CN103933947A - Blood purification material for removing rheumatoid factors, and preparation method thereof - Google Patents
Blood purification material for removing rheumatoid factors, and preparation method thereof Download PDFInfo
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Abstract
The invention provides a blood purification material for removing rheumatoid factors, and a preparation method of the blood purification material, belonging to the technical field of biomedicine. The blood purification material comprises a solid phase carrier and a ligand fixed on the solid phase carrier by chemical coupling, wherein the solid phase carrier is a polysaccharide natural polymer material; the ligand is 1-amino-3-(2-(4-pyridyl)-ethyl sulfydryl)-2-propanol; the ligand fixed on the solid phase carrier by chemical coupling has the density of 1.4-2.8mmol/g dry solid phase carrier. The blood purification material is capable of selectively absorbing the rheumatoid factors in the blood, and is limited in nonspecific adsorption for blood plasma components such as human serum albumin, immunoglobulin G (Ig G) and the like; the blood purification material is low in preparation cost and stable in physicochemical property, and can be used as an absorption filler of a blood purification device for removing the rheumatoid factors in the blood of rheumatoid patients.
Description
Technical field
The present invention relates to a kind ofly for removing blood purification material of 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, be proved with the generation of a series of rheumatoid diseases and develop closely related, as rheumatoid arthritis, systemic loupus erythematosus etc.For these diseases, reduce the concentration of rheumatoid factor in patient blood by the mode of external removing and can obviously improve symptom, disease is played and alleviated and therapeutic action (B.C.McLeod, Introduction to the Third Special Issue:Clinical Applications of Therapeutic Apheresis.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, obtained development and application the eighties, it develops into a kind of important clinical treatment means gradually.Patient's blood is by outside lead body, after purifier in defeated ex vivo, in this process, purifier is for removing metabolic waste, morbid substance, the unnecessary moisture content etc. of blood, unbalance to correct with regulation machine vivo environment, thereby reaches the object for the treatment of disease.
The adsorbent that is applied to blood purification is generally made up of two parts: as the polymer carrier of adsorbent matrix with utilize chemical crosslinking to be fixed on the aglucon 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 at present the macromolecular material of number of different types to can be used as the carrier matrix of blood-purifying adsorbing agent, 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.Aglucon molecule is by covalent bonds on carrier material, and its structure and character have determined the action effect of adsorbent.The adsorbent of removing for antibody class Molecular Adsorption at present mainly adopts two kinds of function aglucons: bion and micromolecular compound type.
Albumin 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 IgG Fc district is had to stronger binding ability.Albumin A is to the mainly ability based on its wide spectrum binding domain-immunoglobulin of the combination of rheumatoid factor.At present, albumin A has been used to the removing of autoantibody and immune complex in clinical middle autoimmune disease patient body as a kind of typical affinity ligand.Prosorba and Immunosorba(Fresenius, Germany) be two kinds of albumin A adsorbents that are 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 come out (CN1367181A).Adopt the adsorbent of albumin A as aglucon for this class, its advantage is to utilize affinity interaction natural between biomolecule, antagonist quasi-molecule has very high selective recognition capability, can realize the scavenging action to a large class autoantibody, therefore can be used for the treatment of the disease that a series of autoantibodies are relevant broad spectrum activity.But its shortcoming is albumin A and will be significantly smaller than the binding ability to IgG type antibody to the binding ability of IgM type antibody.Albumin A Main Function is in the Fc of IgG fragment, but the Fc fragment of IgM concentrates on molecule centre, and degree of exposure is lower, sterically hindered larger in conjunction with albumin A.Therefore, albumin A will, lower than the removal effect to other IgG type autoantibodies, want to obtain comparatively ideal result for the treatment of to 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 from staphylococcus aureus or genetic engineering bacterium, obtain, its production cost is very high.The market price of Prosorba adsorption column is about 1000 Euros at present, and the Immunosorba adsorption column market price of a pair of changeable use is about 10000 Euros.Meanwhile, protein aglucon is unstable in immobilization and preservation process, easily inactivation.Although can regenerate, reuse, also exist and be difficult to the problem of on-line cleaning sterilizing and the hidden danger that aglucon molecule comes off.Above these drawbacks limit the clinical use of albumin A adsorbent in rheumatoid disease treatment.
Except albumin A, also there is report to adopt the affine function base of hot polymerization IgG as 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 preparing by the mode of artificial thermal denaturation is immobilized behind host material 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 screening and designs the little molecular ligand that can be applied to blood purification.
Hydrophobic amino acid is to report at present and apply maximum little molecular radicals for antibody absorption.Japan Asahi medical company has been developed two kinds of sorbent materials taking tryptophan and phenylalanine as aglucon respectively, 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 is 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 do not have considerable non-specific adsorption to albumin in blood plasma and fibrinogen, but the autoantibodies such as Anti-DNA antibody, anti-AchR antibody and anti-C1q antibody are had to 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 sorbent material of histidine as adsorption group that utilize is provided in Chinese patent literature CN1666784A.In addition, also there is the report (Chinese patent literature CN1476908A) that adopts polyaminoacid to be used in this field as adsorption group.Aspect the application study of adsorbent, also have and studies confirm that on a small quantity the adsorbent of this class based on hydrophobic amino acid also exists Adsorption effect to a certain degree to rheumatoid factor.For example, someone has reported that employing phenylalanine is coupled to polyvinylalcohol microsphere bulb matrix as aglucon, the Adsorption (king is superfine, Chinese biological engineering in medicine journal, 2009,28(4) for rheumatoid factor: 561-566).
In addition, inventor place team has also developed the Adsorption of two kinds of little molecular ligands for autoantibody in autoimmune disease patient 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 existing little molecular ligand adsorbent all shows binding ability to a certain degree under laboratory evaluation condition at present, in the experimental evaluation of further simulating clinical treatment, effect is often unsatisfactory.Main cause is, be all for the molecular characterization exploitation of IgG type antibody and the wide spectrum antibody adsorbent of design taking aromatic amino acid and 4-mercaptoethyl pyridine as the adsorbent based on little molecular ligand of representative, and IgM type antibody have significant difference with IgG type antibody in molecular scale and design feature: 1. the molecular weight of IgM is six times of IgG; Although 2. both have the conserved sequence that part is identical, on molecular characterization, still there is larger difference.Therefore also improper to the rheumatoid factor taking IgM as master for the adsorbent of IgG design.Taking 4-mercaptoethyl pyridine as example, 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, will in the face of with the similar problem of albumin A, be rheumatoid factor adsorption capacity a little less than, if treat intensity and improve the clearance of rheumatoid factor by increasing, will cause the loss of a large amount of normal IgG, the risk (J.Ren, the et al. that bring immunologic function to weaken, 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, wherein hydrophobic effect and π-pi-conjugated important function of having brought into play.Be wrapped in intramolecular IgM for Fc fragment, the ability to function of 4-mercaptoethyl pyridine is difficult to effectively embody.Therefore to improve the adsorptive selectivity to IgM antibody-like, realize the selective absorption of rheumatoid factor is removed with regard to needing and carried out brand-new design and the structure optimization of aglucon molecule for the molecular characterization of IgM specially, make 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 removing blood purification material of rheumatoid factor and preparation method thereof.Rheumatoid factor in this blood purification material reply human blood has selective and higher adsorption capacity preferably, and has stable physicochemical properties and relative low production cost.
The present inventor has carried out aglucon MOLECULE DESIGN for the architectural feature of IgM, in conjunction with experiment screening, finally determine a brand-new long-chain polyfunctional compound by computer molecular docking: 1-amido-3-(2-(4-pyridine radicals)-ethyl sulfydryl)-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)-ethyl sulfydryl)-2-propyl alcohol molecule compared with the aglucon using in prior art (benzoic acid, aromatic amino acid, 4-mercaptoethyl pyridine), on the basis of hydrophobic heterocyclic group, introduced multiple electron rich groups (hydroxyl, amido).Molecular docking result shows, 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 fragments are exposed to molecular surface, and the combination of strengthening aglucon molecule and Fab will significantly 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 can be lower to IgM and the needed ligand density of the effective absorption of rheumatoid factor.By reasonable control coupling group density, just can realize the differentiation of adsorbent to IgM and IgG.Based on the factor of above two aspects, on the basis of preferred aglucon, by further optimizing the immobilized reaction condition of aglucon, determine a sorbing material synthetic schemes that is applicable to rheumatoid factor in selective clearing human blood.
Above-mentioned aglucon molecule 1-amido-3-(2-(4-pyridine radicals)-ethyl sulfydryl) structural formula of-2-propyl alcohol is as follows:
Particularly, of the present inventionly form for removing aglucon two parts that the blood purification material of rheumatoid factor is fixed on solid phase carrier by solid phase carrier with by chemical coupling, solid phase carrier is cross-linked polysaccharides, described aglucon is 1-amido-3-(2-(4-pyridine radicals)-ethyl sulfydryl)-2-propyl alcohol, the ligand density being 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, also can bring more non-specific adsorption, the especially combination to IgG simultaneously.The present inventor finds after deliberation, and suitable ligand density is the dry solid phase carrier of 1.4～2.8mmol/g.Wherein, described dry solid phase carrier refers to the rear moisture free solid phase carrier of drying processing.
Should there is good hydrophily and itself should not produce absorption to protein for the solid phase carrier of blood purification material, should there is good blood compatibility simultaneously, can not cause the bad reactions such as clotting mechanism activation.As such solid support material, for example, can enumerate: polysaccharide natural polymer, as agar, agarose, cellulose, glucan, shitosan etc.; Synthesized polymer material, as the polyacrylamide that is substituted or is unsubstituted, polyimides, polyvinyl alcohol etc.; 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 need to have enough permeabilities and metastable space structure, and the molecular weight that sees through in its glue hole is preferably in 150,000 to 5,000,000Da.Because the molecular weight of IgM antibody-like and immune complex thereof is often more than 900,000Da 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)-ethyl sulfydryl) active group that amine groups on-2-propyl alcohol reacts, then react the pyridine radicals by 1-amido-3-(2-(4-by chemical coupling)-ethyl sulfydryl)-2-propyl alcohol is covalently bound to surface of solid phase carriers.
The immobilization of aglucon molecule on solid phase carrier is to rely on can reactive group covalently bound by chemical reaction on amine groups on aglucon molecule and solid phase carrier.Can with above-mentioned aglucon molecule on the group that reacts of amido comprise imidazoles carbamate groups, sulfuryl chlorio, carboxyl, epoxy radicals, haloalkyl, succinimido, trifluoroethyl sulfonic acid mono methoxy etc.If solid phase carrier not containing the active group that can react with amido, needs to adopt some activating reagents to make it produce amido reactive group.This class 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, can also use the standard process for fixation of any amine groups, those skilled in the art can be referring to relevant books 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 aglucon molecule that these can react with amido.Because cyanogen bromide is extremely toxic substance, larger to human body and environmental hazard when 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 carrier activation degree as activating reagent.
In the time using carbonyl dimidazoles to prepare above-mentioned blood purification material as activating reagent, the active group of introducing on solid phase carrier is imidazoles carbamate active group, specifically comprises the steps:
(1) activation solid phase carrier
With carbonyl dimidazoles activation, its addition is the wet solid phase carrier of 0.9～1.5g/10mL; Reaction is carried out in acetone, 15～30 DEG C of reaction 1～2h, and product washs with acetone; By regulating carbonyl dimidazoles can control the activation degree of carrier, and then obtain the thering is suitable ligand density blood purification material of (the dry solid phase carrier of 1.4～2.8mmol/g).Wherein, described wet solid phase carrier refers to the solid phase carrier of 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)-ethyl sulfydryl)-2-propyl alcohol is covalently bound to surface of solid phase carriers, reaction condition is: to the 1-amido-3-(2-(4-pyridine radicals that adds 5-10 times of volume in the suspension of the solid phase carrier that contains equal-volume carbonyl dimidazoles activation and acetone)-ethyl sulfydryl)-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 be by aglucon molecule 1-amido-3-(2-(4-pyridine radicals)-ethyl sulfydryl) amido in-2-propyl alcohol is covalently bound on the active group being generated at carrier surface by carbonyl dimidazoles, thus realize the chemical coupling of aglucon molecule and carrier.
(3) post processing
Use the NaOH solution of pH10～13, the coupling that step (2) is obtained the solid phase carrier of aglucon carry out post processing, the active group that is derived from carbonyl dimidazoles not reacting with aglucon on hydrolysis solid phase carrier.
In the solid phase carrier that step (2) obtains, also may there is the active group that is derived from carbonyl dimidazoles (being imidazoles carbamate active group) not reacting with aglucon.These active groups are the latencies that produce non-specific adsorption, are therefore necessary by above-mentioned post processing, it hydrolysis to be removed.
Application of the present invention refers to the application of above-mentioned blood purification material in the apparatus for purifying blood for the preparation of rheumatoid factor in removing rheumatoid disease human blood, particularly, this material can be used as the adsorption stuffing of apparatus for purifying blood for the removing of rheumatoid disease human blood rheumatoid factor and other IgM type autoantibody and CIC ELISA.
The invention has the beneficial effects as follows that this blood purification materials'use micromolecular compound, as aglucon, has good stability with respect to protein aglucon, do not have the hidden danger of being degraded by proteolytic enzyme.Meanwhile, the ligand structure adopting is simple, synthetic 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 can not be subject to it affect and change yet.In addition, the antibody sorbing material based on micromolecular compound aglucon adopting at present with respect to other, blood purification material of the present invention can show higher IgM adsorptive selectivity, non-specific adsorption to other plasma component such as human serum albumins, IgG is limited, can at utmost reduce the loss of IgG antibody-like in removing rheumatoid factor.
Detailed description of the invention
Describe the present invention in detail below in conjunction with embodiment; but the following examples are only preferably embodiment of the present invention; protection scope of the present invention is not limited to this; any be familiar with those skilled in the art the present invention disclose technical scope in; be equal to replacement or changed according to technical scheme of the present invention and inventive concept thereof, within all should being encompassed in protection scope of the present invention.
One, aglucon molecule 1-amido-3-(2-(4-pyridine radicals)-ethyl sulfydryl)-2-propyl alcohol synthetic
Occurred to make through ammoniacal liquor open loop after substitution reaction by 4-mercaptoethyl pyridine (CAS:2127-05-1) and epoxychloropropane (CAS:106-89-8), concrete grammar is as follows:
The 4-mercaptoethyl pyridine (5g) and the epoxychloropropane (3.33g) that in 25ml dimethyl sulfoxide (DMSO), add equimolar amounts, mix, and then in reaction system, adds 1ml triethylamine, and 25 DEG C are reacted 6 hours.After question response finishes, under stirring condition, reactant liquor is added dropwise in 100ml ammonia spirit (10%), continues reaction 12 hours.Reaction finishes to leave standstill 1 hour afterwards, after solution phase-splitting, collects organic phase, repeatedly cleans employing silicagel column separated product with 0.1M sodium hydroxide solution.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)-ethyl sulfydryl)-2-propyl alcohol coupling density is the Sepharose blood purification material (through carbonyl dimidazoles activation) of the dry glue of 0.75mmol/g
1) getting the cross-linked agarose gel Sepharose CL-6B(glue hole that the rear volume of abundant sedimentation (spending the night) is 10mL is 10 through molecular weight, 000～4,000,000Da), wash several times to remove to remove photoresist and include water 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 is the wet glue of 0.5g/10mL) with above-mentioned gel suspension, gained mixture is stirred to 1h in 15 DEG C of outstanding oars.After reaction finishes, gel cleans several times with 200mL anhydrous propanone, obtains the cross-linked agarose gel with imidazoles carbamate active group.
2) gel of 10mL CDI activation is dispersed in isopyknic acetone, in gel rubber system, press 5 times of volumes interpolation 1-amido-3-(2-(4-pyridine radicals of acetone suspension)-ethyl sulfydryl)-2-propyl alcohol, stirs 2h by gained mixture in 20 DEG C of outstanding oars.After reaction finishes, 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) wet the gel cleaning up cake is joined in the NaOH solution of 50mL pH12, be placed in 30 DEG C of shaking tables and react 10h with remaining active group on sealing gel with the rotating speed concussion of 130rmp.Reactant mixture is got filter cake after suction filtration, and with 50mL deionized water, 50mL1M NaCl solution, the washing of 50mL deionized water, its aglucon coupling density of elementary analysis (nitrogen content) is the dry glue of 0.75mmol/g successively.
Comparative example 2: preparation 1-amido-3-(2-(4-pyridine radicals)-ethyl sulfydryl)-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 the wet glue of 0.8g/10mL, and priming reaction carries out 1h at 30 DEG C; When coupling aglucon, 1-amido-3-(2-(4-pyridine radicals)-ethyl sulfydryl)-2-propyl alcohol consumption is 10 times of volumes, reaction is carried out 2h at 35 DEG C; When hydrolysed residual imidazoles activated group, adopt the NaOH solution of pH13.Its aglucon coupling density of elementary analysis is the dry glue of 1.12mmol/g.
Embodiment 1: preparation 1-amido-3-(2-(4-pyridine radicals)-ethyl sulfydryl)-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 the wet glue of 0.9g/10mL, and priming reaction carries out 2h at 20 DEG C; When coupling aglucon, reaction is carried out 2h at 25 DEG C; When hydrolysed residual imidazoles activated group, adopt the NaOH solution of pH10.Its aglucon coupling density of elementary analysis is the dry glue of 1.4mmol/g.
Embodiment 2: preparation 1-amido-3-(2-(4-pyridine radicals)-ethyl sulfydryl)-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 the wet glue of 1.1g/10mL, and priming reaction carries out 1h at 25 DEG C; When coupling aglucon, 1-amido-3-(2-(4-pyridine radicals)-ethyl sulfydryl)-2-propyl alcohol consumption is 10 times of volumes, reaction is carried out 3h at 25 DEG C; When hydrolysed residual imidazoles activated group, adopt the NaOH solution of pH10.Its aglucon coupling density of elementary analysis is the dry glue of 2.03mmol/g.
Embodiment 3: preparation 1-amido-3-(2-(4-pyridine radicals)-ethyl sulfydryl)-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 the wet glue of 1.2g/10mL, and priming reaction carries out 1h at 25 DEG C; When coupling aglucon, 1-amido-3-(2-(4-pyridine radicals)-ethyl sulfydryl)-2-propyl alcohol consumption is 10 times of volumes, reaction is carried out 3h at 25 DEG C; When hydrolysed residual imidazoles activated group, adopt the NaOH solution of pH13.Its aglucon coupling density of elementary analysis is the dry glue of 2.61mmol/g.
Embodiment 4: preparation 1-amido-3-(2-(4-pyridine radicals)-ethyl sulfydryl)-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 the wet glue of 1.5g/10mL, and priming reaction carries out 1h at 25 DEG C; When coupling aglucon, 1-amido-3-(2-(4-pyridine radicals)-ethyl sulfydryl)-2-propyl alcohol consumption is 10 times of volumes, reaction is carried out 3h at 25 DEG C; When hydrolysed residual imidazoles activated group, adopt the NaOH solution of pH10.Its aglucon coupling density of elementary analysis is the dry glue of 2.82mmol/g.
Comparative example 3: preparation 1-amido-3-(2-(4-pyridine radicals)-ethyl sulfydryl)-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 the wet glue of 1.6g/10mL, and priming reaction carries out 2h at 30 DEG C; When coupling aglucon, 1-amido-3-(2-(4-pyridine radicals)-ethyl sulfydryl)-2-propyl alcohol consumption is 10 times of volumes, reaction is carried out 3h at 25 DEG C; When hydrolysed residual imidazoles activated group, adopt the NaOH solution of pH10.Its aglucon coupling density of elementary analysis is the dry glue of 3.06mmol/g.
Comparative example 4: preparation 1-amido-3-(2-(4-pyridine radicals)-ethyl sulfydryl)-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 the wet glue of 1.8g/10mL, and priming reaction carries out 1h at 30 DEG C; When coupling aglucon, reaction is carried out 2h at 30 DEG C; When hydrolysed residual imidazoles activated group, adopt the NaOH solution of pH10.Its aglucon coupling density of elementary analysis is the dry glue of 3.15mmol/g.
Embodiment 5: preparation 1-amido-3-(2-(4-pyridine radicals)-ethyl sulfydryl)-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)-ethyl sulfydryl)-2-propyl alcohol is dissolved in 10mL carbonate buffer solution (0.5M, pH10), adds 5mL acetone in solution.Take 10g and be scattered in the solution preparing with the wet glue of cellulose microsphere (sulfonic acid chloride group density is greater than the wet glue of 80 μ mol/mL, and it is 100,000～4 that glue hole sees through molecular weight, 000,000Da) of sulfonic acid chloride group, stir 6h in 30 DEG C of outstanding oars.After question response finishes, the gel obtaining washs several times with 100mL acetone, 100mL deionized water, 100mL0.1M hydrochloric acid solution respectively.Coupling has 1-amido-3-(2-(4-pyridine radicals)-ethyl sulfydryl) cellulose microsphere of-2-propyl alcohol is dispersed in the 60mL1M monoethanolamine aqueous solution (pH10) after draining, and stirs 3h in 30 DEG C of outstanding oars.After question response finishes, the cellulose microsphere gel of gained is washed several times by 200mL deionized water.Its aglucon coupling density of elementary analysis is the dry glue of 2.45mmol/g.
Embodiment 6: preparation 1-amido-3-(2-(4-pyridine radicals)-ethyl sulfydryl)-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)-ethyl sulfydryl)-2-propyl alcohol is dissolved in 30mL MES buffer solution (0.1M, pH4.6).(carboxylic group density is greater than the wet glue of 80 μ mol/mL with the wet glue of the chitosan microball of carboxylic group to take 10g, it is 15 that glue hole sees through molecular weight, 000～4,000,000Da) be scattered in the solution preparing, add 2.5g1-ethyl-(3-dimethylaminopropyl) carbonization two amido hydrochlorides (EDCHCl), adjust pH to 4.6, stir 6h in 30 DEG C of outstanding oars.After question response finishes, the gel obtaining washs several times by 100mL deionized water, 100mL0.1M hydrochloric acid, 200mL deionized water respectively.Its aglucon coupling density of elementary analysis is the dry glue of 1.67mmol/g.
Embodiment 7: preparation 1-amido-3-(2-(4-pyridine radicals)-ethyl sulfydryl)-2-propyl alcohol coupling density is the glucan blood purification material (through Epichlorohydrin activation) of the dry glue of 1.92mmol/g
1) (it is 15 that glue hole sees through molecular weight to take 10g sephadex, 000～4,000,000Da) be dispersed in and in 30mL deionized water, obtain gel suspension, in above-mentioned gel suspension, add after 5mL epoxychloropropane, 10mL1M NaOH solution, 0.1g sodium borohydride successively, stir 16h at 30 DEG C of outstanding oars.After reaction finishes, obtained gel is washed several times by 200mL deionized water, obtain the sephadex with epoxide group.
2) getting 10g is 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 of epoxy activation.Get 1g1-amido-3-(2-(4-pyridine radicals)-ethyl sulfydryl)-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 finishes rear gel ethanolic solution and fully washs.
3) gel is dispersed in 50mL1M ethanolamine solutions (pH10.5), stirs 2.5h in 45 DEG C of outstanding oars.After reaction finishes, gel washs several times by 200mL deionized water.Its aglucon coupling 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) take 10g Sepharose CL-6B Ago-Gel (with thiazolinyl active group), after washing several times by 200mL deionized water, be dispersed in 5mL3M NaOH solution and form gel suspension, get 2mL bromopropene and mix with prepared gel suspension, gained mixture is stirred 18 hours in 30 DEG C of outstanding oars.After reaction finishes, gel is washed several times by 100mL anhydrous propanone, 100mL deionized water respectively, obtain the Ago-Gel with thiazolinyl active group through bromopropene activation.
2) get 1g4-MEPHCl(hydrochloride form), be dissolved in the deionized water of 1mL, then the pH value of institute's obtain solution be adjusted to 7 with 10M NaOH solution, then add 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 finishes, gel is washed several times with 100mL acetonitrile, 100mL deionized water, 100mL0.1M hydrochloric acid respectively, obtaining coupling has the Ago-Gel of 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 finishes, gel washs several times by 200mL deionized water.Its aglucon coupling density of elementary analysis is the dry glue of 1.97mmol/g.
Three, the evaluating absorbing of blood purification material to rheumatoid factor in human serum and associated antibodies component
Collect the human serum sample of the rheumatoid factor positive, after mixing for the performance evaluation of blood purification material.After testing, in pooled serum sample, IgG concentration is that 12.4mg/mL, IgM concentration are that 2.1mg/mL, human serum albumins (HAS) concentration are that 42.6mg/mL, rheumatoid factor (RF) content are 948IU/mL.
Get respectively the synthetic blood purification material of embodiment 1-11, use normal saline flushing 3 times, take 0.2g blood purification material and join in the sample bottle that fills 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, taking 1-amido-3-(2-(4-pyridine radicals)-ethyl sulfydryl)-2-propyl alcohol has significant differentiation effect to IgM type antibody in human serum and IgG type antibody as the blood purification material of aglucon; RF is shown to stronger Adsorption ability; By contrast, less to the non-specific adsorption of HSA.Result by table 1 it can also be seen that, the adsorption effect of blood purification material is subject to aglucon coupling Effects of Density larger, along with the increase of ligand density, the clearance of IgG and IgM is all increased thereupon, but for example, along with ligand density further increases (exceeding the dry glue of 3.06mmol/g), absorption to IgG significantly increases, and be there is on the contrary to downward trend in 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, thereby have influence on the absorption combination of IgM type antibody and RF.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)-ethyl sulfydryl) after-2-propyl alcohol, if, in above selected scope, equally also obtaining, ligand density meets expection, comparatively ideal RF removal effect.
In addition, compared with blood purification material of the present invention, although the adsorbent taking 4-mercaptoethyl pyridine as aglucon has also reached 56.7% to the clearance of RF under same experimental conditions, this adsorbent is also considerable to the Adsorption of IgG simultaneously, has reached 84.4%.Although show that 4-mercaptoethyl pyridine aglucon has excellent IgG binding ability, be applied to when RF removes and can cause the loss of significant IgG type antibody, thereby limited its application aspect removal rheumatoid factor.
Claims (4)
1. for removing a blood purification material for rheumatoid factor, it is characterized in that, aglucon two parts that this blood purification material is fixed on solid phase carrier by solid phase carrier with by chemical coupling form; Solid phase carrier is cross-linked polysaccharides, and aglucon is 1-amido-3-(2-(4-pyridine radicals)-ethyl sulfydryl)-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.
2. blood purification material according to claim 1, is characterized in that, described solid phase carrier adopts the form of porous microsphere, and it is 150000～5000000Da that its glue hole sees through molecular weight.
3. a preparation method for the blood purification material described in claim 1 or 2, is characterized in that:
(1) activation solid phase carrier: with carbonyl dimidazoles activation, its addition is the wet solid phase carrier of 0.9～1.5g/10mL, and reaction is carried out in acetone, 15～30 DEG C of reaction 1～2h, product washs with acetone;
(2) coupling aglucon: to the 1-amido-3-(2-(4-pyridine radicals that adds 5-10 times of volume in the suspension of the solid phase carrier that contains equal-volume carbonyl dimidazoles activation and acetone)-ethyl sulfydryl)-2-propyl alcohol, 20-35 DEG C of reaction 2-3h, then uses respectively acetone, 0.1M HCl solution, 0.1M NaOH solution, washed with de-ionized water;
(3) post processing: use the NaOH solution of pH10～13, the coupling that step (2) is obtained the solid phase carrier of aglucon process, the active group that is derived from carbonyl dimidazoles not reacting with aglucon on hydrolysis solid phase carrier.
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 disease human blood rheumatoid factor and other IgM type autoantibody and CIC ELISA as the adsorption stuffing of apparatus for purifying blood.
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| CN110339829A (en) * | 2019-07-19 | 2019-10-18 | 浙江大学 | Using aminobenzene (sulphur) amide pyridine as the chromatography media of functional ligand |
| CN110508263A (en) * | 2019-08-22 | 2019-11-29 | 广州康盛生物科技有限公司 | A kind of adsorbent material and preparation method thereof for blood purification |
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