JP4929635B2 - Maleimide group-containing porous crosslinked polystyrene particles and method for producing the same - Google Patents

Description

  The present invention relates to maleimide group-containing porous cross-linked polystyrene particles and a method for producing the same, and particularly for uses such as chromatography carriers, immunoprecipitation carriers, cell collection beads, DNA hybrid carriers, RNA hybrid carriers, diagnostic agents, and diagnostic agents. The present invention relates to a maleimide group-containing porous crosslinked polystyrene particle that can be suitably used and a method for producing the same.

  There is a specific molecular affinity (affinity) between a general substance and a specific protein, a protein and a specific protein, a protein and its antibody, a peptide and its antibody, and complementary DNA and RNA. Utilizing this principle, it is practiced to purify and collect target biomolecules and cells from a mixture of many substances.

  For this purpose, a ligand substance that specifically binds to the target molecule is immobilized on a particle or substrate support, and a mixed solution containing the target molecule is brought into contact therewith to bind (couple) the target molecule to the ligand. . Next, an excess substance on the carrier is washed away with a washing solution, and then the eluate is brought into contact with the carrier to dissociate the ligand from the target molecule. In this way, the substance containing the target molecule can be purified and recovered.

  Particles are often used for these carriers, and those packed in columns include affinity chromatography, and those handled in batches in tubes include immunoprecipitation carriers and beads for cell collection. It can also be used as a diagnostic or diagnostic agent to confirm the presence or absence of pathogenic substances by detecting affinity reactions on particles. For the same purpose, DNA hybrid carriers and RNA hybrid carriers are also used.

  The method for binding the ligand to the particle carrier is roughly divided into physical adsorption and covalent bonding. The latter is less likely to desorb the ligand than the former, and has a wide range of operating environments. Although there are various covalent bonding methods, a method of fixing to a simple substance using an SH group contained in a ligand is useful. Cysteine contains only the SH group in amino acids, but its presence in proteins is low. Moreover, the SH group is not contained in the nucleic acid of natural DNA or RNA. Therefore, when these molecules or a part thereof is used as a ligand, the part can be selectively bound to a carrier by artificially introducing an SH group at a desired position. For example, by introducing a cysteine residue at the end of a peptide as an epitope which is an antibody reaction site of a protein, the end can be fixed to a carrier.

  Since an SH group easily forms an S—S covalent bond with the same SH group, a ligand immobilization method using this principle is generally used. In Athiom Separose 4B manufactured by Amersham Biosciences, an SH group is introduced into agarose gel particles via glutathione. The epitope peptide of the cysteine terminal can be immobilized therewith by S—S bond, and the anti-peptide antibody can be purified by affinity coupling of the corresponding anti-peptide antibody. However, since the S—S bond is easily reduced to form another bond, the bond between the particle and the ligand cannot be stably maintained.

On the other hand, there is a maleimide group that can be easily covalently bonded to the SH group and that can maintain the bond stably. It is considered that a ligand substance having an SH group can be easily and stably immobilized if a maleimide group is contained in the carrier. There are examples (for example, see Patent Document 1) and products (for example, see Non-Patent Document 1) in which polystyrene particles containing a maleimide group have already been published.
JP-A-11-106391 2001/2002 product catalog, Fuluka, page 909

However, when the epitope peptide of the cysteine terminal is fixed to the particle carrier produced under the conditions described in Patent Document 1 or the product of Non-Patent Document 1, and the corresponding anti-peptide antibody is affinity-coupled, the antibody to be purified The amount was very small compared to the above-mentioned thiol Separose 4B particles, which was not sufficient.
The present invention has been made in view of the above problems, and has a maleimide group that can be easily covalently bonded to the SH group of a ligand molecule and can stably hold the bond, and is excellent in affinity purification characteristics. An object is to provide a group-containing porous crosslinked polystyrene particle and a method for producing the same.

That is, the present invention
<1> A maleimide group-containing porous crosslinked polystyrene particle having a volume average particle diameter of 1-1000 μm, a pore diameter not exceeding the diameter of the particle, and 1-10000 nm.

  <2> The maleimide group-containing porous crosslinked polystyrene particle according to <1>, wherein the maleimide group is bonded to the phenyl group of the polystyrene chain via a spacer molecule.

  <3> The maleimide group-containing porous crosslinked polystyrene particle according to <2> having a group represented by the following chemical formula (1).

(In chemical formula (1), n represents an integer of 1 or more.)

< 4 > A monomer mixture containing styrene, a crosslinking agent, a polymerization initiator, and a diluent that does not cause a polymerization reaction with the styrene and the crosslinking agent is dispersed in an aqueous medium, and the styrene and the crosslinking agent are dispersed. A step of copolymerizing to obtain a diluent-containing crosslinked polystyrene particle, a step of removing a diluent from the diluent-containing crosslinked polystyrene particle to obtain a crosslinked polystyrene particle, a step of introducing a maleimide group into the crosslinked polystyrene particle, It is a manufacturing method of the maleimide group containing porous crosslinked polystyrene particle | grains which have.

< 5 > The method for producing a maleimide group-containing porous crosslinked polystyrene particle according to < 4 >, wherein the diluent is at least one selected from the group consisting of toluene, dodecane, diethylbenzene, isoamyl alcohol, lauryl alcohol, and hexadecane. is there.

< 6 > Content of the said diluent in the said monomer mixture is 30-80 mass% with respect to the total amount of the said styrene, the said crosslinking agent, and the said diluent, As described in < 4 > or < 5 >. This is a method for producing maleimide group-containing porous crosslinked polystyrene particles.

  ADVANTAGE OF THE INVENTION According to this invention, the maleimide group containing porous crosslinked polystyrene particle | grains which have a maleimide group and were excellent in the affinity refinement | purification characteristic, and its manufacturing method can be provided.

Hereinafter, the maleimide group-containing porous crosslinked polystyrene particles of the present invention and the production method thereof will be described in detail.
<Maleimide group-containing porous crosslinked polystyrene particles>
The maleimide group-containing porous crosslinked polystyrene particles of the present invention (hereinafter sometimes referred to as “particles of the present invention”) have a volume average particle diameter of 1 to 1000 μm, and the pore diameter does not exceed the diameter of the particles. In the range and from 1 to 10,000 nm.
The particles of the present invention contain crosslinked polystyrene, which is a copolymer of styrene and a crosslinking agent. Divinylbenzene is mainly used as the cross-linking agent, but in addition, ethylene glycol di (meth) acrylate, diethylene glycol di (meth) acrylate, glycidyl (meth) acrylate, 2-([1′-methylpropylideneamino] carboxyamino It is also possible to use ethyl (meth) acrylate or the like.
The content of the cross-linking agent is variously adjusted depending on the use of the particles. When the total amount of styrene and the cross-linking agent is 100 parts by mass, the content is 0.1 to 50 parts by mass, preferably 0.5 to 25 parts. Mass parts, more preferably 1 to 10 parts by mass are preferred.
If the content of the crosslinking agent is too low, the solvent resistance of the particles of the present invention is lowered, and the particles are dissolved during chemical modification after granulation or when used as affinity particles. On the other hand, if the content of the cross-linking agent is too high, it becomes mechanically brittle and the particles are often broken.

Although the volume average particle diameter of the particle | grains of this invention is adjusted according to a use application, it is required that it is 1-1000 micrometers, Preferably it is 10-100 micrometers. If the volume average particle diameter is less than 1 μm, the gap between the particles may be small in chromatography, and the sample liquid may not pass through. In addition, in batch methods such as immunoprecipitation, it becomes difficult for particles to settle and collect. On the other hand, if the volume average particle diameter is larger than 1000 μm, the gap between the particles is too large, and the sample solution cannot sufficiently come into contact with the particles of the present invention, so that the affinity purification ability is lowered.
Depending on the application, the shape of the particles may be one or two or more of spherical, elliptical, polyhedral, acicular, flat, etc.
The volume average particle diameter of the particles of the present invention is a value measured by the following method.
The diameter of each particle can be measured by an optical microscope, an electron microscope, a pore electrical resistance method, or the like. When the measured n particle diameters are d1, d2,... Dn, the respective volumes V1, V2,. Volume average particle diameter MV is
MV = (d1 ・ V1 + d2 ・ V2 + ・ ・ ・ ・ dn ・ Vn) / (V1 + V2 + ・ ・ ・ ・ Vn)
It is a value calculated by.

The particles of the present invention are porous particles having pores having a diameter not exceeding the diameter of the particles and having a diameter of 1 to 10,000 nm. By making the particles porous, the surface area is increased, the chance of contact between the sample liquid and the particles is increased, and the affinity purification ability is improved. The diameter of the pores is preferably 10 nm to 5000 nm.
If the diameter of the pore is less than 1 nm, the ligand molecule and the molecule to be purified that are fixed to the particle cannot enter the pore, and the effect of the pore is lost. On the other hand, if the diameter of the pores is larger than 10,000 nm, the mechanical strength of the particles decreases and the particles are easily broken.
The diameter of the pores of the particles of the present invention is a value measured by the following method.
There are various methods for measuring pores. In the present invention, a mercury intrusion method and a nitrogen gas adsorption method are employed. The former is a method in which a porous sample is put into mercury and then pressurized, and the entire volume change when mercury enters the pores is measured, and measurement from several nm to several hundred μm is possible. In the latter, a porous sample is put in nitrogen gas and adsorbed, and then nitrogen is released from the sample while degassing. The pore diameter is calculated by measuring the relationship between the supply / recovery amount of gas and the pressure at this time. In the nitrogen gas adsorption method, pores of 1 nm to several tens of nm can be measured. By these two methods, pores of 1 to 10000 nm can be measured.

  The particles of the present invention contain maleimide groups. Maleimide groups are distributed on the surface of the particles and the inner surface of the pores. The content of the maleimide group is adjusted according to the application, but is 0.0001 mmol to 10 mmol, preferably 0.001 mmol to 1 mmol, per 1 g of the particles. When the amount of maleimide groups is less than 0.0001 mmol, a sufficient amount of ligand molecules containing SH groups cannot be immobilized on the particles, and the purification ability may not be improved. On the other hand, if the amount of maleimide groups is too large, excess maleimide groups may inhibit affinity binding and the purification characteristics may deteriorate.

The bonding form of the maleimide group varies depending on the application, but it can be covalently bonded to the phenyl group of the polystyrene chain directly or via a molecule serving as a spacer.
In the present invention, the maleimide group is preferably bonded to the phenyl group of the polystyrene chain via a spacer molecule. A spacer molecule is inserted to increase the distance between the polystyrene chain and the maleimide group. Thereby, the ligand molecule fixed to the maleimide group can be separated from the polystyrene chain. Therefore, the ligand molecule and the molecule having affinity characteristics can be easily bonded without making the polystyrene chain steric hindrance.

Various spacer molecules are used depending on the use of the particles and the types of ligand molecules and molecules to be purified. In particular, ethylene glycol, polyethylene glycol, para-xylene diol, meta-xylene diol, glutathione, dextran, thiodiethanol, bis [4- (2-hydroxyethoxy) phenyl] sulfone, or the like is used by chemically bonding to a polystyrene chain.
Although polyethylene glycol has various molecular weights, those having an average molecular weight of 100 to 1000 are preferably used mainly.

  The particles of the present invention more preferably have a group represented by the following chemical formula (1). In the following chemical formula (1), the maleimide group is bonded to the phenyl group of the polystyrene chain via (poly) ethylene glycol which is a spacer molecule. (Poly) ethylene glycol means ethylene glycol and polyethylene glycol.

  In the chemical formula (1), n represents an integer of 1 or more.

Ligand molecules containing thiol groups (SH groups) can be immobilized on the particles of the present invention. In addition to natural substances such as peptides, proteins, DNA, RNA, saccharides, and fats, synthetic inorganic molecules and synthetic organic molecules such as complexes, pigments, metals, and metal oxides are used as ligand molecules. Among these, at least one selected from the group consisting of peptides, proteins, DNA, and RNA is preferable.
A peptide having 2 to 40 amino acids, preferably 5 to 20 amino acids can be used. Peptides contain cysteines with SH groups, usually preferably only one at either end.
Proteins not containing cysteine, DNA, RNA, saccharides and fats can be used by chemically introducing molecules containing SH groups.
A ligand molecule containing a thiol group is considered to bind to a maleimide group as shown in the following chemical formula (2).

  In the chemical formula (2), R—S— represents a ligand molecule residue.

  The particles of the present invention in which a ligand molecule containing a thiol group is bonded to a maleimide group are suitably used for affinity purification and the like.

<Method for Producing Maleimide Group-Containing Porous Crosslinked Polystyrene Particles>
The method for producing the particles of the present invention is not particularly limited. For example, a monomer containing styrene, a crosslinking agent, a polymerization initiator, and a diluent that does not cause a polymerization reaction with the styrene and the crosslinking agent. Dispersing the mixture in an aqueous medium to copolymerize the styrene and the crosslinking agent to obtain a diluent-containing crosslinked polystyrene particle; and removing the diluent from the diluent-containing crosslinked polystyrene particle to obtain a crosslinked polystyrene particle. The particles of the present invention can be obtained through a step and a step of introducing a maleimide group into the crosslinked polystyrene particles.

-Production of diluent-containing crosslinked polystyrene particles-
Diluent-containing cross-linked polystyrene particles are prepared by, for example, stirring a monomer mixture containing styrene, a cross-linking agent, a polymerization initiator, styrene, a cross-linking agent and a diluent that does not cause a polymerization reaction at a desired ratio in an aqueous solvent at high speed. Thus, it can be obtained by dispersing the particles and then heating to copolymerize styrene and a crosslinking agent. For the production of the diluent-containing crosslinked polystyrene particles, a suspension polymerization method, an emulsion polymerization method, a dispersion polymerization method, a seed polymerization method, a membrane emulsification method, or the like is used.

  As the diluent, a substance that does not cause a polymerization reaction with styrene and a crosslinking agent (a substance that does not participate in a copolymerization reaction) is used. Diluents can be either solid or liquid at room temperature. One type of diluent may be used alone, or two or more types may be mixed and used. Specifically, toluene, dodecane, diethylbenzene, isoamyl alcohol, lauryl alcohol, hexadecane, benzene, xylene, chlorobenzene, cyclohexane, isooctane, chloroform, carbon tetrachloride, dichloroethane, dibutyl phthalate and the like can be used. Among these, it is preferable to use at least one selected from the group consisting of toluene, dodecane, diethylbenzene, isoamyl alcohol, lauryl alcohol, and hexadecane for obtaining suitable affinity characteristics.

Polymerization initiators include organic peroxides such as diacyl peroxide, ketone peroxide and alkyl hydroperoxide, inorganic peroxides such as hydrogen peroxide and ozone, and azobisvaleronitrile (AIBN; Wako Pure Chemical Industries, Ltd.). Available as V-60), dimethyl 2,2′-azobis (2-methylbutyronitrile) (from Wako Pure Chemical Industries, V-6)
01), 2,2′-azobis (2-methylbutyronitrile) (available as V-59 from Wako Pure Chemical Industries) and 2,2′-azobis (2,4-dimethylvaleronitrile) ( Oil-soluble azo organic compound such as V-65 available from Wako Pure Chemical Industries, Ltd., potassium persulfate, ammonium persulfate, 2,2′-azobis (2-amidinopropane) dioic acid salt (from Wako Pure Chemical Industries, Ltd.) V-50), 2,2′-azobis [2-methyl-N—
(2-hydroxyethyl) propionamide] (available as VA-086 from Wako Pure Chemical Industries) and 2,2′-azobis [2- (2-imidazolin-2-yl) propane] dioic acid salt (Wako Pure Chemical) There is a water-soluble azo organic compound such as VA-044 from the company. The polymerization initiator is used in an amount sufficient to initiate the polymerization satisfactorily, and is preferably 0.1 to 5.0% by mass of the oil weight obtained by adding styrene, a crosslinking agent and a diluent.

-Removal of diluent-
Diluent-containing crosslinked polystyrene particles are obtained by the copolymerization reaction. Thereafter, the diluent is removed from the diluent-containing crosslinked polystyrene particles to obtain crosslinked polystyrene particles. As a method for removing the diluent, for example, washing the crosslinked polystyrene particles in a solvent that dissolves the diluent, evaporating the diluent by overheating, evaporating the diluent by lowering the pressure, etc. Or in combination. It is also possible to use washing using Soxhlet extraction. As the washing solvent, methanol, ethanol, isopropyl alcohol, acetone, toluene and the like can be used.

There is also a method for producing crosslinked polystyrene particles without using a diluent. Specifically, fine particles smaller than the final target particle diameter are granulated, and porous particles are obtained by associating these fine particles. About this method, for example, there is a description in JP-A-4-5000726, page 13, lower left column, lines 1 to 6, before the copolymerization, a magnetic substance is added to make the crosslinked polystyrene particles magnetic particles, or a desired color can be obtained. It is also possible to add pigments to form colored particles.

-Classification, drying-
Prior to the next reaction step after granulation of the crosslinked polystyrene particles, the particles can be classified and separated into particles having a desired size or dried. For classification, a mesh, a fluid force such as wind, and a gravity can be used. Moreover, the dry type performed in a gas and the wet type performed in a solvent can be used. Various solvents can be used in the wet process, but those having good particle dispersion in the solvent are preferable. For example, water, methyl alcohol, ethyl alcohol, isopropyl alcohol, other alcohols, acetone or the like can be used alone or in admixture of two or more.

-Introduction of spacer molecules-
Spacer molecules are added to the granulated crosslinked polystyrene particles as necessary. Various chemically possible methods can be applied to this, but there is a method in which a phenyl group of a polystyrene chain is chloromethylated (see Examples below), and then a ligand having an OH group is subjected to Cl elimination etherification reaction. is there. This method is described, for example, in J. Org. Org. Chem. 1981, 46, 3433-3436. If a diol compound having two OH groups is used as a spacer molecule, it can be converted into polystyrene particles containing OH groups after the reaction, which is suitable for the next reaction.

-Classification, drying-
After the spacer molecule addition reaction, before performing the next reaction step, the particles can be classified and separated into particles having a desired size, or dried.

-Introduction of maleimide group-
In order to introduce maleimide groups into the crosslinked polystyrene particles produced as described above, any chemically possible method can be used.
Among them, a method of reacting hydroxymethylmaleimide is effective.
In other words, when there is no spacer molecule, there is a method in which the OH group of hydroxymethylmaleimide is subjected to Cl exchange etherification reaction in the state where the phenyl group of the polystyrene chain is chloromethylated.
Moreover, when there is an OH group-containing spacer, it can be easily bonded to the OH group of hydroxymethylmaleimide by a dehydration condensation etherification reaction. In this reaction, a known acidic or basic etherification catalyst can be used. For example, as the basic catalyst, hydroxides, oxides, carbonates, bicarbonates, and the like of alkali metals and alkaline earth metals can be used, and they can be used alone or in combination. As the acidic catalyst, inorganic acids such as sulfuric acid, hydrochloric acid, nitric acid and phosphoric acid and organic acids such as p-toluenesulfonic acid, trichloroacetic acid and acetic acid can be used, and these may be in the form of hydrates. It can also be used with hydrotalcite solid catalysts.
The step of introducing maleimide groups into the crosslinked polystyrene particles includes the above-described “introduction of spacer molecules” and “introduction of maleimide groups” when introducing spacer molecules into the particles. When not introduced, the above-mentioned “introduction of maleimide group” is included.

-Classification, drying-
After the maleimide group introduction reaction, before performing the next reaction step, the particles can be classified and separated into particles having a desired size or dried.

-Ligand molecule binding-
Ligand molecules containing SH groups can be easily bound to the maleimide groups of the particles of the present invention produced as described above. The reaction is usually carried out by bringing the particles of the present invention into contact with the ligand molecule in a solvent in which the ligand molecule can be dissolved. Water, phosphate buffer, acetate buffer, Tris-HCl buffer, carbonate buffer, glycine-HCl buffer, citrate buffer, HEPES buffer, MOPS buffer, Hanks buffer, etc. can be used as the solvent It is. To the solvent, organic solvents such as dimethylacetamide, methanol, ethanol, acetone, chelating agents such as ethylenediaminetetraacetic acid disodium salt, surfactants such as sodium dodecyl sulfate and polyoxyethylene alkyl ether, EDTA, etc. may be added. Is possible.

  The particles of the present invention can easily and stably immobilize ligand molecules containing SH groups. And since a magnitude | size is 1-1000 micrometers, Preferably it is 10-100 micrometers, even if it fills in a column, sufficient liquid permeability can be ensured. Furthermore, the affinity reaction can be carried out by increasing the particle surface area by making the pore diameter within a range not exceeding the diameter of the particle and 1 to 10,000 nm, preferably 10 to 5000 nm. Further, by arranging a maleimide group on the polystyrene chain via a spacer molecule as shown in chemical formula (1), even better affinity purification characteristics can be realized.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail using an Example, this invention is not limited by the following Example.

Example 1
37.5 parts by mass of styrene, 12.5 parts by mass of divinylbenzene (55% purity product), 25 parts by mass of diethylbenzene and 50 parts by mass of isoamyl alcohol as a diluent, dimethyl 2,2′-azobis (2-methyl) as a polymerization initiator Butyronitrile) 2.0 parts by mass was mixed and dissolved. The ratio of the diluent to the total oil is 20% diethylbenzene and 40% isoamyl alcohol. This mixture was put into a uniformly dispersed solution of 10 parts by mass of calcium carbonate powder, 50 parts by mass of sodium chloride, and 200 parts by mass of water. After emulsification at 6000 rpm for 3 minutes with a mixer, a polymerization reaction was carried out at 70 ° C. for 20 hours in a nitrogen atmosphere. Thereafter, hydrochloric acid was added to decompose calcium carbonate, followed by washing with water, and then washing with ethanol to remove the diluent.
Further, wet classification was performed to select particles having an average diameter of 50 μm, and vacuum drying was performed at 100 ° C. for 12 hours.
A mixture prepared by mixing 9.0 parts by mass of trioxane, 100 parts by mass of chloroform, and 33 parts by mass of chlorotrimethylsilane with 10 parts by mass of the crosslinked porous polystyrene particles produced above was produced and ice-cooled. A solution prepared by dissolving 10 parts by mass of tin tetrachloride in 5 parts by mass of chloroform was added dropwise thereto, and chloromethylation reaction of styrene was performed on ice for 1 hour and at room temperature for 2 hours. After stopping the reaction by injecting methanol, it was washed with an organic solvent and distilled water, and heated and dried under vacuum.
While adding 3.0 parts by mass of the chloromethylated polystyrene particles obtained above, 100 parts by mass of dimethylacetamide, 14 parts by mass of polyethylene glycol 200, and 3.5 parts by mass of sodium methoxide, 80 ° C. For 6 hours to add polyethylene glycol 200 to the polystyrene chain. The particles after the reaction were washed several times with an organic solvent, and heated and vacuum dried.
To 1.0 part by mass of the polyethylene glycol-added polystyrene particles obtained above, 33 parts by mass of toluene, 1.0 part by mass of hydroxymethylmaleimide, and 0.30 part by mass of paratoluenesulfonic acid monohydrate were added. This was reacted for 7 hours at 125 ° C. with stirring to obtain maleimide group-containing polystyrene particles 1 (particles of the present invention).

(Example 2)
1. 45 parts by mass of styrene monomer, 7.5 parts by mass of divinylbenzene (55% purity product), 40 parts by mass of lauryl alcohol as a diluent, and dimethyl 2,2′-azobis (2-methylbutyronitrile) as a polymerization initiator 0 parts by mass was mixed and dissolved. The amount of diluent in the total oil is 45%. This mixture was subjected to suspension polymerization granulation under the same conditions as in Example 1, and the following steps were also performed in the same manner as in Example 1 to produce maleimide group-containing polystyrene particles 2 (particles of the present invention).

(Comparative Example 1)
Maleimide group-containing polystyrene particles 3 were produced in the same manner as in Example 1 except that no diluent was added.
(Comparative Example 2)
A mixture of 37.5 parts by mass of styrene monomer, 12.5 parts by mass of divinylbenzene (55% purity product), 25 parts by mass of diethylbenzene and 50 parts by mass of isoamyl alcohol as a diluent was prepared and dissolved. A polymerization initiator prepared by dissolving 0.25 parts by mass of potassium persulfate, 0.1 part by mass of NaH ₂ PO _{4 and} 2 parts by mass of sodium oleate in 200 parts by mass of water well excluding oxygen was prepared. The two liquids were mixed and emulsified with stirring at 6000 rpm for 3 minutes. Thereafter, polymerization was performed at 70 ° C. for 20 hours. After confirming that the particle diameter was 0.8 μm, it was washed with water and ethanol and then vacuum-dried at 100 ° C. for 12 hours.
Using the crosslinked porous polystyrene particles prepared above, the following operations were performed in the same manner as in Example 1 to prepare maleimide group-containing polystyrene particles 4.

(Comparative Example 3)
As a comparative sample, Amersham Biosciences Thiol Separose 4B (Agarose particles) was used.

<Measurement of volume average particle diameter>
The volume average particle diameter of the above particles was calculated by taking a photograph with an optical microscope and measuring the diameter of 100 particles. The obtained results are shown in Table 1.

<Measurement of pore diameter>
The diameters of the pores of the above particles were determined by the mercury intrusion method (Autopore III 9420 manufactured by MICROMERITICS) for the particles according to Examples 1 and 2 and Comparative Examples 2 and 3, and by the nitrogen adsorption method for the particles according to Comparative Example 1 ( Measured by AUTOSORB-1MP) manufactured by Cantachrome. The obtained results are shown in Table 1.

<Evaluation of affinity purification characteristics>
A 13-residue peptide Phe-Arg-Lys-Lys-Trp-Asn-Lys-Trp-Ala-Leu-Ser-Arg-Cys and antibody serum obtained by placing this in a rabbit body were obtained.
First, each particle was carefully dispersed in a phosphate buffer having a pH of 6.5. They were packed in a Biorad microspin column so that the bed volume was 0.5 ml. To this was added 1 ml of a phosphate buffer containing 100 μg of the above peptide, the upper cap was capped, and the reaction was carried out for 12 hours while shaking at room temperature to immobilize the peptide molecule as a ligand. The top and bottom caps of the column were removed and the liquid was drained.
At this time, the liquid did not escape from the lower part of the column packed with 0.8 μm particles of Comparative Example 2, and pressure was applied from the upper part with the rubber part of the dropper, but the liquid did not pass through the particle part.
5 ml of phosphate buffer was flowed from the upper part of each remaining column to wash away unreacted ligand molecules.
Next, 1 ml of antibody serum was poured from the top and allowed to pass through a column to cause affinity binding between the peptide immobilized on the particles and the antibody contained in the serum.
Again, 5 ml of phosphate buffer was flowed from the top of each column to wash away unbound serum contents.
Thereafter, 1 ml of pH 2.3 glycine hydrochloride buffer was poured from the top to elute the antibody, and the liquid that passed through the column was recovered.
For the samples of Example 1, Example 2 and Comparative Example 3, antibody serum addition, washing, and antibody elution were repeated, and the 20th antibody eluate was also collected.
Each solution collected above was diluted 100-fold with a phosphate buffer and poured onto an ELISA plate on which the 13-residue peptide molecules had been immobilized in advance. Any antibody in the poured solution should bind to the peptide on the plate. After washing the plate, the HRP-conjugated anti-rabbit antibody goat was developed as the secondary antibody, and the TMB solution was used as the chromogenic substrate. The absorbance at 450 nm was measured using a microplate reader (MTP-300 manufactured by Corona Electric Co., Ltd.). The measurement results are shown in Table 1.

  From Table 1, it can be seen that Examples 1 and 2 had high absorbance and excellent affinity purification ability, whereas Comparative Example 1 without pores had low absorbance and low affinity purification ability. Moreover, although the purification ability of the particles of Comparative Example 3 is excellent in the first elution, it is found that the performances of Examples 1 and 2 are not deteriorated after 20 times while they are lowered after 20 times. .

Claims (6)

  A maleimide group-containing porous crosslinked polystyrene particle having a volume average particle diameter of 1-1000 μm, a pore diameter not exceeding the diameter of the particle and 1-10000 nm.   The maleimide group-containing porous crosslinked polystyrene particle according to claim 1, wherein the maleimide group is bonded to the phenyl group of the polystyrene chain via a spacer molecule. The maleimide group-containing porous crosslinked polystyrene particle according to claim 2, which has a group represented by the following chemical formula (1).

(In chemical formula (1), n represents an integer of 1 or more.) A monomer mixture containing styrene, a crosslinking agent, a polymerization initiator, and a diluent that does not cause a polymerization reaction with the styrene and the crosslinking agent is dispersed in an aqueous medium to copolymerize the styrene and the crosslinking agent. To obtain diluent-containing crosslinked polystyrene particles,
Removing the diluent from the diluent-containing crosslinked polystyrene particles to obtain crosslinked polystyrene particles;
Introducing maleimide groups into the crosslinked polystyrene particles;
A method for producing maleimide group-containing porous crosslinked polystyrene particles having 5. The method for producing maleimide group-containing porous crosslinked polystyrene particles according to claim 4 , wherein the diluent is at least one selected from the group consisting of toluene, dodecane, diethylbenzene, isoamyl alcohol, lauryl alcohol, and hexadecane. The maleimide group-containing porous material according to claim 4 or 5 , wherein a content of the diluent in the monomer mixture is 30 to 80% by mass with respect to a total amount of the styrene, the crosslinking agent, and the diluent. A method for producing crosslinked polystyrene particles.