JPH0135670B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an adsorbent for body fluid purification in which a carrier holds a substance having a functional part capable of binding to an adsorbed substance, and a method for producing the same. For more details,
Diseases and phenomena related to the immune system of the body, such as cancer, immunoproliferative syndrome, rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis, allergies, rejection reactions during organ transplants, or kidney diseases such as nephritis, hepatitis, etc. In cases such as liver disease, malignant substances that are expressed in body fluids such as blood and plasma and are thought to be closely related to the cause or progression of the disease are
Regarding adsorbents that adsorb and remove from body fluids, adsorption that adsorbs and removes autoantibodies and/or immune complexes that occur in body fluids, especially in autoimmune diseases such as chronic rheumatoid arthritis, systemic lupus erythematosus, and myasthenia gravis. Regarding materials and their manufacturing methods. Conventionally, activated carbon or activated carbon coated with a hydrophilic polymer has been used as an artificial liver for liver diseases, mainly for body fluid purification treatment. However, as mentioned above, in many diseases, various malignant substances that are closely related to the cause or progression of the disease have become known, and there is a growing need to selectively remove these malignant substances from body fluids. However, adsorbents based on activated carbon have low adsorption selectivity and cannot currently meet this demand. In order to meet the demand for selective adsorption and removal of malignant substances, the present inventors conducted extensive research and found that the carrier retains a special substance that selectively interacts chemically with the adsorbed substance through chemical bonds. He discovered various adsorbents that were
7152, patent application 1982-76776, patent application 1982-159444). Furthermore, the present inventors have proposed a polymer containing a hydrophobic compound and/or a hydroplastic compound bonded to a carrier consisting of a crosslinked copolymer mainly composed of vinyl alcohol units, and a bulin base or a pyrimidine base as a constituent component. At least one low molecular weight substance or derivative thereof containing sugar phosphate as a component and/or at least one low molecular weight substance containing sugar phosphate as a constituent or at least one derivative thereof, and sugar and/or oligosaccharide have extremely high activity against autoantibodies. discovered that it specifically adsorbed immune complexes, and previously filed patent applications (Japanese Patent Applications 112919/1980, 193735/1982, and 14276/1982). The present invention was made as a result of a more detailed study on a binder for a carrier and a substance containing a functional moiety capable of binding to an adsorbed substance (hereinafter referred to as a ligand) in connection with the previous invention. The present invention relates to a method for manufacturing an adsorbent for a binder in an adsorbent for purification. Hitherto, there are no known carriers or binders specifically designed for this purpose, but gels for affinity chromatography are similar. Gels for affinity chromatography often use natural polysaccharides such as agarose, dextran, and cellulose as carriers, and utilize the hydroxyl groups in their sugar chains to bind target ligands using various binding agents. It is used as an affinity adsorbent. There are many known ligand binding methods used in this case, such as the cyanogen halide method, triazine halide method, and bromoacetyl bromide method, but the method using cyanogen halide has an amino group in the molecule. It is most commonly and widely used because biological components such as amino acids, peptides, and proteins can be immobilized by a simple one-step reaction under relatively mild conditions. Since a carrier made of a synthetic polymer having a hydroxyl group also has a hydroxyl group like a natural polysaccharide, it is possible to similarly bind a ligand having an active group such as an amino group using cyanogen halide. However, when an adsorbent in which a ligand is bonded to a carrier having a hydroxyl group via a cyanide halide is used for therapeutic purposes such as body fluid purification and extracorporeal circulation,
It has the following drawbacks. (1) In cases where the ligand is bound via cyanogen halide, the carrier and the ligand are said to be bound by multiple unspecified bonds such as isourea bonds, imidocarbonate bonds, and carbamate bonds. All of these results are relatively unstable both thermally and chemically, making them unsuitable for heat sterilization such as high-pressure steam or radiation sterilization, and also having poor storage stability. (2) Cyanogen halide itself is a cyano compound and is highly toxic, and when used for therapeutic purposes, the toxicity of trace amounts of residue or decomposition products cannot be ignored. (3) Cyanogen halides are extremely reactive and react vigorously with heat generation, making it difficult to control the reaction. Although there is no problem in using it at the laboratory level where small quantities are handled, it is difficult to use on an industrial scale. Not suitable for production. (4) In the reaction between cyanogen halide and the hydroxyl group of the carrier, side reactions occur in addition to the active imidocarbonate production reaction, making it difficult to widely adjust the activity ratio or increase the density of active groups. It is also difficult to quantify the active group density. As a result, it is difficult to control the amount of bound ligand, and it is also difficult to bind the ligand at high density. (5) An adsorbent in which a carrier is bonded to a ligand via cyanogen halide is composed of isourea bonds, imidocarbonate bonds,
It tends to non-specifically adsorb proteins in body fluid components such as blood due to carbamate binding. Furthermore, methods using halogenated triazines and methods using bromoacetyl bromide have almost the same drawbacks as cyanogen halides, and are unsuitable for treatment such as body fluid purification and extracorporeal circulation. In view of the problems of carrier-ligand binders based on the prior art as described above, an object of the present invention is to be generally applicable, to highly actively and specifically adsorb malignant substances in body fluids, and to provide non-specific adsorption of malignant substances in body fluids. The object of the present invention is to provide an adsorbent that exhibits low adsorption, maintains stable activity, is safe, can be easily sterilized, and is suitable for body fluid purification or regeneration, and a method for producing the same. The present inventors have conducted research in line with the above objectives,
On a carrier made of a crosslinked synthetic polymer having hydroxyl groups,
A hydrophobic compound and/or a polymer containing a hydrophobic compound, a low molecular weight substance containing a purine base or a pyrimidine base as a constituent, or at least one derivative thereof, and/or a low molecular weight substance containing a sugar phosphate as a constituent. At least one substance or its derivative, as well as low molecular weight ligands such as sugars and/or oligosaccharides, and high molecular weight ligands such as ptein A, C ₁ q, DNA, RNA, modified γ globulin, and various antibodies, via various binding agents. We evaluated the binding activity for autoantibodies and immune complexes, the nonspecific adsorption of plasma proteins, etc., and whether or not it could be used as an adsorbent for body fluid purification.We found that 2- The present inventors have discovered that an adsorbent formed by bonding various ligands as described above through a bond containing a hydroxytrimethylene group as a constituent element can solve the above problems, and have completed the present invention. That is, in the present invention, a substance containing a functional moiety capable of bonding with an adsorbed substance is bonded to a carrier made of a crosslinked synthetic polymer having a hydroxyl group via a bond containing a 2-hydroxytrimethylene group as a constituent element. The present invention relates to an adsorbent material for body fluid purification characterized in that the carrier is made of a crosslinked synthetic polymer having a hydroxyl group, and contains both a functional group capable of bonding to the carrier in solution and a methyloxirane group. The present invention relates to a method for producing an adsorbent for body fluid purification, which comprises binding a binder and further reacting the methyloxirane group with a substance containing a functional moiety capable of binding to an adsorbent. The carrier used for this purpose is required to have interaction and adsorption characteristics with plasma proteins, such as low non-specific adsorption of plasma proteins and no activation of the complement system or coagulation system. Also, in terms of safety,
It is required to be sterilizable, have physical strength, not cause chipping or scum of the carrier, and be free from eluates from the carrier. Furthermore, when used as an adsorbent for whole blood, interaction with blood cell components, ie, thrombus formation and non-specific adhesive residual blood of blood cell components, etc., becomes a problem. Natural polymeric carriers containing sugars such as dextran, agarose, and cellulose interact with blood cells and plasma components, causing activation of the complement system and coagulation system. On the other hand, synthetic polymer carriers are said to be relatively free from these problems. A carrier made of a crosslinked synthetic polymer having hydroxyl groups solves the above problems and is the most preferred. Due to its hydrophilic nature, a carrier made of a crosslinked synthetic polymer having a hydroxyl group has little interaction with solutes such as proteins in plasma, thereby minimizing nonspecific adsorption. It also has extremely excellent properties such as not interacting with the complement system and coagulation system in plasma. In terms of physical properties, it has heat resistance, enables heat sterilization, and has excellent physical and mechanical strength, which is a characteristic of synthetic polymers. When used as a carrier for whole blood adsorbents,
It has extremely excellent properties such as having little interaction with blood cell components and minimizing thrombus formation, non-specific adhesion of blood cell components, residual blood, etc. The higher the density of hydroxyl groups in the crosslinked synthetic polymer of the present invention, the more hydrophilic it becomes, which is advantageous for minimizing interaction with blood components.
Further, when activated with an activating reagent, the active group density can be maintained at a high level, which is preferable, but on the other hand, the physical and mechanical strength decreases in relation to the crosslinking density (crosslinking agent content). Therefore, the hydroxyl group density is 1
~17meq/g is preferable, more preferably 6~
It is 15meq/g. The hydroxyl group density can be determined by reacting the carrier with acetic anhydride in a pyridine solvent and measuring the amount of acetic anhydride consumed by the reaction with the hydroxyl group or the change in weight of the carrier. 1 g of dry carrier is 1 mmol
The hydroxyl group density when reacting with acetic anhydride is
It is 1meq/g. A crosslinked synthetic polymer having a hydroxyl group can be synthesized by polymerizing a monomer having a hydroxyl group or introducing a hydroxyl group through a chemical reaction of a polymer. It is also possible to synthesize both in combination. As for the polymerization method,
A radical polymerization method can be used. The crosslinking agent may be introduced by copolymerization during polymerization, or may be introduced by chemical reaction between polymers (between polymers or between polymer and crosslinking agent), or both may be used in combination. For example, it can be produced by copolymerizing a vinyl monomer and a vinyl or allyl crosslinking agent. In this case, vinyl monomers include (meth)acrylates such as 2-hydroxyethyl acrylate and 2-hydroxyethyl methacrylate, vinyl esters of carboxylic acids such as vinyl acetate and vinyl propionate, methyl vinyl ether, ethyl vinyl ether, etc. Examples include vinyl ethers, ethylene carbonate, and derivatives thereof. As a crosslinking agent, allyl compounds such as triallyl isocyanurate and triallyl cyanurate, di(meth)acrylates such as ethylene glycol dimethacrylate, diethylene glycol dimethacrylate, and pentaerythritol dimethacrylate, butanediol divinyl ether, Polyvinyl ethers such as diethylene glycol divinyl ether and tetravinylglyoxal, diarylidene pentaerythrite,
Polyallyl ethers such as tetraallyloxyethane and glycidyl acrylates such as glycidyl methacrylate can be used. Furthermore, copolymerized comonomers with other comonomers can also be used, if necessary. In addition, examples of hydrophilic crosslinked polymers having hydroxyl groups include JP-B No. 44-20917, JP-A No. 52-138077,
JP-A-53-1087, JP-A-55-80406, JP-A-55-
The synthetic methods and carriers disclosed in No. 104301 can also be used. An example of a hydrophilic crosslinked polymer having hydroxyl groups whose main constituent is 2-hydroxyethyl methacrylate is Spheron.
(manufactured by Chieko Academy) can be used, but
Cross-linked polyvinyl alcohol is preferred for non-specific adsorption surfaces. Furthermore, the triallylisocyanurate crosslinked product of polyvinyl alcohol obtained by copolymerizing a vinyl ester of carboxylic acid and a vinyl compound (allyl compound) having an isocyanurate ring and hydrolyzing the copolymer has high strength and chemical stability. Provides a particularly good carrier in terms of properties. Although the case of a vinyl copolymer has been exemplified above, the present invention is not limited thereto. As for the shape of the present carrier, any of the shapes such as spherical, granular, filamentous, hollow fiber, and flat membrane shapes can be effectively used.
A spherical or granular shape is particularly preferably used in view of the surface area of the carrier (adsorption capacity as an adsorbent) and the flow surface of body fluid during extracorporeal circulation. Therefore, the known suspension polymerization method is particularly effectively used as a method for synthesizing the carrier. The average particle size of spherical or particulate carrier is 25-2500μ
150~m can also be used, but due to its specific surface area (adsorption ability as an adsorbent) and circulation of body fluids,
Particularly preferred is one with a diameter of 1500 μm. In the present invention, preferably the specific surface area of the crosslinked synthetic polymer having hydroxyl groups is at least 5 m ² /g.
A hard cross-linked synthetic polymer that maintains this property is used. In the present invention, a hard crosslinked synthetic polymer refers to one whose physical property values maintain a certain value or more when external force is applied. Specifically, the diameter of the cross-linked synthetic polymer is
When a container 10 mm long and 50 mm long is packed most densely and water is passed through it, the volume reduction rate of the crosslinked synthetic polymer is 10% or less when the pressure difference between the inlet and outlet of the container is 200 mmHg. Further, when such a crosslinked synthetic polymer is freeze-dried, its specific surface area maintains a value of 5 m ² /g or more. Here, the specific surface area is expressed as the surface occupied by nitrogen gas adsorbed per unit weight of dry crosslinked synthetic polymer. In other words, the specific surface area indicates how effectively the substance constituting the crosslinked synthetic polymer per unit weight forms the surface in a dry state. Generally, a crosslinked synthetic polymer swells in a medium that has an affinity for the crosslinked synthetic polymer, and contracts when dried. In the case of a soft gel in which the pores that are filled with the medium during swelling are maintained only by a network of crosslinks, when the gel dries, the network collapses and the pores almost disappear. In this case, the specific surface area is mostly only for the outside of the particle, so it is generally 1
Since the agarose conventionally known for use in affinity chromatography and exhibiting a low value of m ² /g or less is a soft gel, the pores disappear when dried. Therefore, it is not easy to sterilize, and furthermore, because it has a soft mesh that is easily crushed, it is not possible to flow body fluids at a high flow rate for a long time even when it is packed into a column and used for extracorporeal circulation. . On the other hand, in the case of hard crosslinked synthetic polymers that have a rigid pore structure and can withstand freeze-drying and heat sterilization, the pores will shrink somewhat when dried, but will maintain most of their swollen state. In other words, it has permanent pores, and its specific surface area is higher than that of soft gel, and is at least 5 m ² /g. The specific surface area of the present invention was determined by the most common PET method (BET method) using nitrogen gas. In addition, the sample used for specific surface area measurement must be sufficiently dried, but since the crosslinked synthetic polymer of the present invention is difficult to dry, the sample used for specific surface area measurement must be dried at 60°C or lower after equilibrating the water-wet support with acetone. It was dried under reduced pressure and used for measurement. Water retention capacity of the crosslinked synthetic polymer used in the present invention (hereinafter referred to as
W _R ) is suitably in the range of 0.5 to 16 g/g, preferably in the range of 1.0 to 15.0 g/g. W _R is the amount of physiological saline that can be contained within particles when the crosslinked synthetic polymer is brought into equilibrium with physiological saline, expressed as a value per dry weight of the crosslinked synthetic polymer. In other words, W _R is a measure of the amount of pores in the crosslinked synthetic polymer. When W _R increases, the weight of the part forming the skeleton per unit volume of the crosslinked copolymer in water, that is, the weight of the crosslinked synthetic polymer itself, decreases relatively, and therefore, the weight of the crosslinked synthetic polymer itself decreases in water, and therefore The mechanical strength of the molecule decreases. Furthermore, when W _R becomes smaller, the amount of pores per unit weight (or unit volume) for adsorption becomes smaller, resulting in a decrease in adsorption capacity. Therefore, it is preferable for the carrier for this purpose that W _R be within an appropriate range. W _R is obtained by measuring the weight (W ₂ ) of a sufficiently dried crosslinked synthetic polymer, and then centrifuging the crosslinked synthetic polymer, which has been sufficiently equilibrated with physiological saline, to adhere to the surface of the crosslinked synthetic polymer. After removing the physiological saline, its weight (W ₁ ) can be measured and determined by the following formula. W _R = W ₁ - W ₂ /W ₂ (g/g) The exclusion limit molecular weight (protein) of the carrier is 150,000 (IgG) for the target adsorbent of the present invention, and the molecular weight for immune complexes, especially IgM immune complexes. In this case, it reaches 10 million, so 15 to 10 million is preferable. The most common exclusion limit molecular weight for purposes of this invention is 1 million to 5 million. The carrier-ligand binder used for this purpose is one that can easily bind the carrier and the ligand under mild conditions, that the bond formed is chemically and thermally stable, and that the bond can be easily bonded during storage. that it will not break and will not break when sterilized, preferably by heat sterilization without the use of any chemicals;
The binder itself has low toxicity and is safe; the binder has mild reactivity and is easy to handle in large quantities industrially; it binds to carriers and ligands quantitatively, and the amount of binder can be easily adjusted. It is required that the density of the bound ligand can be adjusted, that high-density binding is possible, and that nonspecific adsorption of protein to the bond itself is small. As a result of studying the bond between the carrier and the ligand that satisfies these requirements, the present inventors successfully solved the above problems with a bond containing a 2-hydroxytrimethylene group derived from an epoxy binder as a constituent element. I found something to do. Methyloxirane derivatives react between their epoxy groups, hydroxyl groups, amino groups, and thiol groups,
Each forms a chemical bond with the 2-hydroxytrimethylene group through an ether bond, an alkylamine bond, or a thioether bond, but this reaction proceeds under mild conditions without side reactions, and the resulting bonds are chemically bonded. It is an extremely stable bond, and it is also a thermally stable bond. In addition, the epoxy group is stable even in water, and there is no intense heat generation during the reaction.
It is also easy to handle in large quantities industrially. Furthermore, when this epoxy group is decomposed in water, it becomes a glycol derivative, which is low in toxicity and safe. Since the 2-hydroxytrimethylene group formed by the reaction of the methyloxirane group is a hydrophilic group containing a hydroxyl group, non-specific adsorption of proteins to this group is minimal and does not pose a problem. Examples of binders containing such methyloxirane groups include epihalohydrins typified by epichlorohydrin, glycidyl acrylates or methacrylates, and bisepoxides. Examples of bisepoxides include 1,
2-bis-(2,3-epoxypropoxy)-ethane, 1,4-bis-(2,3-epoxypropoxy-butane, 1,6-bis-(2,3-epoxypropoxy)-hexane, etc.) 2-
Examples of bonds between a carrier containing a hydroxytrimethylene group and a ligand include those in which the above-mentioned epoxy-based binder is directly bound to the carrier and the ligand, as well as those in which an epoxy-based binder such as epichlorohydrin is bound to the carrier. Later, a polyfunctional compound such as phloroglucinol is bonded, a compound such as divinyl sulfone having a group that can be covalently bonded to the ligand is bonded, and then the ligand is bonded. They may be bonded with a spacer interposed therebetween. Among these epoxy binders, epihalohydrin, which has an asymmetric structure, has a large difference in reactivity between the two functional groups, so when the carrier and the binder react, the two functional groups of the binder react with each other. This is more preferable because crosslinking reactions caused by the reaction of both are less likely to occur. Using epichlorohydrin as an example, a method for bonding a ligand to a carrier made of a crosslinked synthetic polymer mainly composed of vinyl alcohol units will be described. First, the carrier is suspended in a solvent having the same volume as the carrier or about 10 times the volume of the carrier. The solvent may be any hydrophilic or polar solvent that has affinity for the carrier.
It is also preferable to use one that has no reactivity with epichlorohydrin. Specifically, water, an aprotic polar organic solvent such as N,N-dimethylformamide,
Examples include N,N-dimethylacetamide, dimethylsulfoxide, N-methylpyrrolidone, acetone, dioxane, and tetrahydrofuran, but aprotic polar organic solvents are preferred because they provide a higher amount of epoxy group bonding than water. It is also possible to use a mixture of these polar organic solvents with an appropriate amount of water; in this case, the amount of water in the system can be reduced to 1ml.
If the amount is kept within about 0.1 ml per carrier, there is almost no effect of water. Among the above polar organic solvents, solvents with strong polarity, such as N,N-dimethylformamide, N,N-dimethylacetamide, dimethylsulfoxide, and N-methylpyrrolidone, swell the carrier well and have a high bonding amount of epoxy groups. obtained and more preferred. Epichlorohydrin is added to this reaction system in a molar number of about 0.01 to 50 times the molar number of hydroxyl groups in the carrier. As the amount of epichlorohydrin added increases, the amount of binding also increases, but epichlorohydrin added in excess also serves as a solvent. An alkali such as sodium hydroxide is added to drive the reaction. The amount of alkali added is preferably 0.01 to 1 times the number of moles of epichlorohydrin. The reaction temperature can be between 0°C and 100°C, but at too low a temperature the reaction rate is slow, and at a high temperature exceeding 100°C side reactions such as crosslinking tend to occur. Practically speaking, a temperature range of 20°C to 50°C near room temperature is more preferable. The reaction time is selected from several minutes to about 10 hours depending on the reaction temperature and the desired amount of binding. By reacting in this manner, the methyloxirane group is bonded to the hydroxyl group of the carrier via an ether bond. Also, when binding a bisepoxide such as 1,2-bis-(2,3-epoxypropoxy)-ethane to a carrier, it is possible to bind it to a carrier under almost the same conditions as for epichlorohydrin, but in this case, the reaction A small amount of a reducing agent such as sodium borohydride may also be added to the system as a promoter. The amount of bonded epoxy groups in the present invention is determined by the following method. Measure out a certain amount, for example 2 ml, of the epoxy group-bonded carrier and add 5 ml of a 1,3M aqueous sodium thiosulfate solution. The OH ^- group produced by the reaction between the epoxy group and sodium thiosulfate is titrated with 0.1N hydrochloric acid using phenolphthalein as an indicator. Titration is repeated until the coloring of phenolphthalein disappears, and the amount of epoxy groups is determined from the total titration amount. The amount of epoxy groups bonded to the carrier in the present invention can be adjusted arbitrarily from 0 to 500 μmol per ml of carrier, depending on the hydroxyl group density of the carrier and reaction conditions, but for the purpose of the present invention, the amount of epoxy group bonded to the carrier is 0.01 μmol per ml of carrier. ~
The range is preferably 300 μmol, more preferably 1 to
It is 200μmol/ml. The present invention also includes using a vinyl monomer containing an epoxy group, such as glycidyl methacrylate, to introduce the epoxy group during the manufacture of the carrier. The adsorbent of the present invention is produced by bonding a ligand having a functional group having active hydrogen, such as an amino group, a hydroxyl group, or a thiol group, to the carrier to which an epoxy group is bonded as described above. The conditions for ligand binding must be selected depending on the chemical properties of the ligand, but for example, the reaction is carried out in an aqueous solution under basic conditions of about PH 7 to 13. To adjust the pH,
Buffers such as carbonic acid, boric acid, phosphoric acid, etc. can be used. The reaction temperature is 0 depending on the nature of the ligand.
Select a temperature of ~100℃, preferably around 20~60℃,
The reaction time is preferably about 1 to 24 hours. To measure the amount of bound ligand, the method must be selected depending on the type of ligand, but a common method is the so-called residual method, in which the unreacted ligand remaining in the reaction solution is quantified using a spectrophotometer, etc. This is a method of determining the amount of binding from the difference between the amount and the amount of preparation. If the amount of epoxy groups bound to the carrier is in excess compared to the ligand, this excess epoxy group can be blocked with a nucleophile such as glycine, ethanolamine, tris-(hydroxymethyl)-aminomethane, etc. .
Further, in the present invention, an adsorbent may be prepared by first bonding a ligand to a vinyl monomer containing an epoxy group and then copolymerizing it. The adsorbent obtained as described above is thermally stable because the carrier and the ligand are bonded by a stable covalent bond through a bond containing a 2-hydroxytrimethylene group, and even if the ligand itself is stable, For example, high-pressure steam sterilization is possible, and there is no elution of the ligand after sterilization and no accompanying decrease in adsorption capacity. Even if the ligand is unsuitable for heat sterilization, it can be dried and sterilized with ethylene oxide gas because the physical and chemical changes in the carrier and the bond are small due to drying, and there is almost no decrease in adsorption capacity due to sterilization. . Examples of substances to be adsorbed and removed in the present invention include blood waste products such as bilirubin, drug-toxin-conjugated albumin, medium-molecular-weight toxic substances in renal failure, and malignant substances such as hepatitis antigens such as types A and B;
Examples include autoantibodies and/or immune complexes in autoimmune diseases, and particularly autoantibodies and/or immune complexes. To explain in more detail, rheumatoid factor, antinuclear antibodies, anti
DNA antibody, anti-lymphocyte antibody, anti-erythrocyte antibody, anti-platelet antibody, acetylgoline receptor antibody, serum depletion antibody, anti-thyroglobulin antibody, anti-microsome antibody, anti-colon antibody, anti-smooth muscle antibody, anti-epidermal intercellular antibody, Examples include autoantibodies and/or immune complexes thereof such as anti-basement membrane antibodies, anti-proteoglycan antibodies, anti-collagen antibodies, anti-gastric factor antibodies, anti-thyroid microsomal antibodies, anti-microsomal antibodies, anti-colon antibodies, and anti-arterial antibodies. It will be done. The following is an example of a ligand that binds to a carrier made of a crosslinked synthetic polymer having a hydroxyl group in the present invention. For the treatment of systemic lupus erythematosus, adenine,
Homopolymers or copolymers of mono-, di-, and trinucleotides such as guanine, cytosine, uracil, and thymine, and naturally occurring nucleic acids such as DNA and RNA can be used. also present in the blood
Anti-single-stranded for adsorption removal of DNA, RNA, and ENA.
DNA antibody, anti-double-stranded DNA antibody, anti-RNA antibody,
Anti-nucleic acid antibodies such as anti-ENA antibodies, methylated albumin, and basic compounds such as actinomycin D can be used. Furthermore, for the adsorption and removal of immune complexes in the blood, complement components such as CIq, specific proteins such as protein A, and antibodies against immune complexes such as anti-snake chain constant region 2 phase antibodies can be used. . For the treatment of chronic rheumatoid arthritis and malignant rheumatoid arthritis, chemical denaturation (aggregation) methods such as urea, guanidine hydrochloride, mercaptoethanol, surfactants, and organic solvents, and physical denaturation such as heat, ultrasound, and gas bubbling are used. Modified γ modified by (aggregation) method
-Globulin, modified immunoglobulin, aggregated γ-
Antigen-like substances against rheumatoid factors such as globulin, aggregated immunoglobulin, Fc region of immunoglobulin, snake chain constant region 2 of immunoglobulin, and modified products thereof by the above-mentioned modification method, and anti-rheumatoid factor antibodies can be used. can. In addition, for the removal of rheumatoid immune complexes,
Antibodies against complement components such as CIq, specific proteins such as protein A, and immune complexes such as anti-snake chain constant region phase 2 antibodies can be used. For the treatment of Hashimoto's disease, thyroglobulin, a microsomal fraction component of thyroid gland, can be used. For the treatment of myasthenia gravis, neuromuscular acetylcholine receptor fraction components can be used. Glomerular basement membrane components for the treatment of glomerulonephritis, platelet membrane components and platelet granule fraction components for the treatment of idiopathic thrombocytopenic purpura, and transcortisone and anticortisone antibodies for the treatment of Cushing syndrome. Can be used. For the prevention and treatment of hepatitis, hepatitis A virus,
Antibodies against viral surface antigens such as hepatitis B virus can be used. Anti-angiotensin antibodies can be used to treat hypertension, and heparin, anti-lipoproteins, and antibodies can be used to treat hyperlipidemia. For the treatment of immune diseases based on lymphocyte abnormalities, anti-lymphocyte antibodies such as anti-B cell antibodies, anti-sublesser T antibodies, and anti-helper T antibodies can be used. Protein A and anti-immunoglobulin antibodies can be used to treat cancer such as breast cancer. The present invention further uses known lectins such as congnitinin, concanavalin A, and phytohemaagglutinin, which can bind to adsorbed substances such as nucleic acids, amino acids, lipids, sugars, protamines, heparin, antigens, antibodies, enzymes, substrates, and coenzymes. It is also possible to use the following substances. Further, in the present invention, more preferable low molecular weight ligands for adsorbing and removing autoantibodies and/or immune complexes include the following. First of all, hydrophobic compounds and/or low polymers containing hydrophobic compounds are mentioned, and the hydrophobic compounds bound to the carrier and the autoantibodies and/or immune complexes bind based on hydrophobic interactions. be done. The hydrophobic compound as used in the present invention refers to a compound having a solubility in physiological saline of 100 mmol/dl or less (at 25°C), more preferably 30 mmol/dl or less. Compounds whose solubility in physiological saline is greater than 100 mmol/dl are
The hydrophilicity becomes too high and the affinity for autoantibodies and immune complexes decreases, resulting in an extremely low adsorption capacity. Furthermore, an affinity for albumin, which is more hydrophilic, is generated, and albumin is also non-specifically adsorbed, which is undesirable. Among the hydrophobic compounds, compounds having at least one aromatic ring give particularly favorable results. What is an aromatic ring?
It means a cyclic compound with aromatic properties, and any of them can be usefully used, but benzene-based aromatic rings such as benzene, naphthalene, and phenanthrene, pyridine,
6-membered nitrogen-containing rings such as quinoline, acridine, inquinoline, and phenanthridine; 5-membered nitrogen-containing rings such as indole, carbazole, isoindole, indolizine, porphyrin, and 2,3,2′,3′-pyrrolopyrrole; Pyridazine, pyrimidine, sym-triazine, sym-tetrazine, quinazoline, 1,
Polyvalent nitrogen-containing 6-membered rings such as 5-naphthyridine, pteridine, phenazine, pyrazole, iminazole,
Polyvalent nitrogen-containing 5-membered rings such as 1,2,4-triazole, 1,2,3-triazole, tetrazole, benziminazole, imidazole, purine, norharman ring, perimidine ring, benzofuran, isobenzofuran, dibendofuran, etc. Oxygen-containing aromatic rings, sulfur-containing aromatic rings such as bentothiophene, thienothiophene, and thievin; oxygen-containing heteroaromatic rings such as oxazole, isoxazole, 1,2,5-oxadiazole, and benzoxazole;
Hydrophobic low-molecular-weight organic compounds having at least one aromatic ring such as a sulfur-containing heteroaromatic ring such as thiazole, isothiazole, 1,3,4-thiadiazole, and benzothiazole and its derivatives give preferable results. Among these, compounds containing an indole ring such as tryptamine give particularly favorable results. This can be interpreted as a result of the hydrophobicity, tertiary structure, and molecular rigidity of the compound effectively acting on the binding of the compound to autoantibodies or immune complexes. Furthermore, among these hydrophobic compounds, low molecular weight substances or derivatives thereof containing hydrophobic amino acids and their derivatives, purine bases or pyrimidine bases as constituent elements are safe, extremely highly and specifically inhibiting autoantibodies and/or their derivatives. Alternatively, it is practically preferable because it adsorbs immune complexes. What are hydrophobic amino acids and their derivatives?
Tanford, Nozaki (J.Am.Chem.Soc., 184 4240
(1962), J.Biol.Chem. 246 2211 (1971)] [Tanford, Nozaki (Journal of American Chemical Society, 184 , 4240 (1962),
Amino acids and their derivatives with a solubility in physiological saline of 100 mmol/mol or more based on the hydrophobicity scale defined by Journal of Biological Chemistry 246 , 2211 (1971)] are 1500 cal/mol or more.
means a compound below dl. For example, lysine, valine, leucine, tyrosine, phenylalanine,
These include isoleucine, tryptophan and its derivatives. Among these hydrophobic amino acids and their derivatives, aromatic amino acids such as tryptophan, phenylalanine, tyrosine, etc. and their derivatives give particularly good results. Furthermore, the amino acid can be used without any particular limitation on the 1 and d conformations. The low polymer and low molecular weight substance referred to in the present invention refer to a molecular weight of 10,000 or less, more preferably a molecular weight of 10,000 or less.
1000 or less substances. This results in protein A
Compared to natural polymers such as (molecular weight 42,000), it is easier to handle during immobilization and to store after immobilization. Furthermore, even when the substance is eluted from the carrier, a substance with a molecular weight of 10,000 or less has negligible antigenicity to living organisms, is safe, and can be easily sterilized. Low polymers are
It can be obtained by copolymerizing hydrophobic compound monomers alone or with other compounds. As the hydrophobic compound monomer, for example, a vinyl derivative of a compound containing an indole ring such as tributamine, or a hydrophobic amino acid such as tryptophan can be used. The low molecular weight substances that make up purine bases and pyrimidine bases are adenine, cytosine, guanine,
Bases such as uracil, thymine, hypoxanthine, xanthine, adenosine, cytidine, guanosine, uridine, inosine, xanthosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxyuridine, thyminedine, and other nucleosides, adenosine 5'-phosphate, cytidine
5′-phosphate, guanosine 5′-phosphate, inosine
5′-phosphoric acid, uridine 5′-phosphate, these riboses converted into deoxyribose, diphosphoric acid, triphosphoric acid, and those with phosphoric acid at the 2′ and 3′ positions nucleotides such as nucleotides bound to sugars such as glucose or mannose, oligonucleotides with 10 or less nucleotides, nicotinamide adenine dinucleotide (NAD), flavin adenine dinucleotide (FAD), coenzyme A, coenzyme B ₁₂ , etc. Refers to nucleotide coenzymes and all their derivatives. Among these, bases, nucleosides, and nucleotides are particularly preferred, and bases are even better, and among these, the purine bases adenine and guanine are more preferred. Rather than simply immobilizing just one of these, a plurality of types may be immobilized on the carrier. Further, in the present invention, a second ligand particularly suitable for adsorption and removal of autoantibodies and/or immune complexes includes low molecular weight substances containing sugar phosphate as a constituent or derivatives thereof. These ligands are capable of specifically binding autoantibodies such as anti-DNA antibodies and antinuclear antibodies and/or immune complexes thereof. In the present invention, the low molecular weight substances containing sugar phosphate as a component include tetroses such as erythrose and threose, aldopentoses such as arabinose, xylose, lyxose, and ribose, ketopentoses such as xylulose, pentulose, and ribulose, Aldohexoses such as galactose, glycol, talose, and mannose; ketohexoses such as sorbose, tagatose, psicose, and fructose; hexosamines such as N-acetyl-glucosamine; polymonosaccharides such as aldoheptose, ketoheptose, ketooctose, and ketononose; Deoxypentose, 6-deoxyhexose, 2-deoxyhexose, 2,6
- Deoxy sugars such as dideoxyhexose and 3,6-dideoxyhexose, sugar alcohol anhydrides, acidic sugars such as uronic acid, ketoaltonic acid, and ascorbic acid, sugar methyl ethers, branched sugars,
Examples include amino sugars, mono- or di-orthophosphates, mono- or dipyrophosphates, and triphosphates of sialic acids. These sugars are D, L
It can be used regardless of body, threo, or erythro body. Also usable are homo- or hetero-oligosaccharides with a degree of polymerization of 10 or less to which orthophosphoric acid, birophosphoric acid, or triphosphate groups are bonded. Furthermore, homo- or heteropolymers of sugar phosphoric acid having a degree of polymerization of 10 or less can also be used. Derivatives of all these can also be used. Among these, rubose, deoxyribose phosphate esters are particularly preferred, and deoxyribose 3,
More preferred are 5-diorthophosphoric acid and oligomers thereof. A third ligand particularly suitable for removing autoantibodies and/or immune complexes in the present invention includes sugars and/or oligosaccharides. The autoantibodies to which these ligands bind are those directed against antigens present in solid phase within the body, such as body tissues and cells. In these solid-phase antigens, sugars contained in glycoproteins, glycolipids, proteoglycans, etc. often have important immunological functions and antigenicity. Furthermore, in the case of bacterial foreign antigens such as pneumococci, the sugar chains are the main antigens, and antibodies are produced within the body. Unlike other biopolymers such as proteins, sugar chains have a small degree of freedom in molecular chains and are difficult to form relatively complex higher-order structures. Furthermore, compared to amino acids, the three-dimensional structures of various monosaccharides do not differ significantly in their basic skeletons, and in fact, in rheumatic fever and rheumatic heart disease following streptococcal infection, endocardium and type A streptococcal polysaccharides are cross-reactive. Since it acts as an antigen, anti-sugar chain antibodies are likely to cause cross-reactions, and conversely, sugars or their derivatives react with various autoantibodies that use sugar chains as antigens. The sugar and/or oligosaccharide referred to in the present invention is
Monosaccharides and their derivatives that constitute body tissues and cells, surface glycolipids, glycoproteins, proteoglycans, etc. As a monosaccharide, N-acetyl-D with pyranose or furanose structure
- Glucosamine, hexosamines such as N-acetyl-D-galactosamine, D-galactose, D-
Hexoses such as mantose and D-glucose, L
- 6-deoxyhexoses such as fucose and L-rhamnose, pentoses such as D-xylose and D-arabinose, N-acetylneuraminic acid, N-
Sialic acids such as glycolylneuraminic acid are used. Both α-type and β-type isomers can be used without particular limitation. As the oligosaccharide, a single oligomer or two or more types of oligomers of the above monosaccharides can be used regardless of whether they are linear or branched. In particular, dimers to dodecamers give good results. Furthermore, in the present invention, there is no problem even if the above-mentioned sugar and/or oligosaccharide is bound to a carrier in a state in which it is bound to other substances such as lipids, proteins, polypeptides, amino acid nucleic acids, and nucleic acid bases. In the present invention, two or more types of ligands may be bound to a carrier for use, if necessary. The amount of ligand bound to the carrier in the present invention is
0.01 to 1 ml of carrier per type of ligand
A range of 200 μmol is preferred. More preferably 0.05
It is in the range of ~100 μmol/ml. The adsorbent for body fluid purification of the present invention can be used as an adsorption device for body fluid purification by being filled and held in a container equipped with a body fluid outlet/inlet. In FIG. 1, reference numeral 1 shows one example of the adsorption device of the present invention, in which a cap 6 having a body fluid inlet 5 at one end opening of a cylinder 2 is inserted through a packing 4 with a filter 3 stretched inside. A cap 8 having a body fluid outlet 7 is screw-fitted to the other end opening of the cylinder 2 through a packing 4' with a filter 3' stretched inside to form a container. The adsorbent layer 9 is formed by filling and holding an adsorbent in the gap . The adsorbent layer 9 may be filled with the adsorbent of the present invention alone, or may be mixed or laminated with other adsorbents. As other adsorbents, for example, activated carbon having a wide range of adsorption capacities can be used. As a result, a wider range of clinical effects can be expected due to the synergistic effect of the adsorbent. The appropriate volume of the adsorbent layer 9 is about 50 to 400 ml when used for extracorporeal circulation. When this device is used for extracorporeal circulation, there are roughly two methods as follows. One method is to separate blood taken from the body into plasma components and blood cell components using a centrifuge or membrane plasma separator, and then pass the plasma components through the device to purify them.
One method is to return the blood to the body together with blood cell components, and the other method is to directly pass the blood taken out from the body through the device and purify it. In addition, regarding the passage speed of blood or plasma, since the adsorption efficiency of the adsorbent is extremely high, the particle size of the adsorbent can be made coarser, and the degree of packing can be lowered, so the shape of the adsorbent layer can be changed. Regardless, high passing speeds can be provided. Therefore, a large amount of body fluid can be treated. The method for passing body fluids may be either continuous or intermittent, depending on clinical needs or equipment conditions. As described above, the adsorbent of the present invention can adsorb and remove malignant substances in body fluids at a high rate and specifically.
It is extremely compact, convenient and safe. A carrier made of a crosslinked synthetic polymer having a hydroxyl group is used, and a bond containing a 2-hydroxytrimethylene group is used to bond the carrier and the ligand.
In addition to having extremely excellent properties such as low non-specific adsorption of plasma proteins and low interaction with the complement system and coagulation system, it also has excellent physical and mechanical strength and does not chip or crumble due to adsorbent preparation and handling. Very few. Also, since it is hard, body fluids can flow through it at a high flow rate. Furthermore, since the carrier is heat resistant and the bonds connecting the carrier and the ligand are also heat resistant, ordinary sterilization methods (ethylene oxide gas sterilization, heat sterilization such as high-pressure steam, γ-ray sterilization, etc.) can be easily carried out. It also has the effect of being reliably implemented. Furthermore, when used as an adsorbent for whole blood, since the interaction with blood cell components is small, it has the advantage of minimizing thrombus formation, non-specific adhesion of blood cell components, residual blood, etc. The present invention is applicable to the general use of purifying and regenerating body fluids such as autologous plasma, and is applicable to the safe and reliable treatment of diseases related to the immune system of the body, particularly rheumatoid arthritis, systemic lupus erythematosus, etc. It is effective in treating autoimmune diseases. In addition, the adsorbent of the present invention is not only used as a therapeutic device by filling it into a device, but is also extremely effective as an adsorbent for separating and purifying autoantibodies and immune complexes, and as a substrate for measuring these. available for use. Embodiments of the present invention will be explained in more detail with reference to Examples below. Example 1 A homogeneous solution consisting of 1000 g of vinyl acetate, 414 g of triallylisocyanurate (X = 0.30), 1000 g of ethyl acetate, 1000 g of heptane, 70 g of polyvinyl acetate (degree of polymerization 500) and 36 g of 2,2'-azobisisobutyronitrile Mixed liquid, 1% by weight of polyvinyl alcohol, 0.05% by weight of sodium dihydrogen phosphate dihydrate, and 1.5% by weight of disodium hydrogen phosphate dodecahydrate.
Water 4 in which % by weight was dissolved was placed in a flask, stirred thoroughly, and then heated and stirred at 65° C. for 18 hours and then at 75° C. for 5 hours to perform suspension polymerization to obtain a granular copolymer. After filtering, washing with water, and then extracting with acetone, the copolymer was transesterified in a solution consisting of 465 g of caustic soda and 20 g of methanol at 40° C. for 18 hours. The average particle size of the obtained particles is 150μ
It was m. When the hydroxyl group density (qOH) was determined by the above method, it was 7.5 meq/g. This gel was packed into a stainless steel column with an inner diameter of 7.5 mm and a length of 25 cm, and aqueous solutions of dextran and polyethylene glycol with various molecular weights and phosphate buffered salts of albumin, immunoglobulin G, immunoglobulin M, and β-riboprotein were added. When the solution was measured, the molecules were eluted in descending order of molecular weight. The exclusion limit molecular weight of dextran is approximately 6×10 ⁵ , and the exclusion limit molecular weight of protein is approximately 20×
It was 10 ⁶ . Also 0.3M sodium chloride and
When a solution of human γ-globulin and human albumin was run through an aqueous solution containing 0.1M sodium phosphate as a solvent, the recovery rate was almost 100%, and nonspecific adsorption of the gel was very low. . All sample measurements were performed at a flow rate of 1 ml/min. Next, it is transesterified, washed thoroughly with water,
150g of dried gel in dimethyl sulfoxide 1800g
ml and 1200 ml of epichlorohydrin suspended in a solution consisting of 150 ml of 30% aqueous sodium hydroxide solution.
and react at 30°C for 5 hours with stirring. After the reaction was completed, it was filtered through a glass filter and washed with dimethyl sulfoxide (3) and water (20) to obtain an epoxy group-bonded gel. The amount of epoxy group bonded in this gel is 0.11m per ml
It was hot in mol. Using the epoxy group-bonded gel, a hydrophobic compound was bound as a ligand to create an adsorbent. As hydrophobic compounds, 1-phenylalanine, 1-tryptophan, 1-tryptophan ethyl ester, and tryptamine were used, and each was dissolved in a carbonate buffer at pH 9.8 to a concentration of 0.05 mol/. Each of the epoxy bonded gels
Add 300ml of each ligand solution to 200ml and incubate at 50℃ for 16 hours.
After reacting and bonding for a period of time, excess epoxy groups are removed using tris(hydroxymethyl)aminomethane.
Blocking was carried out at 50° C. for 5 hours at 0.1 mol/solution. The amount of immobilized ligand was calculated by determining the amount of ligand remaining in the reaction solution from adsorption at 260 nm for l-phenylalanine and absorption at 280 nm for the other three indole derivatives. After blocking, the adsorbent was repeatedly washed with large amounts of water, washed with physiological saline, drained, and used for experiments. As for the adsorbent bound with l-tryptophan, an adsorption experiment was also conducted on the adsorbent which was subjected to high-pressure steam sterilization at 121°C for 20 minutes. Adsorption experiments were conducted using human chronic rheumatoid arthritis patient plasma 3.
The adsorbent was mixed with 1 volume of adsorbent, incubated at 37°C for 2 hours, and then the adsorbent and plasma were separated, and the plasma after adsorption was analyzed to calculate the amount of adsorption of various components. Globulin and albumin adsorption amounts are measured using A/G test kit [A/GB test Wako, Wako Pure Chemical Industries, Ltd.
Co., Ltd.). The ability to adsorb and remove rheumatoid factors (autoantibodies) and immune complexes as malignant substances in chronic rheumatoid arthritis was measured. Rheumatoid factor was measured using a latex agglutination test and a passive sensitized hemagglutination test. The latex agglutination test is a detection method that measures the tendency of latex particles to agglutinate when patient plasma containing rheumatoid factor is applied to polystyrene latex particles adsorbed with human-gamma-globulin. A plasma dilution series is created and the rheumatoid factor concentration is evaluated at the plasma dilution rate at which latex particles no longer aggregate. Plasma containing a high concentration of rheumatoid factor has a high dilution factor, while plasma with a low concentration has a low dilution factor. The passive sensitization hemagglutination test involves adsorbing rabbit γ-globulin to sheep red blood cells.
The rest is the same as the latex agglutination test. Generally, the passive sensitization hemagglutination test is considered to have higher rheumatoid factor specificity than the latex agglutination test. A dilution series was prepared using glycine saline buffer, and the dilution ratio at which rheumatoid factor was negative in a latex agglutination test was determined. Latex agglutination test
This was done using a kit from the Japan Freeze Drying Research Institute. Similarly, passive sensitization hemagglutination test [RAHA test,
[manufactured by Fuji Organ Pharmaceutical Co., Ltd.] was used for evaluation. The results are expressed as the titer, which is the highest dilution factor that shows a positive result. In addition, immune complexes were measured using polyethylene glycol and complement hemolysis. This method uses polyethylene glycol to precipitate and separate immune complexes.
The amount of immune complexes is evaluated by reacting with complement in the serum of a healthy human, and measuring the amount of remaining complement as the amount of hemolysis of red blood cells bound to antibodies. (1) Pour 0.3ml of sample into a separation tube. 0.2M EDTA50μ
Add and stir. Boric acid buffer (PBS)
Add 50μ and stir. Add 0.1ml of 12.5% PEG (polyethylene glycol) (Mw7500) and stir.
Leave at 4℃ for 90 minutes. (2) Centrifuge at 1700g for 10 minutes at 4℃, and remove the resulting precipitate.
Wash with 1.0ml of 2.5% PEG. Centrifuge at 1700g for 15min.
Drain the supernatant. (3) Add 30μ of GVB ⁺⁺ (gelatin veronal buffer containing divalent cations) at 37°C to dissolve the precipitate. Pool healthy human serum 10μ as complement source
Add. React the immune complex and complement at 37°C for 30 min. (4) Add 1.0 ml of 1.5×10 ⁸ /ml EA (antibody-sensitized red blood cells) and shake at 37° C. for 60 minutes to promote hemolytic reaction due to residual complement. (5) After the reaction, add 6.5ml of saline at 4℃ and centrifuge.
Measure the absorbance (OD ₄₁₄ ) of the supernatant. (6) Calculate the inhibition rate of hemolysis relative to the control (serum from a healthy person) and use the unit as PEG-cc%. [Rejection rate = Control - Specimen absorbance / Control absorbance x 100] [Removal rate = Untreated - Treated plasma inhibition rate / Untreated plasma inhibition rate x 100] The EA is for complement value measurement manufactured by Japan Freeze Drying Institute. Sensitized red blood cells (KW) were used. The results are shown in Table 1 as the removal rate of each component.

【table】

[Table] From this, l-phenylalanine, l-
It is clear that tryptophan, l-tryptophan ethyl ester, and tryptamine efficiently adsorb rheumatoid factors in plasma, and nonspecific adsorption of albumin is also small. Furthermore, no decrease in adsorption capacity was observed due to heat sterilization. In addition, the complement values before and after adsorption were measured, and in both cases the decrease was slight. Comparative Example 1 5 g of the carrier used in Example 1 was suspended in 200 ml of water, and the pH was adjusted to 10 using 2N aqueous sodium hydroxide solution.
-11, add 3 g of cyanogen bromide, and stir for 8 minutes under ice cooling. After the reaction is completed, immediately pass through a glass filter, then wash with water 2,
A cyanogen bromide activated support was obtained. 20ml of the activated carrier
0.05 mol of l-tryptophan/30 ml of a pH 8.4 carbonate buffer solution was added thereto, and the mixture was reacted at room temperature for 4 hours, and then left at 4°C overnight. Remove excess active groups with tris(hydroxymethyl)aminomethane 0.1mol/
Blocking was carried out by reacting with the solution at room temperature for 4 hours. After blocking, the adsorbent was repeatedly washed with large amounts of water, washed with physiological saline, drained, and used for experiments. Regarding the above adsorbent, adsorption experiments similar to those in Example 1 were conducted for both unsterilized material and material that had been autoclaved at 121° C. for 20 minutes to evaluate the adsorption capacity. The results are shown in Table 2.

[Table] From the results of this comparative example, the adsorbent using cyanogen bromide as a binder has a slightly inferior adsorption ability for rheumatoid factors and immune complexes compared to the adsorbent using epichlorohydrin as a binder, and it is also heat sterilized. The fact that the adsorption capacity is slightly reduced by heat sterilization suggests that part of the ligand is cleaved by heat sterilization. Example 2 A homogeneous liquid mixture consisting of 100 g of vinyl acetate, 32.3 g of triallyl isocyanurate (X = 0.25), 100 g of ethyl acetate, 100 g of heptane, 6.6 g of polyvinyl acetate, and 3.3 g of 2,2'-azobisisobutyronitrile. was subjected to suspension polymerization, and the resulting particles were subjected to a transesterification reaction. The average particle size of the gel obtained is
It was 200 μm and q _OH =11 meq/g. The dextran exclusion limit molecular weight of this gel was 6×10 ⁵ . When epoxy groups were bonded to the above gel using epichlorohydrin under the same conditions as in Example 1, the amount of epoxy bonding in the resulting epoxy group-activated gel was 120 μmol/ml. Various concentrations of l-tryptophan were charged and bound to this activated gel to create adsorbents having various tryptophan retention amounts. An adsorption experiment was conducted using rheumatism patient plasma under the same conditions as in Example 1. The results are shown in Table 3.

[Table] By using epichlorohydrin as a binding agent between a carrier made of a cross-linked synthetic polymer having hydroxyl groups and a ligand, it is possible to easily adjust the amount of ligand binding. It is clear that it has particularly excellent ability to remove rheumatoid factors and immune complexes. Example 3 Pentaerythritol dimethacrylate 100
g, 2-hydroxyethyl methacrylate 100g,
A homogeneous mixture of 124 g of ethyl acetate, 124 g of heptanol, 3.1 g of polyvinyl acetate (degree of polymerization 500) and 3.1 g of 2,2'-azobisisobutyronitrile was subjected to suspension polymerization to obtain a granular copolymer. Next, 10 g of the dried gel, thoroughly washed with water, is suspended in a solution consisting of 150 ml of dimethyl sulfoxide and 100 ml of epichlorohydrin, 100 ml of a 30% aqueous sodium hydroxide solution is added, and the mixture is reacted at 25° C. for 3 hours.
The epoxy group-bound gel was obtained by washing with dimethyl sulfoxide and then with sufficient water. Example 1 to the gel
An adsorption experiment was carried out using rheumatism patient plasma under the same conditions as above, with l-phenylalane bound as a ligand. In addition, the same adsorption experiment was performed on samples that had been autoclaved at 121°C for 20 minutes and compared. The results are shown in Table 4.

[Table] This shows that l-phenylalanine bound via 2-hydroxytrimethylene groups to a carrier made of a cross-linked synthetic polymer having hydroxyl groups efficiently adsorbs rheumatoid factor in plasma, and also adsorbs rheumatoid factors in plasma by heat sterilization. No decline in ability was observed. Reference Example 1 50 ml of the l-tributophane-bound adsorbent of Example 1 was placed in a container as shown in FIG. 1 to prepare an apparatus for removing autoantibodies and immune complexes. After the adsorption device was autoclaved at 121° C. for 20 minutes, adsorption experiments of rheumatoid factor and immune complexes were conducted using the experimental system shown in FIG. That is, 250 ml of rheumatism patient plasma (ACD added) 11 is put into a container 10, pumped out at a flow rate of 5 ml per minute by a pump 12, sent to the adsorption device 1, passed through a drip chamber 15 and a sampling port 13, and then pumped out at a flow rate of 5 ml per minute through a drip chamber 15 and a sampling port 13. The tube 14 was arranged so that it could be returned to. After the patient's plasma was circulated for one hour using the above device, the patient's plasma was sampled and the amount of rheumatoid factor and immune complexes in the plasma was measured. The results are shown in Table 5. In all cases, there was relatively little loss of complement before and after circulation.

[Table] Example 4 100 g of vinyl acetate, 24.1 g of triallyl isocyanurate (X = 0.20), 124 g of ethyl acetate, 124 g of heptane, 3.1 g of polyvinyl acetate (degree of polymerization 500) and 2,2'-azobisisobutyro Nitrile 3.1g
A homogeneous mixed liquid consisting of polyvinyl alcohol 1
% by weight, 0.05% by weight of sodium dihydrogen phosphate dihydrate and 400ml of water in which 1.5% by weight of sodium hydrogenphosphate dodecahydrate were dissolved in a flask, stirred thoroughly, and then heated at 65°C for 18 hours. Suspension polymerization was carried out by heating and stirring at 75° C. for 5 hours to obtain a granular copolymer. After filtering, washing with water, and then extracting with acetone, the copolymer was transesterified in a solution consisting of 46.5 g of sodium hydroxide and 2 methanol at 40° C. for 18 hours. The average particle size of the obtained particles is
It was 150 μm. Hydroxyl group density (qOH) using the above method
When it was determined, it was 13meq/g. Next, thoroughly wash 50ml of the above gel with water and
mg of sodium borohydride in 50 ml of 0.5 M aqueous sodium hydroxide solution, and 50 ml of 1,4-bis-(2,3-epoxypropoxy)-butane.
Add and stir. After the reaction was completed, it was filtered through a glass filter and then washed with water from Step 2 to obtain an epoxy group bond. The amount of epoxy group bonded in this gel is 1ml
The amount was 35 μmol per gel. Using the epoxy binding gel, adenine was bound to create an adsorbent. Adjust the pH of adenine to 0.025mol/
Prepare a solution dissolved in 9.8 carbonate buffer, add 40 ml to 20 ml of the epoxy binding gel, and heat at 50°C.
The mixture was allowed to react for 2 hours. Excess active group is 0.1mol/
Blocking was performed with glycine. The fixed amount of adenine is determined by the amount of adenine remaining in the reaction solution at 260 nm.
Calculated from the absorption of The amount of adenine bound in the adsorbent was 31 μmol/ml. After thoroughly washing the adsorbent with water, it was washed with physiological saline, dehydrated, and used for the experiment. In addition, to examine heat resistance, adsorbents that had been autoclaved at 121°C for 20 minutes were also subjected to adsorption experiments. Adsorption experiments were carried out by mixing 3 volumes of plasma from a patient with systemic lupus erythematosus and 1 volume of adsorbent, and incubating at 37°C for 2 hours. Anti-DNA antibody titer was measured using formalin-fixed chicken blood cells.
A positive or negative result is determined based on the presence or absence of an agglutination reaction (at room temperature) that occurs when the sensitized DNA is mixed with a serially diluted solution of treated or untreated patient plasma, and the highest dilution factor that indicates a positive result is determined. Then, the antibody titer was determined. For measurement, use "DNA test" [Fuji Organ Pharmaceutical Co., Ltd.]
A kit manufactured by J.D. Co., Ltd. was used. To determine the antinuclear antibody titer, drop a serially diluted sample (primary antibody) onto a slide glass smeared with cells and perform an antigen-antibody reaction. A peroxidase-labeled anti-human immunoglobulin antibody (secondary antibody) was added dropwise, and the color reaction of the enzyme was observed using an optical microscope. For measurement,
A kit of "Enzyme ANA Test" [manufactured by Medical and Biological Research Institute, Inc.] was used. The antibody titer was expressed using the highest dilution factor indicating positivity. The amount of immune complexes was measured by the method shown in Example 1. The amount of albumin was determined using an albumin measurement method using bromcresol green (BCG), and the reagent used was [A/G B-Test Wako, manufactured by Wako Pure Chemical Industries, Ltd.]. The results are shown in Table 6.

[Table] From Table 6, 2
-Adenine bound through a bond containing a hydroxytrimethylene group is specifically and highly effective against anti-DNA.
It is clear that the adsorbent adsorbs antibodies, antinuclear antibodies, and immune complexes, and that its adsorption capacity is not reduced by heat sterilization. Example 5 Deoxyribose 3,5-diphosphoric acid was bound to the activated gel of Example 1 in the same manner as in Example 5, and excess active groups were blocked with tris(hydroxymethyl)aminomethane. The amount retained was 20 μmol/ml. After thoroughly washing the adsorbent with physiological saline, it was dehydrated and used for the experiment. Anti-double strand-DNA antibody titer
An indirect fluorescent antibody method using Crithidia luciliae was used. The rest was carried out in the same manner as in Reference Example 1.
The results are shown in Table 7.

[Table] From Table 7, deoxyribose 3,5-diphosphoric acid bound to a carrier made of a crosslinked synthetic polymer having a hydroxyl group specifically and highly inhibits double strands.
It is clear that DNA antibodies, antinuclear antibodies, and immune complexes are adsorbed. We also measured the complement value before and after adsorption, and found that the decrease was only slight. Example 6 β-(p-aminophenyl)-ethylated D-(+)-galactose was bound to the activated gel of Example 1 in the same manner as in Example 4, and excess active groups were removed by Tris. Blocking was performed with (hydroxymethyl)aminomethane. The amount retained was 25 μmol/ml. After thoroughly washing the adsorbent with physiological saline, it was dehydrated and used for the experiment. Adsorption experiments were performed on systemic lupus erythematosus patient plasma 3.
and 1 volume of adsorbent were mixed and incubated for 2 hours. The amount of anti-T cell antibodies in the plasma after adsorption was measured by a fluorescent antibody method using normal T cells as a detector. The measurement operations and conditions are as follows. 1. Separate lymphocytes from peripheral blood of healthy individuals using specific gravity centrifugation, and collect T cells using nylon wool column method [“Lymphocyte Function Search Method” Chugai Medical Co., Ltd.
p15, p51 (1980)]. 2. Add 0.1 ml of plasma after the adsorption experiment to 1 x 10 ⁶ isolated healthy human T cells and treat at 4°C for 1 hour. 3. After washing, apply FITC-labeled anti-human immunoglobulin rabbit antiserum and count fluorescence-positive cells. 4 Express this as a percentage of fluorescence-positive cells. The results showed that the patient's plasma before adsorption was 24.4%, and the plasma treated with this adsorbent was 0.3%. In addition, the amount of globulin and albumin in each plasma was measured using an A/G test (A/GB test, Wako Pure Chemical Industries, Ltd.).
There were no major differences. Also, complement (C ₃ , C ₄ )
was measured using a simple immunodiffusion method, but there were no major fluctuations. From this, it is clear that the adsorbent bound to D-(+)-galactose specifically adsorbs anti-lymphocyte antibodies of systemic lupus erythematosus. In addition, this adsorbent was autoclaved at 121℃ for 20 minutes,
When subjected to adsorption experiments, no decrease in adsorption activity was observed due to sterilization. Example 7 Using the carrier used in Example 1 (X=0.30), an adsorbent was prepared in which l-phenylalanine was bound using various binders, and its adsorption performance was examined before and after autoclave sterilization. Adsorbent 1 is the same as in Example 1, using epichlorohydrin as a binder and binding the binder to a carrier in a dimethyl sulfoxide solvent, followed by binding l-phenylalanine, and Adsorbent 2 is the same as Adsorbent 1. In the manufacturing method of
Adsorbent 3 was prepared in the same manner as in Example 4, except that water was used instead of dimethyl sulfoxide as the solvent, and 1,4-bis-(2) was used as the binder. , 3-epoxypropoxy)-butane to which l-phenylalanine is bonded. 1-Phenylalanine is bonded using propoxy)-ethane. Adsorbent material 5 is one in which l-phenylalanine is bonded using cyanogen bromide as a binder in accordance with Comparative Example 1. The adsorption experiment was conducted in the same manner as in Example 1. The results are shown in Table 8.

[Table] From Table 8, it can be seen that the adsorbent in which l-phenylalanine is bonded to a carrier via a bond containing a 2-hydroxytrimethylene group of the present invention has less nonspecific adsorption of albumin, and It can be seen that this is an adsorbent with excellent stability, with almost no deterioration in performance observed.

[Brief explanation of drawings]

FIG. 1 is a sectional view showing an example of an adsorption device using the adsorbent for body fluid purification of the present invention, and FIG. 2 is an explanatory diagram of a model experiment in Reference Example 1. 1... Adsorption device, 2... Cylinder, 3, 3'... Filter, 4, 4'... Packing, 5... Body fluid inlet, 6... Cap, 7... Body fluid outlet, 8
... Cap, 9 ... Adsorbent, 10 ... Container, 1
1... Plasma, 12... Pump, 13... Sampling port, 14... Tube, 15... Drip chamber.

Claims (1)

[Scope of Claims] 1. A substance containing a functional moiety capable of bonding to an adsorbed substance via a bond containing a 2-hydroxytrimethylene group as a component is attached to a carrier made of a crosslinked synthetic polymer having a hydroxyl group. An adsorbent for body fluid purification characterized by being bonded. 2 The bond containing a 2-hydroxytrimethylene group as a constituent is a 2-hydroxytrimethylene group or a structural formula The adsorbent according to claim 1, wherein n is a group represented by 1 to 3. 3. The adsorbent according to claim 1, wherein the crosslinked synthetic polymer is a crosslinked copolymer mainly composed of vinyl alcohol units. 4. Bonding a binder containing both a functional group capable of bonding to the carrier and a methyloxirane group in a solution to a carrier made of a crosslinked synthetic polymer having a hydroxyl group,
A method for producing an adsorbent for body fluid purification, which further comprises reacting the methyloxirane group with a substance containing a functional moiety capable of bonding to an adsorbed substance. 5. The method for producing an adsorbent according to claim 4, wherein the binder containing both a functional group capable of bonding to a carrier and a methyloxirane group is epihalohydrin. 6. The method for producing an adsorbent according to claim 5, wherein the solution is a polar organic solvent.