JPH10328565A - Immunity complex removing device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enable effectively adsorbing immunity complexes in body fluid only by housing in a vessel an adsorbing body for immunity complexes in which specified polyanion compounds (except protein) are fixed on a water insoluble carrier and filters for preventing the adsorbing body from passing through the vessel. SOLUTION: In a vessel 7 in which an inflow port 1 and an outflow port 2 are formed, an adsorbing body 3 is housed, and also filters 4, 5 for preventing the adsorbing body 3 from passing through the vessel 3 are arranged on both the ends thereof. As the adsorbing body 3, an immunity complex in which polyanion compounds of 1000 or more molecular weight (except protein) are fixed on a water insoluble porous carrier is used. The water insoluble porous carrier is preferably that of 400,000 to 60,000,000 replacement limit molecular weight. And the water insoluble porous carrier is preferably formed of compounds having hydroxyl groups. More preferably, on the surface of the water insoluble porous carrier, a functional group used for ligand fixing reaction is made to exist. As the functional group, for example, a hydroxyl group and an amino group are given.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a removing device for removing harmful components from body fluids by adsorption. More specifically, the present invention relates to a removing device for removing an immune complex from a body fluid and suppressing an autoimmune disease.

[0002]

BACKGROUND OF THE INVENTION There are numerous types of viruses, bacteria,
Infectious foreign substances such as molds and parasites are present. Either of these can cause disease in living organisms, and if they grow in the living body without restriction, they will eventually kill the living organism. Therefore, the body has an immune system as a function to protect it. Antibody molecules play a major role in this immune system. That is, when foreign substances such as viruses and bacteria become antigens and stimulate the immune system, antibody molecules that selectively bind to the antigens are produced.

On the other hand, in an autoimmune disease, a humoral or cellular immune response occurs not to an external organism but to an antigen of its own cell or tissue, and an antibody against the self (hereinafter referred to as autoantibody) or an antigen-antibody is produced. A large amount of complexes (hereinafter referred to as immune complexes) formed by reacting specifically with each other are generated. The immune complex in this body fluid is not processed by the physiological exclusion mechanism, but is deposited on tissues such as the glomeruli, synovium, lungs, and blood vessel walls, and directly leads to the appearance of pathological conditions characteristic of autoimmune diseases. Often involved.

[0004] In a typical autoimmune disease, systemic lupus erythematosus (hereinafter, referred to as SLE), a nuclear component of cells,
In particular, antibodies against deoxyribonucleic acid (hereinafter, referred to as DNA) (hereinafter, referred to as anti-DNA antibodies) appear in body fluids, and the autoantibodies themselves bind to antigens to form immune complexes and deposit on tissues, thereby causing tissue damage. A mechanism has been proposed. In the case of SLE, the generated anti-DNA antibody forms an immune complex with the DNA derived from the cells also spilled into the blood and deposits on the blood vessel wall and renal glomerular basement membrane, causing vasculitis and lupus nephritis, respectively. In fact, there are many cases where SLE dies due to renal failure.

[0005] Since various symptoms are caused by the immune complex of the autoantibody thus produced and the corresponding antigen, control of the immune complex is very important for treatment.

Hitherto, steroids, immunosuppressants, immunomodulators, anti-inflammatory agents and the like have been widely used for the treatment of SLE for the purpose of suppressing the production of immune complexes. Among them, steroids are the most commonly used, and a short-term ultra-high dose therapy of steroids called pulse therapy is also often performed. However, since steroids are liable to cause side effects even in small doses, it is obvious that short-term ultra-high dose steroid therapy tends to cause even greater side effects. In addition, these drugs are often used for a long period of time, and in such cases, side effects are more likely to occur, and in some cases, the dosage must be increased gradually due to drug resistance. There are many cases where it is impossible or does not exert a sufficient effect. Especially during the active period of SLE, anti-DN
In spite of the time when suppression of A-antibodies and immune complexes is the most necessary, there are many cases where pulse therapy or powerful therapy using drugs such as immunosuppressive drugs 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 immune complexes in body fluids by extracorporeal circulation. The simplest method is the so-called plasma exchange therapy, which replaces the plasma of a patient containing an immune complex with the plasma of a healthy person. By this method, the immune complex in the blood is greatly reduced, and symptoms have been improved. However, this method requires a large amount of healthy plasma and is not only expensive, but also has not been widely used due to the risk of infection such as serum hepatitis during the treatment.

[0008] In plasma exchange therapy, all components in the plasma are removed and replaced with healthy plasma. On the other hand, in order to selectively remove immune complexes that are pathogenic substances, the molecular size is reduced. Has developed a plasma separation membrane method for separating pathogenic substances. In this method, plasma is separated by a membrane into a high molecular weight fraction and a low molecular weight fraction, the high molecular weight fraction containing the pathogenic substance is discarded, and the low molecular weight fraction containing albumin, the main protein, is separated. Return to the patient, but the immune complex
The main component is a complex of IgG (immunoglobulin G) having a molecular weight of about 160,000 and an antigen, and its molecular weight distribution is wide. Therefore, separation from albumin, normal IgG and IgM (immunoglobulin M) is not always sufficient. However, there are drawbacks such as removal of immune complexes in a large amount when removing immune complexes, and further removal of all proteins having a molecular weight equal to or higher than the pathogenic substance.

[0009] Therefore, there has been a demand for a means for removing immune complexes, which are pathogenic substances, more selectively and with little loss of useful components in body fluids.

[0010]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies in view of such circumstances, and as a result, have found that immunocomplexes capable of selectively adsorbing almost only immunocomplexes almost without losing active ingredients in body fluids. We have found a body removal device and have completed the present invention.

That is, the present invention relates to a container having a fluid inlet and a fluid outlet, a fluid and components contained in the fluid can pass therethrough, but a water-insoluble porous carrier allows a polyanion compound having a molecular weight of 1,000 or more, except for proteins, A filter through which the adsorbent of the immune complex in which is immobilized cannot pass;
And a device for removing an immune complex comprising an adsorbent of the immune complex filled in the container.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION In the present specification, a body fluid refers to blood, plasma, serum, ascites, lymph, intra-articular fluid and fraction components obtained therefrom, and other liquid components derived from living organisms.

In the present invention, the water-insoluble porous carrier is
Polyanion compounds having a molecular weight of 1,000 or more, excluding proteins, are substances that have the property of being insoluble in water for fixing. The water-insoluble porous carrier used in the present invention preferably has large pores of continuous fine pores. That is, the immune complex is mainly composed of IgG and an antigen, and has a molecular weight of
It is expected that some of the macromolecules have a molecular weight of around 1,000,000 or more, and it is necessary that the immune complex can easily enter the porous body in order to adsorb them efficiently. It is.

There are various methods for measuring the pore diameter, and the mercury intrusion method is most often used, but it is difficult to apply the method to a hydrophilic porous body. Exclusion limit molecular weight is often used as a measure of pore diameter instead of this, hydrophilic porous material,
It can be applied to any of the hydrophobic porous bodies. As described in a compendium (eg, Hiroyuki Hatano, Toshihiko Hanai, Experimental High Performance Liquid Chromatography, Chemical Doujinshi) etc., molecules that cannot enter (exclude) pores in gel permeation chromatography Means the molecular weight of the substance having the smallest molecular weight.

It is known that the exclusion limit molecular weight varies depending on the target compound. Generally, globular proteins, dextran, polyethylene glycol and the like have been well examined. It is appropriate to use the values obtained with globular proteins which appear to be similar.

As a result of examination using various water-insoluble porous carriers having different exclusion limits, it was found that the range of pore diameters suitable for the adsorption of the immune complex was from 400,000 to 60,000,000. That is, when a water-insoluble porous carrier having an exclusion limit molecular weight of less than 400,000 is used, the adsorbed amount of the immune complex is small and is not practical. On the other hand, as the exclusion limit molecular weight increases, the amount of adsorbed immune complex increases, but eventually plateaus.If the exclusion limit molecular weight exceeds 60 million, the surface area is too small and the amount of adsorption is not only remarkably reduced, but also the target Adsorption of non-immunocomplexes, ie, non-specific adsorption, increases and the selectivity drops significantly.

Therefore, the water-insoluble porous carrier used in the present invention has a preferable exclusion limit molecular weight of from 400,000 to 60,000, and more preferably from 600,000 to 20,000,000 in view of a larger selective adsorption capacity. Good.

Next, the porous structure of the water-insoluble porous carrier is preferably total porosity rather than surface porosity, and the pore volume is desirably 20% or more from the viewpoint of a large adsorption capacity. The shape of the water-insoluble porous carrier is granular, spherical,
Arbitrary shapes such as a fiber shape, a film shape, and a hollow fiber shape can be selected. When a granular water-insoluble porous carrier is used, the pressure loss is large when the particle size is less than 1 μm, and when the particle size exceeds 5000 μm, the adsorption capacity is small. preferable.

The water-insoluble porous carrier used in the present invention may be either organic or inorganic, but preferably has little adsorption of so-called non-specific adsorption of body fluid components other than the target immune complex. Water-insoluble porous carrier is more hydrophilic than hydrophobic because non-specific adsorption is less.
It is more preferable that they are hydrophilic.

Further, it is advantageous that the surface of the water-insoluble porous carrier has a functional group which can be used for a reaction for immobilizing a ligand. Representative examples of these functional groups include a hydroxyl group, an amino group, an aldehyde group, a carboxyl group, a thiol group, a silanol group, an amide group, an epoxy group, a halogen group, a succinylimide group, and an acid anhydride group. However, it is not limited to these. The water-insoluble porous carrier is particularly preferable when it is made of a compound having a hydroxyl group among the above-mentioned functional groups, since non-specific adsorption is small. It goes without saying that a water-insoluble porous carrier having these functional groups introduced as a spacer can also be used.

Representative examples of the water-insoluble porous carrier used in the present invention include soft porous materials such as agarose, dextran, and polyacrylamide; inorganic porous materials such as porous glass and porous silica gel; polymethyl methacrylate; Examples include, but are not limited to, porous polymer hardgels made from synthetic polymers such as alcohol, styrene, and divinylbenzene copolymers and / or natural polymers such as cellulose.

When the adsorbent used in the present invention is used for extracorporeal circulation treatment, it is necessary to flow a high-viscosity fluid such as blood or plasma at a high speed, so that a hard material having sufficient mechanical strength that does not cause compaction is required. It is preferable to use a water-insoluble porous carrier. That is, as shown in Reference Examples below, the hard water-insoluble porous carrier uniformly fills the water-insoluble porous carrier in a cylindrical column, and the relationship between the pressure loss and the flow rate when the aqueous fluid flows is at least 0.3 kg. / Cm ^{2 in a} linear relationship.

As the anionic functional group of the adsorbent used in the present invention, any functional group can be used as long as the functional group is negatively charged when the pH is around neutral. Typical examples of these are
Examples thereof include, but are not limited to, a carboxyl group, a sulfonic group, a sulfone group, a sulfate group, a silanol group, a phosphate group, and a phenolic hydroxyl group.

The polyanion compound used in the present invention, excluding proteins, is preferred because it has a high affinity for immune complexes and can easily introduce many anionic functional groups into a unit amount of a water-insoluble porous carrier. The polyanionic compound may have one type of anionic functional group or a plurality of types.

Representative examples of the polyanionic compound used in the present invention, except for proteins, include polyacrylic acid,
Polyvinyl sulfate, polyvinyl sulfonic acid, polyvinyl phosphoric acid, polystyrene sulfonic acid, polystyrene phosphoric acid,
Synthetic polyanionic compounds such as polyglutamic acid, polyaspartic acid, polymethacrylic acid, polyphosphoric acid, styrene-maleic acid copolymer, and anionic functional groups such as heparin, dextran sulfate, chondroitin, chondroitin sulfate, phosphomannan, chitin, chitosan Polysaccharides.

The molecular weight fixed on the adsorbent used in the present invention
1000 or more polyanion compounds, except for proteins,
May be one type or two or more types.

The adsorbent used in the present invention refers to a substance in which a polyanion compound having a molecular weight of 1,000 or more, excluding proteins, is fixed on a water-insoluble porous carrier. A method of introducing an anionic functional group into a water-insoluble porous carrier in order to obtain a state in which such a polyanionic compound having a molecular weight of 1,000 or more, except for proteins, or a compound having other anionic functional groups is fixed. Various known methods can be used without any particular limitation. Typical examples of the method include (1) containing an anionic functional group or a functional group which can be easily converted to an anionic functional group. A method of forming an adsorbent by polymerization using a compound as a monomer or a crosslinking agent, (2) a method of immobilizing a compound containing an anionic functional group on a water-insoluble porous carrier, (3) a compound forming an anionic functional group Immobilizing a compound having an anionic functional group on a water-insoluble porous carrier by directly reacting the compound with a water-insoluble porous carrier. Law.

Representative examples of monomers or cross-linking agents 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 Examples include, but are not limited to, esters and styrene sulfonic acids.

The method (2), that is, the method of immobilizing a compound containing an anionic functional group on a water-insoluble porous carrier includes a method by physical adsorption, a method by ionic bonding, and a method by covalent bonding. Yes, any method may be used, but it is important for the preservation and stability of the adsorbent that the anionic functional group-containing compound is not eliminated. desirable.

When the compound having an anionic functional group is fixed by a covalent bond, it is preferable that the compound having an anionic functional group has a functional group which can be used for fixing, in addition to the anionic functional group.

Representative examples of functional groups that can be used for immobilization include amino groups, amide groups, carboxyl groups, acid anhydride groups, succinylimide groups, hydroxyl groups, thiol groups, aldehyde groups, halogen groups, epoxy groups, silanol groups, and the like. But are not limited to these.

There are many anionic functional group-containing compounds having these functional groups, and examples thereof include sulfanilic acid, phosphorylethanolamine, glycine, and taurine, which will be described later.

A typical example of the compound containing a sulfate ester group among the compounds containing an anionic functional group is a sulfate group of a hydroxyl group-containing compound such as alcohol, saccharide or glycol. Partial sulfate compounds of polyhydric alcohols, especially sulfated saccharides, contain both sulfate groups and functional groups required for immobilization, have high biocompatibility and high activity, and furthermore, sulfated polysaccharides are easily prepared. It is particularly preferable because it can be fixed to a water-insoluble porous carrier.

Next, the compound having an anionic functional group is immobilized on the water-insoluble porous carrier by the method (3), ie, by reacting the compound forming the anionic functional group with the water-insoluble porous carrier. As a typical example of the method of introducing an anionic functional group, a reaction of introducing a sulfate group into a hydroxyl group-containing porous carrier can be mentioned. In this case, a sulfate group can be directly introduced by reacting a hydroxyl group-containing water-insoluble porous carrier with a reagent such as chlorosulfonic acid or concentrated sulfuric acid.

The amount of the introduced anionic functional group is preferably 0.01 μmol or more and 10 mmol or less per 1 ml of the adsorbent. 0.
When the amount is less than 01 μmol, the adsorbing ability is not sufficient. When the amount exceeds 10 mmol, non-specific adsorption is too large, and it is difficult to put to practical use. A more preferred anionic functional group introduction amount is 1
It is good to be more than μmol and less than 100μmol.

There are various methods for using the adsorbent used in the present invention for treatment. The simplest method is to draw the patient's blood out of the body, store it in a blood bag, mix the adsorbent used in the present invention with the adsorbent to remove immune complexes, remove the adsorbent through a filter, and remove the blood from the patient. There is a way to return to. Although this method does not require a complicated device, it has a drawback that the amount of processing performed at one time is small, the treatment is time-consuming, and the operation is complicated.

In the following method, the adsorbent is packed in a column, incorporated into an extracorporeal circuit, and adsorbed and removed on-line. That is, a container having an inlet and an outlet for a fluid, the fluid and components contained in the fluid can pass therethrough,
The water-insoluble porous carrier is a polyanion compound having a molecular weight of 1000 or more, except for proteins, except that the adsorbent of the immune complex, which is immobilized, cannot pass through the filter, and the adsorbent of the immune complex filled in the container. A simple and preferable method is to pass a bodily fluid through the immune complex removal device.

FIG. 1 is a schematic sectional view of an embodiment of the immune complex removing apparatus of the present invention. In FIG. 1, (1) and (2) are the inlet and outlet of the respective fluids, (3) is the adsorbent, (4) and (5) are the fluids and the components contained in the fluids. A filter or mesh through which the adsorbent cannot pass, (6) a column, and (7) a container. Here, the filter (4) on the fluid inlet side may not be present.

Hereinafter, the removing apparatus of the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0040]

【Example】

Reference Example Agarose gel (Biogel A5m, Biorad A5m, particle size 50 ~) was attached to a glass cylindrical column (inner diameter 9mm, column length 150mm) equipped with a filter having a pore size of 15μm at both ends.
100 mesh), polymer hard gel (Toyopearl HW65 manufactured by Toyo Soda Kogyo Co., Ltd., particle size 50-100 μm, and Cellulofine GC700 manufactured by Chisso Co., particle size 45-105
μm) were uniformly filled, water was circulated through the column by a peristaltic pump, and the relationship between the flow rate and the pressure loss ΔP was determined. The result is shown in FIG. As is clear from the figure, the agarose gel, which is a soft gel, consolidates above a certain flow rate, and the flow rate does not increase even if the pressure is increased, whereas the hard gels such as Toyopearl and Cellulofine increase the pressure. The flow rate increases almost in proportion to.

Production Example 1 Cellulose CK Gel A3, a porous cellulose gel (trade name, manufactured by Chisso Corporation, exclusion limit molecular weight of globular protein of 5000)
10,000, particle size 45-105μm) 100% 20% NaOH 40g, heptane
120 g and 10 drops of nonionic surfactant Tween 20 (trade name, manufactured by Kao Atlas Co., Ltd.) were added. After stirring at 40 ° C. for 2 hours, epichlorohydrin (50 g) was added, and the mixture was stirred for 2 hours. The gel was washed with water and filtered to obtain a cellulose gel having epoxy groups introduced therein (hereinafter referred to as epoxidized gel).

Comparative Production Example 1 Sulfanilic acid was added to 5 ml of the epoxidized gel obtained in Production Example 1.
A solution adjusted to pH 9.9 by dissolving 17 g in 10 ml of water was added, and the mixture was shaken at room temperature for 24 hours. A 0.5% monoethanolamine aqueous solution was added and shaken to seal unreacted epoxy groups, and sulfanilic acid was added. A fixed cellulose gel was obtained. The amount of anionic functional groups introduced by the immobilized sulfanilic acid was 6.5 μmol / ml of the adsorbent.

Comparative Production Example 2 A solution adjusted to pH 9.6 by dissolving 0.1 g of phosphorylethanolamine in 10 ml of water was added to 5 ml of the epoxidized gel obtained in Production Example 1, and the mixture was shaken at 40 ° C. for 4 hours, and 0.5% An aqueous monoethanolamine solution was added and shaken to seal unreacted epoxy groups to obtain a phosphorylethanolamine-fixed cellulose gel. The amount of anionic functional groups introduced by the immobilized phosphorylethanolamine was 1 ml of adsorbent
4 μmol / mol.

Production Example 1 To 5 ml of the epoxidized gel obtained in Production Example 1 was added 4 g of sodium dextran sulfate having a molecular weight of about 5,000 and a sulfur content of 15% and 5 ml of water, and the mixture was adjusted to pH 9 and shaken at 45 ° C. for 16 hours. Thereafter, the gel was separated by filtration, washed with a 2M aqueous solution of sodium chloride, a 0.5M aqueous solution of sodium chloride and water in this order, added with a 0.5% aqueous solution of monoethanolamine and shaken to seal unreacted epoxy groups, and sodium dextran sulfate was added. A fixed cellulose gel was obtained. The amount of anionic functional groups introduced by the immobilized dextran sulfate was 10 μmol / ml of adsorbent.
l.

Comparative Production Example 3 0.25 g of glycine was added to 5 ml of the epoxidized gel obtained in Production Example 1.
A solution dissolved in 10 ml of water and adjusted to pH 9.8 was added and shaken at room temperature for 24 hours. Thereafter, the gel was separated by filtration, a 0.5% aqueous solution of monoethanolamine was added, and the mixture was shaken to seal unreacted epoxy groups to obtain a cellulose gel on which glycine was fixed. The amount of anionic functional groups introduced by the immobilized glycine was 9 μmol / ml of adsorbent.

Comparative Production Example 4 0.35 g of taurine was added to 5 ml of the epoxidized gel obtained in Production Example 1.
A solution dissolved in 10 ml of water and adjusted to pH 9.0 was added, and the mixture was shaken at room temperature for 24 hours. Thereafter, the gel was separated by filtration, a 0.5% aqueous solution of monoethanolamine was added, and the mixture was shaken to seal unreacted epoxy groups to obtain a cellulose gel in which taurine was fixed. The amount of anionic functional groups introduced by the immobilized taurine was 5 μmol / ml of adsorbent.

EXAMPLE 2 Cellulose CK gel A3,10 similar to that used in Example 1
After washing with water, suction filtration was performed, and dimethyl sulfoxide 6 was added thereto.
ml, 2N-NaOH 2.6 ml, and epichlorohydrin 1.5 ml.
Stirred at 0 ° C. for 2 hours. After the reaction, the gel was separated by filtration and washed with water to obtain a cellulose gel into which an epoxy group was introduced.

6 ml of concentrated ammonia water was added thereto, and the mixture was added at 40 ° C.
After reacting for an hour, an aminated cellulose gel was obtained.

In 5 ml of this gel, 0.2 g of sodium polyacrylate having a molecular weight of 190,000 to 500,000 was dissolved in 10 ml of water to obtain a pH 4.5.
Was added thereto, and 200 mg of 1-ethyl-3- (dimethylaminopropyl) carbodiimide was further added while maintaining the pH at 4.5, followed by shaking at 4 ° C. for 24 hours. After the reaction, the gel was separated by filtration and washed with water to obtain a cellulose gel into which polyacrylic acid had been introduced. The amount of anionic functional groups introduced by the immobilized polyacrylic acid was 14 μmol / ml of adsorbent.

Example 3 A porous cellulose gel was used as a cellulose CK gel A22 (trade name, manufactured by Chisso Corporation, exclusion limit molecular weight of globular protein of 2,000).
10,000, particle size 45-105 μm), Cellulofine GCL-2000m (trade name, manufactured by Chisso Corp., exclusion limit molecular weight of globular protein 30)
100,000, particle size 44-105μm), Cellulofine GCL-1000m
(Trade name, manufactured by Chisso Corp., exclusion limit molecular weight of globular protein: 600,000, particle size: 44-105 μm), Cellulofine GC-700m
(Trade name, manufactured by Chisso Co., Ltd., globular protein exclusion limit molecular weight 400,000, particle size 44-105 μm), Cellulofine GC-200m
(Trade name, manufactured by Chisso Co., Ltd., exclusion limit molecular weight of globular protein: 120,000, particle size: 45 to 105 μm), Cellulofine GCL-90
(Production name, manufactured by Chisso Corp., molecular weight of exclusion limit of globular protein: 35,000, particle size: 45-105 μm) Except for changing to cellulose, dextran sulfate sodium-immobilized cellulose in the same manner as in Production Example 1 and Production Example 1. I got a gel. The amount of anionic functional group introduced by the immobilized dextran sulfate was 16, 18, 30, 24, 30, and 37 μmo per ml of the adsorbent, respectively.
l.

Production Example 4 Epoxy Activated Sepharose CL-6B (trade name, manufactured by Pharmacia Fine Chemicals Co., Ltd., manufactured by Pharmacia Fine Chemicals Co., Ltd., excluding exclusion limit molecular weight of globular protein of 4,000,000, particle size of 45 to 165 μm), which is an epoxidized crosslinked agarose gel, was used. Otherwise, dextran sulfate sodium was immobilized in the same manner as in Example 1. The amount of anionic functional groups introduced by the immobilized dextran sulfate was 20 μmol / ml of adsorbent.

Example Production Example 5 FP-HG which is a hydrophilic porous hard hydrogel containing polymethyl methacrylate as a main component (trade name, Mitsubishi Kasei Corporation)
Made, globular protein exclusion limit molecular weight 4,000,000, particle size 120μ
Except for using m), a gel having sodium dextran sulfate immobilized thereon was obtained in the same manner as in Production Example 1 and Example 1. The amount of anionic functional groups introduced by the immobilized dextran sulfate was 9 μmol / ml of adsorbent.

Example 6 Aminated cellulose gel obtained in the same manner as in Example 2
To 2 g, the same molecular weight as that used in Production Example 1 5000,
4 g of sodium dextran sulfate with 15% sulfur content
A solution dissolved in 8 ml of M phosphate buffer (pH 8.0) was added, and the mixture was shaken at room temperature for 16 hours. After the reaction, 20 mg of NaCNBH ₃ was added, and the mixture was stirred at room temperature for 30 minutes. After heating at 40 ° C. for 4 hours, the gel was separated by filtration and washed with water to obtain a cellulose gel on which dextran sulfate sodium was fixed. The amount of anionic functional groups introduced by the immobilized dextran sulfate was 18 μmol / ml of adsorbent.
l.

Experimental Example 1 Production Examples 1, 2 and 6 and Comparative Production Examples 1-4
The adsorbent obtained above and the carrier CK gel A3 used in Production Example 1 for comparison purposes were washed with a 15 M Tris buffer (pH 7.4), and 0.1 ml of each adsorbent was placed in a polypropylene microtube (capacity: 7 ml). ) And 0.15M Tris buffer (p
H 7.4) was added to bring the total volume to 1 ml. 0.2 ml of the serum containing the immune complex was added thereto, and the mixture was shaken at 25 ° C. for 2 hours.
After completion of the adsorption operation, the gel was sedimented by centrifugation, and the concentration of the immune complex in the collected supernatant was measured using an enzyme-linked immunosorbent assay (ELIS).
A method). That is, the immune complex concentration is C1
Add the diluted sample to the plate coated with q
An antibody reaction was performed, a peroxidase-labeled anti-human immunoglobulin antibody was dropped, and the enzyme color reaction was measured at 486 nm using SLT-210 (trade name, manufactured by Labo Science Co., Ltd.). The measured values were standardized by a standard curve prepared with heat-denatured IgG. Table 1 shows the names of the compounds having an anionic functional group immobilized on each adsorbent, and the concentration of the immune complex in the supernatant of each adsorbent converted to the concentration in the original serum in consideration of dilution.

From Table 1, it can be seen that the adsorbent in which the compound having an anionic functional group is immobilized on the water-insoluble porous carrier adsorbs the immune complex. Further, it can be seen that the adsorbent on which dextran sulfate is immobilized has particularly excellent ability to adsorb the immune complex.

[0056]

[Table 1]

Experimental Example 2 Using the adsorbents obtained in Examples 1 and 3 to 5, the concentration of the immune complex in the supernatant was determined in the same manner as in Experimental Example 1 except that the amount of serum added was 0.1 ml. Was converted to the concentration in the original serum taking into account the dilution. Table 2 shows the names of various water-insoluble porous carriers using the obtained results.
The immunocomplex concentration of the original serum was measured for comparison.
2.2 μg / ml.

From Table 2, it can be seen that the Cellulofine of Example 3 is a water-insoluble porous carrier having an exclusion limit molecular weight of 400,000 or less.
GC-700m, Cellulofine GC-200m and Cellulose GC
It can be seen that the immunocomplex adsorption ability of L-90 is low. Also,
Conversely, even if the exclusion limit molecular weight is too large, 50,000,000, the results of the cellulose CK gel A3 of Example 1 show that the immunocomplex adsorption ability tends to decrease.

[0059]

[Table 2]

[0060]

The removal apparatus of the present invention has the effect of selectively removing immune complexes from body fluids.

[Brief description of the drawings]

FIG. 1 is a schematic cross-sectional view of one embodiment of the immune complex removal device of the present invention.

FIG. 2 is a graph showing the results of examining the relationship between flow rate and pressure loss using three types of gels.

[Explanation of symbols]

 1: Inlet 2: Outlet 3: Adsorbent 4, 5: Filter 7: Container

Claims (3)

[Claims] 1. A container having an inlet and an outlet for a fluid, the fluid and components contained in the fluid can pass therethrough,
The water-insoluble porous carrier is a polyanion compound having a molecular weight of 1000 or more, except for proteins, except that the adsorbent of the immune complex, which is immobilized, cannot pass through the filter, and the adsorbent of the immune complex filled in the container. Immune complex removal device. 2. The removal apparatus according to claim 1, wherein the water-insoluble porous carrier of the adsorbent of the immune complex has a molecular weight exclusion limit of the spherical protein of 400,000 to 60,000,000. 3. The removal device according to claim 2, wherein the water-insoluble porous carrier is made of a compound having a hydroxyl group.