JPH0622633B2 - Adsorbent and removal device using the same - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an anti-lipid antibody adsorbent for adsorbing and removing anti-lipid antibodies from a body fluid or for adsorbing and recovering anti-lipid antibodies, and an anti-lipid antibody removing apparatus using the same. .

[Problems to be Solved by Conventional Techniques and Inventions] Autoimmune diseases are diseases in which, as their name suggests, antibodies against constituent components of their own tissues (hereinafter referred to as autoantibodies) appear, but systemic lupus erythematosus ( In the cases of thrombosis, thrombocytopenia, and intrauterine fetal death, etc. in the following SLE) and related autoimmune diseases, an antibody (hereinafter referred to as an anti-lipid antibody) against the phosphorus finger component of the cell membrane is present in body fluids. It appears and is thought to be closely related to the condition. The mechanism by which the produced autoantibodies are involved in the onset of disease is not always clear, but the mechanism by which the autoantibodies themselves damage the lipids, or the autoantibodies bind to the antigen to form immune complexes and deposit in tissues Therefore, a mechanism that causes an organizational disorder has been proposed. In this case, the generated anti-lipid antibody binds to phospholipids on the platelet membrane and activates or inhibits its function to induce thrombosis or thrombocytopenia, respectively, and placental duct endothelium. A mechanism that binds to phospholipids on the cell membrane to inhibit blood circulation and induces fetal death has been proposed.

Since various symptoms are caused by the anti-lipid antibody thus produced or the immune complex of the antibody and phospholipid on the cell membrane, anti-lipid antibody control is very important for the treatment of the case. is important.

Conventionally, steroids, immunosuppressants, immunomodulators, anti-inflammatory agents and the like have been widely used for the treatment of SLE in order to suppress the production of anti-lipid antibodies. Of these, steroids are most commonly used, and short-term ultra-high dose therapy of steroids called bals treatment is also often used. However, since a steroid is likely to cause a side effect even when administered in a small amount, it is self-evident that a short-term super-high dose therapy of a steroid is likely to cause an even greater side effect. In addition, since these drugs are often used for a long period of time, side effects are more likely to occur in such cases, and it is often necessary to gradually increase the dose due to drug resistance. In many cases, it is impossible to use, or it does not exhibit sufficient effects. In particular, although the time of appearance of the case is the time when the suppression of anti-lipid antibodies is most necessary, it is often the case that a strong therapy using a pulse therapy or a drug such as an immunosuppressant cannot be adopted for the above reasons.

On the other hand, as an approach different from these drug therapies, attempts have been made to directly remove anti-lipid antibodies in body fluids by external circulation. The simplest method is so-called plasma exchange therapy, in which plasma of a patient containing anti-lipid antibody is replaced with plasma of a healthy person. By this method, the anti-lipid antibody in the blood is significantly reduced, and the symptoms are improved. However, this method is not widely used because it requires a large amount of healthy plasma and is not only expensive, but also involves a risk of infection such as serum hepatitis during the therapeutic treatment.

In plasma exchange therapy, all components in plasma are removed and exchanged with healthy plasma. On the other hand, in order to selectively remove an anti-lipid antibody that is a causative agent, the causative agent depends on the molecular size. A plasma separation membrane method for separating blood has been developed. In this method, plasma separates high-molecular-weight fraction and low-molecular-weight fraction with a membrane, discards the high-molecular-weight fraction containing the etiological agent, and removes the low-molecular-weight fraction containing albumin, which is a major protein. Although returned to the patient, the anti-lipid antibody is mainly IgG (immunoglobulin G) with a molecular weight of about 160,000, and because the molecular weight is close to that of albumin (molecular weight of about 60,000), the separation between the two is poor and the anti-lipid antibody is removed. When this is done, albumin is removed in a large amount, and further, all proteins having a molecular weight equal to or higher than that of the pathogenic substance are removed.

Therefore, it has been desired to develop a means for removing the anti-lipid antibody, which is a causative agent, more selectively, with almost no loss of other useful components in the body fluid.

Therefore, the present inventors have conducted intensive studies in view of such actual circumstances, as a result, found an adsorbent capable of selectively adsorbing only anti-lipid antibody with almost no loss of the active ingredient in the body fluid, and to complete the present invention. I arrived.

[Means for Solving the Problems] That is, the present invention relates to an anti-lipid antibody adsorbent comprising a water-insoluble porous material on which a compound having an anionic functional group is immobilized, and a container having a fluid inlet and outlet. A filter capable of passing a fluid and components contained in the fluid, but not an adsorbent of an anti-lipid antibody comprising a compound having an anionic functional group fixed to a water-insoluble porous body,
And an anti-lipid antibody removing device comprising an adsorbent of the anti-lipid antibody filled in the container.

[Examples] In the present specification, the term "body fluid" refers to blood, plasma, serum, ascites fluid, lymph fluid, synovial fluid, and fractionated components obtained from these,
And other liquid components derived from a living body.

The water-insoluble porous body used in the present invention preferably has continuous pores having a large diameter. That is, the anti-lipid antibody is composed of immunoglobulins such as IgG and IgM (immunoglobulin M) and is a macromolecule having a molecular weight of 160,000 to 900,000. Therefore, in order to efficiently adsorb it, the anti-lipid antibody is easily porous. It needs to be able to enter the body.

There are various methods for measuring the pore size, and the mercury porosimetry method is most often used, but it is difficult to apply it to hydrophilic porous bodies. The exclusion limit molecular weight is often used as an alternative measure of the pore size, and can be applied to both hydrophilic porous bodies and hydrophobic porous bodies. Exclusion limit molecular weight is as described in books such as Hiroyuki Hatano and Toshihiko Hanai, Experimental High Performance Liquid Chromatography, Kagaku Dojin, etc.
In gel permeation chromatography, it refers to the molecular weight of the substance having the smallest molecular weight among the molecules that cannot enter (exclude) in the pores.

It is known that the exclusion limit molecular weight differs depending on the target compound, and in general, globular proteins, dextran, polyethylene glycol, etc. have been well investigated,
It is appropriate to use the values obtained with globular proteins (including viruses) that appear to be most similar to anti-lipid antibodies.

As a result of examination using various water-insoluble porous materials having different exclusion limits, unexpectedly, even those having an exclusion limit molecular weight of about 100,000, which is smaller than the molecular weight of the anti-lipid antibody, show a certain adsorption capacity and a large pore size. It has been clarified that the ability is not so high, but rather the ability is reduced or proteins other than the anti-lipid antibody are adsorbed, that is, there is an optimum pore size range. That is, when a water-insoluble porous material having an exclusion limit molecular weight of less than 100,000 is used, the adsorption amount of the anti-lipid antibody is small and it cannot be used practically, but the exclusion limit molecular weight is 100,000 to 150,000 and the molecular weight of the anti-lipid antibody is Even if a water-insoluble porous material close to the above was used, an adsorbent that could be put to practical use was obtained to some extent. On the other hand, as the exclusion limit molecular weight increases, the adsorption amount of the anti-lipid antibody increases, but it eventually reaches the ceiling, and when the exclusion limit molecular weight is 60 million or more, the surface area is too small and the adsorption amount not only decreases remarkably Adsorption of components other than the anti-lipid antibody, that is, non-specific adsorption increases, and the selectivity remarkably decreases.

Therefore, the preferable exclusion limit molecular weight of the water-insoluble porous material used in the present invention is 100,000 or more and 60 million or less, and more preferably 60 or more 30 from the viewpoint of a larger selective adsorption capacity.
It should be less than, 000,000.

Next, with respect to the porous structure of the water-insoluble porous body, total porosity is preferable to surface porosity, and the pore volume is preferably 20% or more from the viewpoint of large adsorption capacity. The shape of the water-insoluble porous body may be any shape such as granular, spherical, fibrous, film-like, hollow fiber-like.
When using a particulate water-insoluble porous material, the pressure loss is large when the particle size is less than 1 μm, and the adsorption capacity is small when it exceeds 5000 μm.
The following is preferable.

The water-insoluble porous material used in the present invention may be either organic or inorganic, but it is preferable that the water-insoluble porous material has a small amount of adsorption of a body fluid component other than the target anti-lipid antibody (so-called non-specific adsorption). Since hydrophilicity is less likely to cause non-specific adsorption, the water-insoluble porous body is preferably hydrophilic rather than hydrophobic, and a water-insoluble porous body made of a compound having a hydroxyl group in the molecule is more preferable.

As a typical example of the water-insoluble porous body used in the present invention,
Agarose, dextran, soft porous materials such as polyacrylamide, porous glass, inorganic porous materials such as porous silica gel, synthetic polymers such as polymethylmethacrylate, polyvinyl alcohol, styrene, and -divinylbenzene copolymer and / or Examples thereof include, but are not limited to, porous polymer hard gels made from natural polymers such as cellulose.

When the adsorbent of the present invention is used for external circulation treatment, an anti-viscous fluid such as blood or plasma needs to flow at a high speed, and therefore a hard water-insoluble porous material having sufficient mechanical strength that does not cause consolidation. It is preferable to use the body. That is, the hard porous body, as shown in the following reference example, uniformly packed the water-insoluble porous body into a cylindrical column, the relationship between the pressure loss and the flow rate when flowing an aqueous fluid is at least 0.3 kg / c
It has a direct relationship up to m ² .

As the anionic functional group used in the present invention, any functional group can be used as long as it is negatively charged near neutral pH. Typical examples thereof include, but are not limited to, a carboxyl group, a sulfonic acid group, a sulfone group, a sulfate ester group, a thiol group, a silanol group, a phosphate ester group, and a phenolic hydroxyl group.

Of these, a carboxyl group, a sulfonic acid group, a sulfuric acid ester group, a thiol group and a phosphoric acid ester group are preferred because of their strong affinity for anti-lipid antibodies.

A compound having an anionic functional group is 1 in the molecule.
It may be a monoanion compound having one anionic functional group or a polyanion compound having a plurality of anionic functional groups. The polyanion compound is preferable because it has a high affinity for the anti-lipid antibody and it is easy to introduce many anionic functional groups into the unit amount of the porous body. Among them, a polyanion compound having a molecular weight of 1000 or more is preferable in terms of affinity and the amount of anionic functional group introduced. The polyanion compound may have one type of anionic functional group or two types.

As a typical example of the polyanion compound used in the present invention,
Synthetic polyanion compounds such as polyacrylic acid, polyvinyl sulfuric acid, polyvinyl sulfonic acid, polyvinyl phosphoric acid, polystyrene sulfonic acid, polystyrene phosphoric acid, polyglutamic acid, polyaspartic acid, polymethacrylic acid, polyphosphoric acid, and styrene-maleic acid copolymer, Examples thereof include, but are not limited to, anionic functional group-containing polysaccharides such as heparin, dextran sulfate, chondroitin, chondroitin sulfate, phosphomannan, chitin and chitosan.

The compound having an anionic functional group fixed to the adsorbent of the present invention may be one type or two or more types.

The adsorbent of the present invention refers to a state in which a compound having an anionic functional group is immobilized on a water-insoluble porous body. There are various methods for introducing an anionic functional group into the adsorbent in order to obtain a fixed state of the compound having such an anionic functional group, and any method may be used. Is (1) a method of forming an adsorbent by polymerization using a compound containing an anionic functional group or a functional group that can be easily converted to an anionic functional group as a monomer or a crosslinking agent, (2) containing an anionic functional group (3) a compound having an anionic functional group in the water-insoluble porous body by directly reacting the compound forming an anionic functional group with the water-insoluble porous body There is a method of fixing.

Of course, an anionic functional group-containing compound originally containing an anionic functional group such as glass, silica, or alumina may be used as the adsorbent.

Representative examples of the monomer or cross-linking agent containing an anionic functional group or a functional group that can be easily converted to an anionic functional group used in the method (1) include acrylic acid and its ester, methacrylic acid and its ester,
Examples thereof include styrene sulfonic acid, but are not limited thereto.

The method of (2), that is, the method of fixing the compound containing an anionic functional group to the water-insoluble porous body, there is a method by physical adsorption, a method by ionic bond, a method by covalent bond, etc. Although a method may be used, in order to use the adsorbent for therapeutic purposes, it is important that the anionic functional group-containing compound does not leave during sterilization or during treatment. preferable.

When the anionic functional group-containing compound is immobilized by a covalent bond, the anionic functional group-containing compound preferably has a functional group that can be used for immobilization in addition to the anionic functional group.

A typical example of a functional group that can be used for immobilization is an amino group,
Examples thereof include, but are not limited to, an amide group, a carboxyl group, an acid anhydride group, a succinylimide group, a hydroxyl group, a thiol group, an aldehyde group, a halogen group, an epoxy group, and a silanol group.

There are many anionic functional group-containing compounds having these functional groups, but sulfanilic acid, 2-aminoethyl hydrogen sulfate, terephthalic acid, phosphorylethanolamine, glycol 6-phosphate, ethanedithiol, etc. described in the examples are This is an example.

Further, among the compounds having an anionic functional group, typical examples of the compound having a sulfuric acid ester group include a sulfuric acid ester of a hydroxyl group-containing compound such as alcohol, sugar and glycol. Among these, polyhydric alcohols are also included. Partial sulfate esterified product, especially the sulfated esterified product of sugar contains both the sulfate ester group and the functional group necessary for immobilization, and has high biocompatibility and activity,
Furthermore, sulfated polysaccharides are particularly preferable because they can be easily immobilized on the water-insoluble porous body.

Next, the method of (3), that is, by reacting a compound forming an anionic functional group with a water-insoluble porous body, by fixing a compound having an anionic functional group to the water-insoluble porous body by anionic functional group A typical example of the method of introducing a group is a reaction of introducing a sulfate ester group into a hydroxyl group-containing porous body. In this case, the sulfate group can be directly introduced by reacting the hydroxyl group-containing water-insoluble porous material with a reagent such as chlorosulfonic acid or concentrated sulfuric acid.

The amount of anionic functional groups introduced is preferably 0.01 μmol or more and 10 mmol or less per 1 ml of the adsorbent. 0.01 μmol
If it is less than 10 mmol, the adsorption capacity is not sufficient, and if it exceeds 10 mmol, non-specific adsorption is too much to make practical use difficult. More preferable amount of anionic functional group introduced is 1 μmol
It is preferably 100 μmol or more.

There are various methods for removing an anti-lipid antibody from a body fluid using the adsorbent of the present invention, and any method may be used, but a container having a fluid inlet and outlet, a fluid and components contained in the fluid Through an anti-lipid antibody adsorbent in which a compound having an anionic functional group is immobilized on a water-insoluble porous material, but not through a filter, and the anti-lipid antibody adsorbent filled in the container. The method of passing the body fluid through the anti-lipid antibody removing device is simple and preferable.

FIG. 2 shows a schematic sectional view of an embodiment of the anti-lipid antibody removing apparatus of the present invention. In FIG. 2, (1) and (2) are inlets and outlets of respective fluids, (3) is the adsorbent of the present invention, (4) and (5) are fluids and components contained in the fluids. A filter or a mesh that can pass but not the adsorbent of the present invention, (6) is a column, and (7) is a container. Here, the filter (4) on the fluid inlet side may not be present.

Hereinafter, the present invention will be described in more detail with reference to Examples.
The invention is not limited to only such embodiments.

Reference example Agarose gel Bioge on a glass column (9 mm inner diameter, 150 mm column length) equipped with filters with a pore size of 15 μm on both ends.
l A5m (Brand name, Bio-Rad, particle size 50-100 mesh), gel made of synthetic polymer, Toyopearl HW65 (Brand name, Toyo Soda Co., Ltd., particle size 50-100 μm), and porous cellulose gel , Cellulofine GC-700 (trade name, manufactured by Chisso Co., Ltd., particle size 45 to 100 μm) are evenly filled, and water is circulated in the column by a peristaltic pump to show the relationship between the flow rate and the pressure loss ΔP. I asked. The results are shown in FIG. As is clear from the figure, the agarose gel, which is a soft gel, causes consolidation at a certain flow rate or higher, and the flow rate does not increase even if the pressure is increased.
For hard gels such as Toyopearl and Cellulofine, the flow rate increases almost in proportion to the increase in pressure.

Production Example 1 CK gel A3 (trade name, Chisso) which is a porous cellulose gel
Co., Ltd., exclusion limit molecular weight of globular protein 50 million, particle size 63 ~
125 μm) 60 ml of water and 80 ml of 2M NaOH are added to 100 ml, and the temperature is 45 ° C.
It was stirred for 1 hour. After stirring, add epichlorohydrin
After adding 25 ml and stirring at 45 ° C. for 2 hours, the reaction was completed, and the gel was filtered and washed with water to obtain an epoxidized cellulose gel (hereinafter referred to as an epoxidized gel).

Example 1 Sulfanilic acid was added to 10 ml of the epoxidized gel obtained in Production Example 1.
Add 0.14 g dissolved in 7.5 ml of water and add 2M Na
After adjusting the pH of the solution to 10 by adding OH, the solution was left at 45 ° C for 20 hours. After the reaction was completed, the gel was separated by filtration and washed with water to obtain a cellulose gel having sulfanilic acid immobilized thereon. The amount of anionic functional group introduced by the immobilized sulfanilic acid was 10 μmol per 1 ml of the adsorbent.

Example 2 2-aminoethyl hydrogensulfate 0.11 instead of sulfanilic acid
A cellulose gel having 2-aminoethyl hydrogen sulfate immobilized thereon was obtained in the same manner as in Example 1 except that g was used. The amount of anionic functional groups introduced by the immobilized 2-aminoethyl hydrogen sulfate was 10 μmol per 1 ml of the adsorbent.

Example 3 Phosphorylethanolamine instead of sulfanilic acid
Except for using 0.11 g, the same procedure as in Example 1 was carried out.
A cellulose gel having phosphorylethanolamine immobilized thereon was obtained. The amount of anionic functional groups introduced by the immobilized phosphorylethanolamine was 10 per 1 ml of the adsorbent.
It was μmol.

Example 4 1,2-ethanedithiol 0.08 g instead of sulfanilic acid
A 1,2-ethanedithiol-immobilized cellulose gel was obtained in the same manner as in Example 1 except that The amount of anionic functional groups introduced by the immobilized 1,2-ethanedithiol was 10 μmol per 1 ml of the adsorbent.

Example 5 10 ml of the epoxidized gel obtained in Production Example 1 had a molecular weight of about 5,000,
5 g of dextran sulfate sodium with 18% sulfur content and 8 ml of water were added, and 2M NaOH was further added to adjust the pH of the solution to 1
After adjusting to 0, it was left at 45 ° C. for 17 hours. After completion of the reaction, the gel was separated by filtration, washed with water, added with 0.5% aqueous monoethanolamine solution and allowed to stand at room temperature for 20 hours to seal unreacted epoxy groups. After completion of the reaction, the gel was separated by filtration and washed with water to obtain a cellulose gel on which dextran sodium sulfate was fixed. The amount of anionic functional group introduced by the immobilized dextran sulfate was 29 μmol per 1 ml of the adsorbent.

Production Example 2 To 40 ml of the epoxidized gel obtained in Production Example 1, a solution prepared by dissolving 0.5 g of ethylenediamine in 30 ml of water was added, and the mixture was left at 45 ° C for 20 hours. After completion of the reaction, the gel is filtered and washed with water to introduce an amino group into a cellulose gel (hereinafter referred to as an aminated gel)
I got it.

Example 6 To 10 ml of the aminated gel obtained in Preparation Example 2 was added 10 ml of water and subsequently 1 g of glucose 6-barium phosphate salt, and
0.5 ml of 2M NaOH was added and the mixture was kept at 45 ° C for 1 hour. To this NaB
H ₄ 0.1 g was added and left at room temperature for 24 hours. After completion of the reaction, the gel was filtered and washed with water to obtain a cellulose having glucose 6-phosphate immobilized. The amount of anionic functional groups introduced by immobilized glucose 6-phosphate is 10 μmol per 1 ml of adsorbent.
Met.

Example 7 10 ml of the aminated gel obtained in Production Example 2 was suspended in N, N-dimethylformamide (DMF) to make the total volume 18 ml. After dissolving 0.27 g of terephthalic acid in it, the condensation reagent (N, N '
-Dicyclohexylcarbodiimide) at room temperature
It was stirred for 24 hours. After completion of the reaction, the gel was filtered off and washed with DMF, ethanol and water in this order to obtain a terephthalic acid-immobilized cellulose gel. The amount of anionic functional groups introduced by fixed terephthalic acid is 10 μmo per 1 ml of adsorbent.
It was l.

Example 8 To 10 ml of the aminated gel obtained in Preparation Example 2 was added 5 g of dextran sodium sulfate of the same kind as used in Example 5 and 8 ml of water, and 0.5 ml of 2M NaOH was further added to the mixture at 45 ° C. for 1 hour.
Kept at. NaBH ₄ 0.1g was added to this and it was left to stand at room temperature for 24 hours. After completion of the reaction, the gel was separated by filtration and washed with water to obtain a cellulose gel having dextran sodium sulfate immobilized thereon. The amount of anionic functional group introduced by the immobilized dextran sulfate was 39 μmol per 1 ml of the adsorbent.

Production Example 3 40 ml of the same kind of porous cellulose gel (CK gel A3) as used in Production Example 1 was suspended in heptane to make the total volume 70 ml. To this, 10 ml of 20% NaOH and 40 drops of the nonionic surfactant Tween 20 (trade name, manufactured by Bio-Rad) were added and shaken at 40 ° C. for 30 minutes. Then epichlorohydrin 10
ml was added and shaken at 40 ° C. for 6 hours. After completion of the reaction, the gel was filtered off and washed with ethanol and water in this order to obtain an epoxidized gel.

Example 9 A cellulose gel on which dextran sodium sulfate was immobilized was obtained in the same manner as in Example 5 except that 10 ml of the epoxidized gel obtained in Production Example 3 was used instead of the epoxidized gel obtained in Production Example 1. The amount of anionic functional group introduced by the immobilized dextran sulfate was 36 μmol per 1 ml of the adsorbent.

Example 10 To 10 ml of the epoxidized gel obtained in Production Example 3, 1 g of polyacrylic acid having an amino group at one end (molecular weight: about 1000) was added to water 5
What was melt | dissolved in ml was added, 1M of 2M NaOH was added to this, and it left at room temperature for 48 hours. After completion of the reaction, the gel was separated by filtration and washed with water to obtain a cellulose gel having polyacrylic acid fixed thereon. The amount of anionic functional groups introduced by the immobilized polyacrylic acid was 560 μmol / ml of the adsorbent. Polyacrylic acid having an amino group at one end is obtained by a low polymerization reaction of acrylic acid using 2-aminoethanethiol as a chain transfer agent and α, α'-azobisisobutyronitrile (AIBN) as an initiator. Was used (Journal of the Chemical Society of Japan, 1977,
No. 1, pages 88 to 92, "Synthesis of 2-hydroxyethyl methacrylate-styrene-based ABA type block copolymer and its structure and wetting", Mitsuo Okano, et al.).

Example 11 Polyacrylic acid having an amino group at one end (molecular weight of about 10
A cellulosic gel on which polyacrylic acid was immobilized was obtained in the same manner as in Example 10 except that 1 g of polyacrylic acid having an amino group at one end (molecular weight of about 10,000) was used instead of 00). The amount of anionic functional groups introduced by the immobilized polyacrylic acid was 5.6 mmol per 1 ml of the adsorbent. Polyacrylic acid having an amino group at one end was synthesized according to a method similar to the method described in Example 10.

Production Example 4 An aminated gel was obtained in the same manner as in Production Example 2 except that 10 ml of the epoxidized gel obtained in Production Example 3 was dissolved in 6 ml of water containing 0.12 g of ethylenediamine.

Example 12 A cellulose gel having dextran sodium sulfate immobilized thereon was obtained in the same manner as in Example 8 except that 10 ml of the aminated gel obtained in Production Example 4 was used instead of the aminated gel obtained in Production Example 2. . The amount of anionic functional group introduced by the immobilized dextran sulfate was 76 μmol per 1 ml of the adsorbent.

Production Example 5 CK gel A22 (trade name, manufactured by Chisso Co., Ltd., a porous cellulose gel, molecular weight exclusion limit of spherical protein 30 million, particle size 53)
Add 10 ml of water and 30 ml of 2M NaOH to 25 ml of ~ 125 μm, and add 40
Shake for 20 minutes at ° C. 12 ml of epichlorohydrin was added to this and shaken at 40 ° C. for 3 hours. After completion of the reaction, the gel was filtered and washed with water to obtain an epoxidized gel.

Production Example 6 CK gel A32 which is a porous cellulose gel (trade name, manufactured by Chisso Corporation, exclusion limit molecular weight of globular protein is 20 million, particle size is 53)
An epoxidized gel was obtained in the same manner as in Production Example 5 except that 15 ml of water, 21 ml of 2M NaOH and 7.1 ml of epichlorohydrin were used in 25 ml of .about.125 .mu.m).

Production Example 7 Cellulofine GCL-2000m which is a porous cellulose gel
(Trade name, manufactured by Chisso Co., Ltd., molecular weight exclusion limit for globular proteins)
3 million, particle size 44-105 μm) 25 ml, water 25 ml, 2M NaOH 15 ml
And Production Example 5 except that 5 ml of epichlorohydrin was used
An epoxidized gel was obtained in the same manner as in.

Production Example 8 Cellulofine GCL-1000m which is a porous cellulose gel
(Trade name, manufactured by Chisso Co., Ltd., molecular weight exclusion limit for globular proteins)
600,000, particle size 44 ~ 105μm) 25ml, water 25ml, 2M NaOH 11.75
Epoxidized gel was obtained in the same manner as in Production Example 5, except that 4 ml of epichlorohydrin and 4 ml of epichlorohydrin were used.

Production Example 9 Cellulofine GC-700m which is a porous cellulose gel
(Trade name, manufactured by Chisso Co., Ltd., molecular weight exclusion limit for globular proteins)
400,000, particle size 44-105μm) 25ml, water 25ml, 2M NaOH 8.75m
An epoxidized gel was obtained in the same manner as in Production Example 5, except that 1 ml of epichlorohydrin and 3 ml of epichlorohydrin were used.

Production Example 10 Cellulofine GC-200m which is a porous cellulose gel
(Trade name, manufactured by Chisso Co., Ltd., molecular weight exclusion limit for globular proteins)
Production Example 5 except that 120,000, particle size 44 to 105 μm) 25 ml, water 25 ml, 2M NaOH 7 ml and epichlorohydrin 2.5 ml were used.
An epoxidized gel was obtained in the same manner as in.

Example 13 To 20 ml of the epoxidized gel (CK gel A22) obtained in Production Example 5,
10 g of the same type of dextran sulfate sodium as used in Example 5 and water were added to bring the total volume to 33 ml. The pH of the solution was adjusted to 10.0 by adding 2M NaO, and the mixture was left standing at 45 ° C for 17 hours. After completion of the reaction, the gel was separated by filtration, washed with water, added with 0.5% aqueous monoethanolamine solution and allowed to stand at room temperature for 20 hours to seal unreacted epoxy groups. After completion of the reaction, the gel was separated by filtration and washed with water to obtain a cellulose gel having dextran sodium sulfate immobilized thereon. The amount of anionic functional group introduced by the immobilized dextran sulfate was 31 μmol per 1 ml of the adsorbent.

Example 14 To 20 ml of the epoxidized gel (CK gel A32) obtained in Production Example 6,
Dextran sulfate sodium was prepared in the same manner as in Example 13 except that 10 g of dextran sulfate sodium of the same kind as that used in Example 5 and water were added to make the total volume 36 ml, and the pH of the solution was adjusted to 9.9. A fixed cellulose gel was obtained. The amount of anionic functional groups introduced by the immobilized dextran sulfate was 27 per 1 ml of the adsorbent.
It was μmol.

Example 15 To 20 ml of the epoxidized gel (GCL-2000m) obtained in Production Example 7,
Dextran sulfate sodium was added in the same manner as in Example 13 except that 9.3 g of dextran sulfate sodium of the same type as that used in Example 5 and water were added to make a total volume of 35 ml, and the pH of the solution was adjusted to 9.3. A cellulosic gel was obtained. The amount of anionic functional groups introduced by the immobilized dextran sulfate was 30 per 1 ml of the adsorbent.
It was μmol.

Example 16 To 20 ml of the epoxidized gel (GCL-1000m) obtained in Production Example 8,
Dextran sulfate sodium was added in the same manner as in Example 13 except that 9.3 g of dextran sulfate sodium of the same type as that used in Example 5 and water were added to make a total volume of 35 ml, and the pH of the solution was adjusted to 9.3. A cellulose gel in which was fixed was obtained. The amount of anionic functional group introduced by the immobilized dextran sulfate was 1 ml of the adsorbent.
It was 30 μmol.

Example 17 To 20 ml of the epoxidized gel (GC-700m) obtained in Production Example 9, 9.0 g of sodium dextran sulfate of the same kind as used in Example 5 and water were added to bring the total volume to 36 ml, and the pH of the solution was adjusted. A cellulose gel having dextran sodium sulfate immobilized thereon was obtained in the same manner as in Example 13 except that the concentration was adjusted to 9.3. The amount of anionic functional groups introduced by the immobilized dextran sulfate was 30 per 1 ml of the adsorbent.
It was μmol.

Example 18 To 20 ml of the epoxidized gel (GC-200m) obtained in Production Example 10, 9.0 g of sodium dextran sulfate of the same kind as used in Example 5 and water were added to make the total volume 36 ml, and the pH of the solution was adjusted. A cellulose gel having dextran sodium sulfate immobilized thereon was obtained in the same manner as in Example 13 except that the concentration was adjusted to 9.2. The amount of anionic functional groups introduced by the immobilized dextran sulfate was 32 per 1 ml of the adsorbent.
It was μmol.

Example 19 The adsorbents obtained in Examples 1 to 12 were treated with 0.5M phosphate buffer (pH
After washing with 7.4), 0.1 ml of each adsorbent was placed in a polypropylene microtube (volume: 1.5 ml), and 0.5 M phosphate buffer (pH 7.4) was added to make the total volume 1 ml. 0.2 ml of serum containing anti-lipid antibody was added thereto and shaken at 37 ° C. for 2 hours. After completion of this adsorption operation, the gel was precipitated by centrifugation, and the anti-lipid antibody titer in the collected supernatant was measured by enzyme-linked immunosorbent assay (ELISA method). The anti-lipid antibody titer was determined by adding a diluted sample to a cardiolipin-coated plate, performing an antigen-antibody reaction, adding a peroxidase-labeled anti-human immunoglobulin antibody, and performing an enzyme color reaction CS-930.
(Trade name, manufactured by Shimadzu Corporation). Table 1 shows the compound name having an anionic functional group fixed to each adsorbent, and the anti-lipid antibody titer in the supernatant after the adsorption operation with respect to the anti-lipid antibody titer in the original serum of each adsorbent as a percentage. Is shown as a relative antibody titer.

It can be seen from Table 1 that the adsorbent having dextran sulfate or polyacrylic acid immobilized thereon has a particularly excellent anti-lipid antibody adsorption capacity.

Example 20 The relative antibody titer was determined in the same manner as in Example 19 except that the adsorbents obtained in Examples 5 and 13 to 18 were used and the added serum amount was 0.15 ml. The obtained results are shown in Table 2 together with the names of various water-insoluble porous materials.

From Table 2, it can be seen that the adsorbents using Cellufine GC-700m and Cellufine GC-200m, which are porous bodies with exclusion molecular weights of 400,000 or less, have a slightly higher anti-lipid antibody adsorption capacity. On the contrary, it can be seen that the anti-lipid antibody adsorbing capacity tends to decline even if the exclusion limit molecular weight is too high, at 50 million.

[Advantages of the Invention] The adsorbent of the present invention and the removal device using the same have the effect of selectively removing anti-lipid antibodies from body fluids.

[Brief description of drawings]

FIG. 1 is a graph showing the results of investigating the relationship between flow velocity and pressure loss using three types of gels, and FIG. 2 is a schematic cross-sectional view of an embodiment of the anti-lipid antibody removing device of the present invention. is there. (Main symbols in the drawing) (1): Inlet (2): Outlet (3): Adsorbent (4), (5): Filter (7): Container

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location G01N 33/53 S 8310-2J 33/543 Z 9217-2J

Claims (5)

[Claims] 1. An adsorbent for an anti-lipid antibody comprising a compound having an anionic functional group immobilized on a water-insoluble porous body. 2. The anionic functional group comprises at least one selected from the group consisting of a sulfuric acid ester group, a sulfonic acid group, a carboxyl group, a thiol group and a phosphoric acid ester group. An adsorbent of the described anti-lipid antibody. 3. The adsorbent for an anti-lipid antibody according to claim 1, wherein the compound having an anionic functional group is a polyanion compound having a plurality of anionic functional groups in one molecule. 4. The adsorbent for an anti-lipid antibody according to claim 1, wherein the water-insoluble porous body comprises a compound having a hydroxyl group. 5. A container having a fluid inlet and a fluid outlet,
The fluid and the components contained in the fluid can pass through, but the adsorbent of the anti-lipid antibody obtained by immobilizing the compound having an anionic functional group on the water-insoluble porous body cannot pass through the filter, and the filter is filled in the container. An anti-lipid antibody removing device comprising an adsorbent of the anti-lipid antibody.