JPH035821B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an adsorbent that specifically adsorbs and removes autoantibodies and/or immune complexes that are thought to be closely related to various diseases caused by biological immune function. As is well known, autoantibodies and/or immune complexes expressed in the blood are associated with autoimmune diseases such as cancer, immunoproliferative syndrome, rheumatoid arthritis, and systemic lupus erythematosus, as well as allergies, rejection reactions during organ transplants, etc. It is thought to have a close relationship with the cause or progression of diseases and phenomena related to the body's immune function. Therefore, by specifically adsorbing and removing the above-mentioned autoantibodies and/or immune complexes from body fluid components such as blood and plasma, it is possible to prevent the progression of the above-mentioned diseases, alleviate symptoms, and even cure them. 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 low molecular weight substances or derivatives thereof containing purine bases, pyrimidine bases or sugar phosphates as constituents bound to insoluble carriers. We discovered that at least one species adsorbs autoantibodies and immune complexes with extremely high activity, and in particular specifically adsorbs anti-DNA antibodies, antinuclear antibodies, and immune complexes of systemic lupus erythematosus, and we have previously applied for a patent. (Special Show 56-76776). 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 pores, 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 cannot be restored to their original state even if they are redispersed 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,
Nonspecific 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. The object of the present invention is to be generally applicable, in view of the problems of carriers based on the prior art as described above,
An adsorbent that highly actively and specifically adsorbs autoantibodies and/or immune complexes, maintains stable activity, is safe, and can be easily sterilized, making it suitable for body fluid purification or regeneration. We aim to provide the following. The present inventors have conducted research in line with the above objectives,
Low molecular weight substances containing purine bases, pyrimidine bases or sugar phosphates or their derivatives are bound to various carriers to achieve binding activity for autoantibodies and immune complexes, non-specific adsorption of plasma proteins, and use for whole blood. As a result of evaluating the properties, it was surprisingly discovered that a copolymer containing vinyl alcohol units as a main component gave extremely good results as a carrier, leading to the completion of the present invention. That is, the present invention provides a carrier made of a crosslinked copolymer mainly composed of vinyl alcohol units,
The present invention relates to an adsorbent for autoantibodies and/or immune complexes, which is characterized in that at least one of low molecular weight substances or derivatives thereof containing purine bases, pyrimidine bases, or sugar phosphates as constituents is bound thereto. The carrier used for this purpose is required to have low non-specific adsorption of plasma proteins and to have interaction and adsorption characteristics with plasma proteins that do not activate 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 the amount of non-specific adhesive residual blood of blood cell components, 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. The present inventors investigated synthetic polymer carriers and found that a carrier made of a copolymer containing vinyl alcohol units as a main component successfully solves the above-mentioned unresolved problems. Due to its hydrophilic nature, the carrier made of a crosslinked copolymer whose main component is vinyl alcohol units has little interaction with solutes such as proteins in plasma.
Reduce non-specific adsorption 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~
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. 1g of dry carrier is 1mmol
The hydroxyl group density when reacting with acetic anhydride is
It is 1meq/g. A crosslinked polymer mainly composed of vinyl alcohol units can be synthesized by polymerizing a monomer having a hydroxyl group or introducing a hydroxyl group through a chemical reaction of the polymer. It is also possible to synthesize both in combination. As 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. Examples of the vinyl monomer in this case include vinyl esters of alboxylic acids such as vinyl acetate and vinyl propionate, and vinyl ethers such as methyl vinyl ether and ethyl vinyl ether. Examples of crosslinking agents include allyl compounds such as triallyl isocyanurate and triallyl cyanurate, di(meth)acrylates such as ethylene glycol dimethacrylate and diethylene glycol dimethacrylate, butanediol divinyl ether, diethylene glycol divinyl ether, and tetravinyl. Polyvinyl ethers such as glyoxal, polyallyl ethers such as diarylidene pentaerythrite and tetraallyloxyethane, and glycidyl acrylates such as glycidyl methacrylate can be used. Furthermore, copolymerized comonomers with other comonomers can also be used, if necessary. In the case of vinyl copolymers, triallyl isocyanurate of vinyl alcohol is obtained by copolymerizing a vinyl ester of carboxylic acid with a vinyl compound having an isocyanurate ring (allyl compound), and then cross-hydrolyzing the copolymer. A crosslinked product provides a particularly good carrier in terms of strength and chemical stability. Although the case of a vinyl copolymer has been exemplified above, the present invention is not limited thereto. In the present invention, any shape such as spherical, granular, filamentous, hollow fiber, flat membrane, etc. can be effectively used, but
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 carriers is 25~
2500 μm can be used, but due to its specific surface area (adsorption ability as an adsorbent) and body fluid circulation,
Particularly preferred are those with a diameter of 150 to 1500 μm. In the present invention, it is preferable that the crosslinked copolymer has a specific surface area of at least 5 m ² /g. The specific surface area is expressed as the surface area 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. In the case of soft gels, where the pores filled with medium during swelling are maintained only by a network of crosslinks,
When it dries, the mesh collapses and most of the pores disappear. In this case, the specific surface area is mostly only for the outside of the particle, so it is generally 1 m ² /g.
Indicates a low value of: Since the agarose conventionally known for use in affixine chromatography is a soft gel, its pores disappear when it dries. Therefore, sterilization cannot be performed easily.
Furthermore, since it has a soft mesh that is easily crushed, it can be used 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 (crosslinked copolymer) that has a firm pore structure and can withstand freeze-drying and heat sterilization.
In this case, although the pores shrink somewhat when dried, they maintain most of their swollen state. In other words, it had permanent pores, and the specific surface area showed a value higher than that of a soft gel, at least 5 m ² /g. 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 dry, 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
_W2 ) 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 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, and therefore, the weight of the crosslinked copolymer itself decreases in water. 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. _WR measures the weight (W ₂ ) of a crosslinked copolymer that has been sufficiently dried in advance, and then centrifuges 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, is 150,000 (IgG). 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 substance to be adsorbed and removed in the present invention is anti-DNA
Antibodies (eg, native DNA antibodies, double-strand DNA antibodies, single-strand DNA antibodies), antinuclear antibodies and other antinuclear antigen antibodies, autoantibodies such as antigenic antigen antibodies, and/or immune complexes thereof. As the target disease, any disease in which any of the above substances appears can be applied. Specifically, systemic lupus erythematosus (SLE), mixed connective tissue disease (MCTD), systemic sclerosis, Siegren's syndrome, dermatomyositis (polymyositis), Keihei syndrome,
Chronic rheumatoid arthritis, malignant rheumatoid arthritis, Hashimoto's disease,
Applicable to chronic hepatitis, diabetes, bronchiectasis, etc. The low molecular weight substances constituting the purine bases and pyrimidine bases used in the present invention include bases such as adenine, cytosine, guanine, uracil, thymine, hypoxanthine, xanthine, adenosine, cytidine, guanosine, uridine, inosine, xanthosine, deoxy Nucleosides such as adenosine, deoxycytidine, deoxyguanosine, deoxyuridine, thyminedine, deanosine 5'-phosphate, cytidine 5'-phosphate, guanosine 5'-phosphate, inosine 5'-phosphate, uridine 5'-phosphate ,
and these liposes turned into deoxyliposes, diphosphates, triphosphates, and
Nucleotides such as those with phosphates attached at the 2' and 3' positions, nucleotides with sugars such as glucose or mannose attached, oligonucleotides with 10 or less nucleotides, nicotinamide adenine dinucleotide (NAD), flavin adenine dinucleotide (FAD), coenzyme A, coenzyme
Refers to nucleotide coenzymes such as _B12 , and all derivatives thereof. 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. The low molecular weight substances containing sugar phosphate as a component used in the present invention include tetroses such as erythrose and threose, aldopentoses such as arabinose, xylose, lyxose, and lipose, and ketopentoses such as xylulose, pentulose, and ligulose. , aldohexoses such as galactose, glucose, talose, and mannose, ketohexoses such as sorbose, tagatose, psicose, and fructose, N-acetyl-
Hexosamines such as glucosamine, polysaccharides such as aldoheptose, ketoheptose, ketooctose, and ketononose, 2-deoxypentose, 6
-Deoxy sugars such as deoxyhexose, 2-deoxyhexose, 2,6-dideoxyhexose, and 3,6-dideoxyhexose, sugar alcohol anhydrides, acidic sugars such as uronic acid, ketoaltonic acid, and ascorbic acid, sugar methyl ethers ,
Examples include branched sugars, amino sugars, mono- or di-orthophosphates, mono- or dipyrophosphates, and triphosphates of sialic acids. These sugars can be used regardless of their D, L, threo, or erythro forms. Furthermore, a homo- or hetero-oligosaccharide having a degree of polymerization of 10 or less to which an orthophosphoric acid, pyrophosphoric acid, or triphosphate group is bonded can also be used. 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, phosphoric acid esters of lupose and deoxylipose are particularly preferred, and 3,5 diorthophosphoric acid of deoxylipose and oligomers thereof are more preferred. In the present invention, a low molecular weight substance refers to a substance with a molecular weight of 1
It is a substance with a molecular weight of 1,000 or less, more preferably a substance with a molecular weight of 1,000 or less. This allows protein A (molecular weight
Compared to natural polymers such as 4200), it is easier to handle during immobilization and to store after immobilization. Furthermore, even if 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. The above-mentioned low molecular weight substances can be bonded to a carrier made of a crosslinked copolymer mainly composed of vinyl alcohol units, using any known method such as covalent bonding, ionic bonding, physical adsorption, embedding, or precipitation and insolubilization on the polymer surface. However, in view of the elution properties of the bound substance, it is preferable to use it after it is fixed and insolubilized by covalent bonding. Therefore, known methods for activating immobilized enzymes and carriers commonly used in affinity 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 low molecular weight substance. For example, by reacting and bonding the hydroxyl group of the carrier with the isocyanate group on one side of hexamethylene diisocyanate,
This can be carried out by reacting and bonding the remaining isocyanate groups with amino groups, hydroxyl groups, thiol groups, carboxyl groups, etc. of the conjugate.
As for the spacer length, a range from no spacer to 20 atoms in the spacer gives particularly preferable results. In the present invention, the amount of the ligand bound to the carrier is 0.1 μmol or more per 1 g (dry weight) of the carrier.
in the range of 1000μmol, more preferably 0.5μmol
or 100 μmol. Ligand (low molecular weight substance) to be bound to the carrier
As the moiety, a hydroxyl group, an amino group, a phosphoric acid group, etc. can be used. The mechanism of action of this adsorbent is thought to be similar to that of an antigen-antibody reaction, but it can be used internally without any problems without using biological polymers or similar synthetic polymers, which are the original antigens. It is surprising that small molecules that are metabolized in the body exhibit sufficient adsorption and removal ability. It is preferable that the adsorbent of the present invention be used as an adsorbent, which is filled in a container having a fluid inlet/outlet so that bodily fluids can pass through while coming into contact with the adsorbent.
A concrete example of this is shown in FIGS. 1 and 2. FIG. 1 shows an adsorbent 1, which has a cap 6, which has body fluid inlet/outlet ports 5, 5' at both end openings of a cylinder 2 via packings 4, 4' with filters 3, 3 placed inside.
6' are fitted with screws 7, 7', and an adsorbent 8 is filled and held in the gap between the filters at both ends. As the adsorbent, the adsorbent of the present invention may be filled alone, or may be mixed or laminated with other adsorbents. The appropriate volume of the adsorbent layer is about 50 to 400 ml when used for extracorporeal circulation. When using the adsorber made of the adsorbent of the present invention for extracorporeal circulation, there are roughly the following two methods.
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 an adsorbent using the adsorbent material of the present invention. One method is to purify the blood and then return it to the body together with the blood cell components.The other method is to directly pass the blood taken out from the body through an adsorbent made of the adsorbent of the present invention and purify it. In addition, regarding the passage speed of blood or plasma, the adsorption performance of the adsorbent is very high, so the particle size of the adsorbent can be made coarser, and the degree of filling can be lowered, so the shape of the adsorbent layer can be changed. Regardless, a high passing speed can be provided. Therefore, a large amount of body fluid can be treated. The method for passing body fluids may be continuous or intermittent depending on clinical needs or the installation status of the equipment. As described above, the adsorbent of the present invention can absorb anti-DNA antibodies (native DNA antibodies, double-strand DNA antibodies, single-strand DNA antibodies, etc.) in body fluids.
Antibodies), autoantibodies such as antinuclear antibodies, and immune complexes are adsorbed and removed with high efficiency and specificity, and it is extremely compact, simple, and safe. Because a cross-linked copolymer whose main component is vinyl alcohol units is used as a carrier, it has extremely excellent properties such as less non-specific adsorption of plasma proteins and less interaction with the complement system and coagulation system.
It has excellent physical and mechanical strength, and there is very little chipping or flaking due to adsorbent preparation and handling. Also, since it is hard, liquid can flow at a high flow rate. In addition, it is heat resistant, so it can be used using normal sterilization methods (ethylene oxide gas sterilization, heat sterilization such as high-pressure steam, γ
It also has the effect of easily and reliably carrying out sterilization (wire sterilization, etc.). Furthermore, when used as an adsorbent for whole blood, the interaction with blood cell components is small, so there is no risk of thrombus formation or non-specific adhesion of blood cell components.
It has the advantage of minimizing 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, particularly for the treatment of autoimmune diseases such as systemic lupus erythematosus. It is valid. 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 anti-DNA antibody,
Separation of autoantibodies and immune complexes such as antinuclear antibodies,
It can also be used extremely effectively as an adsorbent for purification and as a substrate for these measurements. Hereinafter, embodiments of the present invention will be explained in more detail with reference to Examples. 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
A homogeneous mixed liquid consisting of polyvinyl alcohol 1
wt%, sodium dihydrogen phosphate dihydrate 0.05 wt% and disodium hydrogen phosphate dodecahydrate
Add 400ml of water in which 1.5% by weight was dissolved into a flask, stir thoroughly, and heat at 6.5℃ for 18 hours.
Suspension polymerization was carried out by heating and stirring at °C for 5 hours to obtain a granular copolymer. After filtration, washing with water, and extraction 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 solutions of albumin, immunoglobulin G, immunoglobulin M, and β-riboprotein were added. When the salt 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 ⁵ . 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. Adenine (manufactured by Yamasa Soy Sauce Co., Ltd.) was bound to the activated gel by a conventional method, and excess active groups were blocked with glycine. The amount retained is
After treatment with 6N hydrochloric acid at room temperature for 24 hours, the ligand was released and calculated from the absorption at 260 nm. The adsorbent was thoroughly washed with physiological saline, dehydrated, and used in the experiment. The adsorption experiment was conducted by mixing 3 volumes of systemic lupus erythematosus patient plasma and 1 volume of adsorbent, and incubating the mixture at 37°C for 3 hours. Also, as a control,
Unactivated Sepharose 4B was used. Anti-DNA antibody titer was measured using formalin-fixed chicken blood cells.
Positive or negative results are 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 positivity is determined. Then, the antibody titer was determined. For the measurement, a "DNA Test" kit (manufactured by Fuji Organ Pharmaceutical Co., Ltd.) was used. To measure antinuclear antibody values, serially diluted samples (primary antibodies) are dropped onto a slide glass smeared with cells, and an antigen-antibody reaction is performed. 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. To measure the amount of immune complexes, a method was used in which immune complexes were precipitated with polyethylene glycol (PEG) and combined with complement hemolysis reaction. The operating method and conditions for this method were as follows. (1) Pour 0.3ml of sample into a separation tube, then
Add 50μ of 0.2NEDTA and stir, then add 50μ of boric acid buffer and stir. 12.5% PEG
Add 0.1ml of (molecular weight 6000) and stir at 4℃, 90℃.
Let stand for a minute. (2) Centrifuge at 1700g at 4℃ for 10 minutes to reduce the precipitate to 2.5%.
Wash with 1.0ml of PEG. Centrifuge at 1700g for 15 minutes and discard the supernatant. (3) Add 30μ of GVB ⁺⁺ (gelatin veronal buffer containing divalent cations) at 37°C to dissolve the precipitate. Add 10 µl of pooled healthy human serum as a complement source. The immune complex and complement are allowed to react at 37°C for 30 minutes. (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 6.5 ml of physiological saline at 4°C, centrifuge, and measure the absorbance (OD ₄₁₄ ) of the supernatant. (6) Calculate the inhibition rate of hemolysis against healthy human serum as a control. The unit is PEG-CC%. Rejection rate = (control) - (analyte absorbance) / (control absorbance)
×100 Removal rate = (untreated) - (treated plasma inhibition rate) / (untreated plasma inhibition rate) ×100 For EA, sensitized red blood cells (KW) for complement value measurement manufactured by Japan Freeze Drying Institute were used. The amount of albumin was measured 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 ratio of the decreased amount of albumin to the patient's plasma after the adsorption experiment was defined as the removal rate. The results are shown in Table-1. From Table 1, it is clear that adenine bound to the crosslinked copolymer mainly composed of vinyl alcohol units adsorbs anti-DNA antibodies, anti-nuclear antibodies, and immune complexes specifically and at a high rate.

[Table] Example 2 Deoxyribose 3,5-diphosphate was bound to the activated gel of Example 1 by a conventional method ("Experimental and Applied Affinity Chromatography").
P48, 1976 published by Kodansha Scientific Books), the usual active groups were blocked with trihydroxymethylaminomethane. Retention amount is 60μmol/g
It was hot. After thoroughly washing the adsorbent with physiological saline, it was dehydrated and used for the experiment. The anti-ds-DNA antibody titer was measured using an indirect fluorescent antibody method using Crithidia luciliae. The rest was carried out in the same manner as in Example 1. The results are shown in Table-2. Table 2 shows that deoxyribose 3,5-diphosphoric acid bound to a crosslinked copolymer mainly composed of vinyl alcohol units specifically and highly binds anti-double-stranded DNA antibodies, antinuclear antibodies, and immune complexes. It is clear that it adsorbs . We also measured the complement value before and after adsorption, and found that the decrease was only slight.

[Table] Example 3 The adsorbents of Examples 1 and 2 were placed in a container (2.5 ml each, 5 ml total) as shown in Figure 1, an adsorber was created, and an adsorption experiment was conducted using the experimental system shown in Figure 2. Ivy. That is, 20 ml of systemic lupus erythematosus patient plasma 10 is put into a container 9, and the pump 11 pumps the plasma every minute.
The tube 13 was arranged so that it was pumped out at a flow rate of 0.5 ml, sent to the adsorber 1, passed through the drip camber 14 and the sampling port 12, and returned to the container 9. After circulating plasma for 1 hour using the above device,
Plasma was sampled and protein components in the plasma were measured. The results are shown in Table-3.

【table】 [Brief explanation of the drawing]

FIG. 1 is a cross-sectional view showing an example of an adsorber using the adsorbent of the present invention, and FIG. 2 is an explanatory diagram of a model experiment in the example. 1... Adsorption remover, 2... Cylinder, 3, 3'... Filter, 4, 4'... Packing, 5, 5'... Body fluid inlet/outlet, 6, 6'... Cap, 7, 7'... Screw, 8...
Adsorbent, 9... Container, 10... Plasma, 11... Pump,
12...Sampling port, 13...Tube, 14...
Drippet yambar.

Claims (1)

[Scope of Claims] 1. At least one of a low molecular weight substance or a derivative thereof containing a purine base, a pyrimidine base, or a sugar phosphoric acid as a constituent on a carrier made of a crosslinked copolymer containing vinyl alcohol units as a main constituent.
An adsorbent for autoantibodies and immune complexes characterized by the binding of species. 2. The crosslinked copolymer containing vinyl alcohol units as a main component is a crosslinked polyvinyl alcohol obtained by hydrolyzing a copolymer of a vinyl ester of carboxylic acid and a vinyl compound having an isocyanurate ring. The adsorbent according to item 1.