JP5940788B2 - Selective separation material, method for producing the same, and blood treatment device - Google Patents

Description

  The present invention relates to a selective separation material, a method for producing the same, and a blood treatment device.

  For the purpose of removing a specific component from blood, a substance having affinity for the specific component, that is, a separation material in which a ligand is fixed to a water-insoluble carrier is widely used clinically. These separating materials are used in such a manner that the blood of the patient is once taken out of the body, the blood itself or the plasma separated by the plasma separator is flowed through the separating material, processed, and then returned to the patient.

  For this reason, when the ligand is eluted from the separation material, there is a risk that the ligand directly enters the patient and causes various physiological effects. Therefore, a separation material having a large ligand elution amount cannot be clinically used from the viewpoint of safety. That is, in putting these separation materials into practical use, the most important technical issue is to strengthen the bond between the water-insoluble carrier and the ligand and reduce the amount of the eluted ligand.

  The separation material is also required to have blood compatibility (biocompatibility) in which proteins, cells (for example, leukocytes and platelets) in blood or plasma, and other blood components are difficult to adsorb on the surface. In particular, in a selective separation material, in order to selectively adsorb a specific component, it is desired that the separation material itself does not nonspecifically adsorb components other than the target component.

  Therefore, in order to improve the hydrophilicity and hydrophobicity of the surface of the medical material, a method of irradiating with radiation and ultraviolet rays, a method of plasma processing by arc, direct current glow, high frequency, microwave, corona discharge, etc., and UV-ozone A method is widely used in which a radical generated by a treatment method or the like is used as a starting point and a radical polymerizable monomer is allowed to act thereon to form a graft polymerization layer on the surface of a medical material.

  On the other hand, N-methacryloyloxyethyl-N, N-dimethylammonium-α-N-methylcarboxybetaine (hereinafter also referred to as “CMB”) having a glycine-type amphoteric group has been proposed as a polymer for enhancing biocompatibility. (For example, Patent Documents 1, 2, and 3). There is a report that a copolymer of CMB and a polymerizable monomer has excellent biocompatibility such as a small interaction with biological components such as proteins and blood cells (Patent Document 4).

  However, in order to improve biocompatibility, when a hydrophilic polymer such as CMB is highly contained on the substrate surface, biological components such as proteins and blood cells are difficult to adsorb, and at the same time, the ligand itself cannot be bound. There's a problem.

  As a method for immobilizing an antibody on a substrate containing a hydrophilic polymer, a method using a buffer containing about 1M dipotassium hydrogen phosphate is disclosed (Patent Document 5). Non-Patent Document 1 describes a method of using a buffer containing about 0.5 M ammonium sulfate in order to promote chemical bonding between an epoxy group and an antibody.

JP-A-9-95474 JP-A-9-95586 JP-A-11-222470 JP 2007-130194 A JP 2008-128854 A

Wheatly JB, et. al. , J Chromatogr A .; 849 (1): 1-12 (1999).

  As described above, when producing a selective separation material that selectively removes or recovers a target substance from blood as well as the cell separation material, the safety of leakage of the ligand from the separation material is extremely small. is important. That is, it is necessary to substantially achieve the immobilization of the ligand by chemical bonding and sufficiently reduce the physical bonding of the ligand. Further, in the selective separation material, in order to selectively adsorb specific components, a substrate that does not cause nonspecific adsorption of components other than the target component to the substrate itself is desired. One example is the use of a substrate containing a high amount of hydrophilic polymer.

  In the method described in Patent Document 5, the amount of antibody immobilized is insufficient for clinical use. The method described in Non-Patent Document 1 cannot be applied as a chemical fixing method to a substrate containing a high amount of hydrophilic polymer. Moreover, after fixing a ligand to a base material, the method of coating the base-material surface with a hydrophilic polymer is also implemented, but in this case, the problem that the coated polymer elutes and the performance of the ligand decreases. There's a problem.

  Further, in the selective separation material, it is also important to control the orientation of the ligand so that the ligand can efficiently bind to the target substance. For example, when an antibody is used as a ligand, the antibody needs to be immobilized such that the Fc part of the antibody binds to the base material and the Fab part, which is a binding site with the antigen, faces outward from the base material surface. When the antibody is immobilized so that the Fc part is directed outward from the substrate surface, the efficiency of binding to the target substance is reduced, and in addition, there is a problem that platelet adsorption to the Fc part and complement activation occur. Arise.

  As a method for controlling the orientation of the antibody, there is a method of specifically adsorbing with the Fc part of the antibody using Protein G / A or biotin / avidin. However, Protein G / A cannot be used clinically because it has a high affinity for antibodies in human blood and the antibody used as a ligand is replaced with an antibody in blood. Further, even when biotin / avidin is used, since the binding between biotin and avidin is not a chemical bond, there is a problem of leakage of the antibody from the separating material and it is not clinically used.

  The present inventor has excellent biocompatibility such as low non-specific interaction with biological components such as proteins and blood cells, and binds the ligand firmly and allows specific interaction with the immobilized ligand. Ligand immobilization base material comprising a copolymer of CMB and a polymerizable monomer having an electrophilic functional group bonded to at least the surface of a water-insoluble carrier as a base material for ligand immobilization capable of exhibiting high adsorption performance based on Have found that this problem can be solved, and the applicant has filed a patent application (Japanese Patent Application No. 2010-083455). However, the specification of the prior application did not describe the control of the orientation of the ligand to be immobilized, and a higher usage efficiency of the ligand was desired. Further, although not completely the same as the polymerization conditions of the monomers described in the specification of the prior application, when the present inventors copolymerized the monomers, as the CMB molar composition ratio of the substrate surface increases, Although platelet adsorption is suppressed, it has been found that the ligand becomes difficult to be immobilized and the ability to remove the target cells is reduced. It has also been clarified that the ligand immobilization method described in the specification of the prior application cannot control the orientation of the antibody (see Comparative Examples 4 and 7, etc. of the present application).

  The present invention has been made in view of the above circumstances, and an object thereof is to provide a selective separation material having excellent biocompatibility and having a sufficient amount of antibodies chemically bonded with good orientation. . Another object of the present invention is to provide a method for producing a selective separation material having excellent biocompatibility and having a sufficient amount of antibodies chemically bonded with good orientation.

［一般式（１）中、Ｒ^１はＨ又はＣＨ_３、Ｒ^２は求電子官能基を有する有機基を示す。］
［５］
前記求電子官能基がエポキシ基である、［４］に記載の選択的分離材。
［６］
前記水不溶性担体が、多孔膜又は粒子である、［１］〜［５］のいずれかに記載の選択的分離材。
［７］
血液の導入口及び導出口を有する容器と、
該容器の内部に充填された［１］〜［６］のいずれかに記載の選択的分離材と、を備える、血液処理器。
［８］
［１］〜［６］のいずれかに記載の選択的分離材の製造における抗体固定化用基材の使用であって、
前記抗体固定化用基材が、水不溶性担体と、該水不溶性担体の少なくとも表面に結合したポリマーと、を有し、
前記ポリマーが、親水性モノマー及び疎水性モノマーの共重合体であり、かつ前記親水性モノマーに由来する構造単位のモル組成比が、全構造単位に対して、０．３０〜０．６０である、前記使用。
［９］
水不溶性担体と、該水不溶性担体の少なくとも表面に結合したポリマーとを有する抗体固定化用基材に対し、濃度が０．６Ｍ以上のアンチカオトロピックイオンの存在下、酸性ｐＨの条件下で抗体を結合させる工程を備え、
前記ポリマーが、親水性モノマー及び疎水性モノマーの共重合体であり、かつ前記親水性モノマーに由来する構造単位のモル組成比が、全構造単位に対して、０．３０〜０．６０である、［１］〜［６］のいずれかに記載の選択的分離材を製造する製造方法。 The present invention relates to the following [1] to [9].
[1]
A water-insoluble carrier, a polymer bound to at least the surface of the water-insoluble carrier, and an antibody bound to the polymer,
The polymer is a copolymer of a hydrophilic monomer and a hydrophobic monomer, and the molar composition ratio of structural units derived from the hydrophilic monomer is 0.30 to 0.60 with respect to all the structural units. ,
The selective separation material, wherein the fixed amount per unit surface area of the antibody is 1.50 mg / m ² or more, and the anti-Fab / anti-Fc ratio value indicating antibody orientation is 6 or more.
[2]
The selective separation material according to [1], wherein the antibody has a chemical fixation rate of 80% or more.
[3]
The hydrophilic monomer is at least one selected from the group consisting of N-methacryloyloxyethyl-N, N-dimethylammonium-α-N-methylcarboxybetaine, sulfobetaine and polyethylene glycol monomethacrylate [1] Or the selective separation material as described in [2].
[4]
The selective separation material according to any one of [1] to [3], wherein the hydrophobic monomer is a polymerizable monomer represented by the following general formula (1).

[In General Formula (1), R ¹ represents H or CH ₃ , and R ² represents an organic group having an electrophilic functional group. ]
[5]
The selective separation material according to [4], wherein the electrophilic functional group is an epoxy group.
[6]
The selective separation material according to any one of [1] to [5], wherein the water-insoluble carrier is a porous membrane or particles.
[7]
A container having a blood inlet and outlet, and
A blood treatment device comprising: the selective separation material according to any one of [1] to [6] filled in the container.
[8]
Use of the antibody immobilization substrate in the production of the selective separation material according to any one of [1] to [6],
The antibody immobilization substrate comprises a water-insoluble carrier and a polymer bound to at least the surface of the water-insoluble carrier;
The polymer is a copolymer of a hydrophilic monomer and a hydrophobic monomer, and the molar composition ratio of structural units derived from the hydrophilic monomer is 0.30 to 0.60 with respect to all structural units. , Said use.
[9]
An antibody is immobilized on an antibody immobilization substrate comprising a water-insoluble carrier and a polymer bound to at least the surface of the water-insoluble carrier in the presence of an antichaotropic ion having a concentration of 0.6 M or more under acidic pH conditions. Comprising the step of combining,
The polymer is a copolymer of a hydrophilic monomer and a hydrophobic monomer, and the molar composition ratio of structural units derived from the hydrophilic monomer is 0.30 to 0.60 with respect to all structural units. The manufacturing method which manufactures the selective separation material in any one of [1]-[6].

  According to the present invention, it is possible to provide a selective separation material having excellent biocompatibility and having a sufficient amount of antibodies chemically bonded with good orientation. Moreover, according to the present invention, it is possible to provide a method for producing a selective separation material having excellent biocompatibility and having a sufficient amount of antibodies chemically bonded with good orientation.

It is a schematic cross section of the selective separation material according to an embodiment. It is a schematic cross section of the selective separation material according to an embodiment. It is a schematic cross section of the blood processing apparatus which concerns on one Embodiment.

  Hereinafter, although this invention is demonstrated in detail based on embodiment, this invention is not limited to these embodiment.

  The selective separation material according to this embodiment includes a water-insoluble carrier, a polymer bound to at least the surface of the water-insoluble carrier, and an antibody bound to the polymer.

(Water-insoluble carrier)
In the present specification, the water-insoluble carrier means a carrier that is insoluble in water. For example, when it comes into contact with blood, it does not dissolve in the blood.

  Examples of the material forming the water-insoluble carrier according to the present embodiment include organic polymer compounds such as natural polymers, synthetic polymers, and regenerated polymers, inorganic compounds typified by glass and metal, and organic / inorganic hybrid compounds. There is no particular limitation.

  From the viewpoint of workability, organic polymer materials are particularly preferred. For example, polyalkylene terephthalates, polycarbonates, polyurethanes, poly (meth) acrylates, polyacrylonitrile, polyvinyl alcohol, ethylene / vinyl alcohol copolymers (Eval), ethylene / vinyl acetate copolymer, polyvinyl acetal, polystyrene, polysulfones, cellulose and cellulose derivatives, polyphenylene ethers, polyethylene, polypropylene, polyvinyl fluoride, polyvinyl chloride, polyvinylidene fluoride, and the like. Examples thereof include a copolymer of monomers, and one or more alloys and blends of the above polymers.

  As the water-insoluble carrier, all known carriers for medical separation materials (medical adsorbents) can be used, but porous membranes or particles are preferred, and porous particles, nonwoven fabrics, or hollow fiber membranes are body fluids during extracorporeal circulation. It is more preferable because of its excellent flowability. Moreover, when a target adsorption component is a cell, a nonwoven fabric or particle | grains are preferable. Hereinafter, the selective separation material when the target adsorption component is a cell is also referred to as “cell selective separation material”.

  Examples of the porous membrane include non-woven fabrics, woven fabrics, knitted fabrics, and meshes manufactured using fibrous materials (solid fibers and hollow fibers) obtained from these materials. In addition, from the state in which an organic polymer material or an inorganic polymer material is thermally melted, in a solution state dissolved with a solvent, or in a state of plasticization with a plasticizer, a foaming method, a phase separation method (thermally induced phase separation method or A sheet-like membrane (flat membrane) and an empty fiber membrane that can be obtained by a wet phase separation method), a stretching method, a sintering method, or the like can also be used.

  The porous membrane may have any structure as long as it has communication holes and the target component is adsorbed by contact with an affinity ligand present on at least the surface portion of the communication holes. The communication hole is a hole communicating from one membrane surface of the support porous membrane to the opposite membrane surface, and if the liquid can pass through the communication hole, the pore surface of the membrane The shape and the structure inside the film may be anything.

  In particular, in the case of a cell selective separation material, preferred are nonwoven fabrics, woven fabrics, knitted fabrics, meshes and the like manufactured from various fibrous materials from the viewpoint of permeability of cell suspension and cell trapping properties. In particular, a nonwoven fabric is a porous film that is preferably used. In the case of a nonwoven fabric shape, the average fiber diameter (average fiber diameter) is preferably 0.1 μm to 50 μm. Furthermore, the average fiber diameter is preferably 0.5 μm to 40 μm, more preferably 1 μm to 30 μm, still more preferably 1 μm to 20 μm, and even more preferably 2 μm to 10 μm. When the average fiber diameter is less than 0.1 μm, the mechanical strength of the cell selective separation material tends to decrease. On the other hand, if it exceeds 50 μm, the adsorption surface area becomes small and the cell trapping property tends to decrease.

  The particles may be nonporous or porous depending on the target adsorption component. In the case of a porous material, the pore size may be selected depending on the target adsorption component. An average particle diameter of 25 μm or more and 2500 μm or less can be used, but in view of its specific surface area (adsorption ability as a separating material) and fluidity of body fluid, a particle diameter of 50 μm or more and 1500 μm or less is preferable.

〔polymer〕
The polymer according to this embodiment is a copolymer of a hydrophilic monomer and a hydrophobic monomer. The copolymer may be any of a random copolymer, a block copolymer, a periodic copolymer, and an alternating copolymer.

<Hydrophilic monomer>
In the present specification, the “hydrophilic monomer” means a monomer having a polymerizable functional group (for example, a functional group having an unsaturated bond such as a carbon-carbon double bond) and a hydrophilic functional group. The hydrophilic functional group may be any functional group that can form a hydrogen bond with a water molecule, and examples thereof include a hydroxyl group, a carboxyl group, an amino group, an alkylene glycol residue, and a betaine group. The hydrophilic functional group is preferably a functional group that is ionized in water and positively or negatively charged.

The hydrophilic monomer is at least one selected from the group consisting of N-methacryloyloxyethyl-N, N-dimethylammonium-α-N-methylcarboxybetaine (CMB), sulfobetaine and polyethylene glycol monomethacrylate. Is preferred. Among these, CMB represented by the following formula (2) is more preferable as the hydrophilic monomer because it is superior in the orientation of the antibody.

  CMB can be easily prepared with high purity by the methods described in JP-A-9-95474, JP-A-9-95586, JP-A-11-222470, and the like.

<Hydrophobic monomer>
In the present specification, the “hydrophobic monomer” means a monomer having a polymerizable functional group (for example, a functional group having an unsaturated bond such as a carbon-carbon double bond) and an electrophilic functional group. A monomer having no hydrophilic functional group is preferable. The electrophilic functional group means a functional group that reacts electrophilically, and specific examples of the electrophilic functional group include an epoxy group, an isocyanate group, and an aldehyde group.

一般式（１）中、Ｒ^１はＨ又はＣＨ_３、Ｒ^２は求電子官能基を有する有機基を示す。 As the hydrophobic monomer, a polymerizable monomer represented by the following general formula (1) is preferable.

In general formula (1), R ¹ represents H or CH ₃ , and R ² represents an organic group having an electrophilic functional group.

  The polymerizable monomer having an epoxy group in the molecule as an electrophilic functional group is not particularly limited, but glycidyl acrylate, glycidyl methacrylate, glycidyl acrylamide, allyl glycidyl ether, methacryl glycidyl ether, glycidyl sorbate, glycidyl metaitaconate, ethyl Examples thereof include glycidyl maleate and glycidyl vinyl sulfonate, and the polymerizable monomers having an isocyanate group as an electrophilic functional group in the molecule include acryloyloxyethyl isocyanate, acryloyloxymethyl isocyanate, acryloyl isocyanate, methacryloyl isocyanate, methacryloyl ethyl isocyanate. A polymerizable monomer having an aldehyde group in the molecule as an electrophilic functional group Examples thereof include cinnamaldehyde, crotonaldehyde, acrolein, methacrolein, etc. Among them, a polymerizable monomer having an epoxy group as an electrophilic functional group in the molecule because of availability, cost, and ease of handling. Among them, glycidyl methacrylate is particularly preferable.

<Polymer>
From the viewpoint that the polymer according to the present embodiment exhibits excellent biocompatibility, the molar composition ratio of the structural unit derived from the hydrophilic monomer is a structure derived from the structural unit derived from the hydrophilic monomer and the hydrophobic monomer. What is necessary is just 0.30-0.60 with respect to the sum total (namely, all structural units). When this molar composition ratio is less than 0.30, the hydrophobicity of the polymer becomes too high, and nonspecific adsorption of biological components such as proteins and blood cells tends to increase. On the other hand, when the molar composition ratio exceeds 0.60, the amount of antibody binding tends to decrease. The molar composition ratio is preferably 0.30 to 0.50, and more preferably 0.40 to 0.50.

「＊」は他の構造単位との結合を示す。
また、疎水性モノマーに由来する構造単位とは、例えば、疎水性ポリマーが上記一般式（１）で表される重合性モノマーである場合、以下の構造単位を意味する。

「＊」は他の構造単位との結合を示す。 The structural unit derived from the hydrophilic monomer means, for example, the following structural unit when the hydrophilic polymer is CMB.

“*” Represents a bond with another structural unit.
Moreover, the structural unit derived from a hydrophobic monomer means the following structural units, for example, when a hydrophobic polymer is a polymerizable monomer represented by the general formula (1).

“*” Represents a bond with another structural unit.

［一般式（２）中、ｎ及びｍは正の整数を示し、ｍ／（ｎ＋ｍ）の値は０．３０以上０．６０以下である。また、一般式（２）中、Ｒ^１はＨ又はＣＨ_３、Ｒ^２は求電子官能基を有する有機基、Ｒ^３は

を示す。］ When the hydrophilic polymer is CMB and the hydrophobic polymer is a polymerizable monomer represented by the general formula (1), the polymer can be represented by the following general formula (2).

[In general formula (2), n and m show a positive integer, and the value of m / (n + m) is 0.30 or more and 0.60 or less. In the general formula (2), R ¹ is H or CH ₃ , R ² is an organic group having an electrophilic functional group, and R ³ is

Indicates. ]

  The manner in which the polymer (copolymer) is bonded to at least the surface of the water-insoluble carrier is not particularly limited as long as it is a chemical bond such as a covalent bond, a coordinate bond, an ionic bond, and a hydrogen bond. Any binding mode may be used. Among these, a covalent bond is preferable because elution of the polymer is sufficiently reduced.

  As a method for bonding the polymer to the water-insoluble carrier, any known method such as a graft method and an insolubilized precipitation method can be used. Therefore, a method of performing surface modification by a known method such as graft polymerization of a polymer compound or a monomer thereof using radiation or plasma, or covalent bonding (Japanese Patent Laid-Open Nos. 1-249063 and 3-502094). Can be suitably used.

  When the radiation irradiation graft polymerization method is used, various known methods can be used. For example, examples of the ionizing radiation for introducing the active site on the carrier include α rays, β rays, γ rays, accelerated electron rays, X rays, and ultraviolet rays. Is preferred. The irradiation amount of the accelerated electron beam or γ-ray can be arbitrarily changed depending on the nature of the carrier, the nature of the polymerizable monomer (hydrophilic monomer and hydrophobic monomer), the amount of the fixed polymer, and the like, but preferably 10 kGy to 200 kGy. As a method for graft polymerization of a water-insoluble carrier and a polymerizable monomer, a simultaneous irradiation method in which radiation is irradiated in the coexistence of a water-insoluble carrier and a polymerizable monomer, or irradiation of only a water-insoluble carrier in advance, and then a polymerizable monomer Any of the pre-irradiation methods in which a water-insoluble carrier is brought into contact with each other can be used, but the pre-irradiation method is preferable because it hardly generates a side reaction other than graft polymerization. The polymerizable monomer concentration is usually 1% by mass or more and 20% by mass or less, and the reaction temperature is in the range of 5 ° C. or more and 40 ° C. or less. The reaction between the polymerizable monomer and the water-insoluble carrier can be carried out, but is limited to this range. It may be set appropriately. The solvent for the reaction between the polymerizable monomer and the water-insoluble carrier may be a solvent-free solvent such as water, methanol, ethanol, other alcohol or acetone, or a mixed solvent thereof, in which the polymerizable monomer is dissolved and dispersed. Can be used. When using the radiation irradiation graft polymerization method, the graft ratio (G) of the copolymer to the water-insoluble carrier is preferably 5% to 300%, more preferably 20% to 200%, and more preferably 50% to 150%. The following are particularly preferred: If the graft ratio is less than 5%, the surface of the water-insoluble carrier is likely to be insufficiently coated with the graft chains, and the surface of the water-insoluble carrier may not be sufficiently modified, and the amount of ligand (antibody) immobilized may be insufficient. May be sufficient. On the other hand, if the graft ratio exceeds 300%, the physical properties of the water-insoluble carrier itself may be lost, which is not preferable in designing the separating material.

The graft ratio (G) referred to here is a value represented by the following formula (4).
G (%) = [(weight of water-insoluble carrier after grafting−weight of water-insoluble carrier before grafting) / (weight of water-insoluble carrier before grafting)] × 100 (4)

  After the polymerization reaction, the excess polymerizable monomer and the ungrafted polymer produced by the chain transfer reaction may be sufficiently washed away with a suitable solvent.

[Base material for antibody immobilization]
A substrate in which the above-described polymer is bonded to at least the surface of the above-described water-insoluble carrier can be suitably used as an antibody (ligand) immobilization substrate. The antibody immobilization substrate according to this embodiment can immobilize an antibody by covalent bonding between an electrophilic functional group in a structural unit derived from a hydrophobic polymer and the antibody.

  Nonwoven fabric type antibody immobilization substrates having an epoxy group as an electrophilic functional group are very effective in the production of cell selective separation materials. An epoxy group is formed by immobilizing glycidyl methacrylate or a derivative thereof (having 2 to 5 methylene groups connecting an epoxy group and an ester group) on a nonwoven fabric (for example, polyethylene or polypropylene nonwoven fabric) by a radiation-induced graft polymerization method. When the antibody is immobilized on the introduced one, a cell selective separation material that expresses the original high affinity of the antibody without any problem and sufficiently captures the target cell can be obtained.

〔antibody〕
The antibody used for the selective separation material according to the present embodiment is not particularly limited as long as it is chemically bonded to the electrophilic functional group. Specific examples of antibodies include mouse antibodies, rabbit antibodies, sheep antibodies, goat antibodies, rat antibodies, hamster antibodies, pig antibodies, bovine antibodies, human antibodies, and chimeric antibodies thereof. Further, it is a ligand of F (ab ′) ₂ , Fab, Fab ′, and other peptides or modified peptides thereof having an amino acid sequence capable of forming the complementarity determining region of the variable region of the heavy chain or light chain of the antibody. May be.

[Selective separator]
FIG. 1 is a schematic view of a selective separation material according to an embodiment. A selective separating material 100 shown in FIG. 1 includes a water-insoluble carrier 1 and a copolymer (polymer) 2 of a hydrophilic monomer and a hydrophobic monomer bonded to at least the surface of the water-insoluble carrier 1. The copolymer 2 is bonded to the water-insoluble carrier 1 at the end of the polymer main chain 10, and the hydrophilic group 20 derived from the hydrophilic monomer and the hydrophobic monomer derived from the hydrophobic monomer are formed on the surface of the selective separation material 100. An electronic functional group 30 is present. The antibody 40 is bound to the electrophilic functional group 30.

  FIG. 2 is a schematic view of a selective separation material according to another embodiment. In the selective separating material 110 shown in FIG. 2, the copolymer 2 and the water-insoluble carrier 1 are bonded through a portion corresponding to the polymer main chain 10. The bond between the copolymer 2 and the water-insoluble carrier 1 is a bond through the end of the polymer main chain 10 as shown in FIG. 1, or a bond through the entire polymer main chain 10 as shown in FIG. Not only that, it is only necessary that they are bonded via a part of the polymer main chain 10.

Selective separation material according to the present embodiment, a fixed amount of antibody per unit surface area of the 1.50 mg / ^{m 2} or more, preferably 1.70mg / ^{m 2} or more, 2.00 mg / ^{m 2} or more It is more preferable that Although there is no restriction | limiting in particular in the upper limit of fixed amount, Usually, it is 2.50 mg / m < ² > or less. The selective separation material according to this embodiment preferably has an antibody chemical fixation rate of 80% or more, and more preferably 90% or more. The antibody chemical immobilization rate can be determined from the amount of antibody immobilized per unit surface area before and after washing the selective separation material with 2% sodium dodecyl sulfate (hereinafter referred to as “SDS”).

  In the selective separation material according to the present embodiment, the anti-Fab / anti-Fc ratio value indicating antibody orientation is 6 or more, preferably 6.5 or more, and more preferably 7 or more. The upper limit of the anti-Fab / anti-Fc ratio value is not particularly limited, but is usually 10 or less. The value of the anti-Fab / anti-Fc ratio means the ratio of the binding amount of the anti-Fab antibody to the selective separation material and the binding amount of the anti-Fc antibody. This means that the amount of binding is greater. That is, among the antibodies immobilized on the selective separation material, it means that the ratio of antibodies in which the Fab portion capable of binding to the antigen is immobilized in an orientation outward from the substrate surface is high.

  The selective separation material according to this embodiment can specifically adsorb and separate proteins and cells (for example, leukocytes and platelets) in blood or plasma. Therefore, the selective separation material according to the present embodiment can be suitably used for blood purification or as an adsorbent for blood purification.

[Method for producing selective separating material]
The method for producing a selective separation material according to the present embodiment is an antibody in the presence of an anti-chaotropic ion having a high concentration of 0.6 M or higher and an acidic pH with respect to the antibody immobilization substrate described above. At least the step of bonding. The antibody immobilization substrate and the antibody are bound by a covalent bond between the electrophilic functional group in the structural unit derived from the hydrophobic polymer and the antibody.

  As a method for immobilizing an antibody on a substrate, a salt having a high antichaotropic ionic strength may be used for the purpose of enhancing the hydrophobic interaction between the antibody and the substrate (hydrophobic). For example, in Non-Patent Document 1, a relatively low concentration of ammonium sulfate of about 0.5M is used when an antibody is immobilized on an epoxy group. Patent Document 5 describes the use of a solution containing about 1M dipotassium hydrogen phosphate as a method for immobilizing an antibody on a substrate containing a hydrophilic polymer. Furthermore, there is a report that the hydrophobicity of the Fc part of an antibody can be increased by lowering the pH (Clinical Chemistry, 36, 492-496, 1990). However, as a method for chemically immobilizing an antibody to a substrate containing a high amount of hydrophilic polymer, there is a problem that a sufficient amount of antibody cannot be chemically immobilized only by increasing the salt concentration or lowering the pH.

  In general, proteins are sensitive to pH and salt. For example, it has been reported that an antibody is denatured and cannot be refolded in a buffer solution having a pH of 3 or less (Clinical Chemistry, 36, 492-496, 1990). In addition, if the concentration of a strong salt as an antichaotropic ion such as ammonium sulfate is too high, the protein may be aggregated or denatured. From such a background, the ligand is generally immobilized under neutral or alkaline conditions. For example, when immobilizing a ligand to an epoxy group, it is generally performed under neutral to alkaline conditions, and when immobilizing a ligand to a thiol group or an amino group, it is performed under neutral conditions. Generally done. In other words, immobilization under acidic conditions is not recommended at present, and antibody immobilization has not been performed under conditions of high salt concentration and low pH where the antibody may be inactivated. It was.

  According to the method for producing a selective separation material according to this embodiment, the amount of chemical immobilization to a polymer containing a high amount of structural units derived from hydrophilic monomers can be increased, and the antibody can be immobilized with good orientation. Is possible. Although this mechanism is not necessarily clear, the present inventors consider as follows. In other words, the hydrophobic interaction between the antibody immobilization substrate and the antibody is improved by binding the antibody immobilization substrate and the antibody under acidic pH conditions in the presence of a high concentration of anti-chaotropic ions. In addition, it is possible to increase the amount of chemical fixation to a polymer containing a high amount of structural units derived from hydrophilic monomers. Furthermore, the hydrophobicity of the Fc part can be increased in the structure of the antibody due to the influence of high concentration of antichaotropic ions and acidic pH, and the antibody can be immobilized on the antibody immobilization substrate with good orientation. On the other hand, in the method described in Patent Document 5, a solution containing about 1M dipotassium hydrogen phosphate is a solution having a pH of 8 to 9 and a high pH, and the reaction between the substrate and the antibody includes a nitrophenyl ester group and an antibody. It is considered that the N-terminal in the amino acid of the amino acid or the amino group of the lysine side chain which is present at random is bound. Therefore, in the immobilization method described in Patent Document 5, the orientation of the antibody is not improved.

<Anti-chaotropic ion>
In the method for producing a selective separation material according to the present embodiment, the antibody immobilization substrate and the antibody are bound in the presence of a high concentration of antichaotropic ions.

Anti-chaotropic ions generally have a function of strengthening hydrogen bonds with water molecules, and a function of strengthening hydrophobic interactions. In the production method according to the present embodiment, examples of the antichaotropic ion that can be suitably used include PO ₄ ³⁻ , SO ₄ ²⁻ , CH ₃ CO ₂ ⁻ , NH ₄ ⁺ , K ⁺ , and Na ^+. Can be mentioned. These antichaotropic ions may be used alone or in combination of two or more.

  Examples of the anti-chaotropic salt that generates anti-chaotropic ions include, for example, ammonium phosphate, potassium phosphate, sodium phosphate, potassium hydrogen phosphate, dipotassium hydrogen phosphate, sodium hydrogen phosphate, disodium hydrogen phosphate, hydrogen phosphate Ammonium, diammonium phosphate, dipotassium phosphate, ammonium dihydrogen phosphate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, ammonium sulfate, potassium sulfate, sodium sulfate, potassium hydrogen sulfate, dipotassium hydrogen sulfate, sodium hydrogen sulfate , Disodium hydrogen sulfate, ammonium hydrogen sulfate, ammonium dihydrogen sulfate, potassium dihydrogen sulfate, sodium dihydrogen sulfate, ammonium acetate, potassium acetate, sodium acetate, potassium hydrogen acetate, dipotassium hydrogen acetate, sodium hydrogen acetate Um, sodium hydrogen acetate disodium, acid ammonium hydrogen acetate dihydrogen ammonium, potassium acetate dihydrogen acetate dihydrogen sodium, and the like. These antichaotropic salts may be used alone or in combination of two or more.

  The concentration of the antichaotropic ion is preferably 0.6M or more, and more preferably 1M or more. Moreover, although there is no restriction | limiting in particular in the upper limit of the density | concentration of an antichaotropic ion, Usually, it is 2M or less.

<PH>
In the method for producing a selective separation material according to this embodiment, the antibody immobilization substrate and the antibody are bound under acidic pH conditions. The pH is preferably 2.6 to 6.0. Moreover, it is more preferable that it is 3.0-5.0, and it is still more preferable that it is 3.0-4.0.

[Blood processor]
The blood processing apparatus according to the present embodiment includes a container having an inlet (inlet port) for introducing blood into the inside and an outlet (outlet port) for discharging the blood inside to the outside, And the above-mentioned selective separation material filled. Since the selective separation material can selectively adsorb a target component, it can be preferably used for the purpose of removing a specific component from blood, for example. Specific examples include a blood component adsorber for direct blood perfusion and a specific cell adsorber.

  FIG. 3 is a schematic cross-sectional view of a blood processing apparatus according to an embodiment. The blood processing device 200 includes a container 50 and a plurality of selective separation materials 120 filled in the container 50. Header caps 60 a and 60 b are provided at both ends of the container 50. The header cap 60a serves as an inlet (inlet) for introducing blood into the inside, and the header cap 60b serves as an outlet (outlet) for discharging blood to the outside. The blood flowing into the blood processing device 200 from the direction of the arrow F from the inlet of the header cap 60a comes into contact with the selective separation material 120, so that the components in the blood serving as the antigen of the antibody 40 are adsorbed. Thereby, when it flows out from the outlet of the header cap 60b, the above components are removed from the blood.

  EXAMPLES Hereinafter, although this invention is demonstrated more concretely based on an Example and a comparative example, this invention is not limited to these.

[Example 1]
(A) The method (1) non-woven fabric made of polypropylene as γ-irradiation carrier (average fiber diameter 3.8 .mu.m, basis weight ^{80 g} / m 2) and 0.108M ² encapsulated in low oxygen permeability bag together with a deoxygenating agent sufficiently oxygen After removing γ-rays of 25 kGy at −78 ° C.

(2) Grafting reaction 68.9 g of N-methacryloyloxyethyl-N, N-dimethylammonium-α-N-methylcarboxybetaine (CMB) and 21.1 mL of glycidyl methacrylate (GMA) were dissolved in 400 mL of methanol. Nitrogen was bubbled through at 60 ° C. for 60 minutes.
The above-mentioned nonwoven fabric was quickly put into a pressure-resistant glass container, and after reducing the pressure, the above-mentioned solution was drawn and reacted at 40 ° C. for 1 hour. After the reaction, the removed nonwoven fabric was washed with dimethylformamide and methanol, and vacuum dried at 40 ° C. to obtain a ligand-immobilized nonwoven fabric (ligand-immobilized substrate).

(3) Production of Cell Adsorption Filter (Selective Separation Material) The ligand-immobilized nonwoven fabric obtained above was cut into a circle having a diameter of 0.68 cm (hereinafter referred to as “circular nonwoven fabric substrate A”). Next, the obtained circular shape was added to 400 μL of citrate phosphate buffer (pH 4.0) (hereinafter referred to as “anti-CD4 solution”) in which anti-human CD4 monoclonal antibody (16 μg) and ammonium sulfate (105.7 mg) were dissolved. Four nonwoven fabric base materials A were soaked at 37 ° C. for 16 hours, and the monoclonal antibody was fixed to the circular nonwoven fabric base material A. Thereafter, the circular non-woven substrate A (hereinafter referred to as “antibody-immobilized circular non-woven fabric A”) on which the monoclonal antibody is immobilized is converted into a phosphate buffered saline solution (hereinafter referred to as “PBS (−)) that does not contain calcium and magnesium. "). Next, the antibody-immobilized circular non-woven fabric A is immersed in a 0.2% polyoxyethylene sorbitan monolaurate / PBS (−) solution (hereinafter referred to as “Tween 20 solution”) at room temperature for 2.5 hours for blocking. It was. Thereafter, the antibody-immobilized circular nonwoven fabric A was washed with 2 ml of PBS (−) to prepare a cell adsorption filter.

(4) Washing of Cell Adsorption Filter Four cell adsorption filters were soaked at 95 ° C. for 5 minutes with 2% sodium dodecyl sulfate / PBS (−) (hereinafter referred to as “SDS solution”). After repeating this twice, the cell adsorption filter was washed with 2 ml of PBS (−).

(5) Production of cell adsorber A 1 ml container having an inlet and an outlet was filled with the four cell adsorption filters obtained above and a PBS (-) solution as a filling liquid to produce a cell adsorber.

(6) Cell adsorbability of cell adsorber From the inlet of the cell adsorber obtained above, 8 ml of ACD-A-added human fresh blood (blood: ACD-A = 8: 1) was flowed at 0.2 ml with a syringe pump. The solution was fed at / min. 8 mL of the post-cell adsorption solution recovered from the outlet of the cell adsorber was collected. Thereafter, the solution after cell adsorption was recovered from the outlet of the cell adsorber.

(7) Production of Orientation Evaluation Base Material The ligand-immobilized nonwoven fabric was cut into a circle having a diameter of 0.48 cm (hereinafter referred to as “circular nonwoven fabric-like substrate B”). Next, 400 μL of citrate phosphate buffer (pH 4.0) in which human IgG (16 μg) and ammonium sulfate (105.7 mg) are dissolved (hereinafter referred to as “IgG solution”) is obtained in a circular nonwoven fabric base material B8 sheets were immersed for 16 hours at 37 ° C., and the human IgG was fixed to the circular nonwoven fabric substrate B. Thereafter, the circular nonwoven fabric substrate B on which human IgG was immobilized (hereinafter referred to as “antibody-immobilized circular nonwoven fabric B”) was washed with PBS (−). Next, the antibody-immobilized circular nonwoven fabric B was immersed in a 0.2% Tween 20 solution at room temperature for 2.5 hours for blocking. Thereafter, the antibody-immobilized circular nonwoven fabric B was washed with 2 ml of PBS (−) to prepare an orientation evaluation base material.

(B) Results (1) Surface analysis by X-ray photoelectron spectroscopy (XPS) The surface of the non-woven fabric for ligand immobilization obtained above was analyzed by X-ray photoelectron spectroscopy (XPS ESCA). When the relative element concentrations of carbon, oxygen, and nitrogen obtained from XPS are C ₁ , C ₂ , and C ₃ , CMB relative molar concentration (X), GMA relative molar concentration (Y) existing on the substrate surface, carrier The constituent polymer relative molar concentration (Z) is represented by the following formulas (1) to (3), respectively.
X = x / (x + y + z) (1)
Y = y / (x + y + z) (2)
Z = z / (x + y + z) (3)
_{However, x = C 3, y =} (C 2 -4x) / 3, z = - a {C 1 (10x + 7y) } / A, A is a unit carbon composition of the carrier constituent polymers, e.g., polyethylene In the case of A = 2, in the case of polypropylene, A = 3.
From the relative molar concentration obtained above, the molar composition ratio R of CMB in the copolymer is expressed by the following formula (4).
R = m / (n + m) = X / (X + Y) (4)
As a result of calculating the CMB molar composition ratio R based on these calculation formulas, the molar composition ratio R of CMB was 0.48.

(2) Specific surface area measurement The specific surface area of the non-woven fabric for immobilizing the ligand obtained above is measured by the BET method using the automatic specific surface area / pore distribution measuring device (MICRIMERICS TRISTAR 3000 manufactured by SHIMADZU). did. As a result, the specific surface area was 0.40 m ² / g.

(3) Quantification of antibody immobilization amount of cell adsorption filter The immobilization amount of the anti-human CD4 monoclonal antibody in the cell adsorption filter obtained above is BCA protein assay reagent (Micro BCA (registered trademark) Protein Assay Reagent Kit manufactured by PIERCE). 23235), the absorbance was measured with a microplate spectrophotometer (SPECTRA MAX340PC, analysis software SOFT max PRO manufactured by Molecular Devices) and quantified. As a result, the cell absorption filter before washing the cells adsorption filter after 1.85 mg ^/ m 2, SDS washing and 1.65 mg / ^{m 2} is immobilized, were chemically fixed rate 89.1% .

(4) CD4 positive cell removal rate The number of CD4 positive cells in ACD-A-added human fresh blood before cell adsorption and the solution after cell adsorption by flow cytometry using a flow cytometer (FACSCALIBUR manufactured by BECTON DICKINSON) Was measured, and the adsorption rate of the cells to the cell adsorber was calculated. As a result, the adsorption rate of CD4 positive cells was 98.3%. The platelet recovery rate was 94.7%.

(5) Quantification of antibody immobilization amount of substrate for orientation evaluation The quantification of immobilization amount of human IgG in the substrate for orientation evaluation obtained above is (3) quantification of antibody immobilization amount of cell adsorption filter. The same was done. As a result, 1.63 mg / m ² was immobilized on the alignment evaluation substrate before washing, and 1.57 mg / m ² was immobilized on the alignment evaluation substrate after SDS washing, and the chemical fixation rate was 96.5. %Met.

(6) Orientation Evaluation Monoclonal Antibody to IgG (Fab) (PE) conjugated (manufactured by Sigma) (hereinafter referred to as “anti-Fab antibody”) 0.1 mg / ml solution is already added to one orientation evaluation base material. Monoclonal Antibody to Human IgG (Fc) -PE (manufactured by Sigma) (hereinafter referred to as “anti-Fc antibody”) (50 μl) was added to each sheet, and light-shielded for 1 hour. Then, it was washed once with 0.2% Tween 20 solution and three times with PBS (−). The secondary antibody (anti-PE antibody) was bound, and the amount of secondary antibody binding was quantified by fluorescence measurement using a fluorescence plate reader (Biosearch CytoFluor II). As a result, the ratio of the anti-Fab antibody binding amount to the anti-Fc antibody binding amount to human IgG immobilized on the orientation evaluation substrate (anti-Fab / anti-Fc ratio) was 7.35. A higher anti-Fab / anti-Fc ratio means a higher ratio of human IgG immobilized in an orientation capable of binding to the antigen.

[Example 2]
The anti-CD4 solution was replaced with 400 μl of citrate phosphate buffer (pH 3.0) in which anti-human CD4 monoclonal antibody (16 μg) and ammonium sulfate (52.9 mg) were dissolved, and the anti-IgG solution was replaced with IgG (16 μg). Preparation and evaluation of cell adsorption filter and orientation evaluation substrate in the same manner as in Example 1, except that 400 μl of citrate phosphate buffer (pH 3.0) in which ammonium sulfate (52.9 mg) was dissolved was used. Went.
As a result of quantifying the antibody immobilization of cells absorption filter, the cells absorption filter before washing the cells adsorption filter after 2.21 mg ^/ m 2, SDS washing 2.17 mg / ^{m 2} is immobilized, The chemical fixation rate was 98.1%.
As a result of measuring the cell adsorption rate to the cell adsorber, the CD4 positive cell adsorption rate was 96.9%, and the platelet recovery rate was 96.3%.
As a result of quantifying the amount of antibody immobilized on the alignment evaluation substrate, 2.01 mg / m ² was obtained for the alignment evaluation substrate before washing, and 1.82 mg / m ² was obtained for the alignment evaluation substrate after SDS washing. It was immobilized, and the chemical fixation rate was 90.4%.
As a result of the orientation evaluation of the substrate for orientation evaluation, the anti-Fab / anti-Fc ratio was 7.23.

[Example 3]
The anti-CD4 solution was replaced with 400 μl of citrate phosphate buffer (pH 3.0) in which anti-human CD4 monoclonal antibody (16 μg) and ammonium sulfate (105.7 mg) were dissolved, and the anti-IgG solution was replaced with IgG (16 μg). Preparation and evaluation of cell adsorption filter and orientation evaluation substrate in the same manner as in Example 1 except that 400 μl of citrate phosphate buffer (pH 3.0) in which ammonium sulfate (105.7 mg) was dissolved was used. Went.
As a result of quantifying the antibody immobilization of cells absorption filter, the cells absorption filter before washing the cells adsorption filter after 1.99mg ^/ m 2, SDS washing 1.64 mg / ^{m 2} is immobilized, The chemical fixation rate was 82.4%.
As a result of measuring the cell adsorption rate to the cell adsorber, the CD4 positive cell adsorption rate was 93.1%, and the platelet recovery rate was 93.9%.
As a result of quantifying the amount of antibody immobilized on the alignment evaluation substrate, 1.75 mg / m ² was obtained for the alignment evaluation substrate before washing, and 1.56 mg / m ² was obtained for the alignment evaluation substrate after SDS washing. It was immobilized, and the chemical fixation rate was 89.2%.
As a result of the orientation evaluation of the substrate for orientation evaluation, the anti-Fab / anti-Fc ratio was 7.10.

[Example 4]
The anti-CD4 solution was replaced with 400 μl of citrate buffer (pH 3.5) in which anti-human CD4 monoclonal antibody (16 μg) and potassium dihydrogen phosphate (54.4 mg) were dissolved, and the anti-IgG solution was replaced with IgG ( 16 μg) and a cell adsorption filter and a substrate for orientation evaluation in the same manner as in Example 1 except that 400 μl of citrate buffer (pH 3.5) in which potassium dihydrogen phosphate (54.4 mg) was dissolved was used. Were prepared and evaluated.
As a result of quantifying the antibody immobilization of cells absorption filter, the cells absorption filter before washing the cells adsorption filter after 1.75 mg ^/ m 2, SDS washing 1.74 mg / ^{m 2} is immobilized, The chemical fixation rate was 99.5%.
As a result of measuring the cell adsorption rate to the cell adsorber, the CD4 positive cell adsorption rate was 98.5%, and the platelet recovery rate was 93.7%.
As a result of quantifying the amount of antibody immobilized on the alignment evaluation substrate, 1.62 mg / m ² was obtained for the alignment evaluation substrate before washing, and 1.61 mg / m ² was obtained for the alignment evaluation substrate after SDS washing. It was immobilized and the chemical fixation rate was 99.2%.
As a result of the orientation evaluation of the substrate for orientation evaluation, the anti-Fab / anti-Fc ratio was 8.21.

[Example 5]
The anti-CD4 solution was replaced with 400 μl of citrate buffer (pH 3.5) in which anti-human CD4 monoclonal antibody (16 μg) and potassium dihydrogen phosphate (81.7 mg) were dissolved, and the anti-IgG solution was replaced with IgG ( 16 μg) and a cell adsorption filter and a substrate for orientation evaluation in the same manner as in Example 1 except that 400 μl of citrate buffer (pH 3.5) in which potassium dihydrogen phosphate (81.7 mg) was dissolved was used. Were prepared and evaluated.
As a result of quantifying the antibody immobilization of cells absorption filter, the cells absorption filter before washing the cells adsorption filter after 2.01mg ^/ m 2, SDS washing 1.90mg / ^{m 2} is immobilized, The chemical fixation rate was 94.7%.
As a result of measuring the cell adsorption rate to the cell adsorber, the CD4 positive cell adsorption rate was 96.3%, and the platelet recovery rate was 95.3%.
As a result of quantifying the antibody immobilization of orientation evaluation substrate, the orientation evaluation substrate before cleaning to 1.89 mg ^/ m 2, the orientation evaluation substrate after SDS washing 1.80 mg / ^{m 2} It was immobilized, and the chemical fixation rate was 95.1%.
As a result of the orientation evaluation of the substrate for orientation evaluation, the anti-Fab / anti-Fc ratio was 7.24.

[Example 6]
A cell adsorption filter and a substrate for orientation evaluation were prepared and evaluated in the same manner as in Example 3 except that the graft reaction was performed by the following method and the surface analysis by XPS was performed by the following method.
(Graft reaction)
Sulfobetaine (SB) (145.4 g) and glycidyl methacrylate (GMA) (26.3 mL) were dissolved in methanol to obtain 500 mL of a reaction solution. Using this reaction solution, a ligand-immobilizing nonwoven fabric (ligand-immobilizing substrate) was obtained in the same manner as in Example 1.
(Surface analysis by XPS)
The surface of the non-woven fabric for immobilizing a ligand obtained above was analyzed by XPS method. When the relative element concentrations of carbon, oxygen and nitrogen obtained from XPS are C ₁ , C ₂ and C ₃ , respectively, the SB relative molar concentration (X), GMA relative molar concentration (Y) existing on the substrate surface, carrier The constituent polymer relative molar concentration (Z) is represented by the following formulas (5) to (7), respectively.
X = x / (x + y + z) (5)
Y = y / (x + y + z) (6)
Z = z / (x + y + z) (7)
_{However, x = C 3, y =} (C 2 -5x) / 3, z = - a {C 1 (11x + 7y) } / A, A is a unit carbon composition of the carrier constituent polymers, e.g., polyethylene In the case of A = 2, in the case of polypropylene, A = 3.
From the relative molar concentration obtained above, the molar composition ratio R of SB in the copolymer is expressed by the following formula (8).
R = m / (n + m) = X / (X + Y) (8)
As a result of calculating the SB molar composition ratio R based on these calculation formulas, the molar composition ratio R of SB was 0.40.
(Production and evaluation of cell adsorption filter and orientation evaluation substrate)
As a result of quantifying the antibody immobilization of cells absorption filter, the cells absorption filter before washing the cells adsorption filter after 2.05 mg ^/ m 2, SDS washing 1.95 mg / ^{m 2} is immobilized, The chemical fixation rate was 95.0%.
As a result of measuring the cell adsorption rate to the cell adsorber, the CD4 positive cell adsorption rate was 90.2%, and the platelet recovery rate was 96.8%.
As a result of quantifying the amount of antibody immobilized on the alignment evaluation substrate, 1.99 mg / m ² was obtained for the alignment evaluation substrate before washing, and 1.90 mg / m ² was obtained for the alignment evaluation substrate after SDS washing. It was immobilized, and the chemical fixation rate was 95.3%.
As a result of the orientation evaluation of the substrate for orientation evaluation, the anti-Fab / anti-Fc ratio was 6.12.

[Example 7]
A cell adsorption filter and a substrate for orientation evaluation were prepared and evaluated in the same manner as in Example 3 except that the graft reaction was performed by the following method and the surface analysis by XPS was performed by the following method.
(Graft reaction)
Polyethylene glycol monomethacrylate (PEGMM) 152.8 g and glycidyl methacrylate (GMA) 26.3 mL were dissolved in 500 mL of methanol to obtain a reaction solution. Using this reaction solution, a ligand-immobilizing nonwoven fabric (ligand-immobilizing substrate) was obtained in the same manner as in Example 1.
(Surface analysis by XPS)
The surface of the non-woven fabric for immobilizing a ligand obtained above was analyzed by XPS method. In the XPS analysis, first, in order to open the epoxy group on the surface of the non-woven fabric for ligand immobilization, the non-woven fabric for ligand immobilization was treated with 2N (2N) sulfuric acid at 50 ° C. for 10 hours, and then before the epoxy group was opened. The surface of the non-woven fabric for ligand immobilization and the non-woven fabric for immobilization of ligand after ring opening of epoxy group was analyzed. The relative element concentrations of carbon and oxygen obtained from XPS are measured, and the relative element concentrations of carbon and oxygen of the non-woven fabric for immobilizing the ligand before epoxy group ring opening are C ₄ and C ₅ , respectively. When the relative element concentrations of carbon and oxygen of the non-woven fabric base material are C ₆ and C ₇ , respectively, C ₄ + C ₅ = 1 and C ₆ + C ₇ = 1. Further, the PEGMM relative molar concentration (X) and the GMA relative molar concentration (Y) existing on the substrate surface are represented by the following formulas (9) and (10), respectively.
X = x / 12 (9)
Y = C ₇ -C ₅ (10)
_However, x = C 5 _-y, represented by _{y = 3 (C 7 -C 5} ). From the relative molar concentration obtained above, the molar composition ratio R of PEGMM in the copolymer is expressed by the following formula (11).
R = m / (n + m) = X / (X + Y) (11)
As a result of calculating the PEGMM molar composition ratio R based on these calculation formulas, the molar composition ratio R of PEGMM was 0.35.
(Production and evaluation of cell adsorption filter and orientation evaluation substrate)
As a result of quantifying the antibody immobilization of cells absorption filter, the cells absorption filter before washing the cells adsorption filter after 2.20 mg ^/ m 2, SDS washing 2.05 mg / ^{m 2} is immobilized, The chemical fixation rate was 93.3%.
As a result of measuring the cell adsorption rate to the cell adsorber, the CD4 positive cell adsorption rate was 98.7%, and the platelet recovery rate was 92.1%.
As a result of quantifying the amount of antibody immobilized on the alignment evaluation substrate, 1.81 mg / m ² was obtained for the alignment evaluation substrate before washing, and 1.79 mg / m ² was obtained for the alignment evaluation substrate after SDS washing. It was immobilized, and the chemical fixation rate was 99.0%.
As a result of the orientation evaluation of the substrate for orientation evaluation, the anti-Fab / anti-Fc ratio was 6.53.

[Example 8]
A cell adsorption filter and a substrate for orientation evaluation were prepared and evaluated in the same manner as in Example 3 except that the graft reaction was performed by the following method.
(Graft reaction)
28.3 g of N-methacryloyloxyethyl-N, N-dimethylammonium-α-N-methylcarboxybetaine and 26.3 mL of glycidyl methacrylate were dissolved in 500 mL of methanol to obtain a reaction solution. Using this reaction solution, a ligand-immobilizing nonwoven fabric (ligand-immobilizing substrate) was obtained in the same manner as in Example 1. As a result of surface analysis by XPS, the molar composition ratio R of CMB was 0.30. Moreover, it was 0.47 m < ² > / g when the specific surface area was measured.
(Production and evaluation of cell adsorption filter and orientation evaluation substrate)
As a result of quantifying the antibody immobilization of cells absorption filter, the cells absorption filter before washing the cells adsorption filter after 2.20 mg ^/ m 2, SDS washing 2.16 mg / ^{m 2} is immobilized, The chemical fixation rate was 98.0%.
As a result of measuring the cell adsorption rate to the cell adsorber, the CD4 positive cell adsorption rate was 98.1%, and the platelet recovery rate was 93.2%.
As a result of quantifying the amount of antibody immobilized on the alignment evaluation substrate, 2.13 mg / m ² was obtained for the alignment evaluation substrate before washing, and 1.84 mg / m ² was obtained for the alignment evaluation substrate after SDS washing. It was immobilized, and the chemical fixation rate was 86.3%.
As a result of the orientation evaluation of the substrate for orientation evaluation, the anti-Fab / anti-Fc ratio was 6.51.

[Example 9]
A cell adsorption filter and a substrate for orientation evaluation were prepared and evaluated in the same manner as in Example 3 except that the graft reaction was performed by the following method.
(Graft reaction)
43.1 g of N-methacryloyloxyethyl-N, N-dimethylammonium-α-N-methylcarboxybetaine and 26.3 mL of glycidyl methacrylate were dissolved in 500 mL of methanol to obtain a reaction solution. Using this reaction solution, a ligand-immobilizing nonwoven fabric (ligand-immobilizing substrate) was obtained in the same manner as in Example 1. As a result of the surface analysis by XPS, the molar composition ratio R of CMB was 0.33. Moreover, it was 0.53 m < ² > / g when the specific surface area was measured.
(Production and evaluation of cell adsorption filter and orientation evaluation substrate)
As a result of quantifying the antibody immobilization of cells absorption filter, the cells absorption filter before washing the cells adsorption filter after 2.22mg ^/ m 2, SDS washing 2.14 mg / ^{m 2} is immobilized, The chemical fixation rate was 96.2%.
As a result of measuring the cell adsorption rate to the cell adsorber, the CD4 positive cell adsorption rate was 99.5%, and the platelet recovery rate was 98.3%.
As a result of quantifying the amount of antibody immobilized on the alignment evaluation substrate, 2.07 mg / m ² was obtained for the alignment evaluation substrate before washing, and 1.84 mg / m ² was obtained for the alignment evaluation substrate after SDS washing. It was immobilized, and the chemical fixation rate was 88.8%.
As a result of the orientation evaluation of the substrate for orientation evaluation, the anti-Fab / anti-Fc ratio was 7.92.

[Example 10]
A cell adsorption filter and a substrate for orientation evaluation were prepared and evaluated in the same manner as in Example 3 except that the graft reaction was performed by the following method.
(Graft reaction)
N-methacryloyloxyethyl-N, N-dimethylammonium-α-N-methylcarboxybetaine (103.3 g) and glycidyl methacrylate (15.8 mL) were dissolved in 350 mL of methanol to obtain a reaction solution. Using this reaction solution, a ligand-immobilizing nonwoven fabric (ligand-immobilizing substrate) was obtained in the same manner as in Example 1. As a result of the surface analysis by XPS, the molar composition ratio R of CMB was 0.56. Moreover, it was 0.36 m < ² > / g when the specific surface area was measured.
(Production and evaluation of cell adsorption filter and orientation evaluation substrate)
As a result of quantifying the antibody immobilization of cells absorption filter, the cells absorption filter before washing the cells adsorption filter after 1.50 mg ^/ m 2, SDS washing 1.21 mg / ^{m 2} is immobilized, The chemical fixation rate was 80.4%.
As a result of measuring the cell adsorption rate to the cell adsorber, the CD4 positive cell adsorption rate was 99.3%, and the platelet recovery rate was 92.6%.
Results of measurement of the antibody-immobilized amount of alignment evaluation substrates, the orientation evaluation substrate before cleaning to 1.52 mg ^/ m 2, the orientation evaluation substrate after SDS washing 1.25 mg / ^{m 2} The immobilization rate was 82.3%.
As a result of the orientation evaluation of the substrate for orientation evaluation, the anti-Fab / anti-Fc ratio was 8.37.

[Comparative Example 1]
The graft reaction was carried out by the following method, the anti-CD4 solution was replaced with 400 μl of PBS (−) (pH 7.4) in which anti-human CD4 monoclonal antibody (16 μg) and ammonium sulfate (26.4 mg) were dissolved, and The cell adsorption filter and orientation evaluation were performed in the same manner as in Example 1 except that the IgG solution was replaced with 400 μl of PBS (−) (pH 7.4) in which IgG (16 μg) and ammonium sulfate (26.4 mg) were dissolved. Substrate preparation and evaluation were performed.
(Graft reaction)
26.3 mL of glycidyl methacrylate was dissolved in 500 mL of methanol to obtain a reaction solution. Using this reaction solution, a ligand-immobilizing nonwoven fabric (ligand-immobilizing substrate) was obtained in the same manner as in Example 1. As a result of surface analysis by XPS, the molar composition ratio R of CMB was 0. Moreover, it was 0.51 m < ² > / g when the specific surface area was measured.
(Production and evaluation of cell adsorption filter and orientation evaluation substrate)
As a result of quantifying the antibody immobilization of cells absorption filter, the cells absorption filter before washing the cells adsorption filter after 2.32 mg ^/ m 2, SDS washing 2.24mg / ^{m 2} is immobilized, The chemical fixation rate was 96.4%.
As a result of measuring the cell adsorption rate to the cell adsorber, the CD4 positive cell adsorption rate was 69.7%, and the platelet recovery rate was 8.7%.
As a result of quantifying the amount of antibody immobilized on the alignment evaluation substrate, 2.01 mg / m ² was obtained for the alignment evaluation substrate before washing, and 1.85 mg / m ² was obtained for the alignment evaluation substrate after SDS washing. It was immobilized, and the chemical fixation rate was 92.0%.
As a result of the orientation evaluation of the substrate for orientation evaluation, the anti-Fab / anti-Fc ratio was 3.17.

[Comparative Example 2]
A cell adsorption filter and a substrate for orientation evaluation were prepared and evaluated in the same manner as in Comparative Example 1 except that the graft reaction was performed by the following method.
(Graft reaction)
21.5 g of N-methacryloyloxyethyl-N, N-dimethylammonium-α-N-methylcarboxybetaine and 26.3 mL of glycidyl methacrylate were dissolved in 500 mL of methanol to obtain a reaction solution. Using this reaction solution, a ligand-immobilizing nonwoven fabric (ligand-immobilizing substrate) was obtained in the same manner as in Example 1. As a result of the surface analysis by XPS, the molar composition ratio R of CMB was 0.26. Moreover, it was 0.50 m < ² > / g when the specific surface area was measured.
(Production and evaluation of cell adsorption filter and orientation evaluation substrate)
As a result of quantifying the antibody immobilization of cells absorption filter, the cells absorption filter before washing the cells adsorption filter after 1.43 mg ^/ m 2, SDS washing 1.26 mg / ^{m 2} is immobilized, The chemical fixation rate was 87.6%.
As a result of measuring the cell adsorption rate to the cell adsorber, the CD4 positive cell adsorption rate was 78.2%, and the platelet recovery rate was 50.5%.
As a result of measuring the amount of antibody immobilized on the alignment evaluation substrate, 2.03 mg / m ² was obtained for the alignment evaluation substrate before washing, and 1.72 mg / m ² was obtained for the alignment evaluation substrate after SDS washing. It was immobilized, and the chemical fixation rate was 84.6%.
As a result of the orientation evaluation of the substrate for orientation evaluation, the anti-Fab / anti-Fc ratio was 3.15.

[Comparative Example 3]
A cell adsorption filter and a substrate for orientation evaluation were prepared and evaluated in the same manner as in Example 3 except that the graft reaction was performed by the following method.
(Graft reaction)
N-methacryloyloxyethyl-N, N-dimethylammonium-α-N-methylcarboxybetaine (207.14 g) and glycidyl methacrylate (15.8 mL) were dissolved in 300 mL of methanol to obtain a reaction solution. Using this reaction solution, a ligand-immobilizing nonwoven fabric (ligand-immobilizing substrate) was obtained in the same manner as in Example 1. As a result of the surface analysis by XPS, the molar composition ratio R of CMB was 0.63. Moreover, it was 0.32 m < ² > / g when the specific surface area was measured.
(Production and evaluation of cell adsorption filter and orientation evaluation substrate)
As a result of quantifying the antibody immobilization of cells absorption filter, the cells absorption filter before washing the cells adsorption filter after 1.25 mg ^/ m 2, SDS washing 0.85 mg / ^{m 2} is immobilized, The chemical fixation rate was 68.0%.
As a result of measuring the cell adsorption rate to the cell adsorber, the CD4 positive cell adsorption rate was 75.8%, and the platelet recovery rate was 99.8%.
As a result of measuring the amount of antibody immobilized on the alignment evaluation substrate, 1.37 mg / m ² was obtained for the alignment evaluation substrate before washing, and 0.79 mg / m ² was obtained for the alignment evaluation substrate after SDS washing. It was immobilized, and the chemical fixation rate was 57.7%.
As a result of the orientation evaluation of the substrate for orientation evaluation, the anti-Fab / anti-Fc ratio was 8.52.

[Comparative Example 4]
The anti-CD4 solution was replaced with 400 μl of PBS (−) (pH 7.4) in which anti-human CD4 monoclonal antibody (16 μg) and ammonium sulfate (26.4 mg) were dissolved, and the anti-IgG solution was replaced with IgG (16 μg) and ammonium sulfate. A cell adsorption filter and a substrate for orientation evaluation were prepared and evaluated in the same manner as in Example 1 except that 400 μl of PBS (−) (pH 7.4) in which (26.4 mg) was dissolved was used.
As a result of quantifying the antibody immobilization of cells absorption filter, the cells absorption filter before washing the cells adsorption filter after 0.95 mg ^/ m 2, SDS washing 0.66 mg / ^{m 2} is immobilized, The chemical fixation rate was 69.5%.
As a result of measuring the cell adsorption rate to the cell adsorber, the CD4 positive cell adsorption rate was 49.5%, and the platelet recovery rate was 95.4%.
As a result of quantifying the amount of antibody immobilized on the alignment evaluation substrate, 0.87 mg / m ² was obtained for the alignment evaluation substrate before washing, and 0.63 mg / m ² was obtained for the alignment evaluation substrate after SDS washing. It was immobilized, and the chemical fixation rate was 72.4%.
As a result of the orientation evaluation of the substrate for orientation evaluation, the anti-Fab / anti-Fc ratio was 4.01.

[Comparative Example 5]
The anti-CD4 solution was replaced with 400 μl of citrate phosphate buffer (pH 3.0) in which anti-human CD4 monoclonal antibody (16 μg) and ammonium sulfate (26.4 mg) were dissolved, and the anti-IgG solution was replaced with IgG (16 μg). Preparation and evaluation of cell adsorption filter and orientation evaluation substrate in the same manner as in Example 1 except that 400 μl of citrate phosphate buffer (pH 3.0) in which ammonium sulfate (26.4 mg) was dissolved was used. Went.
As a result of quantifying the antibody immobilization of cells absorption filter, the cells absorption filter before washing the cells adsorption filter after 1.33 mg ^/ m 2, SDS washing 0.52 mg / ^{m 2} is immobilized, The chemical fixation rate was 38.8%.
As a result of measuring the cell adsorption rate to the cell adsorber, the CD4 positive cell adsorption rate was 50.1%, and the platelet recovery rate was 100.0%.
As a result of quantifying the amount of antibody immobilized on the alignment evaluation substrate, 1.15 mg / m ² was obtained for the alignment evaluation substrate before washing, and 6.58 mg / m ² was obtained for the alignment evaluation substrate after SDS washing. It was immobilized, and the chemical fixation rate was 57.2%.
As a result of the orientation evaluation of the substrate for orientation evaluation, the anti-Fab / anti-Fc ratio was 5.03.

[Comparative Example 6]
The anti-CD4 solution was replaced with 400 μl of PBS (−) (pH 7.4) in which anti-human CD4 monoclonal antibody (16 μg) and ammonium sulfate (105.7 mg) were dissolved, and the anti-IgG solution was replaced with IgG (16 μg) and ammonium sulfate. A cell adsorption filter and a substrate for orientation evaluation were prepared and evaluated in the same manner as in Example 1 except that 400 μl of PBS (−) (pH 7.4) in which (105.7 mg) was dissolved was used.
As a result of quantifying the antibody immobilization of cells absorption filter, the cells absorption filter before washing the cells adsorption filter after 1.05 mg ^/ m 2, SDS washing 0.83 mg / ^{m 2} is immobilized, The chemical fixation rate was 79.0%.
As a result of measuring the cell adsorption rate to the cell adsorber, the CD4 positive cell adsorption rate was 64.2%, and the platelet recovery rate was 100.0%.
As a result of quantifying the amount of antibody immobilized on the alignment evaluation substrate, 0.93 mg / m ² was obtained for the alignment evaluation substrate before washing, and 0.85 mg / m ² was obtained for the alignment evaluation substrate after SDS washing. It was immobilized, and the chemical fixation rate was 91.4%.
As a result of the alignment evaluation of the substrate for alignment evaluation, the anti-Fab / anti-Fc ratio was 4.92.

[Comparative Example 7]
The anti-CD4 solution was replaced with 400 μl of PBS (−) (pH 7.4) in which anti-human CD4 monoclonal antibody (16 μg) and ammonium sulfate (26.4 mg) were dissolved, and the anti-IgG solution was replaced with IgG (16 μg) and ammonium sulfate. A cell adsorption filter and a substrate for orientation evaluation were prepared and evaluated in the same manner as in Example 9, except that 400 μl of PBS (−) (pH 7.4) in which (26.4 mg) was dissolved was used.
As a result of quantifying the antibody immobilization of cells absorption filter, the cells absorption filter before washing the cells adsorption filter after 1.13 mg ^/ m 2, SDS washing 0.81 mg / ^{m 2} is immobilized, The chemical fixation rate was 71.7%.
As a result of measuring the cell adsorption rate to the cell adsorber, the CD4 positive cell adsorption rate was 72.0%, and the platelet recovery rate was 76.5%.
As a result of quantifying the amount of antibody immobilized on the alignment evaluation substrate, 1.05 mg / m ² was obtained for the alignment evaluation substrate before washing, and 0.94 mg / m ² was obtained for the alignment evaluation substrate after SDS washing. It was immobilized, and the chemical fixation rate was 89.5%.
As a result of the orientation evaluation of the substrate for orientation evaluation, the anti-Fab / anti-Fc ratio was 2.81.

[Comparative Example 8]
The anti-CD4 solution was replaced with 400 μl of PBS (−) (pH 7.4) in which anti-human CD4 monoclonal antibody (16 μg) and ammonium sulfate (26.4 mg) were dissolved, and the anti-IgG solution was replaced with IgG (16 μg) and ammonium sulfate. A cell adsorption filter and a substrate for orientation evaluation were prepared and evaluated in the same manner as in Example 8, except that 400 μl of PBS (−) (pH 7.4) in which (26.4 mg) was dissolved was used.
As a result of quantifying the antibody immobilization of cells absorption filter, the cells absorption filter before washing the cells adsorption filter after 1.25 mg ^/ m 2, SDS washing 0.98 mg / ^{m 2} is immobilized, The chemical fixation rate was 78.4%.
As a result of measuring the cell adsorption rate to the cell adsorber, the CD4 positive cell adsorption rate was 78.2%, and the platelet recovery rate was 68.4%.
As a result of quantifying the amount of antibody immobilized on the alignment evaluation substrate, 1.13 mg / m ² was obtained for the alignment evaluation substrate before washing, and 0.92 mg / m ² was obtained for the alignment evaluation substrate after SDS washing. It was immobilized, and the chemical fixation rate was 81.4%.
As a result of the orientation evaluation of the substrate for orientation evaluation, the anti-Fab / anti-Fc ratio was 2.20.

[Comparative Example 9]
The anti-CD4 solution was replaced with 400 μl of PBS (−) (pH 7.4) in which anti-human CD4 monoclonal antibody (16 μg) and ammonium sulfate (26.4 mg) were dissolved, and the anti-IgG solution was replaced with IgG (16 μg) and ammonium sulfate. A cell adsorption filter and a substrate for orientation evaluation were prepared and evaluated in the same manner as in Example 6 except that the solution was replaced with 400 μl of PBS (−) (pH 7.4) in which (26.4 mg) was dissolved.
As a result of quantifying the antibody immobilization of cells absorption filter, the cells absorption filter before washing the cells adsorption filter after 0.72 mg ^/ m 2, SDS washing 0.62 mg / ^{m 2} is immobilized, The chemical fixation rate was 86.0%.
As a result of measuring the cell adsorption rate to the cell adsorber, the CD4 positive cell adsorption rate was 15.0%, and the platelet recovery rate was 85.2%.
As a result of quantifying the amount of antibody immobilized on the alignment evaluation substrate, the alignment evaluation substrate before washing was 0.35 mg / m ² , and the alignment evaluation substrate after SDS washing was 0.14 mg / m ^2. It was immobilized, and the chemical fixation rate was 39.5%.
As a result of the orientation evaluation of the substrate for orientation evaluation, the anti-Fab / anti-Fc ratio was 3.15.

[Comparative Example 10]
The anti-CD4 solution was replaced with 400 μl of PBS (−) (pH 7.4) in which anti-human CD4 monoclonal antibody (16 μg) and ammonium sulfate (26.4 mg) were dissolved, and the anti-IgG solution was replaced with IgG (16 μg) and ammonium sulfate. A cell adsorption filter and a substrate for orientation evaluation were prepared and evaluated in the same manner as in Example 7, except that 400 μl of PBS (−) (pH 7.4) in which (26.4 mg) was dissolved was used.
As a result of quantifying the antibody immobilization of cells absorption filter, the cells absorption filter before washing the cells adsorption filter after 0.28 mg ^/ m 2, SDS washing 0.28 mg / ^{m 2} is immobilized, The chemical fixation rate was 99.1%.
As a result of measuring the cell adsorption rate to the cell adsorber, the CD4 positive cell adsorption rate was 75.5%, and the platelet recovery rate was 98.4%.
As a result of quantifying the amount of antibody immobilized on the alignment evaluation substrate, 0.50 mg / m ² was obtained for the alignment evaluation substrate before washing, and 0.30 mg / m ² was obtained for the alignment evaluation substrate after SDS washing. It was immobilized, and the chemical fixation rate was 60.4%.
As a result of the orientation evaluation of the substrate for orientation evaluation, the anti-Fab / anti-Fc ratio was 3.68.

[Comparative Example 11]
The anti-CD4 solution was replaced with 400 μl of citrate phosphate buffer (pH 3.0) in which anti-human CD4 monoclonal antibody (16 μg) and sodium chloride (23.4 mg) were dissolved, and the anti-IgG solution was replaced with IgG (16 μg ) And sodium citrate phosphate solution (23.4 mg) dissolved in citrate phosphate buffer (pH 3.0) 400 μl, except that the cell adsorption filter and orientation evaluation substrate were prepared in the same manner as in Example 1. And evaluated.
As a result of quantifying the antibody immobilization of cells absorption filter, the cells absorption filter before washing the cells adsorption filter after 2.11 mg ^/ m 2, SDS washing 1.51 mg / ^{m 2} is immobilized, The chemical fixation rate was 71.3%.
As a result of measuring the cell adsorption rate to the cell adsorber, the CD4 positive cell adsorption rate was 95.1%, and the platelet recovery rate was 94.9%.
As a result of quantifying the amount of antibody immobilized on the alignment evaluation substrate, 2.06 mg / m ² was obtained for the alignment evaluation substrate before washing, and 1.35 mg / m ² was obtained for the alignment evaluation substrate after SDS washing. It was immobilized, and the chemical fixation rate was 65.3%.
As a result of the orientation evaluation of the substrate for orientation evaluation, the anti-Fab / anti-Fc ratio was 5.61.

[Comparative Example 12]
The anti-CD4 solution was replaced with 400 μl of citrate phosphate buffer (pH 3.0) in which anti-human CD4 monoclonal antibody (16 μg) and sodium chloride (46.7 mg) were dissolved, and the anti-IgG solution was replaced with IgG (16 μg ) And sodium citrate (46.7 mg) dissolved in citrate phosphate buffer (pH 3.0) 400 μl, the same method as in Example 1 was used to prepare a cell adsorption filter and orientation evaluation substrate. And evaluated.
As a result of quantifying the antibody immobilization of cells absorption filter, the cells absorption filter before washing the cells adsorption filter after 2.15 mg ^/ m 2, SDS washing 1.42 mg / ^{m 2} is immobilized, The chemical fixation rate was 66.1%.
As a result of measuring the cell adsorption rate to the cell adsorber, the CD4 positive cell adsorption rate was 98.4%, and the platelet recovery rate was 95.9%.
As a result of measuring the amount of antibody immobilized on the alignment evaluation substrate, 1.94 mg / m ² was obtained for the alignment evaluation substrate before washing, and 1.13 mg / m ² was obtained for the alignment evaluation substrate after SDS washing. It was immobilized, and the chemical fixation rate was 58.2%.
As a result of the orientation evaluation of the substrate for orientation evaluation, the anti-Fab / anti-Fc ratio was 5.79.

The evaluation results of Examples 1 to 10 and Comparative Examples 1 to 12 are summarized in Table 1 below.

  DESCRIPTION OF SYMBOLS 1 ... Water-insoluble carrier, 2 ... Polymer (copolymer), 10 ... Polymer main chain, 20 ... Hydrophilic group, 30 ... Electrophilic functional group, 40 ... Antibody, 50 ... Container, 60a, 60b ... Header cap, F ... blood flow direction, 100, 110 ... specific separating material, 200 ... blood processing device.

Claims (9)

A water-insoluble carrier, a polymer bound to at least the surface of the water-insoluble carrier, and an antibody bound to the polymer,
The polymer is a copolymer of a hydrophilic monomer and a hydrophobic monomer, and the molar composition ratio of structural units derived from the hydrophilic monomer is 0.30 to 0.60 with respect to all the structural units. ,
The fixed amount per unit surface area of the antibody is at 1.50 mg / ^{m 2} or more, and Ri der value 6 or more anti-Fab / anti-Fc ratios showing antibody orientation,
Said said polymer antibody, you are bound by covalent bond between the electrophilic functional groups in the structural unit derived from the hydrophobic monomer and said antibody, specific separation materials.   The specific separation material according to claim 1, wherein the chemical immobilization ratio of the antibody is 80% or more.   The hydrophilic monomer is one or more selected from the group consisting of N-methacryloyloxyethyl-N, N-dimethylammonium-α-N-methylcarboxybetaine, sulfobetaine and polyethylene glycol monomethacrylate. Or the specific separation material of 2. The specific separation material according to any one of claims 1 to 3, wherein the hydrophobic monomer is a polymerizable monomer represented by the following general formula (1).

[In General Formula (1), R ¹ represents H or CH ₃ , and R ² represents an organic group having an electrophilic functional group. ]   The specific separating material according to claim 4, wherein the electrophilic functional group is an epoxy group.   The specific separation material according to claim 1, wherein the water-insoluble carrier is a porous membrane or particles. A container having a blood inlet and outlet, and
A blood treatment device comprising: the specific separation material according to any one of claims 1 to 6 filled in the container. Use of a substrate for immobilizing an antibody in the production of the specific separation material according to any one of claims 1 to 6,
The antibody immobilization substrate comprises a water-insoluble carrier and a polymer bound to at least the surface of the water-insoluble carrier;
The polymer is a copolymer of a hydrophilic monomer and a hydrophobic monomer, and the molar composition ratio of structural units derived from the hydrophilic monomer is 0.30 to 0.60 with respect to all structural units. , Said use. An antibody is immobilized on an antibody immobilization substrate comprising a water-insoluble carrier and a polymer bound to at least the surface of the water-insoluble carrier in the presence of an antichaotropic ion having a concentration of 0.6 M or more under acidic pH conditions. Comprising the step of combining,
The polymer is a copolymer of a hydrophilic monomer and a hydrophobic monomer, and the molar composition ratio of structural units derived from the hydrophilic monomer is 0.30 to 0.60 with respect to all structural units. The manufacturing method which manufactures the specific separation material as described in any one of Claims 1-6.

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