JPH0113861B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an adsorbent for removing autoantibodies from blood, plasma, and other body fluid components (hereinafter collectively referred to as body fluids). More specifically, by specifically adsorbing and removing autoantibodies that frequently appear in the body fluids of autoimmune patients and are thought to be closely related to the cause and progression of the disease, The purpose of the present invention is to provide a simple adsorbent that purifies and regenerates body fluids, prevents the progression of diseases, alleviates symptoms, and accelerates healing.

Conventionally, autoimmune diseases such as systemic lupus erythematosus (SLE) and myasthenia gravis (MG) were considered intractable diseases, and no effective treatments were available. Recently, these autoimmune diseases have been treated with plasma exchange therapy, in which the patient's plasma containing antigens, antibodies, immune complexes, etc. is replaced with other fresh frozen plasma, and it has been confirmed that it can significantly prevent the progression of the disease, reduce symptoms, or cure the disease. I'm getting old.

However, plasma exchange therapy requires a large amount of fresh frozen plasma and cannot be widely used due to supply constraints. Because it uses someone else's plasma, it has fatal drawbacks such as a high risk of infection with Hb antigens.

Similarly, removal using an ultrafiltration membrane has been proposed as a method to non-specifically remove malignant substances, but it is difficult to separate sharply due to molecular weight, and relatively useful substances such as albumin are also removed. Furthermore, there were problems such as a decrease in overspeed due to clogging of the membrane, and fluctuations in cut-off molecular weight, so that it was not always sufficient.

In view of the problems of the conventional techniques for removing autoantibodies from body fluids as described above, the present inventors have developed a method that is simple, can be widely used, is safe, has high activity, and can be used to specifically remove autoantibodies from plasma, etc. As a result of intensive research aimed at providing an adsorbent that regenerates and purifies body fluids by adsorbing self-pores from body fluids, various substances were bound to hydrophilic carriers and the binding activity for autoantibodies was evaluated, and a truly surprising result was discovered. discovered that a hydrophilic carrier bound to sugars and/or oligosaccharides adsorbs autoantibodies with extremely high activity and specificity, and filed a patent application (Japanese Patent Application Laid-Open No. 134164/1983).

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 exhibit significant nonspecific adsorption of plasma proteins and, when used for whole blood, cause thrombus formation, so they are not used.

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,
To provide an adsorbent that highly actively and specifically adsorbs autoantibodies, maintains stable activity, is safe, can be easily sterilized, and is suitable for body fluid purification or regeneration. It is.

The present inventors have conducted research in line with the above objectives,
When sugars and/or oligosaccharides were bound to various carriers and their binding activity to autoantibodies, nonspecific adsorption of plasma proteins, and usability to whole blood were evaluated, it was surprisingly found that vinyl alcohol units were the main component. It was discovered that the polymer as a constituent component gives extremely good results as a carrier, and the present invention was completed.

That is, the present invention relates to an autoantibody adsorbent characterized in that sugars and/or oligosaccharides are bonded to a carrier made of a crosslinked polymer whose main component is vinyl alcohol units.

The carrier used in the present invention is required to have interaction characteristics and adsorption characteristics with plasma proteins, such as low non-specific adsorption of plasma proteins and not activating 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 such as dextran, agarose, and cellulose are used for blood cells,
Interacts with plasma components, activating the complement system and coagulation system. On the other hand, synthetic polymer carriers
It is said that these problems do not occur relatively.

The present inventors investigated synthetic polymer carriers and found that a carrier made of a polymer whose main constituent is vinyl alcohol units successfully solves the above-mentioned unresolved problems.

Due to its hydrophilic nature, the carrier made of a crosslinked polymer whose main constituent is vinyl alcohol units has a small 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 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 polymer of the present invention, the more its hydrophilicity increases, which is advantageous for minimizing interaction with blood components, and also increases the activity when activated with an activation reagent. This is preferable because the group density can be maintained at a high level, but on the other hand, the physical and mechanical strength decreases due to the relationship with 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. 1 g of dry carrier is 1 mmol
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 carboxylic acids such as vinyl acetate and vinyl propionate, and vinyl ethers such as methyl vinyl ether and ethyl vinyl ether.

As a crosslinking agent, allyl compounds such as triallyl isocyanurate and triallyl cyanurate, polyvalent (meth)acrylates such as ethylene glycol dimethacrylate, diethylene glycol dimethacrylate, and pentaerythritol di, tri, and tetraacrylate; Polyvinyl ethers such as butanediol divinyl ether, diethylene glycol divinyl ether, and tetravinylglyoxal, 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, triallylisocyanurate crosslinking 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. The body 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.

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
m can be used, but from the comparative area (adsorption capacity as an adsorbent) and the 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. 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 is crushed and the pores almost disappear.

In this case, the specific surface area is almost only the value on the outside of the particle, so the agarose conventionally known for affinity chromatography, which generally has a low value of 1 m ² /g or less, is a soft gel, so it cannot be dried. The pores disappear due to Therefore, it is not easy to sterilize, and furthermore, it has a soft mesh that is easily crushed, so even when packed in a column and used for extracorporeal circulation, body fluids cannot be passed through it at a high flow rate for a long period 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
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 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 union 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, the polymerization (W ₁ ) can be measured and determined by the following equation.

W _R = W ₁ - W ₂ /W ₂ (g/g) The exclusion limit molecular weight (protein) of the carrier is that the molecular weight of the self-carrier, which is the target adsorption substance of the present invention, is about 150,000 to 900,000, and The molecular weight of immune complexes generated from autoantibodies sometimes reaches 10 million, so 150,000 to 10 million is preferable. The most commonly used exclusion limit molecular weight for purposes of the present invention is 150,000 to 2,000,000.

The autoantibodies that are particularly targeted for removal in the present invention are autoantibodies against antigens present in solid phase in 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 produced polymers 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 are expected to react with various autoantibodies that use sugar chains as antigens.

To explain in more detail the autoantibodies against self-body components targeted by the present invention, anti-lymphocyte antibodies, anti-platelet antibodies, anti-erythrocyte antibodies, anti-acetylcholine receptor antibodies, anti-smooth muscle antibodies, anti-epidermal intercellular antibodies, anti- These are autoantibodies such as interstitial membrane antibodies, anti-proteoglycan antibodies, anti-collagen antibodies, anti-gastric factor antibodies, anti-thyroid microsomal antibodies, anti-microsomal antibodies, anti-colon antibodies and arterial antibodies, and are used regardless of their immunoglobulin class. be able to.

The sugars used in the present invention are monosaccharides and their derivatives that constitute glycolipids, glycoproteins, proteoglycans, etc. on the surface of body tissues and cells. Examples of monosaccharides include N-acetyl-D-glucosamine, N-
Hexosamines such as acetyl-D-galactosamine, hexoses such as D-galactose, D-mannose, D-glucose, 6-deoxyhexoses such as L-fucose and L-rhamnose, pentoses such as D-xylose and D-arabinose, N −
Sialic acids such as acetylneuraminic acid and N-glycolylneuraminic acid are used. These are alpha type,
Any β-type isomer 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, the present invention uses the above-mentioned sugars and/or oligosaccharides as agents against autoantibodies, and the sugars and/or oligosaccharides are used as agents for other substances such as lipids, proteins, polypeptides, amino acids, nucleic acids, and nucleobases. There is no problem even if the compound is bound to a carrier and allowed to act. as needed,
It is also possible to use two or more types of sugars and/or oligosaccharides bound to the same carrier.

Any known method such as covalent bonding, ionic bonding, physical adsorption, embedding, or precipitation and insolubilization on the carrier surface can be used to immobilize sugars or oligosaccharides on the surface of the carrier. Therefore, it is preferable to use it after being fixed and insolubilized by covalent bonding. Therefore, generally known methods for activating immobilized enzymes, carriers used in affinity chromatography, and methods for binding to sugars or oligosaccharides can be used. Furthermore, if necessary, a molecule (spacer) of arbitrary length can be introduced between the carrier and the sugar or oligosaccharide. For example, the hydroxyl group of agarose and the isocyanate group on one side of hexamethylene diisocyanate may be reacted and bonded, and the remaining isocyanate group may be reacted and bonded with the amino group, hydroxyl group, etc. of sugar or oligosaccharide. can.

The amount of sugar or oligosaccharide bound to the carrier in the present invention ranges from 0.1 mg to 30 mg per ml of carrier. More preferably, it is in the range of 0.5 mg to 15 mg. If the retained amount is less than 0.1 mg/ml, the amount of autoantibody adsorbed will be extremely reduced, and if it exceeds 30 mg/ml, the adsorption specificity will decrease, which is not preferable.

The autoantibody adsorbent of the present invention can be filled and held in a container equipped with a body fluid outlet and used as an adsorption device as shown in FIG.

In FIG. 1, reference numeral 1 shows an example of an autoantibody adsorption device using the autoantibody 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. A cap 6 having a body fluid inlet 5 is screwed into the cylinder 2, and a cap 8 having a body fluid outlet 7 is screwed into the opening at the other end of the cylinder 2 through a packing 4' having a filter 3' on the inside. A container is formed by filling and holding the adsorbent in the gap between the filters 3 and 3' to form an adsorbent layer 9.
It is formed by forming.

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 an autoantibody adsorption device 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.The plasma components are passed through the device, purified, and then separated into blood cells. One method is to return the blood to the body together with its components, and the other method is to directly pass blood taken from the body through the device for purification.

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 adsorbs and removes autoantibodies in body fluids at a high rate and specifically.
It is extremely compact, convenient 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, body fluids can flow through it at a high flow rate. In addition, it has heat resistance, so it can be sterilized using liquid flow 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,
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. Furthermore, since anti-sugar chain antibodies are likely to cause cross-reactions, the adsorbent of the present invention has the effect of adsorbing a wide range of anti-sugar chain autoantibodies against various solid-phase antigens.

The present invention is applicable to the general use of purifying and regenerating body fluids such as autologous plasma, and is applicable to safe and reliable treatment of diseases related to the body's immune function, particularly idiopathic thrombocytopenic purpura and hemolytic anemia. , pernicious anemia, myasthenia gravis, pemphigus, Hashimoto's disease, Graves' disease, Gutsudpasture syndrome, gastritis, systemic lupus erythematosus vasculitis, and other autoimmune diseases due to cross-reactions after infection, and rejection after organ transplantation. Effective in treating reactions.

Furthermore, the adsorbent of the present invention can be used not only as a therapeutic device by filling an apparatus, but also as an adsorbent for separating and purifying autoantibodies and as a base material for measuring these.

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
A homogeneous mixed liquid consisting of polyvinyl alcohol and
Weight%, sodium dihydrogen phosphate dihydrate 0.005
Weight% 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 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-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 3
×10 ⁵ , the exclusion limit molecular weight of protein is approximately 18 × 10 ⁶
It was hot. Also 0.3M sodium chloride and 0.1M
When a solution of human-gamma-globulin and human albumin was run through an aqueous solution containing sodium phosphate as a solvent, the recovery rate was almost 100%, and non-specific adsorption of the gel was extremely 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.

Separately, D-(+)-lactose (4-O-β-D-
β- to galactopyranosyl-D-glucose)
(p-aminophenyl)-ethylamine is reacted to form β-(p-aminophenyl)-ethylated D-(+).
-Lactose was synthesized. This was reacted and bonded to the above-mentioned activated gel by a conventional method ("Experiments and Applications of Affinity Chromatography", p. 46, published by Kodansha Scientists, 1976). The retained amount was 3.7 mg/ml gel in terms of D-(+)-lactose. Excess remaining active groups were blocked with ethanolamine. After blocking with ethanolamine, the adsorbent was mixed with PH4, 0.1M acetic acid buffer, PH8.5, 0.1M
After repeated washing with boric acid buffer, the sample was washed with physiological saline, drained, and used for an adsorption experiment.

The adsorption experiment was performed on systemic lupus erythematosus patient plasma 5
and 1 volume of adsorbent were mixed and incubated for 3 hours. Separately, a similar adsorption experiment was conducted using unactivated gel as a control. When handling these,
Hardly any chips or debris of the adsorbent were observed. The amount of anti-T cell antibodies in plasma after adsorption was determined to be
Measurement was performed using a fluorescent antibody method using 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 patient plasma before adsorption was 23.6%, control was 21.6%, and plasma treated with this adsorbent was 0.2%.
It was hot. Furthermore, the amount of globulin and albumin in each plasma was measured using an A/G test (A/GB test, Wako Pure Chemical Industries, Ltd.), but no major differences were found. Furthermore, complement C ₃ and C ₄ were measured by simple immunodiffusion method.
There were no major changes. From this, it is clear that the adsorbent bound to D-(+)-lactose specifically adsorbs anti-lymphocyte antibodies of systemic lupus erythematosus.

Furthermore, this adsorbent was sterilized in an autoclave and subjected to adsorption experiments, but no decrease in adsorption activity was observed due to sterilization.

Example 2 Same as Example 1 except that D-(+)-lactose was changed to D-(+)-galactose. The amount retained was 3.2 mg/ml gel.

The results showed that patient plasma before adsorption was 23.6%, control was 21.6%, and plasma treated with this adsorbent was 1.2%.
It was hot. It is clear from this that the adsorbent bound to D-(+)-galactose adsorbs anti-lymphocyte antibodies at a high rate.

Example 3 5 ml of the adsorbent of Example 1 was placed in a container as shown in FIG. 1 to prepare an apparatus for adsorbing anti-lymphocyte antibodies.
An anti-lymphocyte antibody adsorption experiment was conducted using the experimental system shown in FIG.

50 ml of systemic lupus erythematosus patient peripheral blood (with ACD) 11 is put into a container 10, pumped out at a flow rate of 10 ml per minute by a pump 12, and sent to an anti-lymphocyte antibody adsorption device 1, a drip chamber 15 and a sampling port 13. The tube 14 is arranged so that the liquid is returned to the container 10 through the air.

After circulating blood for 1 hour using the above device,
Blood was sampled, centrifuged to separate plasma, and anti-lymphocyte antibodies in the plasma were measured. The concentration was 23.6% for untreated plasma, while it was 0.4% for plasma using this adsorbent. Furthermore, there was almost no decrease in white blood cells and platelets before and after circulation.

Example 4 Preparation of adsorbent was carried out by converting D-(+)-lactose to D-
It is the same as Example 1 except for changing to (+)-mannose. The retained amount was 3.3 mg/ml gel in terms of D-(+)-mannose.

The adsorption experiment was the same as in Example 1 except that the plasma of a patient with systemic lupus erythematosus was changed to the plasma of a patient with myasthenia gravis. The amount of anti-acetylcholine receptor antibody in the plasma after adsorption was measured by the Con A method. The measurement method and conditions are as follows.

1 Fill the tip of the mini-column with cotton and add Con A-Sef Arrows (manufactured by Pharmacia, Sweden).
Fill 0.4ml of buffer (Triton
Wash with 4 ml of buffer (1000 to 0.2%).

2 Pre-incubate 250μ of a solution prepared by diluting the rat acetylcholine receptor compartment 50 times with buffer and 40μ each of healthy human plasma before and after adsorption at 4°C overnight.

3 Add 50μ (0.2 pmol) of ^125- labeled neurotoxin to the above pre-incubated solution and incubate at 37℃ for 4 hours.
time to react.

4 Pass the reaction solution through a previously prepared Con A column and wash the column twice with 2 ml of buffer.

5 Next, measure the radioactivity per column using a gamma counter.

The rat acetylcholine receptor compartment was prepared according to Almon's method (Science vol. 186,
p55, 1974), the neurotoxin was prepared by the method of Hayashi et al. (metabolic
15, p. 209, 1976), and ¹²⁵ I labeling was performed by the chloramine-T method of Hunter et al. (Handbook of Experimental Immunology, p. 17, 1973, published by Blackwell Scientific).

In this method, if there is an anti-acetylcholine receptor antibody in the plasma, the amount of ^125- labeled neurotoxin bound will be reduced, and the radioactivity of the Con A column will be reduced.
That is, the amount of anti-acetylcholine receptor antibody and radioactivity are inversely proportional.

When the results are taken as the radioactivity of healthy human plasma as 100%,
The plasma concentration before adsorption was 21%, the control unactivated gel-treated plasma was 32%, and the adsorbent of the present invention was 91%.

It is clear from this that the adsorbent to which D-(+)-mannose is bound adsorbs anti-acetylcholine receptor antibodies at a high rate.

Example 5 Preparation of adsorbent was carried out by converting D-(+)-lactose to D-
Same as Example 1 except for changing to (+)-glucose. The retained amount was 3.4 mg/ml gel in terms of D-(+)-glucose.

The adsorption experiment was the same as in Example 1, except that the plasma of a patient with systemic lupus erythematosus was changed to the plasma of a patient with Gutsudpasture syndrome. Measurement of anti-renal basement membrane antibodies in plasma after adsorption is performed by measuring anti-renal basement membrane antibodies that bind to the patient's renal basement membrane collected by biopsy using the amount of fluorescence from the fluorescently labeled anti-immunoglobulin antibodies bound to the antibodies. evaluated (fluorescent antibody method). The method and conditions are as follows.

1. After acid washing (PH2.5 glycine-hydrochloride buffer), 300 μg of renal basement membrane collected from a patient is suspended in 20 μg of phosphate buffered saline (PBS).

2 Add 20μ of test plasma to the suspension and incubate at 37°C for 2 hours.

3 After washing, rinse 3 times with PBS and resuspend at 20μ.

4 Add a solution of a fluorescently labeled anti-human immunoglobulin antibody (manufactured by Miles, lyophilized product, 100 mg, immunized to rabbits) in 2 ml of PBS to the above suspension.
Add 20μ and incubate at 37°C for 2 hours.

5 After washing, rinse three times with PBS and evaluate the fluorescence intensity using a fluorescence microscope.

As a result, a clear decrease in fluorescence intensity was observed in the plasma treated with the adsorbent of the present invention compared to the patient's plasma before the adsorbent and the non-activated gel treated plasma as a control.

[Brief explanation of drawings]

FIG. 1 is a sectional view showing an example of an autoantibody adsorption device using the autoantibody adsorbent of the present invention, and FIG. 2 is an explanatory diagram of a model experiment in an example. 1... Autoantibody adsorption 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. An adsorbent for autoantibodies, characterized in that sugars and/or oligosaccharides are bonded to a carrier made of a crosslinked polymer mainly composed of vinyl alcohol units. 2. The crosslinked polymer whose main component is vinyl alcohol units is 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.