JPH0215222B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a globulin-based compound that specifically adsorbs and removes immunoglobulins and/or immunoglobulin complexes that are thought to be closely related to various diseases caused by the immune system of the body. Regarding adsorbents. As is well known, immunoglobulins and/or immunoglobulin complexes expressed in blood are
Closely related to the cause or progression of autoimmune diseases such as cancer, immunoproliferative syndrome, rheumatoid arthritis, and systemic lupus erythematosus, or diseases and phenomena related to the immune system of the body such as allergies and rejection reactions during organ transplants. It is believed that Therefore, by specifically adsorbing and removing the above-mentioned immunoglobulin and/or immunoglobulin complex from body fluid components such as blood and plasma, the progression of the above-mentioned diseases can be prevented, symptoms can be alleviated, and even cured. It was hoped that this would be accelerated. As a result of intensive research in accordance with the above request, the present inventors have surprisingly found that a hydrophobic compound bound to an insoluble carrier and/or an oligomer containing a hydrophobic compound adsorbs immunoglobulin with extremely high activity. In particular, we discovered that it specifically adsorbs autoantibodies and immune complexes, and filed a patent application (Japanese Patent Application No. 7152/1982). The present invention was made as a result of a more detailed study on carriers with respect to the previous invention, and relates to improvements in carriers. Hitherto, no carrier specifically designed for this purpose is known. Therefore, there was no choice but to repurpose a carrier that is generally known for use in affinity chromatography. Known carriers include natural polymer carriers such as agarose carriers, dextran carriers, and cellulose carriers, polyacrylamide carriers, and glass carriers. However, natural polymeric carriers have the following drawbacks when used for therapeutic purposes. (1) There are many operational restrictions due to insufficient mechanical strength. For example, the carrier may be destroyed during preparation of the adsorbent such as activation or immobilization, or the carrier may break or crumble during transportation or use. (2) Since it is a soft gel, when it is packed into a column and used for extracorporeal circulation, it is not possible to flow body fluids containing substances to be removed at a high flow rate. When a high viscosity, high solute concentration liquid such as body fluid is flowed at a high flow rate, since it is a soft gel, the filling volume decreases, causing clogging and a decrease in flow rate, which may eventually stop flowing. (3) Since it is a soft gel and does not have permanent bores, it cannot be easily sterilized, which is an essential requirement for adsorbents for extracorporeal circulation therapy. For example, in the case of chemical sterilization such as ethylene oxide gas sterilization, the material is sterilized by freeze-drying, but the pores are destroyed by freeze-drying and do not return to their original state even if they are re-dispersed in an aqueous medium. During freeze-drying, its volume decreases to about half, and even when it is redispersed in an aqueous medium, it only returns to about 80% of its original volume, and its adsorption capacity usually decreases. Some methods include adding additives to protect the pores during freeze-drying, but to prevent the additives from entering body fluids, they must be thoroughly washed before use. Furthermore, heat sterilization such as high-pressure steam sterilization cannot be used because it destroys the pores. Similarly, radiation sterilization cannot be used as it destroys the skeleton and pores. (4) Furthermore, when natural polymer carriers are used for extracorporeal circulation therapy, they are said to activate the complement system and the coagulation system, resulting in leukopenia, thrombocytopenia, etc., and are therefore not very desirable. Although polyacrylamide carriers have the advantage of being relatively physically and chemically stable,
Specific adsorption of plasma proteins occurs, and residual toxicity of acrylamide cannot be ignored. Although glass carriers are physically and chemically stable, they are not used because they cause significant nonspecific adsorption of plasma proteins and cause thrombus formation when used with whole blood. In view of the problems of carriers based on the prior art as described above, an object of the present invention is to be generally applicable,
Adsorbs immunoglobulins and/or immunoglobulin complexes with high activity and specificity, maintains stable activity, is safe, can be easily sterilized, and is suitable for body fluid purification or regeneration. The aim is to provide materials. The present inventors have conducted research in line with the above objectives,
Hydrophobic compounds and/or polymers containing hydrophobic compounds were bound to various carriers, and the binding activity for immunoglobulins and immunoglobulin complexes, nonspecific adsorption of plasma proteins, and usability for whole blood were evaluated. What is really surprising is that vinyl alcohol units are the main constituent,
The present inventors have discovered that a copolymer crosslinked with a vinyl compound having an isocyanurate ring gives extremely good results as a carrier, and has completed the present invention. That is, the present invention provides a carrier in which a hydrophobic compound and/or a polymer containing a hydrophobic compound is bonded to a carrier consisting of a copolymer mainly composed of vinyl alcohol units and crosslinked with a vinyl compound having an isocyanurate ring. The present invention relates to an adsorbent for immunoglobulins and/or immunoglobulin complexes, characterized in that: 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. As a result of studies on synthetic polymer carriers, the present inventors found that a carrier consisting of a copolymer mainly composed of vinyl alcohol units and cross-linked with a vinyl compound having an isocyanurate ring successfully solved the above unresolved problems. I found a solution to this. Due to its hydrophilic nature, a carrier consisting of a copolymer mainly composed of vinyl alcohol units and cross-linked with a vinyl compound having isocyanurate rings has a small interaction with solutes such as proteins in plasma, and does not cause non-specific adsorption. Reduce to a minimum. 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 adsorbent for whole blood, it has extremely excellent properties such as minimal interaction with blood cell components, minimizing thrombus formation, non-specific adhesion of blood cell components, and residual blood. . The higher the density of hydroxyl groups in the crosslinked copolymer of the present invention, the more its hydrophilicity increases, which is advantageous in minimizing interaction with blood components, and also increases the hydroxyl group density when activated with an activation reagent. This is preferable because the active group density can be maintained at a high level, 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 5 to 17
meq/g is preferred, more preferably 6 to 15 meq/g
It is 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. 1g dry carrier
When reacted with 1 mmol of acetic anhydride, the hydroxyl group density is 1 meq/g. A copolymer containing vinyl alcohol units as a main component and crosslinked with a vinyl compound having an isocyanurate ring can be produced by copolymerization of a vinyl monomer and an allyl crosslinking agent. Examples of the vinyl monomer in this case include vinyl esters of carboxylic acids such as vinyl acetate and vinyl propionate, and vinyl ethers such as methyl vinyl ether and ethyl vinyl ether. As the allylic crosslinking agent, triallyl isocyanurate, triallyl cyanurate, etc. can be used. Furthermore, copolymerized comonomers with other comonomers can also be used, if necessary. A copolymer whose main constituent is vinyl alcohol units and crosslinked with a vinyl compound having an isocyanurate ring is produced by copolymerizing a vinyl ester of carboxylic acid and a vinyl compound having an isocyanurate ring (allyl compound). A crosslinked polyvinyl alcohol triallyl isocyanurate obtained by hydrolysis provides a particularly good carrier in terms of strength and chemical stability. An example of a method for producing a copolymer having vinyl alcohol units as a main component and crosslinked with a vinyl compound having an isocyanurate ring used in the present invention is as follows:
It is also described in Japanese Unexamined Patent Publication No. 190003/1983. 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 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, a crosslinked copolymer having a specific surface area of at least 5 m ² /g is used. The specific surface area is expressed as the surface occupied by nitrogen gas adsorbed per unit weight of the dry crosslinked copolymer. In other words, the specific surface area indicates how effectively the substance constituting the crosslinked copolymer per unit weight forms the surface in a dry state. Generally, a crosslinked copolymer swells in a medium that is compatible with the crosslinked copolymer and shrinks when dried. For soft gels whose medium-filled bores are maintained only by a network of crosslinks during swelling;
When it dries, the mesh collapses and the boa almost disappears. The specific surface area in this case is generally 1 m ² /g since it is almost only the value on the outside of the particle.
Since the agarose conventionally known for use in affinitai chromatography, which exhibits the following low values, is a soft gel, its bores disappear when it dries. Therefore, it is not easy to sterilize, and it has a soft mesh that is easily crushed, so it is difficult to sterilize it when packed in a column and used for extracorporeal circulation.
Body fluids cannot flow at high flow rates for long periods of time. On the other hand, it is a hard gel (cross-linked copolymer) that has a firm bore structure and can withstand freeze-drying and heat sterilization.
In this case, although the bore shrinks somewhat when dried, it maintains most of its swollen state. In other words, it had permanent bores, and the specific surface area showed a value higher than that of the soft gel, at least 5 m ² /g or more. The specific surface area of the present invention was determined by the most common BET method using nitrogen gas. In addition, the sample used for specific surface area measurement must be sufficiently dried, but since the crosslinked copolymer 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-wetted carrier with acetone. It was dried under reduced pressure and used for measurement. Water retention capacity of the crosslinked copolymer 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 the particles when the crosslinked copolymer is brought into equilibrium with physiological saline, expressed as a value per dry weight of the crosslinked copolymer. In other words, W _R is a measure of the amount of pores within the crosslinked copolymer. As W _R increases, the weight of the part that forms the skeleton per unit volume of the crosslinked copolymer in water, that is, the weight of the crosslinked copolymer itself, decreases relatively. The mechanical strength of the coalescence is reduced. Furthermore, as W _R becomes smaller, the amount of pores per unit weight (or unit volume) effective for adsorption decreases, 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 measured in advance by measuring the weight (W ₂ ) of a sufficiently dried crosslinked copolymer, and then centrifuging the crosslinked copolymer that has been sufficiently equilibrated with physiological saline so that it adheres to the surface of the crosslinked copolymer. 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 adsorbed substances targeted by the present invention are immunoglobulins and/or immunoglobulin complexes, and to explain in more detail, ordinary immunoglobulins, rheumatoid factors, anti-nuclear antibodies, anti-DNA antibodies,
anti-lymphocyte antibodies, anti-erythrocyte antibodies, anti-platelet antibodies,
acetylcholine receptor antibody, serum depletion antibody,
Immunoglobulins including autoantibodies such as anti-thyroglobulin antibodies, anti-microsome antibodies, anti-colon antibodies, immunoglobulin derivatives such as reduction products of immunoglobulins, chemically modified products, and other substances between immunoglobulins or immunoglobulins, especially They are complexes with antigens and antigen-like substances (hereinafter collectively referred to as globulin compounds). Among these, autoantibodies and immune complexes, which are closely related to the cause and progression of autoimmune diseases, are particularly preferred as adsorbent substances targeted by the present invention. The hydrophobic compound 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. A compound having a solubility in physiological saline of more than 100 mmol/dl becomes too hydrophilic and has a reduced affinity for globulin compounds, resulting in a reduced 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,
Nitrogen-containing 6-membered rings such as quinoline, acridine, isoquinoline, and phenanthridine; nitrogen-containing 5-membered rings such as indole, carbazole, isoindole, indolizine, porphyrin, 2,3,2',3'-pyrrolopyrrole, and 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 five-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;
A hydrophobic low-molecular organic compound 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 gives 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, steric structure, and molecular rigidity of the compound acting effectively in the bonding between the globulin compound and the compound. In addition, as a result of intensive research in search of inexpensive hydrophobic compounds that can be used more safely and practically, the present inventors found that hydrophobic amino acids and their derivatives adsorb globulin compounds at an extremely high rate and specifically. , found that it can be removed. What are hydrophobic amino acids and their derivatives?
Tanford, Nozaki (J.Am.Chem.Soc., 184 4240
(1962), J.Biol.Chqm. 246 2211 (1971))
Chemical Society, 184 , 4240 (1962), Journal of Biological Chemistry
246, 2211 (1971)], the solubility in physiological saline is 100 mmol/dl for amino acids and their derivatives of 1500 cal/mol or more.
It means the following compounds. Examples include lysine, valine, leucine, tyrosine, phenylalanine, isoleucine, tryptophan and derivatives thereof. Among these hydrophobic amino acids and their derivatives, tryptophan and its derivatives give particularly good results. Also, amino acids are l, d
The conformation of can be used without particular limitation. Further, as a result of intensive research, the present inventors have surprisingly found that among hydrophobic compounds, non-dissociable, amphoteric, and cationic compounds when bound to the carrier are globulin-based compounds. was found to be able to adsorb more selectively. That is, anionic compounds have an affinity for albumin, contrary to the expected result since albumin molecules are more electronegative than globulin molecules.
The adsorption amount of globulin compounds decreased. For example, phenylalanine methyl ester, phenylalanine, and tyrosine do not have much difference in their hydrophobicity, but when their amino groups are covalently bonded to a carrier and their immunoglobulin adsorption ability is evaluated, phenylalanine methyl ester > An example of this can be seen in the fact that the adsorption capacity of phenylalanine and tyrosine decreases. Furthermore, non-aromatic ring compounds also showed similar results. In particular, a more hydrophilic compound having a sulfonic acid group as an anionic group had more affinity for albumin than a globulin compound, and acted as an albumin adsorbent. This is presumed to be due to the interaction between the charge of the compound, albumin, and the charge of the microenvironment within the molecule. Based on the above experimental results, the mechanism of action of the present invention is based on the hydrophobic interaction force (Van der
This is thought to be based on the Waals force. It is also presumed that the interaction force between charges (Coulomb force) as a long-distance force acts secondarily. The polymer containing the hydrophobic compound of the present invention has a molecular weight of 10,000 or less, more preferably a molecular weight of 1,000 or less. This makes it easier to handle during immobilization and to store after immobilization compared to natural polymers such as protein A (molecular weight 42,000). Furthermore, even if the substance is eluted from the carrier, a polymer with a molecular weight of 10,000 or less has negligible antigenicity to living organisms and is safe, and can be easily sterilized. The polymer is
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. Methods for bonding a hydrophobic compound or/and a polymer containing a hydrophobic compound to a carrier made of a crosslinked copolymer mainly composed of vinyl alcohol units include covalent bonding, ionic bonding, physical adsorption, embedding, or polymerization. Any known method such as precipitation and insolubilization on the combined surface can be used, but in view of the elution properties of the bound substance, it is preferable to use it after fixation and insolubilization by covalent bonding. Therefore, known methods for activating immobilized enzymes and carriers commonly used in acquisition chromatography can be used. Examples of activation methods include cyanogen halide method, epichlorohydrin method, bisepoxide method,
Examples include the halogenated triazine method, the bromoacetyl bromide method, the ethyl chloroformate method, and the 1,1'-carbonyldiimidazole method. The activation method of the present invention comprises an amino group of a conjugate,
It is sufficient that it can undergo a substitution and/or addition reaction with a nucleophilic reactive group having active hydrogen such as a hydroxyl group, a carboxyl group, a thiol group, and is not limited to the above examples. Further, if necessary, a molecule (spacer) of any length may be introduced between the crosslinked copolymer (carrier) and the hydrophobic compound or/and the polymer containing the hydrophobic compound. For example, the hydroxyl group of the carrier and the isocyanate group on one side of hexamethylene diisocyanate are reacted and bonded, and the remaining isocyanate group is reacted and bonded with the amino group, hydroxyl group, thiol group, carboxyl group, etc. of the bond. can do. As for the spacer length, a range from no spacer to 20 atoms in the spacer gives particularly preferable results. In addition, in the present invention, if necessary, 2
It is also possible to use more than one kind of hydrophobic compound or/and a polymer containing a hydrophobic compound in combination. In the present invention, the amount of the hydrophobic compound and/or the polymer containing the hydrophobic compound bound to the carrier is preferably in the range of 0.1 to 30 mg per ml of the carrier. More preferably, it is in the range of 0.5 to 15 mg. Retention amount is 0.1
If it is less than mg/ml, the adsorption amount of globulin compounds will be extremely reduced, and if it is more than 30 mg/ml, the adsorption specificity will be reduced, which is not preferable. It is preferable that the adsorbent of the present invention is used as an adsorption device by being filled and held in a container equipped with an inlet and outlet for body fluids. In FIG. 1, reference numeral 1 shows an example of an adsorption device using the globulin-based compound adsorbent of the present invention, in which a packing 4 with a filter 3 stretched inside is inserted into an opening at one end of a cylinder 2. Body fluid inlet 5
A cap 8 is screw-fitted with a cap 6 having a body fluid outlet 7 through a packing 4' having a filter 3' inside the opening at the other end of the cylinder 2.
are screw-fitted to form a container, and an adsorbent layer 9 is formed by filling and holding an adsorbent in the gap between the filters 3 and 3'. 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, adsorbents for other malignant substances (antigens) such as DNA, activated carbon having a wide range of adsorption capacity, etc. 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 using the above device 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 and adsorption device of the present invention adsorb and remove globulin compounds in body fluids at a high rate and specifically, and are extremely compact, simple, and safe. Because a copolymer containing vinyl alcohol units as the main component and cross-linked with a vinyl compound having an isocyanurate ring is used as a carrier, non-specific adsorption of plasma proteins is small, and interaction with the complement system and coagulation system is also small. In addition to having excellent properties, it has excellent physical and mechanical strength, and is suitable for the preparation of adsorbents.
There are very few chips and chips due to handling. Also, since it is hard, body fluids can flow through it at a high flow rate. Furthermore, since it is heat resistant, it also has the effect that ordinary sterilization methods (ethylene oxide gas sterilization, heat sterilization such as high-pressure steam, γ-ray sterilization, etc.) can be carried out easily and reliably. 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 suitable for safe and reliable treatment of diseases related to the body's immune function, especially autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. Effective in treating diseases. In addition, the adsorbent of the present invention can be used not only as a therapeutic device by filling it into a device, but also as an adsorbent for separating and purifying immunoglobulins, autoantibodies, immunoglobulin derivatives, and immune complexes, and for measuring these. It can also be used extremely effectively as a base material. Embodiments of the present invention will be explained in more detail with reference to Examples below. Example 1 100 g of vinyl acetate, 24.1 g of triallylisocyanurate (X = 0.20), 124 g of ethyl acetate, 124 g of heptane, 3.1 g of polyvinyl acetate (degree of polymerization 500), and 3.1 g of 2,2'-azobisisobutyronitrile. g
A homogeneous liquid mixture consisting of 1% by weight of polyvinyl alcohol, 0.05% by weight of sodium dihydrogen phosphate dihydrate, and disodium hydrogen phosphate dodecahydrate
Add 400ml of water in which 1.5% by weight was dissolved into a flask, stir thoroughly, and then heat at 6.5℃ for 18 hours, then for a further 75 minutes.
Suspension polymerization was carried out by heating and stirring at °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 caustic soda and 2 methanol at 40° C. for 18 hours. The average particle size of the obtained particles was 150 μm. When the hydroxyl group density (qOH) was determined by the above method, it was 13 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-lipoprotein 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 3×10 ⁵ , and the exclusion limit molecular weight of protein is approximately 18×
It was 10 ⁶ . Also 0.3M sodium chloride and
When a solution of human-gamma-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 non-specific adsorption of the gel was very low. . All sample measurements were performed at a flow rate of 1 ml/min. Next, 50 c.c. of the gel that has been transesterified and thoroughly washed with water is suspended in 200 ml of water, 3 g of cyanogen bromide is added, and the mixture is stirred. The pH was maintained at 10 to 11 using a 2N aqueous sodium hydroxide solution, and the reaction was carried out for 8 minutes. After the reaction was completed, it was immediately filtered through a glass filter and then washed with 2 portions of water to obtain an activated gel. A hydrophobic compound was bound to the activated gel by a conventional method, and excess active groups were blocked with ethanolamine. The retained amount is determined by reacting and bonding the primary amino group of the remaining hydrophobic compound with 4-phenylspiro[furan-2(3H), 1'-phthalane]-3,3'-dione ("Flurum" manufactured by Rochie).
Measured by fluorescence from 475 to 490 nm (excitation wave 390 nm),
It was calculated by subtracting the amount of physical adsorption from a separate experiment using an unactivated gel. The adsorbent after ethanolamine blocking is
After repeated washing with PH4-0.1M acetic acid buffer and PH8.5-0.1M boric acid buffer, the sample was washed with physiological saline, drained, and used for experiments. In the adsorption experiment, 3 volumes of human plasma and 1 volume of adsorbent were mixed and incubated at 37°C for 3 hours. The amount of globulin and albumin after adsorption was measured using an A/G test kit [A/GB Test Wako, manufactured by Wako Pure Chemical Industries, Ltd.]. In addition, an adsorption experiment was conducted using unactivated gel as a control. The results are shown in Table 1 with the adsorption rate when the control adsorption rate is 0%.
It was shown to. From Table 1, it is clear that the hydrophobic compound bonded to the crosslinked copolymer mainly composed of vinyl alcohol units adsorbs globulin specifically and at a high rate. [Table] [Table] Example 2 The same experiment as in Example 1 was conducted except that l-tributophane methyl ester was used as the hydrophobic compound and human rheumatoid arthritis patient plasma was used instead of human plasma. The ability to adsorb and remove rheumatoid factor (autoantibody) and immune complexes, which are malignant substances in 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. 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. The operating method and conditions for this method are as follows. (1) Pour 0.3ml of sample into a separation tube. 0.2M EDTA50μl
Add and stir. Boric acid buffer (PBS) 50μ
Add and stir. Add 0.1ml of 12.5% PEG (polyethylene glycol) Mw7500) and stir.
Leave at ℃90mm. (2) Centrifuge at 4°C, 1700g, 10mm, and remove the resulting precipitate.
Wash with 1.0ml of 2.5% PEG. Centrifuge at 1700g for 15min.
Drain the supernatant. (3) Add 30 μl 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 min 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 PED-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. Furthermore, globulin was measured by the method used in Example 1.
An unactivated gel was used as a control, and the results are shown in Table 2 as removal rates when the adsorption rate of the control was set to 0%. [Table] From the above, it is clear that l-tryptophan methyl ester bound to the carrier of the present invention efficiently adsorbs globulin compounds in plasma, particularly rheumatoid factors and immune complexes. In addition, the complement values before and after adsorption were measured, and in both cases the decrease was slight. Example 3 5 ml of the adsorbent of Example 2 was placed in a container as shown in FIG. 1 to prepare a globulin removal device. Globulin adsorption experiments were conducted using the experimental system shown in FIG. That is, 25 ml of healthy human blood (heparin-added 1200 U/100 ml blood) 11 is placed in a container 10, pumped out at a flow rate of 1 ml per minute by a pump 12, sent to the immunoglobulin removal device 1, drip chamber 15 and sampling. A tube 14 is arranged so that the liquid is returned to the container 10 through the port 13. In addition, 250 U of hevarin was added to the blood. After circulating blood for 1 hour using the above device,
Blood was sampled and the amount of globulin in plasma was measured. The results are shown in Table-3. In all cases, there was a relatively small decrease in white blood cells and platelets before and after circulation. In addition, there was relatively little thrombus formation and residual blood. [Table] Example 4 Hydrophobic compounds: l-tryptophan, l-
An experiment similar to Example 1 was conducted except that phenylalanine and adenine were used in myasthenia gravis patient plasma instead of human plasma. The adsorption rate of anti-acetylcholine receptor antibody (autoantibody), which is a malignant substance in myasthenia gravis, was measured by Lindstrom's two-antibody method. The results are shown in Table 4 as the removal rate when the control is set as 0. Table 4 shows that the adsorbent in which l-tryptophan, l-phenylalanine, and adenine are bound to the carrier of the present invention selectively and highly adsorbs anti-acetylcholine receptor, which is an autoantibody for myasthenia gravis. That is clear. 【table】

[Brief explanation of drawings]

FIG. 1 is a cross-sectional view showing one example of the globulin compound adsorption apparatus of the present invention, and FIG. 2 is an explanatory diagram of a model experiment in an example. 1... Globulin compound removal device, 2...
Cylinder, 3, 3'... Filter, 4, 4'... Packing, 5... Body fluid inlet, 6... Cap, 7...
...Body fluid outlet, 8...Cap, 9...Adsorbent.

Claims (1)

[Scope of Claims] 1. A hydrophobic compound and/or a polymer containing a hydrophobic compound is bonded to a carrier consisting of a copolymer mainly composed of vinyl alcohol units and crosslinked with a vinyl compound having an isocyanurate ring. An adsorbent for immunoglobulins and/or immunoglobulin complexes, characterized in that: 2 A crosslinked copolymer mainly composed of vinyl alcohol units and crosslinked with a vinyl compound having an isocyanurate ring is obtained by hydrolyzing a copolymer of a vinyl ester of carboxylic acid and a vinyl compound having an isocyanurate ring. The adsorbent according to claim 1, which is polyvinyl alcohol.