JP6126327B2 - Adsorbent for IgM-type antibody adsorption and IgM-type antibody removal system - Google Patents

Description

  The present invention relates to an adsorbent for IgM type antibody adsorption and an IgM type antibody removal system.

  In recent years, many ABO blood group-incompatible living donor kidney transplants have been implemented as one method for expanding donor indications. In blood type incompatible kidney transplantation, IgM type antibodies (anti-A antibody, anti-B antibody, etc.) that are natural antibodies present in the recipient body react with blood type antigens expressed in transplanted renal vascular endothelial cells. Therefore, how to avoid the abolition of transplanted kidney function early after transplantation, which can be caused by so-called antibody-associated rejection (AMR), was the biggest problem.

  In contrast, by exchanging plasma before surgery, anti-A and anti-B antibodies, which are IgM antibodies, are removed, and further, splenectomy is performed, so that ABO blood type-incompatible living donor kidney transplantation is planned. It has been reported that it can be safely implemented (Non-Patent Document 1). In Japan, the double filtration plasma separation and exchange method (DFPP) was performed in 1989 to remove IgM-type anti-A and anti-B antibodies, and spleenctomy to remove blood type incompatible kidneys. It has been reported that transplantation is performed and stable results are obtained (Non-patent Document 2). Furthermore, plasma is passed through a column packed with a resin in which blood group A and B antigens are immobilized, and anti-A and anti-B antibodies, which are IgM antibodies, are removed, and blood type incompatible kidney transplantation is performed by splenectomy. It has been reported that good results can be obtained by carrying out (Non-patent Document 3).

On the other hand, as a product for removing immune complexes and IgG antibodies, tryptophan and phenylalanine Imusoba of Asahi Kasei Kuraray Medical to ligand -TR ^TM and Imusoba -PH ^TM is known (Non-Patent Document 4). Patent Document 1 describes that a ligand having a thienyl group can remove an anti-desmoglein antibody (IgG type antibody) that is a pathogen of pemphigus.

JP 2008-194304 A

Alexandre GPJ, De Bruyer M et al. : Splenectomy as a prequesity for successful human ABO-incompatible real translation. Transplant Proc 17: 138-143, 1985. Takahashi K, Tanabbe K, Ooda S et al. : Prophylactic use of a new immunosuppressive agent, deoxyspergualin, inoaients withkidneytransplantorbidoporbidpremandoroporbidoporbid. Transplant Proc 26: 1078-1082, 1991. Ota K, Takahashi K, Ashishi T, Sonda T, Oka T, Ueda S, Amemiya H, Shiramizu T, Okazaki H, Akiyama N, Hasegawa A, KawaTau. : Multicentre trial of ABO-incompatible kidney translation. Japan Byossorb ABO-incompatible kidney transform study group. Transpl Int. 1992; 5 Suppl 1: S40-3. Yoshida M, Tamura Y, Yamada Y, Yamawaki N, Yamashita Y. et al. : Immusorba TR and Immusorba PH: Basics of Design and Features of Functions. Therapeutic apheresis, vol2, pages 185-192 (1998).

  Non-Patent Document 1 describes a method of removing anti-A antibody and anti-B antibody, which are IgM antibodies, by exchanging plasma, but in this method, it is necessary to supplement plasma other than that of the patient. That is, since plasma (or albumin) donated by a healthy person is used for plasma supplementation, there are side effects similar to those of blood transfusion. Especially regarding viral infections, hepatitis B, hepatitis C, AIDS (acquired) Although immunodeficiency syndrome) and adult T-cell leukemia have been tested and confirmed to be negative, the possibility of infection with these viruses is not zero and there is a risk of infection, and the cost of medical expenses There was a drawback of being expensive.

  Non-Patent Document 2 describes a method for removing anti-A antibody and anti-B antibody, which are IgM antibodies, by performing double filtration plasma separation (DFPP). In this method, blood cells and plasma components are separated by a primary membrane, and then the separated plasma components are further removed by a secondary membrane. However, in this method, when the antibody component is removed, fibrinogen, which is an important component of the blood coagulation factor, is also removed, so that the blood coagulation ability is reduced and there is a risk of bleeding. In addition, in order to avoid the risk of bleeding, it may be necessary to replenish the fibrinogen preparation after the operation, and in this case, there was also a risk of infection by an unknown virus present in the fibrinogen preparation.

  Non-Patent Document 3 describes a method of removing anti-A antibody and anti-B antibody, which are IgM antibodies, by passing plasma through a column packed with a resin in which blood type A antigen and B antigen are immobilized. . According to this method, it is presumed that the adsorption of other plasma components such as fibrinogen can be suppressed and the IgM antibody can be specifically removed, but the A antigen and B antigen immobilized on the resin are Since it is mainly composed of sugar chains, its safety and stability are poor, and there is a disadvantage that it is very expensive to produce this antigen.

  Non-Patent Document 4 describes that an IgG type antibody or an IgM type antibody is adsorbed by using a column packed with a polyvinyl alcohol resin to which tryptophan or phenylalanine is immobilized as a ligand. However, these columns cannot sufficiently adsorb IgM-type antibodies and have the disadvantages of adsorbing fibrinogen.

  Patent Document 1 describes that an anti-desmoglein antibody of an IgG type antibody is adsorbed, but there is no mention of the adsorption of an IgM type antibody, and further, regarding the suppression of fibrinogen adsorption. There is no mention or suggestion.

  In view of the above situation, the present invention is an IgM type that adsorbs an IgM type antibody selectively and at a high rate from blood components such as plasma while suppressing the adsorption of fibrinogen. An object is to provide an adsorbent for antibody adsorption and an IgM-type antibody removal system.

  As a result of intensive studies to solve the above problems, the present inventors have found that an adsorbent in which a ligand composed of a compound having a thienyl group and a carboxyl group is immobilized on a water-insoluble carrier suppresses the adsorption of fibrinogen. In addition, the inventors have found that IgM-type antibodies are selectively and highly adsorbed and are excellent in safety and stability, and the present invention has been completed.

That is, the present invention relates to the following.
[1]
A water-insoluble carrier;
A ligand immobilized on the water-insoluble carrier,
The ligand has a monovalent group consisting of the remaining atomic group excluding one hydrogen atom bonded to the carbon atom constituting the heterocyclic ring from thiophene or a derivative thereof, and a carboxyl group.
Adsorbent for IgM type antibody adsorption.
[2]
The adsorbent for IgM antibody adsorption according to [1], wherein the monovalent group and the carboxyl group are bonded via 5 or less atoms.
[3]
The adsorbent for IgM-type antibody adsorption according to [1] or [2], wherein the monovalent group is a thienyl group.
[4]
The adsorbent for IgM antibody adsorption according to any one of [1] to [3], wherein the ligand comprises a compound having a thienyl group and a carboxyl group.
[5]
The adsorbent for IgM antibody adsorption according to any one of [1] to [4], wherein the ligand has 5 to 13 carbon atoms.
[6]
The adsorbent for IgM type antibody adsorption according to any one of [1] to [5], wherein the ligand is at least one selected from the group consisting of thienylglycine, thienylalanine and thienylserine.
[7]
The adsorbent for IgM type antibody adsorption according to any one of [1] to [6], which is for adsorbing an IgM type antibody selected from blood type anti-A antibody and anti-B antibody.
[8]
The adsorbent for IgM type antibody adsorption according to any one of [1] to [7], wherein the amount of the immobilized ligand is 10 μmol to 200 μmol per 1 mL gel of the water-insoluble carrier.
[9]
The adsorption for IgM antibody adsorption according to any one of [1] to [8], wherein the water-insoluble carrier is a particulate porous body, and the exclusion limit molecular weight of the porous body is 150,000 to 10 million. Wood.
[10]
The adsorbent for IgM-type antibody adsorption according to any one of [1] to [9], wherein Pm / Pf ≧ 3 when the adsorption rate of fibrinogen is Pf and the adsorption rate of IgM-type antibody is Pm.
[11]
A container having an inlet port through which blood or plasma flows in and an outlet port through which blood or plasma flows out;
A blood or plasma IgM antibody removal system comprising an extracorporeal circulation module comprising the IgM antibody adsorption adsorbent according to any one of claims 1 to 7 filled in the container.
[12]
In the production of an IgM type antibody removal system in blood or plasma,
Use of an adsorbent comprising a water-insoluble carrier and a ligand comprising a compound having a thienyl group and a carboxyl group immobilized on the water-insoluble carrier.

  The adsorbent for IgM-type antibody adsorption according to the present invention suppresses the adsorption of fibrinogen in plasma and selectively adsorbs IgM-type antibody at a high rate, and the adsorbent is safe and stable in storage. Excellent in properties. In addition, by using an extracorporeal circulation module encapsulating the adsorbent, removal of IgM autoantibodies during ABO-incompatible kidney transplantation, liver transplantation, and bone marrow transplantation, and macroglobulinemia using IgM autoantibodies as a causative agent, And IgM autoantibodies can be safely removed from patients with high IgM syndrome.

It is a schematic cross section showing one embodiment of an extracorporeal circulation module using an adsorption material for IgM type antibody adsorption.

  The present invention will be described in detail below.

  The adsorbent for adsorbing IgM antibodies of the present invention is extremely effective as an adsorbent for IgM antibodies because it can selectively adsorb IgM antibodies at a high rate and at the same time suppress adsorption of fibrinogen and the like. IgM type antibody is the largest protein among immunoglobulins composed of 5 subunits and having a molecular weight of about 900,000 and a sedimentation constant of 19S, and is also called macroglobulin. Each subunit is composed of an H chain (two μ chains) and an L chain (χ, λ chain) lacking a hinge region, and is linked to each other by an SS bond formed between CH3 and CH4 domains. A pentamer structure is constituted by one J chain. There are two subclasses of μ chain, μ1 and μ2. IgM type antibodies are expressed on the cell surface as B cells differentiate, and it has become clear that they control various functions involved in the immune system such as B cell differentiation, antibody production, and apoptosis. IgM type antibodies are the main antibodies against the A and B antigens derived from the ABO blood group of erythrocytes.

  There are five classes of immunoglobulins. IgG and IgM are one of these classes. Different classes of immunoglobulins have different structures and different physical / chemical properties. For example, IgG-type antibodies exist as monomers, and IgM exists as a pentamer, so the molecular weights differ greatly. In addition, IgG-type antibodies have high affinity with protein A and protein G, while IgM-type antibodies show very low affinity. Because it has such a different structure and different physical / chemical properties, it can be applied to adsorption / removal of IgM antibody even if it is a ligand that can adsorb / remove IgG type antibody. Usually not considered. Although Patent Document 1 describes that a ligand having a thienyl group can adsorb and remove IgG type antibodies, there is no description or suggestion about adsorption and removal of IgM type antibodies. That is, the property of adsorption removal ability of IgM antibody possessed by a ligand having a thienyl group and a carboxyl group (and the ability to suppress fibrinogen adsorption at the same time) is an unknown attribute that cannot be predicted at all from the description of the cited document 1. The adsorbent for IgM-type antibody adsorption of the present invention provides a new application using this unknown attribute.

  The adsorbent for IgM-type antibody adsorption of the present invention includes a water-insoluble carrier and a ligand immobilized on the water-insoluble carrier.

  The ligand according to the present invention has a monovalent group consisting of the remaining atomic group obtained by removing one hydrogen atom bonded to the carbon atom constituting the heterocyclic ring from thiophene or a derivative thereof, and a carboxyl group.

  In this specification, “derivative of thiophene” means a thiophene having a substituent, or a sulfur atom constituting a heterocyclic ring is coordinated between a lone pair of a sulfur atom and an electron-accepting functional group. It means thiophene forming a bond.

  Examples of the substituent include an alkyl group, an alkoxy group, an aryl group, an aryloxy group, an amino group, a halogen atom, an acyl group, an amide group, a hydroxy group, a cyano group, and a nitro group. When a plurality of substituents are present, they may be the same or different. Especially, as a substituent, an alkyl group is desirable and a methyl group, an ethyl group, and a propyl group are preferable.

  Examples of the electron-accepting functional group include a hydrogen atom, an ammonium ion, and a monovalent metal ion.

  The ligand is preferably a compound in which a monovalent group and a carboxyl group are bonded via 5 or less atoms. In addition, a compound in which a monovalent group and a carboxyl group are bonded via one or two carbon atoms is more preferable.

As the monovalent group, a thienyl group is preferable. Moreover, as a ligand, the compound which has a thienyl group and a carboxyl group is preferable. The number of thienyl groups contained in the ligand is not particularly limited, but is preferably 1 to 5 in view of the chemical stability and blood compatibility of the ligand. Further, since the ligand has a carboxyl group, adsorption of fibrinogen can be suppressed. In this specification, “thienyl group” refers to a residue obtained by removing one hydrogen atom from thiophene (chemical formula: C ₄ H ₄ S).

  The number of carbon atoms of the ligand is preferably 5 or more and 13 or less, and more preferably 5 or more and 8 or less. When the number of carbon atoms is 5 or more, the adsorptivity to the IgM antibody is sufficiently exhibited. Moreover, when the number of carbon atoms of the ligand is 13 or less, the hydrophobicity of the ligand becomes moderate, and nonspecific adsorption that other proteins in plasma are adsorbed can be sufficiently suppressed. Good compatibility. Further, it is preferable because solubility and storage stability at the time of ligand fixation are good. Furthermore, it is more preferable that the number of carbon atoms of the ligand is 8 or less because the above-described effect becomes more remarkable.

  The ligand preferably has one or more monovalent groups obtained by removing one hydrogen atom from ammonia effective for immobilization on a carrier and adsorption of an IgM type antibody.

  Specific examples include thienylglycine, thienylalanine, and thienylserine, which are amino acids containing a thienyl group. Without a carboxyl group, the adsorption of fibrinogen increases and an important blood coagulation factor is lost during extracorporeal circulation, resulting in a risk of bleeding and the like. Although the mechanism of suppression of fibrinogen adsorption by having a carboxyl group is not limited to this, the present inventors have attributed that the isoelectric point of fibrinogen is relatively on the negative side. thinking.

  The amino acids and amino acid derivatives described in this specification are L-forms unless otherwise specified.

  The water-insoluble carrier in the present invention means a carrier that is not soluble in water. For example, when it comes into contact with blood, it does not dissolve in the blood.

  As a carrier used for immobilizing a low molecular ligand, an amino group, a carboxyl group, a hydroxyl group, etc. that have a hydrophilic surface and can be used to form a covalent bond with the low molecular ligand. Those having a reactive functional group are preferred. The water-insoluble carrier is preferably a porous material having a large effective surface area that can be adsorbed (porous material).

  The carrier can be in any shape such as particulate, fiber, sheet, hollow fiber, etc. However, in consideration of the amount of ligand retained and the handling property as an adsorbent, the carrier is preferably particulate.

  The average particle diameter of the particulate carrier can be from 25 μm to 2500 μm, but considering the specific surface area (adsorption capacity as an adsorbent) and the fluidity of the body fluid, those having a particle diameter of 50 μm to 1500 μm are preferable.

  As the carrier, organic or inorganic porous materials such as cellulose gel, dextran gel, agarose gel, polyacrylamide gel, porous glass, vinyl polymer gel, etc. can be used. For carriers used in ordinary affinity chromatography Any of these materials can be used.

Examples of these carriers include Asahi Kasei Microcarrier (Asahi Kasei Co., Ltd.), CM-Cellulofine (registered trademark) CH (exclusion limit protein molecular weight: about 3 × 10 ⁶ , sold by Seikagaku Corporation), and the like. Cellulose-based carriers, all porous activated gels described in JP-B-1-44725, and polyvinyl alcohol such as CM-Toyopearl (registered trademark) 650C (exclusion limit protein molecular weight: 5 × 10 ⁶ , manufactured by Tosoh Corporation) System carrier, polyacrylamide type carrier such as CM-Trisacryl M (CM-Trisacryl M) [exclusion limit protein molecular weight: 1 × 10 ⁷ , Pharmacia-LKB, Sweden], Sepharose CL-4B ( SepharoseCL-4B) [exclusion limit protein molecular weight: 2 × ¹⁰ 7, Swedish country Pharmacia -LKB (Pharmacia-LKB) Co. Ltd.] organic carrier such as agarose based carrier, such as, and CPG-10-1000 [exclusion limit protein molecular weight: 1 × 10 ^8, the average pore diameter: 100 nm, USA Electro - New Inorganic supports such as porous glass such as those manufactured by Electro-nucleonics Co., Ltd.

  As the water-insoluble carrier, a particulate porous body is preferable, and for example, porous polymer particles can be used. The particulate porous body can immobilize a ligand, and the exclusion limit molecular weight (protein) is preferably 150,000 to 10,000,000. The most preferred exclusion limit molecular weight for the purposes of the present invention is between 1 and 5 million.

  The polymer composition may be a known polymer that can take a porous structure, such as a polyamide-based, polyester-based, polyurethane-based, or vinyl-based polymer. Particularly, a vinyl compound-based porous material that has been hydrophilized with a hydrophilic monomer. Sex polymers give favorable results.

  The method for immobilizing the ligand on the water-insoluble carrier is not limited, but a covalent bond is preferred. For example, when the carrier has a carboxyl group, it is converted to a succinimideoxycarbonyl group by reacting with N-hydroxysuccinimide, and this is reacted with a ligand at the amino group (active ester method). Can be mentioned. When the carrier has an amino group or a carboxyl group, a method of condensing the carboxyl group or amino group of the ligand in the presence of a condensation reagent such as dicyclohexylcarbodiimide (condensation method), Examples include a method of bonding using a compound having two or more functional groups. In addition, when the carrier has a hydroxyl group, a cyanogen halide such as cyanogen bromide is allowed to act on the carrier, and a method of reacting with the amino group portion of the ligand or an epoxide such as epichlorohydrin is allowed to act on the carrier, Examples thereof include a method of reacting with the amino group part or hydroxyl part of the ligand.

  Furthermore, if necessary, a molecule (spacer) having an arbitrary length can be introduced between the water-insoluble carrier and the ligand. Examples of the spacer molecule include a polymethylene chain and a polyethylene glycol chain. For example, the hydroxyl group of agarose and the isocyanate group on one side of hexamethylene diisocyanate can be reacted and combined, and the remaining isocyanate group and the amino group, hydroxyl group, carboxyl group, etc. of the ligand can be reacted and combined.

  The amount of ligand bound to the carrier in the present invention, that is, the amount of ligand retained (the amount of immobilized ligand) is in the range of 10 μmol to 200 μmol, more preferably in the range of 10 μmol to 100 μmol, per 1 mL gel of the carrier. . When the retention amount is less than 10 μmol, the adsorption ability of the IgM antibody tends to decrease, and when it exceeds 200 μmol, nonspecific adsorption of useful plasma components tends to occur.

  As a method for measuring the amount of IgM-type antibody adsorbed to the adsorbent for IgM-type antibody adsorption, a plasma component is added to the adsorbent for IgM-type antibody adsorption, the IgM-type antibody is adsorbed, and the amount of IgM-type antibody in the plasma decreased The method of measuring a rate is mentioned. For example, the carrier on which the ligand is immobilized can be immersed in plasma for a certain period of time, and then the rate of decrease in IgM antibody can be determined by quantification using an immunological technique. Alternatively, plasma is allowed to flow through a column packed with a carrier on which a ligand is immobilized, and then the rate of decrease in IgM antibody in the plasma before and after passing through the column is determined by an immunological technique. Can also be determined quantitatively.

  The above-mentioned adsorbent for adsorbing IgM antibodies can suppress the adsorption of fibrinogen and simultaneously adsorb IgM antibodies selectively and at a high rate. Here, in the above-mentioned adsorbent for IgM-type antibody adsorption, when the adsorption rate of fibrinogen is Pf and the adsorption rate of IgM-type antibody is Pm, the value of Pm / Pf is usually 3 or more. The upper limit of the value of Pm / Pf is not particularly limited, but is usually 100 or less.

  The adsorbent for IgM-type antibody adsorption of the present invention can be used by being filled and held in a container having an inlet port through which blood or plasma flows and an outlet port through which blood or plasma flows out.

  FIG. 1 is a schematic cross-sectional view showing an embodiment of an extracorporeal circulation module using an adsorbent for IgM-type antibody adsorption. The extracorporeal circulation module 1 shown in FIG. 1 is screwed into a cylindrical end of a cylinder 2 having a cap 6 having an inlet port 5 through which blood or plasma flows through a packing 4 with a filter 3 on the inside. A cap 8 having an outlet port 7 for allowing blood or plasma to flow out through a packing 4 ′ with a filter 3 ′ on the inside of the other end opening 2 is screwed to form a container, and the filters 3 and 3 ′. The adsorbent layer 9 is formed by filling and holding the IgM-type adsorbing adsorbent of the present invention in the gap. The extracorporeal circulation module 1 can be used, for example, for removing IgM type antibodies (anti-A antibody and anti-B antibody) that increase at the time of ABO blood group-incompatible living body kidney transplantation.

  The adsorbent layer 9 may be filled with the adsorbent for IgM-type antibody adsorption of the present invention alone, or may be mixed with other adsorbents and filled. Moreover, you may fill so that the adsorption material for IgM type | mold antibody adsorption of this invention and another adsorption material may laminate | stack. As other adsorbents, for example, adsorbents of other malignant substances (antigens) such as DNA, activated carbon having a wide range of adsorption ability, and the like can be used. As a result, a wide range of clinical effects due to the synergistic effect of the adsorbent can be expected. The volume of the adsorbent layer 9 is suitably about 50 mL to 400 mL when used for extracorporeal circulation.

  The extracorporeal circulation module 1 can be used as an IgM type antibody removal system for removing IgM type antibodies in blood or plasma.

  When the system for removing IgM type antibody in blood or plasma of the present invention is used in extracorporeal circulation, there are two main methods. For one thing, the blood extracted from the body is separated into a plasma component and a blood cell component using a centrifuge or a membrane plasma separator, and then the plasma component is passed through the IgM antibody removal system to be purified. Then, the blood cell component is returned to the body together with the blood cell component, and the other one is a method of purifying the blood taken out from the body directly by passing it through the IgM type antibody removal system.

  In addition, with regard to the blood or plasma passage speed, since the adsorption rate of the adsorbent for IgM antibody adsorption is very high, the particle size of the adsorbent can be coarsened and the filling degree can be lowered. Regardless of the shape, a high passage rate can be provided so that large amounts of blood or plasma can be processed. As a method of passing blood or plasma, it may be continuously passed or used intermittently depending on clinical needs or according to the equipment status of the equipment.

  The IgM type antibody removal system can be manufactured by filling an adsorbent into a container having an inlet port through which blood or plasma flows in and an outlet port through which blood or plasma flows out. The adsorbent is an adsorbent comprising a water-insoluble carrier and a ligand made of a compound having a thienyl group and a carboxyl group, which is immobilized on the water-insoluble carrier. Moreover, it is preferable to use the IgM type | mold antibody adsorption adsorbent of this invention as an adsorbent.

  The adsorbent for IgM antibody adsorption and the IgM antibody removal system of the present invention may be used as an adsorbent for IgM autoantibody adsorption and an IgM autoantibody removal system. As used herein, “autoantibody” means an antibody produced against autologous cells or tissues (including transplanted ones). That is, for example, the IgM type antibody that increases during blood type ABO incompatible transplantation is an IgM type autoantibody. The adsorbent for IgM type antibody adsorption and the IgM type antibody removal system of the present invention are also suitable for adsorption and removal of such IgM type autoantibodies. Therefore, IgM type autoantibodies (anti-A antibodies) that increase during blood type ABO incompatible transplantation using the adsorbent for adsorbing IgM type (auto) antibodies and the IgM type (auto) antibody removal system of the present invention Adsorbing and removing (anti-B antibody) is an effective treatment.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these.

[Examples 1 to 4]
1. Production method of adsorbent Spherical porous body made of polyvinyl acetate (Asahi Glass Co., Ltd., average particle size of 100 μm, exclusion limit molecular weight of about 1 million or more, the amount of pullulan having a molecular weight of 40,000 that can be filled in 1 mL of resin is 0.20 mL 100 g / mL or more) is charged into 1 L of dimethyl sulfoxide (Wako Pure Chemical Industries, Ltd.), 120 g of sodium hydroxide (Wako Pure Chemical Industries, Ltd.) 120 g, epichlorohydrin (Wako Pure Chemical Industries, Ltd.) 780 mL, hydrogenated Epoxy groups were introduced by reacting 750 mg of sodium boron (manufactured by Wako Pure Chemical Industries, Ltd.) at 30 ° C. for 5 hours. After the reaction, it was washed with methanol (manufactured by Wako Pure Chemical Industries, Ltd.) and then washed with pure water to obtain an activated porous body (hereinafter also referred to as “activated carrier”). The amount of epoxy groups introduced into the obtained activated porous material was confirmed by a titration method. Specifically, after dropping 2 drops of 1% phenolphthalein ethanol solution (Wako Pure Chemical Industries, Ltd.) into 4 mL of 1.3 mmol / L sodium thiosulfate aqueous solution (Wako Pure Chemical Industries, Ltd.) and 2 mL of activated carrier. Then, add 0.1N hydrochloric acid at 70 ° C. until red coloration is not confirmed, and calculate the amount of epoxy groups according to the formula of “activation amount (epoxy group amount) = hydrochloric acid titration amount / resin amount × 100”. Asked. As a result, the amount of the introduced epoxy group was 110 μeq / mL gel or more.

2. Ligand immobilization reaction Next, a pH 9.3 carbonate buffer (Wako Pure Chemical Industries, Ltd., sodium carbonate / sodium hydrogen carbonate) was used as a solvent, and for Examples 1 to 3, an immobilized amount of 50 μeq / mL gel was obtained. The ligand was dissolved as This ligand solution and the above-mentioned activated carrier were reacted at 50 ° C. for 16 hours to covalently bond the amino group and the epoxy group of the activated carrier to obtain an adsorbent. As a ligand, thienylglycine (L-α- (3-THIENYL) GLYCINE) is used in Example 1, thienylserine (β- (2-THIENYL-DL-SERINE)) is used in Example 2, and , Thienylalanine (β- (2-THIENYL) -D-ALANINE)) was used (the ligands of Examples 1 to 3 were all manufactured by SIGMA). The amount of ligand immobilized is determined by measuring the absorbance spectrum of the ligand solution at a wavelength of 200 nm to 280 nm, and using the wavelength showing the highest absorbance, the amount immobilized per 1 mL gel (eq / mL gel) from the difference before and after the immobilization reaction. Calculated as

3. Adsorption experiment by column method 2.5 mL of Laboratory Column (manufactured by MOLECULAR BIOTECHNOLOGY) was filled with 1.0 mL of adsorbent. Then, 3.0 mL of plasma obtained by centrifuging from healthy human blood to which CPD was added to blood at a ratio of 1: 8 (volume ratio) was 10 at a flow rate of 0.3 mL / min from the inlet of the column with a syringe pump. The plasma was separated and all the plasma flowing out from the column outlet was collected.

4). Analysis The IgM concentration and the fibrinogen concentration were measured for healthy human plasma (original plasma) before flowing through the column and plasma collected from the column. The concentration of IgM was measured by the immunoturbidimetric method, and the fibrinogen concentration was measured by the thrombin clotting time method. The amount of adsorption was calculated as the difference between the concentration of IgM and fibrinogen in the original plasma (concentration in the original plasma) and the concentration of IgM and fibrinogen in the plasma that was discharged from the column and collected (the concentration in plasma after the adsorption reaction). The adsorption performance was calculated by the following equation: adsorption rate (%) = (1−plasma concentration after adsorption reaction / original plasma concentration) × 100.

[Comparative Examples 1 to 6 and Reference Example]
Comparative Examples 1 to 6 were performed in the same manner as in Examples 1 to 3 except that a compound containing no thienyl group and a carboxyl group was used as a ligand. Moreover, the reference example was performed by the method similar to Examples 1-3 except having used the activated support | carrier which does not fix a ligand. As a ligand, in Comparative Example 1, glycine (manufactured by SIGMA) is used, in Comparative Example 2, alanine (manufactured by SIGMA) is used, in Comparative Example 3, serine (manufactured by SIGMA) is used, and in Comparative Example 4, 2-thiophene is used. Ethylamine (2-THIOPHENEETHYLAMINE) (manufactured by Wako Pure Chemical Industries, Ltd.), phenylalanine (manufactured by SIGMA) was used in Comparative Example 5, and tryptophan (manufactured by SIGMA) was used in Comparative Example 6.

[result]
The results of Examples 1 to 4, Comparative Examples 1 to 6 and Reference Example (no ligand) are shown together in Table 1. The adsorbents containing ligands having both thienyl groups and carboxyl groups in Examples 1 to 3 had lower fibrinogen adsorption rates and higher IgM antibody adsorption rates than Comparative Examples 1 to 6. . That is, it can be seen that IgM antibody is selectively adsorbed from plasma.

  The adsorbent for adsorbing IgM antibodies of the present invention is useful as a therapeutic instrument for extracorporeal circulation adsorption therapy because it suppresses fibrinogen adsorption and can selectively adsorb IgM antibodies at a high rate.

  DESCRIPTION OF SYMBOLS 1 ... Extracorporeal circulation module, 2 ... Cylinder, 3, 3 '... Filter, 4, 4' ... Packing, 5 ... Inlet port, 6 ... Cap, 7 ... Outlet port, 8 ... Cap, 9 ... Adsorption for IgM antibody adsorption Wood.

Claims (12)

A water-insoluble carrier;
A ligand immobilized on the water-insoluble carrier,
The ligand has a monovalent group consisting of the remaining atomic group excluding one hydrogen atom bonded to the carbon atom constituting the heterocyclic ring from thiophene or a derivative thereof, and a carboxyl group.
Adsorbent for IgM antibody adsorption. The monovalent group and said carboxyl group is linked via a 5 atoms or less, IgM antibody-adsorbing adsorbent according to claim 1. The monovalent group is a thienyl group, IgM antibody-adsorbing adsorbent according to claim 1 or 2. Said ligand, a compound having a thienyl group and a carboxyl group, IgM antibody-adsorbing adsorbent according to any one of claims 1-3. The number of carbon atoms of the ligand is 13 from five, IgM antibody-adsorbing adsorbent according to any one of claims 1-4. Wherein said ligand is at least one selected from the group consisting of thienyl glycine, thienylalanine and Chieniruserin, IgM antibody-adsorbing adsorbent according to any one of claims 1-5. It is for the adsorption of IgM antibody selected from blood anti-A antibody and anti-B antibodies of, IgM antibody-adsorbing adsorbent according to any one of claims 1-6. The amount of immobilized the ligand is a said 10μmol~200μmol per water-insoluble carrier 1mL gel, IgM antibody-adsorbing adsorbent according to any one of claims 1 to 7. The adsorption for IgM type antibody adsorption according to any one of claims 1 to 8, wherein the water-insoluble carrier is a particulate porous body, and the porous body has an exclusion limit molecular weight of 150,000 to 10,000,000. Agent . The adsorption rate of fibrinogen Pf, the adsorption rate of IgM antibody is taken as Pm, is Pm / Pf ≧ 3, IgM antibody-adsorbing adsorbent according to any one of claims 1-9. A container having an inlet port through which blood or plasma flows in and an outlet port through which blood or plasma flows out;
Either the IgM antibody-adsorbing adsorbent according to paragraph (1) consists of extracorporeal circulation module comprising a blood or plasma IgM antibody removal system according to claim 7 filled in the container. In the production of an IgM type antibody removal system in blood or plasma,
A water-insoluble carrier, the water-insoluble carrier is immobilized, the use of IgM antibody-adsorbing adsorbent comprising a ligand comprising a compound having a thienyl group and a carboxyl group.

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