JP2012093290A - Particle for medical use and method of capturing physiologically active substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a particle for medical use having an excellent capability of fixing a biotin-binding protein compound and being quickly and easily recovered by general-purpose equipment such as a desktop small-sized centrifuge; and to provide a method of capturing physiologically active substances using the medical particles.SOLUTION: In this particle for medical use having a function of capturing a biologically active substance, a polymer is immobilized on the surface of a particulate substrate, and includes: a first repetition unit having a hydrophilic group at a side chain; and a second repetition unit having biotin derivative at the terminal of the side chain. This particle can selectively capture the biologically active substance including the biotin-binding protein and suppress adsorption and binding of unnecessary biologically active substances and fluorescent substance other than that.

Description

The present invention relates to a medical particle and a method for capturing a physiologically active substance.

It has been known for a long time that biotin causes a specific binding reaction with a biotin-binding protein, and its binding power is as strong as 1 million times or more that of an antigen-antibody reaction, and it exhibits a binding property almost similar to an irreversible reaction. Taking advantage of this property, the biotin-biotin binding protein reaction is often used for detection reactions in the biochemical field.

Since biotin is a molecule with a molecular weight of 244 and has a carboxyl group at the molecular end, it can be easily bound to a substance having an amino residue by active esterification with hydroxysuccinimide or the like, and biotin can be labeled. A molecular recognition reaction using an avidin-biotin bond has been widely used.

On the other hand, a biotin-binding protein that has been used for a long time is avidin, and avidin is a protein derived from raw egg white. In addition, after streptavidin has been purified from Streptomyces avidini, which is a microorganism, it has become easy to obtain and has become widely used. In addition, neutravidin has been developed that suppresses non-specific adsorption caused by interaction with sugar chains such as lectins by deglycosylating avidin. As a functional protein group.

Various systems for immobilizing biological materials and immunoassay using the biotin-biotin-binding protein group reaction as described above have been developed more easily, but most of them are biotin-binding proteins. A method has been taken to bond the group to the substrate.

A system in which biotin is immobilized on a substrate is often used for immobilizing biotin and binding a biotin-binding protein group thereon.
In many cases, biotin-binding proteins have multiple binding sites for biotin in one molecule (for example, four biotin-binding sites in one streptavidin molecule). After the biotin-binding protein is bound, the bioactive substance containing biotin is further captured.
For example, Patent Document 1 describes a method of immobilizing biotin on water-insoluble polymer particles. In this method, casein molecules are bound to water-insoluble polymer particles, and biotin is immobilized using the casein molecules as a binder.

On the other hand, in Patent Document 2, a biotin molecule is immobilized on a slide glass, and a biotin molecule on which a probe molecule of DNA is further immobilized with a biotin molecule via the biotin molecule is base-biotin-avidin-biotin-probe. A method for producing a biomolecule microarray immobilized in this order is described.
In this method, too, no countermeasures against non-specific adsorption are taken as in Patent Document 2, and the increase in the book ground value due to non-specific adsorption and the deterioration of the S / N ratio due to the case where the target object is a contaminant. There was a drawback that it could not cope.

JP 63-192186 A JP 2002-153272 A

An object of the present invention is to provide a medical particle that has an excellent ability to immobilize a biotin compound and can be quickly and easily collected by general-purpose equipment such as a desktop small centrifuge and a method for capturing a physiologically active substance using the medical particle. It is to provide.

（８）前記粒子状の基材は、無機材料で構成されている（１）ないし（７）のいずれか１項に記載の医療用粒子。
（９）前記無機材料が、無機酸化物である（８）に記載の医療用粒子。
（１０）前記無機酸化物が、酸化ケイ素である（９）に記載の医療用粒子。
（１１）前記粒子状の基材表面にシランカップリング剤を介して導入された重合性官能基が、前記重合体合成時に重合体中に取り込まれることにより、前記粒子状の基材と前記重合体とが共有結合で固定化されている（１）ないし（１０）のいずれか１項に記載の医療用粒子。
（１２）（１）ないし（１１）のいずれか１項に記載の医療用粒子を用いることを特徴とする、生理活性物質の捕捉方法。
Such an object is achieved by the present invention described in the following (1) to (12).
(1) Medical particles having a function of capturing a physiologically active substance,
The polymer is immobilized on the surface of the particulate substrate,
The medical particle, wherein the polymer has a first repeating unit having a hydrophilic group in a side chain and a second repeating unit having a biotin derivative at a terminal of the side chain.
(2) The medical particle according to (1), wherein the second repeating unit is obtained by binding a biotin derivative to a side chain end of a polymer of an ethylenically unsaturated polymerizable monomer having an alkylene glycol residue.
(3) The medical particle according to (1) or (2), wherein the polymer is a random copolymer of the first repeating unit and the second repeating unit.
(4) The copolymerization ratio (first repeat unit / second repeat unit) of the first repeat unit and the second repeat unit is 97/3 to 50/50 (1) to (3) The medical particles according to any one of the above.
(5) The biotin derivative according to any one of (1) to (4), wherein the biotin derivative is at least one kind of biotin terminal carboxyl group-introduced biotin which is a terminal amino group-modified biotin.
(6) The medical particle according to any one of (1) to (5), wherein the first repeating unit is a polymer of an ethylenically unsaturated polymerizable monomer having an alkylene glycol residue.
(7) The medical particle according to (6), wherein the ethylenically unsaturated polymerizable monomer having an alkylene glycol residue is a monomer represented by the following formula (1).

(8) The medical particle according to any one of (1) to (7), wherein the particulate base material is composed of an inorganic material.
(9) The medical particle according to (8), wherein the inorganic material is an inorganic oxide.
(10) The medical particle according to (9), wherein the inorganic oxide is silicon oxide.
(11) When the polymerizable functional group introduced into the surface of the particulate base material via a silane coupling agent is taken into the polymer during the synthesis of the polymer, the particulate base material and the weight The medical particle according to any one of (1) to (10), wherein the coalescence is immobilized by a covalent bond.
(12) A method for capturing a physiologically active substance, comprising using the medical particles according to any one of (1) to (11).

ADVANTAGE OF THE INVENTION According to this invention, the capture | acquisition method of the medical particle | grains and bioactive substance which is excellent in the fixability of a biotin compound, and can be collect | recovered rapidly and easily with general purpose equipment, such as a desktop small centrifuge, can be obtained.

Hereinafter, the method for capturing medical particles and physiologically active substances of the present invention will be described.
The medical particles of the present invention are medical particles having a function of capturing a physiologically active substance, and a polymer is immobilized on the surface of a particulate base material, and the polymer is attached to a side chain. It has the 1st repeating unit which has a hydrophilic group, and the 2nd repeating unit which has a biotin derivative at the terminal of a side chain, It is characterized by the above-mentioned.
The method for capturing a physiologically active substance of the present invention is characterized by using the medical particles described above.

The medical particles of the present invention have a function of capturing a physiologically active substance. Specifically, the present invention relates to a medical particle having a function of capturing a biotin-binding protein and uses a biotin-biotin-binding protein binding reaction. More specifically, particles capable of selectively recovering biomolecules that specifically bind to a bioactive substance by capturing a bioactive substance containing a biotin-binding protein on a base material to which a biotin derivative is bound. To get.
Here, examples of the physiologically active substance include biomolecules including a biotin-binding protein, for example, antigens, antibodies, peptides, DNA, RNA, sugars, lectins and the like bound to a biotin-binding protein.

In addition, many biotin-binding proteins have multiple binding sites for biotin (for example, four biotin-binding sites in one molecule of streptavidin). After binding biotin, the bioactive substance containing biotin can be selectively recovered by capturing bioactive substance containing biotin in the biotin-binding protein-bound particle. Is what you get.
Here, examples of the physiologically active substance include biomolecules including biotin, such as biotinylated antigen, antibody, peptide, DNA, RNA, sugar, lectin and the like.

The particulate base material is not particularly limited, and any organic material or inorganic material can be used. Examples of organic materials include porous agarose particles (trade name: Sepharose) and dextran particles (trade name: Sephadex) used as carriers for affinity chromatography, as well as polyacrylamide gel (trade name: Bio-Gel P, Biorad), polystyrene, ethylene-maleic anhydride copolymer, polymethyl methacrylate and the like.

Examples of the inorganic material include gold, silver, platinum, palladium, iridium, rhodium, osmium, iron, copper, cobalt, and alloys and inorganic oxides thereof. Among these, inorganic oxides are preferable because the strength of the particles themselves is high. Among these, silicon oxide is most preferable because it is easy to handle.

The average particle diameter of the particulate substrate is appropriately selected according to the purpose and application. In particular, in the case of an inorganic substance, the particle diameter can be easily controlled as compared with a method of producing organic particles by emulsion polymerization or suspension polymerization in which it is difficult to control the particle diameter.
Specifically, the particle diameter of the substrate used for the medical particles of the present invention is preferably one having an average particle diameter of about several nm to 100 μm, although it varies depending on the application. In particular, 100 nm to 50 μm is preferable, and 1 μm to 40 μm is most preferable. When the average particle size of the substrate is within the above range, the balance between the amount of the physiologically active substance captured and the good handling is particularly excellent.
Such an average particle diameter can be measured with a particle size distribution meter, for example.

A polymer is fixed on the surface of the particulate substrate. This polymer makes it possible to bind a biotin derivative efficiently and easily to a particulate substrate. Furthermore, non-specific adsorption of another substance (such as a protein other than the detection target) that reacts with the biotin derivative is effectively suppressed.

The polymer has a first repeating unit having a hydrophilic group in the side chain and a second repeating unit having a biotin derivative at the end of the side chain. That is, the first repeating unit having a hydrophilic group in the side chain suppresses non-specific adsorption of proteins other than the detection target, and the second repeating unit having a biotin derivative at the end of the side chain has a biotin-binding physiologically active substance. It can be captured.

The first repeating unit having a hydrophilic group in the side chain is derived from, for example, an ethylenically unsaturated polymerizable monomer having an alkylene glycol residue, an ethylenically unsaturated polymerizable monomer having a phosphorylcholine group, or the like. Among these, those derived from an ethylenically unsaturated polymerizable monomer having an alkylene glycol residue are preferred. Thereby, the nonspecific adsorption | suction of the protein contained in serum can be suppressed significantly.

Specifically, the ethylenically unsaturated polymerizable monomer having an alkylene glycol residue is composed of a chain of a (meth) acryl group and an alkylene glycol residue Y having 1 to 10 carbon atoms, as represented by the following formula (1). A compound is preferred.

Carbon number of alkylene glycol residue Y in said Formula (1) is 1-10, Preferably it is 1-6, More preferably, it is 2-4, More preferably, it is 2-3, Most preferably Is 2. When the carbon number is within the above range, it is particularly excellent in suppressing nonspecific adsorption.
Further, the repeating number q of the alkylene glycol residue Y is not particularly limited, but is preferably an integer of 1 to 100, more preferably an integer of 2 to 100, still more preferably 2 to 95. It is an integer, most preferably an integer of 20 to 90. When the repeat number q is within the above range, it is particularly excellent in suppressing nonspecific adsorption.
In addition, when the repeating number q is 2 or more and 100 or less, the carbon number of the alkylene glycol residue Y to be repeated may be the same or different.

Specific examples of the ethylenically unsaturated polymerizable monomer having an alkylene glycol residue include methoxypolyethylene glycol (meth) acrylate, ethoxypolyethylene glycol (meth) acrylate, 2-hydroxyethyl (meth) acrylate, and 2-hydroxypropyl. (Meth) acrylate and mono-substituted ester thereof, 2-hydroxybutyl (meth) acrylate and mono-substituted ester thereof, glycerol mono (meth) acrylate, (meth) acrylate having polypropylene glycol as a side chain, 2-methoxy Ethyl (meth) acrylate, 2-ethoxyethyl (meth) acrylate, methoxydiethylene glycol (meth) acrylate, ethoxydiethylene glycol (meth) acrylate, ethoxypoly Ji glycol (meth) acrylate and the like, methoxy polyethylene glycol methacrylate or ethoxy polyethylene glycol methacrylate is preferred because it and availability is less nonspecific adsorption of components other than the physiologically active substance of interest.

The biotin derivative of the second repeating unit having a biotin derivative at the end of the side chain is preferably a molecule that specifically reacts with a biotin-binding protein bound to a physiologically active substance.
Specifically, the biotin-binding protein is preferably avidin, streptavidin, or neutravidin. This is because the interaction between biotin and the avidins is very strong and highly specific. In addition, since biotin, which is a low molecule, can be easily introduced into a physiologically active substance, it can be a powerful tool for immobilizing substances.

The second repeating unit is derived from, for example, an ethylenically unsaturated polymerizable monomer having an alkylene glycol residue in the side chain or an ethylenically unsaturated polymerizable monomer having a glycidyl group in the side chain, and further at the tip of the side chain. What bound the biotin-binding protein mentioned above is mentioned. Among these, those obtained by binding a biotin-binding protein to a unit derived from an ethylenically unsaturated polymerizable monomer having an alkylene glycol residue in the side chain are preferable. Thereby, nonspecific adsorption | suction can be suppressed more.

The second repeating unit may be a unit in which a biotin-binding protein is immobilized using W of a unit derived from a monomer represented by the following formula (2).

W in Formula (2) is an active ester, specifically, for example, p-nitrophenyl active ester group, N-hydroxysuccinimide active ester group, phthalimide active ester group, 5-norbornene-2, 3- Examples thereof include a dicarboximide active ester group, among which a p-nitrophenyl active ester group or an N-hydroxysuccinimide active ester group is preferable, and a p-nitrophenyl active ester group is most preferable. Thereby, the reactivity of a biotin derivative and an active ester can be improved, and it becomes easy to arrange a biotin derivative at the end of the side chain of the second repeating unit.

The copolymerization ratio (first repeating unit / second repeating unit) between the first repeating unit and the second repeating unit of the polymer is not particularly limited, but is 97/3 to 50/50. Particularly preferred is 95/5 to 70/30. When the copolymerization ratio is within the above range, non-specific adsorption is particularly effectively suppressed and the biotin compound capturing effect is excellent.
The copolymerization ratio can be calculated, for example, by evaluating the elemental composition by X-ray photoelectron spectroscopy (XPS).

The polymer is preferably a random copolymer including a first repeating unit and a second repeating unit. Thereby, since the biotin derivative which exists in the terminal of the side chain of the 2nd repeating unit disperses, it can work effectively.

Although the weight average molecular weight of the said polymer is not specifically limited, It is preferable that it is 5000-1 million, and it is especially preferable that it is 10000-100,000. When the weight average molecular weight is within the above range, handling during synthesis is good, and nonspecific adsorption can be effectively suppressed.

In the medical particle of the present invention, a polymer composed of the first repeating unit, the second repeating unit, and a unit having a silane coupling agent in the side chain is bonded to the base material via the silane coupling agent. May be. This can prevent the polymer from detaching from the beads.

As the unit having a silane coupling agent in the side chain, methacryloxypropyldimethylmethoxysilane, methacryloxypropyldimethylethoxysilane, methacryloxypropylmethyldimethoxysilane, methacryloxypropylmethyldiethoxysilane, methacryloxypropyltrimethoxysilane, Those derived from methacryloxypropyltriethoxysilane are mentioned.

As a specific method for forming a polymer bonded to particles by a silane coupling agent, in the case of an inorganic oxide, it can be easily bonded by a coupling reaction with a hydroxyl group present on the surface.

By using the silane coupling agent, it is possible to prevent the polymer from detaching from the beads. Therefore, in the use as medical particles, the polymer is prevented from being dissolved in repeated heat treatments and washing steps. Furthermore, it is possible to provide chemically and physically stable medical particles in which deterioration of non-specific adsorption that occurs with the desorption of the polymer is suppressed.
In addition, since non-specific adsorption to proteins other than the detection target is small and the detection target can be captured on the particle surface due to the high reactivity of the biotin derivative and the biotin-binding protein, the S / N ratio is low. High medical particles can be provided.

(Production of polymer fixed beads)
The production of the polymer-fixed beads will be described. The method for synthesizing the beads of the present invention is not particularly limited, but for ease of synthesis, a silane coupling agent having a polymerizable functional group is immobilized on the surface of the bead particles, and the biotin-binding protein is immobilized. An ethylenically unsaturated polymerizable monomer having a functional group to be converted, an ethylenically unsaturated polymerizable monomer having an alkylene glycol residue, and a mixture containing the beads may be radically polymerized in a solvent in the presence of a polymerization initiator. preferable.
Any solvent may be used as long as each ethylenically unsaturated polymerizable monomer can be dissolved. Examples thereof include 2-butanone, methanol, ethanol, t-butyl alcohol, benzene, toluene, tetrahydrofuran, dioxane, dichloromethane, and chloroform. be able to. These solvents are used alone or in combination of two or more.
The polymerization initiator may be any ordinary radical initiator, such as 2,2′-azobisisobutylnitrile (hereinafter referred to as “AIBN”), 1,1′-azobis (cyclohexane-1-carbonitrile), and the like. Examples thereof include organic peroxides such as azo compounds, benzoyl peroxide, and lauryl peroxide.
The chemical structure of the polymer compound of the present invention may be in any form such as random, block or graft.

(Immobilization of biotin derivatives)
In the present invention, the biotin derivative immobilized on the substrate is preferably a biotin molecule that reacts specifically with a biotin-binding protein derivative, and particularly preferably a terminal amino group-modified biotin. For example, molecules such as Biotinyl-3,6,9-trioxaundecanamine, Amine-PEO-Biotyne, Amine-PEG2-Biotyne, Amine-PEG3-Biotyn and the like can be mentioned. These molecules are characterized by having a spacer such as PEO or PEG between biotin and the terminal amino group, which is a certain distance from the substrate when biotin is immobilized. Thus, the aim is to eliminate the lack of binding due to steric hindrance in the biotin-biotin binding protein interaction.

In the present invention, when the biotin derivative is immobilized on the beads, a method of attaching a liquid in which the biotin derivative is dissolved or dispersed is preferable. As the liquid in which the biotin derivative is dissolved or dispersed, a water-soluble solvent, an organic solvent, or a mixture of both solvents can be used depending on the reagent. In the case of a water-soluble solvent, the pH is preferably 5.0 to 11.0, and more preferably pH 6.0 to 10. Outside this range, the substrate, polymer and biotin derivative may be denatured.

In addition, when an organic solvent is used, it is not particularly limited as long as it does not react with the functional group that binds the biotin derivative of the second repeating unit, but polar solvents such as methanol and ethanol alcohols, dimethyl sulfoxide, etc. Nonpolar solvents such as toluene and benzene can be used, but it is preferable to use alcohols or dimethyl sulfoxide since it is necessary that the beads and the polymer substance on the surface of the beads are not affected.
When the immobilization reaction of the biotin derivative to the beads is completed, the beads are washed to remove unreacted substances. Although it does not specifically limit as a cleaning agent, Pure water, various buffer solutions, alcohol, such as methanol and ethanol, etc. are preferable.

(Capture of physiologically active substances)
A physiologically active substance to be captured using a biotin derivative immobilized on a bead needs to be bound to a biotin-binding protein.
On the other hand, after binding only a biotin-binding protein to a biotin derivative immobilized on a bead, it is also possible to capture a physiologically active substance further bound to the biotin derivative.

(Structure of bioactive substance captured by modification of biotin-binding protein)
The physiologically active substance to be captured needs to be modified with a biotin-binding protein.
This reaction can be easily prepared by using a commercially available bifunctional crosslinking agent.
Commercially available bifunctional crosslinking agents include, for example, glutaraldehyde, Pierce Biotechnology's BASED, DSS, N- [κ-Maleimidoundecanoyloxy] -Sulfosuccinimide Ester, N-Succinimidyl Iodoacetate, Succinimidyl-6-[(β- Maleimidopropionamido) Hexanoate] can be preferably used.

(Structure of bioactive substance captured by biotin modification)
First, as a first step, it is necessary to capture the biotin derivative on the biotin derivative immobilized on the bead surface. This capture reaction can be easily produced under general biotin-avidin reaction conditions.
Subsequently, by introducing biotin or a biotin derivative into the physiologically active substance to be captured by a general method, the target biologically active substance can be effectively immobilized by the biotin-binding protein captured on the substrate surface. Is possible. In order to enhance the reactivity, it is desirable that the biotin or biotin derivative is introduced at the end or outside of the physiologically active substance. Advances in the synthesis of bioactive substances have made it possible to introduce biotin or biotin derivatives at any position, and at positions that do not affect the important sites for developing bioactivity. The physiologically active substance can be immobilized on the surface of the carrier.
As a general method for introducing biotin or a biotin derivative, a bioactive substance can be labeled with biotin using a commercially available biotin labeling kit (for example, Dojin Labling Kit, manufactured by Dojin Chemical).

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

Example 1
(Production of biotin beads)
After adding 13 g of methacryloxypropyldimethylmethoxysilane (SIM 6486.5 manufactured by Gelest Co.) to a mixed solution of 50 mL of acetic acid aqueous solution of pH 3.0 and 50 mL of ethanol, hydrolyzing the silane coupling agent, silica beads (average particle size) 10 μg of 5 μm, 70 μm pore size, SMB70-5 manufactured by Fuji Silysia Chemical Ltd. was added and stirred at 70 ° C. for 2 hours, and then silica beads were collected from the reaction solution by suction filtration and heated at 100 ° C. for 1 hour. Then, after dispersing with ethanol and shaking well, the supernatant was removed by centrifugation and dried.

Polyethylene glycol methyl ether methacrylate (also known as methoxy polyethylene glycol methacrylate, hereinafter referred to as PEGMA475, manufactured by Aldrich) having a number average molecular weight Mn of about 475, p-nitrophenyloxycarbonylethylene glycol methacrylate (MEONP) is dissolved in ethanol, and monomer A mixed solution was prepared. The total monomer concentration is 0.2 mol / L, and the respective molar ratios are 90:10, 80:20, and 70:30 in the order of PEGMA475 and MEONP. AIBN was added there so that it might become 0.004 mol / L, and it stirred until it became uniform. Thereafter, 10 g of silica beads treated with the above methacryloxypropyldimethylmethoxysilane were added and reacted at 70 ° C. for 22 hours in an argon gas atmosphere. Next, silica beads were collected from the reaction solution by centrifugation, dispersed in dimethyl sulfoxide, shaken well, and then collected by suction filtration and dried.

About 100 mg of the obtained silica beads were mixed with a 0.5 mg / mL biotin solution (EZ-Link Biotin-LC-PEO-Amine manufactured by PIERCE was diluted with DMSO), treated at 37 ° C. for 4 hours, and then centrifuged to obtain a supernatant. The sample was removed three times, dispersed with pure water, and the supernatant was removed by centrifugation three times, and further 0.1 mol / L 2-aminoethanol (solvent: pH 9.5, 0.05 mol / L Tris-HCl). Buffer) for 1 hour at room temperature to inactivate MEONP. The supernatant was removed by centrifugation, then dispersed with pure water, and the operation of removing the supernatant by centrifugation was performed three times and dried. Moreover, the biotin was not fixed but the bead which produced only a polymer coat and inactivation by the above-mentioned method was produced.

(Measurement of biotin beads and inactivated beads)
270 μL of HRP-labeled streptavidin (CHEMICON INTERNATIONAL STREPTAVIDIN, HRP CONJUGATED diluted in PBS) adjusted to 5 μg / mL was added to each 1 mg of the obtained silica beads (biotin-immobilized beads and beads inactivated only), After stirring at room temperature for 30 minutes, the supernatant was removed by centrifugation. Next, PBS containing Triton × 100 at a concentration of 0.05% was added and dispersed well, and then the supernatant was removed by centrifugation five times, and a filter unit (Ultrafree-MC manufactured by MILLIPORE) was used. The silica beads were recovered. After reacting TMBZ and HRP-labeled streptavidin on the surface of the silica beads by adding a TMBZ solution (prepared and used from a color kit for peroxidase manufactured by Sumitomo Bakelite Co., Ltd.) to the recovered silica beads and stirring for 15 minutes at room temperature Then, a reaction stop solution (used from a peroxidase coloring kit manufactured by Sumitomo Bakelite Co., Ltd.) was added to stop the reaction. Thereafter, the silica beads and the reaction solution were separated by a filter unit, and the absorbance at 450 nm in the reaction solution was measured. This absorbance mainly reflects the amount of HRP-labeled streptavidin captured specifically on silica beads when biotin-immobilized beads are used in the above operation, and mainly when inactivated beads are used. The amount of HRP labeled streptavidin adsorbed non-specifically on the silica beads is reflected.

(Comparative Example 1)
In the same manner as the preparation of the avidin beads, the ratio of PEGMA475 and MEONP was changed to 99: 1 and 20:80 in this order. As above, specific capture evaluation and non-specific adsorption amount evaluation of HRP-labeled streptavidin were performed on biotinylated beads and inactivated beads.

(Comparative Example 2)
The silica beads (average particle size 5 μm, pore size 70 mm, SMB70-5 manufactured by Fuji Silysia Chemical Ltd.) were used as they were without the treatment of Example 1, and HRP-labeled streptavidin was used for biotinylated beads and inactivated beads. Specific capture evaluation and non-specific adsorption amount evaluation were performed.

Table 1 shows the absorbance at 450 nm of HRP-labeled streptavidin for the biotin-immobilized beads and inactivated beads of Example 1 and Comparative Examples 1 and 2. As is clear from the table, the particles of the present invention were compared with the beads of Comparative Examples 1 and 2 in terms of absorbance (1) due to HRP-labeled streptavidin for biotin-immobilized beads and HRP-labeled streptoid for inactivated beads. It can be seen that the ratio ((1) / (2)) to the absorbance (2) by avidin is large, and the target streptavidin can be captured more selectively.

Table 1

(Example 2)
(Preparation of avidin-immobilized beads)
About 37 mg of each biotinylated bead prepared in Example 1 was mixed with 1 mL of a 50 μg / mL avidin solution (NeutrAvidin ™ Biotin-Binding Protein manufactured by PIERCE diluted with phosphate buffer (PBS)) and treated at 37 ° C. for 4 hours. Next, the supernatant is removed by centrifugation, and then dispersed with a phosphate buffer solution (PBST) containing Tween 20 at a concentration of 0.05%, and the supernatant is removed by centrifugation three times, and further dispersed with PBS. The operation of removing the supernatant by centrifugation was performed once.

(Measurement of avidin-immobilized beads)
270 μL of 5 μg / mL biotin-labeled HRP solution (Zymed Laboratories, Inc. Biotinylated Peroxidase diluted 200-fold with PBS) that binds specifically to 1 mg of the obtained silica beads, or 5 μg / mL of HRP After adding 270 μL of the antibody solution and stirring at room temperature for 30 minutes, the supernatant was removed by centrifugation. Next, PBS containing Triton × 100 at a concentration of 0.05% was added and dispersed well, and then the supernatant was removed by centrifugation five times, and a filter unit (Ultrafree-MC manufactured by MILLIPORE) was used. The silica beads were recovered. Biotin-labeled HRP specifically captured on the surface of TMBZ and silica beads by adding a TMBZ solution (prepared and used from a color development kit for peroxidase manufactured by Sumitomo Bakelite Co., Ltd.) to the collected silica beads and stirring for 15 minutes at room temperature, or After reacting with the non-specifically adsorbed HRP-labeled antibody, a reaction stop solution (used from a peroxidase coloring kit manufactured by Sumitomo Bakelite Co., Ltd.) was added to stop the reaction. Thereafter, the silica beads and the reaction solution were separated by a filter unit, and the absorbance at 450 nm in the reaction solution was measured. When the biotin-labeled HRP solution is added in the above operation, the absorbance mainly reflects the amount of biotin-labeled HRP specifically captured on the silica beads, and when the HRP-labeled antibody solution is input. , Mainly reflecting the amount of HRP-labeled antibody adsorbed non-specifically to silica beads.

(Comparative Example 3)
The same operation as in Example 2 was performed on the inactivated beads prepared in Example 1.

  Table 2 shows the absorbance at 450 nm in the specific capture evaluation and the nonspecific adsorption amount evaluation for the beads of Example 2 and Comparative Example 3. As is apparent from the table, the particles of the present invention are compared with the silica beads of Comparative Example 3 in the ratio of the absorbance (1) by biotin-labeled HRP and the absorbance (2) by HRP-labeled antibody ((1) / ( 2)) is large, indicating that the target protein can be captured more selectively.

Table 2

Claims (12)

A medical particle having a function of capturing a physiologically active substance,
The polymer is immobilized on the surface of the particulate substrate,
The medical particle, wherein the polymer has a first repeating unit having a hydrophilic group in a side chain and a second repeating unit having a biotin derivative at a terminal of the side chain.   2. The medical particle according to claim 1, wherein the second repeating unit is obtained by binding a biotin derivative to a side chain end of a polymer of an ethylenically unsaturated polymerizable monomer having an alkylene glycol residue.   The medical particle according to claim 1 or 2, wherein the polymer is a random copolymer of the first repeating unit and the second repeating unit.   The copolymerization ratio (first repeating unit / second repeating unit) between the first repeating unit and the second repeating unit is 97/3 to 50/50. The medical particles described.   The medical particle according to any one of claims 1 to 4, wherein the biotin derivative is at least one kind of biotin derivative which is a terminal amino group-modified biotin by introducing an amino group into a biotin terminal carboxyl group.   The medical particle according to any one of claims 1 to 5, wherein the first repeating unit is a polymer of an ethylenically unsaturated polymerizable monomer having an alkylene glycol residue. The medical particle according to claim 6, wherein the ethylenically unsaturated polymerizable monomer having an alkylene glycol residue is a monomer represented by the following formula (1).

  The medical particle according to any one of claims 1 to 7, wherein the particulate substrate is made of an inorganic material.   The medical particle according to claim 8, wherein the inorganic material is an inorganic oxide.   The medical particle according to claim 9, wherein the inorganic oxide is silicon oxide.   When the polymerizable functional group introduced into the surface of the particulate base material via a silane coupling agent is taken into the polymer during the synthesis of the polymer, the particulate base material and the polymer are The medical particle according to any one of claims 1 to 10, which is immobilized by a covalent bond.   A method for capturing a physiologically active substance, wherein the medical particles according to any one of claims 1 to 11 are used.