 3.15mmol/g
Concrete steps are with embodiment 1, and difference is: CDI consumption increases to 1.8g/10mL and to wet glue, and priming reaction carries out 1h at 30 DEG C; During coupling aglucon, react and carry out 2h at 30 DEG C; During hydrolysed residual imidazoles activated group, adopt the NaOH solution of pH10.Its ligand cou density of elementary analysis is the dry glue of 3.15mmol/g.
Embodiment 5: preparation 1-amido-3-(2-(4-pyridine radicals)-ehtylmercapto)-2-propyl alcohol coupling density is the cellulose blood purification material (through the coupling of sulfonic acid chloride group) of the dry glue of 2.45mmol/g
Take 1g1-amido-3-(2-(4-pyridine radicals)-ehtylmercapto)-2-propyl alcohol is dissolved in 10mL carbonate buffer solution (0.5M, pH10), adds 5mL acetone in solution.Taking 10g is scattered in the solution prepared with the cellulose microsphere of the sulfonic acid chloride group glue (sulfonic acid chloride groups density is greater than 80 μm of ol/mL and wets glue, and glue hole is 100,000 ~ 4,000,000Da through molecular weight) that wets, and stirs 6h in 30 DEG C of outstanding oars.After question response terminates, the gel obtained washs several times with 100mL acetone, 100mL deionized water, 100mL0.1M hydrochloric acid solution respectively.Coupling has 1-amido-3-(2-(4-pyridine radicals)-ehtylmercapto) cellulose microsphere of-2-propyl alcohol is dispersed in (pH10) in 60mL1M aqueous ethanolamine after draining, and stirs 3h in 30 DEG C of outstanding oars.After question response terminates, the cellulose microsphere gel 200mL deionized water of gained is washed several times.Its ligand cou density of elementary analysis is the dry glue of 2.45mmol/g.
Embodiment 6: preparation 1-amido-3-(2-(4-pyridine radicals)-ehtylmercapto)-2-propyl alcohol coupling density is the shitosan blood purification material (through carboxylic group coupling) of the dry glue of 1.67mmol/g
Take 1g1-amido-3-(2-(4-pyridine radicals)-ehtylmercapto)-2-propyl alcohol is dissolved in 30mL MES buffer solution (0.1M, pH4.6).Take 10g and wet with the chitosan microball of carboxylic group that (carboxylic group density is greater than 80 μm of ol/mL and wets glue glue, glue hole is 15 through molecular weight, 000 ~ 4,000,000Da) be scattered in the solution prepared, add 2.5g1-ethyl-(3-dimethylaminopropyl) carbonization two amido hydrochloride (EDCHCl), adjust pH to 4.6, stir 6h in 30 DEG C of outstanding oars.After question response terminates, the gel obtained washs several times by 100mL deionized water, 100mL0.1M hydrochloric acid, 200mL deionized water respectively.Its ligand cou density of elementary analysis is the dry glue of 1.67mmol/g.
Embodiment 7: preparation 1-amido-3-(2-(4-pyridine radicals)-ehtylmercapto)-2-propyl alcohol coupling density is the glucan blood purification material (through Epichlorohydrin activation) of the dry glue of 1.92mmol/g
1) (glue hole is 15 through molecular weight to take 10g sephadex, 000 ~ 4,000,000Da) be dispersed in 30mL deionized water and obtain gel suspension, after adding 5mL epoxychloropropane, 10mL1M NaOH solution, 0.1g sodium borohydride successively in above-mentioned gel suspension, stir 16h at 30 DEG C of outstanding oars.After reaction terminates, obtained gel 200mL deionized water is washed several times, obtains the sephadex with epoxide group.
2) get 10g to be dispersed in the mixed solution (volume ratio of ethanol and carbonate buffer solution is 65/35) that 50mL is made up of carbonate buffer solution (0.05M, pH9) and ethanol through the sephadex that epoxy activates.Get 1g1-amido-3-(2-(4-pyridine radicals)-ehtylmercapto)-2-propyl alcohol is dissolved in 1mL deionized water, joins in mixed solution after adjusting pH to 9, stirs 16h at 45 DEG C of outstanding oars.Reaction terminates rear gel ethanolic solution and fully washs.
3) by gel dispersion in 50mL1M ethanolamine solutions (pH10.5), stir 2.5h in 45 DEG C of outstanding oars.After reaction terminates, gel 200mL deionized water is washed several times.Its ligand cou density of elementary analysis is the dry glue of 1.92mmol/g.
Comparative example 5: preparation 4-mercaptoethyl pyridine (4-Mercaptoethylpyridine, 4-MEP) coupling density is the agarose blood purification material (through bromopropene activation) of the dry glue of 1.97mmol/g
1) 10g Sepharose CL-6B Ago-Gel (with thiazolinyl active group) is taken, after washing several times by 200mL deionized water, be dispersed in 5mL3M NaOH solution and form gel suspension, get 2mL bromopropene to mix with prepared gel suspension, gained mixture is stirred 18 hours in 30 DEG C of outstanding oars.After reaction terminates, gel is washed several times by 100mL anhydrous propanone, 100mL deionized water respectively, obtain through the Ago-Gel of bromopropene activation with thiazolinyl active group.
2) 1g4-MEPHCl(hydrochloride form is got), be dissolved in the deionized water of 1mL, then by 10M NaOH solution, the pH value of institute's obtain solution be adjusted to 7, then added 6mL phosphate buffer (0.1M, pH7) and 3mL acetonitrile.Ago-Gel after activation is added in the solution of this preparation and forms gel suspension, stir 12 hours in 30 DEG C of outstanding oars.After reaction terminates, gel is washed several times with 100mL acetonitrile, 100mL deionized water, 100mL0.1M hydrochloric acid respectively, obtain the Ago-Gel that coupling has 4-MEP, this Ago-Gel is dispersed in the 60mL1M mercaptoethanol aqueous solution (pH10) after draining, and 30 DEG C of outstanding oars stir 3 hours.After reaction terminates, gel 200mL deionized water is washed several times.Its ligand cou density of elementary analysis is the dry glue of 1.97mmol/g.
Three, blood purification material is to the evaluating absorbing of rheumatoid factor in human serum and associated antibodies component
Collect the human serum sample of rheumatoid factor positive, for the performance evaluation of blood purification material after mixing.After testing, in pooled serum sample, IgG concentration is 12.4mg/mL, IgM concentration is 2.1mg/mL, human serum albumins (HAS) concentration is 42.6mg/mL, rheumatoid factor (RF) content is 948IU/mL.
The respectively blood purification material of Example 1-11 synthesis, with normal saline flushing 3 times, takes 0.2g blood purification material and joins in the sample bottle filling 1mL serum (material and serum volume ratio are 1:5), 37 DEG C of incubation 2h.Detect IgG, IgM, HSA and RF concentration in absorption serum sample, calculate clearance.Concrete outcome is as shown in table 1.
Table 1
Evaluation result shows, with 1-amido-3-(2-(4-pyridine radicals)-ehtylmercapto)-2-propyl alcohol is that the blood purification material of aglucon has IgM type antibody in human serum and IgG type antibody and distinguishes effect significantly; Stronger Adsorption ability is shown to RF; By contrast, less to the non-specific adsorption of HSA.Be it can also be seen that by the result of table 1, the adsorption effect of blood purification material is larger by ligand cou Effects of Density, along with the increase of ligand density, the clearance of IgG and IgM is increased all thereupon, but along with ligand density further increases (such as more than the dry glue of 3.06mmol/g), the absorption of IgG is significantly increased, and downward trend is existed on the contrary to the clearance of IgM and RF, illustrate ligand density acquire a certain degree after blood purification material to molecular weight, but there is stronger effect in the IgG that serum-concentration is larger, more binding site can be occupied by it, thus the absorption having influence on IgM type antibody and RF combines.Consider the removal effect of blood purification material to RF, choosing the dry glue of 1.4-2.8mmol/g is comparatively ideal aglucon coupling density.Simultaneously, from evaluation result, except cross-linked agarose gel, other polysaccharide carrier material (embodiment 9-11) coupling 1-amido-3-(2-(4-pyridine radicals)-ehtylmercapto) after-2-propyl alcohol, if ligand density, in above selected scope, equally also can obtain and meet expection, comparatively ideal RF removal effect.
In addition, compared with blood purification material of the present invention, although also reach 56.7% to the clearance of RF with the adsorbent that 4-mercaptoethyl pyridine is aglucon under same experimental conditions, this adsorbent is also considerable to the Adsorption of IgG simultaneously, reaches 84.4%.Although show that 4-mercaptoethyl pyridine aglucon has excellent IgG binding ability, be applied to the loss that can cause significant IgG type antibody when RF removes, thus limit it and removing the application in rheumatoid factor.
Claims (4)
1. for removing a blood purification material for rheumatoid factor, it is characterized in that, this blood purification material forms by solid phase carrier with by aglucon two parts that chemical coupling is fixed on solid phase carrier; Solid phase carrier is cross-linked polysaccharides, and aglucon is 1-amido-3-(2-(4-pyridine radicals)-ehtylmercapto)-2-propyl alcohol, be fixed on solid phase carrier by chemical coupling, ligand density is the dry solid phase carrier of 1.4 ~ 2.8mmol/g; Moisture free solid phase carrier after described dry solid phase carrier refers to drying process.
2. blood purification material according to claim 1, is characterized in that, described solid phase carrier adopts the form of porous microsphere, and its glue hole is 150000 ~ 5000000Da through molecular weight.
3. a preparation method for the blood purification material described in claim 1 or 2, is characterized in that:
(1) activated solid support: with carbonyl dimidazoles activation, its addition is that 0.9 ~ 1.5g/10mL wets solid phase carrier, and reaction is carried out in acetone, 15 ~ 30 DEG C of reaction 1 ~ 2h, product acetone washs; Described wet solid phase carrier refers to the solid phase carrier removing non-binding state water through vacuum filtration;
(2) coupling aglucon: the 1-amido-3-(2-(4-pyridine radicals adding 5-10 times of suspension volume in the suspension of the solid phase carrier activated containing equal-volume carbonyl dimidazoles and acetone)-ehtylmercapto)-2-propyl alcohol, 20-35 DEG C of reaction 2-3h, then uses acetone, 0.1M HCl solution, 0.1M NaOH solution, washed with de-ionized water respectively;
(3) post processing: use the NaOH solution of pH10 ~ 13, to the coupling that step (2) obtains, the solid phase carrier of aglucon processes, the active group being derived from carbonyl dimidazoles that hydrolysis solid phase carrier does not react with aglucon.
4. the application of the blood purification material described in a claim 1 or 2, it is characterized in that, described blood purification material is used for the removing of rheumatoid factor and other IgM type autoantibody and CIC ELISA in rheumatoid disease human blood as the adsorption stuffing of apparatus for purifying blood.
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