JPH0245065A - Adsorbent for cleaning body fluid of beta2-microglobulin - Google Patents

Abstract

PURPOSE:To selectively adsorb beta2-microglobulin with high efficiency by fixing collagen having a specific or higher isoelectric point to the surface of a water insoluble carrier. CONSTITUTION:A blood-compatible polymer includes all of known blood- compatible materials if the materials can fix the collagen exhibiting >=9.5 isoelectric point. An acrylate polymer, acrylamide polymer, gelatin, etc., are exemplified in terms of the prevention of the generation of microparticles, i.e., the ease of application on the water insoluble material, safety and sterilizing effect. The granular carrier is preferably porous particles. The average pore size of the fine pores of the porous particles is preferably in a 20-500Angstrom range. While known methods are usable as the method of fixing the collagen to the water insoluble carrier, immobilizing enzyme, a method of activating known carriers used in affinity-chromatography and a method of bonding ligand are usable.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention is an adsorption system for body fluid purification that selectively adsorbs and removes malignant substances related to diseases from body fluids such as blood, plasma, serum, ascites, and script. Regarding materials.

More specifically, β2- is increased in the body fluids of patients with renal failure and malignant tumors, and causes symptoms such as palmistry canal syndrome, amyloidosis, pop-finger/shoulder/knee joint disorders, skin pruritus, and kidney damage.
This invention relates to a microglobulin adsorbent.

As the years have passed, abnormalities such as lost tube syndrome have become apparent. In recent years, this causative substance is β2, which is relatively difficult to remove by dialysis.
- Microglobulin, and its accumulation in the body has been shown to cause various symptoms.

Conventionally, hemofiltration and diafiltration have been used for the purpose of removing such medium-molecular-weight substances, but the removal rate is low, and effective removal requires a large amount of fluid replacement.

In addition, increasing the removal rate can be achieved by increasing the membrane bore, but if the bore becomes slightly larger, albumin, a useful protein, will leak out, and controlling the bore size will not effectively selectively remove medium molecular weight substances. The current situation is that this cannot be done. On the other hand, recently, adsorbents for β2-microglobulin have been published in Japanese Patent Application Laid-open Nos. 62-204761 and 62-2400.
No. 68 and Japanese Unexamined Patent Publication No. 62-261367.

(Prior Art) It has been approximately 10 years since hemodialysis was performed on patients with renal failure (Problem to be solved by the invention) The purpose of the present invention is to solve the problems based on the therapeutic polymer membrane technology as described above. - Can be widely used in boat fishing, selectively adsorbs medium molecular weight substances, especially β8-microglobulin, with high efficiency, has low non-specific adsorption, especially albumin adsorption, and does not require fluid replacement, making it safe The objective is to provide an adsorbent that can be easily sterilized in one day and is suitable for purifying or regenerating body fluids such as whole blood or plasma.

(Means for Solving the Problems) As a result of intensive research in line with the above objectives, the present inventors found that a water-insoluble carrier having a specific collagen on the surface
We have discovered that microglobulins can be selectively adsorbed with surprisingly high efficiency. When this matter was investigated in more detail, it was found that β,-microglobulin can be adsorbed selectively and with high efficiency only when the isoelectric point of collagen is 9.5 or higher, thereby completing the present invention. reached.

That is, the present invention is an adsorbent for β2-microglobulin for body fluid purification, which has collagen having an isoelectric point of 9.5 or more on the surface of a water-insoluble carrier.

The β2-microglobulin adsorbent for body fluid purification of the present invention is an adsorbent having the above-mentioned specific collagen on its surface, and is not limited to the following examples, but more specifically: Preferably, the above-mentioned specific collagen (these are referred to as ligands) is immobilized on the surface of a water-insoluble carrier by some method (eg, epochlorohydrin method, cyanide halide method). Furthermore, it is more preferable that the immobilized ligand does not cover the surface of the water-insoluble carrier in a flat manner, but extends in a long manner. The reason for this is thought to be that the surface area of the adsorbent of the present invention that can come into contact with β2-microglobulin becomes larger, which allows the adsorbent of the present invention to adsorb β2-microglobulin more efficiently. One example is becoming.

The β2-microglobulin referred to in the present invention is β2-microglobulin that is usually measured by enzyme immunoassay or the like in clinical tests, and more specifically, it has the following physical property values.

Sedimentation constant 1.63 Partial specific volume 0.72-0.73 I11/g molecule l 11000-12000 Nitrogen content
16-17% The β,-microglobulin targeted by the present invention includes β2
- It includes microglobulin itself and complexes with other proteins, and also includes partially mutated amino acid sequences of β, -microglobulin.

The isoelectric point in the present invention specifies the collagen on the surface of a water-insoluble carrier, and is based on the principle of isoelectric focusing [for example, Biochemistry Experiment Course 1. Protein Award Chemistry I, P
305-P312. Japanese Biochemistry Edition.

The isoelectric point of the present invention, as measured by Tokyo Kagaku Dojin Publishing (March 30, 1977), must be 9°5 or higher, preferably 9°8 to 12°.
．． 0, more preferably 10°0 to 11.0.

When the isoelectric point is smaller than 9.5, the interaction between collagen and β2-microglobulin becomes weak, and the adsorption ability of β2-microglobulin of the adsorbent having collagen on the surface becomes low.

These things mean that collagen has a stronger positive charge in neutral electrolyte fluids such as blood and body fluids, that is, collagen has anionic groups (such as carboxyl groups).
cationic groups (primary, secondary, tertiary,
By having more ionic groups (such as quaternary amino groups, which are positively charged in neutral electrolyte fluids such as blood and body fluids), the ionic interaction between β-microglobulin and collagen is enhanced. It is thought that this increases the adsorption ability of the adsorbent having collagen on its surface for β2-microglobulin, and improves the adsorption selectivity for substances other than the target substance such as albumin.

In the present invention, collagen refers to all collagens having an isoelectric point of 9.5 or higher as shown above. Examples of the collagen of the present invention include skin, bones, thighs, blood vessels, basement membranes, placenta, muscles, cartilage, etc. of various animals such as humans, cows, and pigs.
Various types of collagen obtained by treatment with enzymes, acids, alkalis, etc. [For example, (Collagen Metabolism and Diseases, PIIO
~P133. Yutaka Nagai, edited by Dai Fujimoto, Kodansha, 19
Published April 1, 1982) (Collagen, P196-20
6. Haruhiko Noda, Yutaka Nagai, Daisanbu Fujimoto (eds., Nankodo, published May 1973)), and includes atelocollagen in which the telopeptide at the terminal of the collagen molecule has been cleaved] can be mixed with methanol, ethanol, or isopropyl. Examples include those chemically modified with organic alcohol compounds such as alcohol. Among these, methylated atelocollagen Type I, in which the side chain carboxyl group (-COOH) of aspartic acid and glutamic acid constituting atelocollagen Type I is treated with methanol and methylated (-COOCIh), gives particularly favorable results. .

In the present invention, the water-insoluble carrier may be cellulose-based, tonyl polymer-based, polyacrylamide-based, polyhydroxyethylmethylacrylate-based, glass-based, etc., as long as it can immobilize collagen with an isoelectric point of 9.5 or higher as shown above. All organic polymer compounds such as silica or inorganic compounds can be used, but when β2-microglobulin is selectively adsorbed with higher efficiency and used in whole blood where blood cells such as platelets coexist. It is preferred that the water-insoluble carrier has at least a two-layer structure of a water-insoluble material having a contact angle of at least 20 degrees and a blood-compatible polymer.

The above contact angle is the contact angle of air bubbles on a solid surface in water, and is described by W, C, Hamilton, J, Co.
11oid Interface Sci, 40.
219-222 (1972) (Double Sea Hamilton Journal of Colloid Interface Science, 40.219-222 (1972)
)) J, D, Andrade, J, Polys, S
ci, Po1y+m, Symp, 66, 313-3
36 (1979) (J.D. Andrade,
Journal of Polymer Science
Symposium, 66, 313-336 (1979)
] Refers to the contact angle measured according to the principle and method shown in [ ]. The conventionally well-known method of measuring the contact angle of a droplet on a solid surface in air is difficult to use as a physical property value for water-absorbing materials because the contact angle value changes over time. .

In addition, as samples, sheet and film-like molded products were produced, and the contact angle was measured at least 10 times at a temperature of 25°C, and the average value was taken as the value of the contact angle of the material.

As a water-insoluble material with a contact angle of 20 degrees or more, if the contact angle measured by the method shown above is 20 degrees or more,
It includes all inorganic compounds and organic polymer compounds, but
Organic polymeric materials are preferably used as body fluid purification materials in terms of safety and adsorption affinity for eluates.

The organic polymer material used preferably has a contact angle of 20 degrees or more, more preferably 30 degrees or more, particularly preferably 40 degrees or more, in view of its adsorption affinity with β2-microglobulin.

Preferred organic polymer materials include polyolefin compounds such as polyethylene, polypropylene, and polytetrafluoroethylene, polymers of vinyl compounds such as polystyrene, polymethacrylate ester, and polyacrylate ester, and polyamide compounds such as nylon 6.66.
Examples include polyester compounds such as polyethylene terephthalate. Among these, homopolymers such as methacrylate esters, acrylate esters, styrene and styrene derivatives, or copolymers with comonomers and crosslinking agents are preferably used. In particular, methyl methacrylate or crosslinked polymer particles containing styrene as a main component are preferably used. As the crosslinking agent, any known crosslinking agent can be used, and examples thereof include divinylbenzene, ethylene glycol di(meth)acrylate, polyethylene glycol di(meth)acrylate, and the like.

The blood-compatible polymer has an isoelectric point of 9.
If collagen showing 5 or more can be fixed, all known blood-compatible materials are included in -a, but from the viewpoint of prevention of generation of fine particles, that is, ease of coating on water-insoluble materials, safety, and sterilization, ( meth)acrylic acid ester polymer,
Acrylamide polymer, polyvinylpyrrolidone polymer, polyvinyl alcohol polymer, ethylene-vinyl alcohol polymer, ethylene-vinyl acetate polymer,
Examples include cellulose nitrate and gelatin.

In order to prevent the generation of fine particles and further improve blood compatibility, a polymer having a nitrogen-containing basic functional group is particularly preferably used as the blood compatible polymer.

The above-mentioned "nitrogen-containing basic functional group" is a functional group that has a positive charge on a nitrogen atom and can become a cation in an acidic aqueous solution. Examples of such functional groups include primary amino groups, secondary amino groups, tertiary amino groups, quaternary ammonium groups, and nitrogen-containing aromatic ring groups such as pyridyl groups and imidazolinyl groups.

Therefore, examples of the polymer having a nitrogen-containing basic functional group used in the present invention include vinylamine; 2-vinylpyridine, 4-vinylpyridine, 2-methyl-5-
Vinyl derivatives of nitrogen-containing aromatic compounds such as vinylpyridine, 4-vinylimidazole, N-vinyl-2-ethylimidazole, N-vinyl-2-methylimidazole; dimethylaminoethyl (meth)acrylate, diethylaminoethyl (meth)acrylate , dimethylaminopropyl (meth)acrylate, 3-dimethylamino-2-
Acrylic acid and methacrylic acid derivatives such as hydroxypropyl (meth)acrylate; Acrylic acid amide and methacrylic acid amide derivatives such as N-dimethylaminoethyl (meth)acrylic amide and N-diethylaminoethyl (meth)acrylic amide; Polymers containing styrene derivatives such as p-dimethylaminomethylstyrene and p-diethylaminoethylstyrene; and derivatives of the above-mentioned vinyl compounds converted into quaternary ammonium salts with alkyl halides and the like can be mentioned.

Particularly preferred among these are polymers containing diethylaminoethyl (meth)acrylate, diethylaminoethyl (meth)acrylate, p-dimethylaminomethylstyrene, p-diethylaminoethylstyrene, and the like.

Further, the polymer having a nitrogen-containing basic functional group used in the present invention is preferably a copolymer of a vinyl compound and a monomer having a nitrogen-containing basic functional group, and the nitrogen content thereof is 0.
The nitrogen content is preferably 0.05 to 3.5% by weight, and more preferably, the nitrogen content is 0.1 to 2.5% by weight. The nitrogen content herein refers to the weight percent of the nitrogen atoms in the functional group in the prepolymer.

Examples of the vinyl compounds mentioned above include alkyl (meth)acrylates such as 2-hydroxyethyl methacrylate, methyl (meth)acrylate, ethyl (meth)acrylate, and n-butyl (meth)acrylate, (meth)acrylamide, and N-methyl Examples include amides such as (meth)acrylamide, N-vinylpyrrolidone, vinyl acetate, and styrene.

Furthermore, copolymers of vinyl compounds and monomers having nitrogen-containing basic functional groups include block copolymers, graft copolymers, random copolymers, etc.; The polymer preferably has a microdomain structure with an average length of 100 to 100 microns in view of its blood compatibility.

Hereinafter, as for the method of manufacturing the adsorbent of the present invention, a method of fixing collagen to a water-insoluble carrier will be exemplified, but it goes without saying that the present invention is not limited to this example.

The shape of the water-insoluble carrier may be any known shape such as particulate, fibrous, hollow fiber, or membrane, but from the viewpoint of the amount of collagen retained in the present invention and ease of handling as an adsorbent, particulate , fibrous ones are preferred.

The average particle size of the spherical or particulate carrier is 25 to 2500 μm
However, from the viewpoint of its specific surface area (adsorption capacity as an adsorbent) and body fluid circulation surface, those with a diameter of 50 to 1500 μm are particularly preferable.

The specific surface area of the carrier is preferably 5 rrf/g or more, preferably 55 rrf/g or more.

Porous particles are preferred as the particulate carrier.

The porous particles used in the present invention are those that can immobilize the collagen of the present invention on their surfaces, and further have β2
- In order to increase the adsorption efficiency of microglobulin, it is preferable to introduce β2-microglobulin into the pores of the porous particles, so the average pore size of the pores of the porous particles should be in the range of 20 to 5000 particles. It is preferable that there be.

When a fibrous carrier is used, the fiber diameter is preferably in the range of 0.02 denier to 10 denier, more preferably 0.1 denier to 5 denier. If the fiber diameter is too large, the adsorption amount and adsorption rate of β2-microglobulin compounds will be reduced, and if it is too small, activation of the coagulation system, blood cell adhesion, and clogging will easily occur.

As the fibrous carrier to be used, generally known fibers such as regenerated cellulose fibers, nylon, acrylic, and polyester can be used.

In the present invention, collagen can be immobilized on a water-insoluble carrier by any known method such as covalent bonding, ionic bonding, physical adsorption, embedding, or precipitation insolubilization on the surface of a water-insoluble carrier. From the viewpoint of elution properties, it is preferable to use it after being immobilized and insolubilized by covalent bonding. Therefore, an immobilized enzyme, a known method for activating a carrier used in affinity chromatography, and a method for binding a ligand can be used.

Examples of activation methods include halogenated cyanide method, epichlorohydrin method, bisepoxide method, halogenated triazine method, bromoacetyl bromide method, ethyl chloroformate method, 1,1'-carbonyldiimidazole method, etc. can. The activation method of the present invention is not limited to the above examples as long as it can perform a substitution and/or addition reaction with a nucleophilic reactive group having active hydrogen such as an amino group, a hydroxyl group, a carboxyl group, or a thiol group of the ligand. However, in consideration of chemical stability, thermal stability, etc., a method using an epoxide is preferable, and an epichlorohydrin method is particularly recommended.

Two or more types of collagen used in the present invention may be bound to the carrier.

In the above, in the present invention, a method of binding collagen after activating a water-insoluble carrier has been described in detail as a method of fixing collagen to a water-insoluble carrier, but the present invention is not limited to this. . For example, after coating an insoluble substance with a polymer capable of binding collagen,
A method of binding collagen or a method of coating an insoluble substance with a polymer having collagen can also be used.

In this case, if necessary, the polymer used for the coating can also be post-crosslinked. Alternatively, a method of activating collagen and then binding it to a carrier can also be adopted.

Further, if necessary, a molecule (spacer) of arbitrary length, such as an aminoethyl group, an aminopentyl group, an aminooctyl group, an aminododecyl group, etc., can be introduced between the water-insoluble carrier and the collagen. That is, the present invention exhibits its effects due to the presence of collagen on the surface of the adsorbent, and is not dependent on the manufacturing method.

The method of using the β2-microglobulin adsorbent of the present invention will be exemplified below, but it goes without saying that the present invention is not limited to this example.

The adsorbent of the present invention may be used alone, or may be mixed or stacked with other body fluid purifying materials. Examples of other body fluid purification materials include activated carbon used in adsorption type artificial kidneys, hollow fiber membranes and flat membranes used in dialysis type artificial kidneys and filtration type artificial kidneys. As a result, a wider range of clinical effects can be expected due to the synergistic effect of the adsorbent. The adsorbent volume is 50 to 600 Jd when used for extracorporeal circulation.
The degree is appropriate.

When the adsorbent of the present invention is used in extracorporeal circulation, there are three methods as follows: One method is to pass blood taken from the body directly through a nuclear device to purify it, and the other is to use a centrifuge or deformed plasma separator to separate blood from the body into plasma. After separating the plasma components into blood cell components, the plasma components are passed through the device, purified, and then returned to the body together with the blood cell components.
In this method, blood extracted from the body is passed through a body fluid purifier such as an adsorption type artificial kidney, a see-through type artificial kidney, or a filtration type artificial kidney, and then passed through the device for purification. Conversely, the blood may be passed through the device and then through the body fluid purifier.

In addition, regarding the passage speed of blood or plasma, since the adsorption efficiency of the adsorbent is very 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. High passing speeds can be achieved regardless of the Therefore, a large amount of body fluid can be treated.

The method for passing body fluids such as blood and plasma may be continuous or intermittent, depending on clinical needs or equipment conditions. good.

(Effects of the Invention) As described above, the adsorbent of the present invention can absorb β in body fluids.
2- Microglobulin is highly efficiently and specifically adsorbed and removed, and it is simple and safe.

The present invention can be used for general purposes to purify and regenerate body fluids such as autologous blood and plasma, and can be used to treat body fluids that increase in the body fluids of patients with renal failure or malignant tumors, leading to prepalmist syndrome, amyloidosis, and stroke. Amyloid protein, especially β2, is a medium molecular weight substance that causes finger, shoulder, and knee joint disorders, skin pruritus, bone disorders, etc.
- It can be used effectively and safely for adsorption and removal of microglobulin.

(Example) Next, the present invention will be described in more detail with reference to Examples.

Example 1 In order to examine the binding ability between collagen and β2-microglobulin on the surface of the β2-microglobulin adsorbent of the present invention, an enzyme immunoassay method commonly used in clinical tests was used.・Plate) The binding ability between β2-microglobulin fixed on the surface and collagen was evaluated. The evaluation method and conditions are described below.

(1) Immobilization of β2-microglobulin on the surface of ELISA plate β2-microglobulin (manufactured by Sigma, USA) purified from human urine was dissolved in PBS (phosphate buffered saline). , 10 μg β1-microglobulin/
A dPBs solution was prepared. 100 μE of this solution is applied to the Elaza plate (trade name: Imielon), which is used for enzyme immunoassay.

600, manufactured by Greiner, West Germany) at 4℃, 24
I left it for a while.

(2) Immobilization of bovine serum albumin to the free ELISA plate surface where βf-microglobulin is not immobilized (blocking operation) In order to suppress the binding between the free ELISA plate surface and collagen, perform the following operation. .

Added 10°C 1g β2-microglobulin solution 10
After removing 01Il by suction, add 200μ of 0.5% bovine serum albumin PBS solution! Add to the Eliza plate,
Leave at 25°C for 12 hours.

(3) Removal of unfixed and free bovine serum albumin from the ELISA plate After removing the above bovine serum albumin solution from the ELISA plate by suction, add 100μ of PBS! Added. After that, remove the PBS by suction and add 100μ of PBS again! Added. The operation of adding PBS and removing it by suction is called a washing operation.Here, the washing operation was repeated three times and
Remove bovine serum albumin that is not fixed on the plate.

(4) Collagen bound to β2-microglobulin fixed on the surface of ELISA plate in PBS
to prepare a 0.1/III PBS solution. After the operation (3), remove the PBS remaining on the ELISA plate by suction, and remove the collagen O, lng/
Add 100 μl of dPBs solution to each ELISA plate and leave at 25° C. for 2 hours.

(5) Removal of free collagen that is not bound to β,-microglobulin fixed on the surface of the ELISA plate After removing the collagen PBS solution by suction, perform the washing operation with PBS shown in (3) three times. , removes free collagen that is not bound to β2-microglobulin.

(6) Measurement of β2-microglobulin immobilized on the surface of the ELISA plate and not bound to collagen This operation is performed in the same manner as in a normal enzyme immunoassay.

(a) After the procedure in (5), add 100 uN of anti-human β2-microglobulin rabbit antibody to the ELISA plate, leave it at 37°C for 11 hours, and then wash with PBS in the same manner as in (3). By this operation, β
An antigen-antibody reaction between 2-microglobulin and anti-human β2-microglobulin is performed.

(b) Next, enzyme-labeled (peroxidase-labeled) anti-rabbit IgG (Vectastin ABC kit)
Add 100 μi of 2-microglobulin and perform antigen antibody with anti-human β2-microglobulin rabbit antibody and anti-rabbit IgG.

(C) Next, 2,2゛-azinobis(3-ethylbenthiazoline)-6-sulfonic acid (molecular weight 514, manufactured by Wako Pure Chemical Industries, Ltd.) and hydrogen peroxide solution are added, and after 0 color development, color is developed by peroxidase reaction. , the absorbance is measured at a wavelength of 405 nm.

This absorbance value correlates with the amount of anti-human β2-microglobulin antibody that binds to β2-microglobulin. In addition, considering the influence of collagen itself on this evaluation system,
Operations (2) to (6) were performed separately without adding β2-microglobulin to the ELISA plate.

Also, do not add collagen to ELISA plates,
That is, only operations (1), (2), (3), and (6) were performed separately.

By performing the above operations, β2 that was fixed on the surface of the ELISA plate and not bound to collagen was obtained.
- The ratio of microglobulin (Y) is [C]; the amount of anti-human β2-microglobulin antibody bound (D) when all operations from (1) to (5) are performed; Amount of anti-human β2-microglobulin antibody bound (E) when performing all operations (2) to (5) without adding microglobulin to the ELISA plate; without adding collagen, that is, (4)
, without performing the operation in (5), (1), (2),
The amount of anti-human β2-microglobulin antibody bound when performing operations (3) and (6) can be expressed as follows.

That is, the smaller Y is, the higher the binding ability between β2-microglobulin and collagen fixed on the surface of the ELISA plate is.

Table 1 below shows the results obtained using this evaluation method.

Table 1 Methylated atelocollagen types used in this experiment
Atelocollagen Type-1, which is obtained by treating bovine dermal collagen with pepsin and purifying it, is treated with methanol, and the 10,000 groups of Atelocollagen Type-T are converted into -COOCHj.

Comparative Example 1 Using the same evaluation method as in Example 1, the following samples were evaluated. The results are shown in Table 2.

Table 2 Atelocollagen Type-1 used in this experiment is obtained by treating bovine dermal collagen with pepsin, and succinylated atelocollagen Type-1 is obtained by treating atelocollagen Type-1 with succinic anhydride. The amino group of atelocollagen Type-1 is succinylated.

The results of Example 1 and Comparative Example 1 show that collagen with an isoelectric point of 9.5 or higher exhibits strong binding ability to β2-microglobulin.

Example 2 A sheet of methylmethacrylate-divinylbenzene copolymer (80:20% by weight) and particles of 420 to 800μ were prepared as a water-insoluble material, and the contact angle of air bubbles in water was measured. , 2% wt/v methanol solution of 2-hydroxyethyl methacrylate-diethylaminoethyl methacrylate copolymer was prepared, and the above particles 30IIt1 were immersed in 100 d of this solution for 5 minutes (with occasional stirring). After removing the excess solution by suction on the glass filter, the solution is dried with nitrogen on the glass filter for 20 minutes while balancing the amount of nitrogen gas to be fed and the amount of nitrogen gas to be sucked. Next, it was dried in a vacuum dryer at room temperature and at 755 mmHg or higher for 24 hours. By this operation, a water-insoluble carrier having a two-layer structure consisting of methyl methacrylate/divinylbenzene copolymer and 2-hydroxyethyl methacrylate/diethylaminoethyl methacrylate is obtained.

This water-insoluble carrier is heat-treated at 125° C. for 145 minutes, suspended in ethanol, and then washed with water. It is then dehydrated and suspended in dimethyl sulfoxide. Next, the operation of removing dimethyl sulfoxide and suspending the water-insoluble carrier in dimethyl sulfoxide is repeated. The water-insoluble carrier 301d obtained after this operation was treated with dimethyl sulfoxide 36
d, and add 24 m of epichlorohydrin and 50 m of epichlorohydrin to this.
% Sodium Hydroxide 3. Od was added and the activation reaction was carried out with stirring at 30°C for 5 hours. After the reaction, the mixture was washed with methanol, water, and dehydrated under suction.

The obtained activated water-insoluble carrier 30d was mixed with methylated atelocollagen Type-1 (Koken Co., Ltd.) solution (2/xt
f1. pH8,0) Suspended at 150m, 25°C148
Immobilization reaction of methylated atelocollagen was performed while shaking for hours.

Next, after washing alternately with 0.1M sodium carbonate buffer and IM sodium citrate buffer, PB
S. Washed thoroughly with physiological saline to obtain an adsorbent.

The retained amount of methylated atelocollagen is determined by converting the primary amino group of methylated atelocollagen to 4-phenylspiro[furan-2(3H), 1'-phthalane]-3,3'-dione ("Fulham■" manufactured by Roche). It was calculated by reacting with

In the adsorption experiment, plasma of a patient with renal failure, a water-insoluble carrier, and an adsorbent were mixed at a volume ratio of 24:1, then shaken at 25°C for 1 hour, and β2-microglobulin and albumin in the plasma before and after were quantified. The RIA method was used for β2-microglobulin, and the BCG method was used for albumin. The results are shown in Table 3.

Comparative Example 2 Using the same water-insoluble carrier and activation method as in Example 2, atelocollagen Type-I (manufactured by Kouken Co., Ltd.) solution (2
(mg/IIi, pH 8,0), and the adsorption experiment was also conducted in the same manner as in Example 2. The results are shown in Table 4.

Table 4 Alhumin; 4. b g/42 From the results of Example 2 and Comparative Example 2, the adsorbent having collagen with an isoelectric point of 9.5 or more on the surface of the water-insoluble carrier has a high adsorption ability for βt-microglobulin, and , it can be seen that there is adsorption selectivity for albumin.

Claims (1)

[Claims] An adsorbent for β_2-microglobulin for body fluid purification, characterized by having collagen having an isoelectric point of 9.5 or more on the surface of a water-insoluble carrier.