WO2006036003A1

WO2006036003A1 - Microparticle having, linked thereto, substance having biospecific affinity and use thereof

Info

Publication number: WO2006036003A1
Application number: PCT/JP2005/018399
Authority: WO
Inventors: Yukio Nagasaki; Tomoko Jomura; Yoshinori Katsuyama; Tadahito Takahashi; Kazunori Kataoka
Original assignee: Yukio Nagasaki; Tomoko Jomura; Yoshinori Katsuyama; Tadahito Takahashi; Kazunori Kataoka
Priority date: 2004-09-28
Filing date: 2005-09-28
Publication date: 2006-04-06
Also published as: JPWO2006036003A1

Abstract

Microparticle having, linked to its surface, one constituent member of a substance having a pair of biospecific affinities, wherein further a polymer derivative containing a polyethylene glycol segment is directly linked to the surface. Moreover, there is disclosed use of the microparticle in purification of bio-related substances, etc. By the use of the microparticle, there can be provided a highly efficient method of bio-related substance purification, etc.

Description

Fine particles bound with a substance having biospecific affinity and use thereof

Technical field

The present invention provides a pair of bio-specific affinities.

For example, a member of a substance having a substance, for example, an antibody, an antigen, a fine particle bound with a nucleic acid, etc., which is contained in a biological sample.

On the other hand, the present invention relates to use for detecting, separating and purifying members, and to the fine particles. Background art

Conventionally, specific target substances in samples such as blood, body fluids, crushed fluid obtained from biological tissues, extracts, cell-free protein synthesis solutions (hereinafter collectively referred to as “biological samples”) can be efficiently There are a wide variety of methods, means and approaches for detecting or separating and purifying in high yield.

As an example of a general approach, when using specific affinity for a separation carrier, for example, an antibody capable of specifically binding to a target drug, a sequence complementary to a nucleic acid having the target sequence When the target substance is a cell, the factor or the antibody that specifically binds to the cell or the ligand is immobilized on a water-insoluble solid phase carrier. The separated single unit is used. Usually, after such a separation carrier is packed in a column, it has an affinity for the antibody, ligand, etc. and can bind specifically. Is added to the column to bring the substance into contact with a separation carrier to form a complex of the substance with the antibody, ligand, etc., and washed with a buffer or the like as appropriate. Subsequently, the complex is dissociated and the substance is eluted and obtained, for example, with a buffer with high ionic strength, a surfactant solution, or a buffer with very high or low pH. Affinity chromatographies that contain are widely used. This affinity chromatography is very selective and effective, utilizing specific interactions between the target substance and the solid phase, eg, ligand. It is a purification method.

However, this method has a poor recovery rate and is very time consuming. In some cases, purified biomolecules may be denatured and lose their activity. There was a drawback of requiring the use of special facilities.

On the other hand, as an alternative method, the above-mentioned complex is formed on a microparticle carrier such as microsphere, and the separation carrier is recovered by centrifugation or the like, and if necessary, washed with a buffer or the like as appropriate. Later, if necessary, separation by affinity, for example, by placing the complex in a buffer with very high ionic strength or a buffer with very high or low pH. There is also a method of dissociating and eluting the target substance bound on the carrier.

Using such a fine particle carrier, the target substance, for example, a specific protein is separated and recovered, and is examined by electrophoresis, Western plot, etc. This method is called immunoprecipitation, and is a very useful means for detecting trace proteins from cell extracts. However, in this method, impurities are often non-specifically adsorbed on the surface of the microsphere used as a solid phase carrier, resulting in poor separation efficiency.

In recent years, detection methods and separation methods using such fine particle carriers have attracted attention for the purpose of eliminating the complexity of implementing conventional techniques related to detection and separation of biological materials and improving separation efficiency. Various fine particles have been proposed. For example, a microsphere described in Japanese Patent Application Laid-Open No. 10-1950 599 (or EP 787988A2, hereinafter referred to as Document 1), Japanese Patent Application Laid-Open No. 1 1 1 1 9 1 5 0 9 No. 1 (hereinafter referred to as Document 2) or Japanese Patent Application Laid-Open No. 11 1 1 9 1 5 10 (hereinafter referred to as Document 3), the present inventors, etc. Is bound to a latex particle at one end of a polyoxyethylene chain described in WO 2 0 4/0 5 6 8 9 5 A1 (hereinafter referred to as Reference 4) Examples thereof include latex particles that can carry ligand or the like on the other end of the chain.

Document 1 describes a microsphere in which a physicoactive substance is bound to a styrene-glycidyl methacrylate polymer via a spacer. It has been suggested that this microsphere has an advantage in that it can efficiently and specifically bind to a substance in a biological sample having affinity for the substance having the physiological activity. Reference 2 describes magnetic particles that consist of a magnetic material coated with a metal oxide that is different from the magnetic material, with nucleic acid adsorbed on the surface. Document 3 describes magnetic polymer particles in which particles made of a magnetic metal or metal compound are coated with a polymer, on which a specific antibody is adsorbed on the surface. Further, in Reference 3, such a magnetic particle is further added to a phosphate physiological saline containing 0.5% ushi serum albumin (BSA) and 0.1% polyethylene glycol. Also listed is a microparticle that has been shaken and dispersed in a buffer solution and then gently shaken at room temperature for 30 minutes to adsorb the antibody and block the particle surface with albumin. .

However, for example, the microparticles described in any one of References 1 to 3 are used from biological samples other than the target, that is, those of interest, proteins, lipids, inorganic salts, etc. In order to purify specific substances, it is possible to purify non-specific adsorption of coexisting molecules on the surface of fine particles along with specific reactions, and at the same time, the particles may easily aggregate and settle. In some cases, the rate dropped.

Disclosure

Fine particles obtained by blocking the surface of the particles in Reference 3 with albumin can suppress the nonspecific adsorption to some extent, and the particles described in Reference 4 further suppress the nonspecific adsorption. be able to. However, the fine particles described in Reference 3 can further suppress the non-specific adsorption, or the fine particles described in Reference 4 can be obtained by another preparation method. Or have a different structure, but the same or better than the fine particles Providing microparticles with specific adsorption characteristics will contribute to the advancement of this technical field.

For example, a ligand or an antibody described in Reference 1, Reference 2, or Reference 3 (also including polymer particles containing a magnetic substance described in the reference cited in Reference 3). In some cases, the fine particles bound through a spacer are made of polyethylene glycol (sometimes abbreviated as “PE G”) so that the chains can move freely in an aqueous medium. It was found that the above-mentioned non-specific adsorption can be significantly reduced when a polymer derivative containing a glycol segment is further bound to the surface of the fine particles. This is also advantageous in that it can be easily produced from commercially available fine particles.

Thus, the present invention

As a first aspect, a fine particle in which one member of a substance having a pair of bio-specific affinity is bound to the surface directly or via a spacer. In addition, a polymer derivative containing a polyethylene glycol segment is directly bonded to the surface, and the polyethylene glycol chain in the derivative can freely move in an aqueous medium. In a sample and an aqueous medium, the microparticles in the form contain the other member of the substance having the biospecific affinity, and the substance suspected of non-specifically binding to the microparticles. Contacting with, and

Selectively forming one member on the microparticle with the other member; A method of forming a specific binding pair between one member of the pair of substances having biospecific affinity and the other member;

Or

The microparticles contain the other member of the substance having biospecific affinity, and the microparticles in the sample suspected of having non-specific binding to the microparticles. Use for a carrier to form a selective bond between the other member and the one member on the microparticle,

I will provide a.

As another aspect of the present invention, there are also provided a method for producing specific fine particles of the fine particles by modifying conventional fine particles, and specific fine particles.

Detailed description of the invention

A substance having a pair of biospecific affinity referred to in the present invention is not limited, but is an antibody and an antigen (or hapten), a drug (or ligand) and its receptor (expressing a receptor). A single-stranded RNA or a polynucleotide with its complementary nucleotide arrangement IJ, an enzyme and its substrate, and a sugar and a lectin, etc. Can be mentioned. In the fine particles used in the present invention, a compound or a substance that is a member of any one of the pair forming between the substances is physically or chemically applied to the surface depending on the characteristics of the fine particle surface. Combined. Bonding may also be accomplished via a spacer. Such spacers may be, for example, ethylene dust as described in Table 1. Using a functional group of a compound that has been modified so that both ends have a functional group, such as cold diglycidyl ether, a member of a pair of substances having biospecific affinity is bound to a microparticle, in particular a covalent bond. It may be. The ethylene glycol can be an oligoethylene glycol such as di-, tri- or tetra-ethylene glycol, and the functional group is replaced by a glycidyl group, a hydroxy group, It can be an amino group, a carboxyl group or a sulfo group. When a high molecular weight molecule such as an antibody is bound to a microparticle, the antibody can be bound directly to the microparticle as long as the binding properties of the molecule to the antigen are not adversely affected.

Such fine particles are selected from the group consisting of the above-mentioned literature 1, literature 2, literature 3 and the known fine particles cited in them, and can be used for the purpose of the present invention. Any fine particles, if any, are included. Therefore, by citing, the matters described in Documents 1 to 3 are the contents of this specification. For the purpose of the present invention, such fine particles have an average particle size of 0.02 μη! ~ 5 mm, preferably 0.0 5 μπ! ~ 1 0 0 μπι.

According to the present invention, the fine particles may be directly bonded to a polymer-derived polymer containing a polyethylene glycol segment (physical, for example, ionic bond, hydrogen bond, hydrophobic bond, etc. Or by chemistry, in particular by covalent bonds, and is in a form in which the polyethylene glycol chains in the derivatives are free to move in aqueous media. In aqueous media, buffer solutions, biological samples such as blood, urine, body fluids, cell debris-containing buffers, These aqueous solutions, diluents, aqueous suspensions, and the like that meet the purpose of the present invention, such as cell extracts. Polyethylene glycol is a form in which the recall chain can move freely in such an aqueous medium. The term “polyethylene glycol chain that is not bonded to (or non-attached to) the fine particles is in the aqueous medium. W; preferably in a state in which the brush structure composed of a polyethylene glycol chain can substantially cover the surface of the fine particles that are not bonded to the members bonded to the fine particles. It means being in a state. It is well known in the art that a polyethylene glycol chain having a plurality of polyethylene glycol chains on its surface can have a brush structure in a k-type medium. It is used in such a well-known sense.

The polyethylene glycol derivative has a polyethylene glycol segment having a degree of polymerization of 2 to 10 O 0 0, preferably 4 0 to 5 0 0, and a polymer length on the surface of the fine particles. It has a residue or a molecular part that constitutes a site or region that can be bound so that the recall chain can be held or fixed in such a way as to freely move in an aqueous medium. Such a site or region is a segment made of origo or polyamine, and can be a block copolymer of polyethylene glycol and polyamine. The polyamine segment can be a polymer segment represented by the following general formulas (1) and (2):

General formula (1):

Wherein, m represents an integer of 1 ~ 1 0, n is 1: Represents an integer of L 0 0, and 1 E及Pi 1 _2, independently of one another, also a hydrogen atom is properly carbon atoms Is an alkyl group of 1 to 5.

General formula (2):

Where X + y is an integer from 1 to 30 and R ¹ ~! I ⁴ each independently represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms.

Fine particles (hereinafter also referred to as PEG-modified fine particles) having such a polyethylene dallic derivative bound to the surface, and particularly preferred are one member of a pair of substances having biospecific affinity directly. Alternatively, a polymer derivative containing a polyethylene glycol segment is bonded to the surface through a spacer and the polyethylene glycol chain moves in an aqueous medium. Latex particles that are directly bonded fine particles, and in which the fine particles are embedded in a magnetic material or in a surface layer A polymer having a carboxyl group on the latex surface, and a polymer derivative having a polyethylene glycol segment and a polyamine segment, which is a polyethylene glycol segment. Having a polymerization degree of 2 to 100 and a polyamine segment is represented by the general formula (2);

Where X + y is! ~ Is an integer up to 30, R ¹ ~! ⁴ may include fine particles, each of which is independently a hydrogen atom or an alkylene quinole having 1 to 5 carbon atoms.

For example, the PEG-modified microparticles are bound to the surface of one of a pair of substances having biospecific affinity, which are separated in an aqueous medium, directly or via a spacer. Contacting the polymerized microparticles with a polymer derivative containing a polyethylene glycolate segment in the aqueous medium, and binding the polymer derivative to the surface of the microparticles, and a pair of biospecific affinities One member of the substance having the above can be conveniently produced by a method of modifying the surface of the fine particles bonded to the surface directly or via a spacer. In this modification method, the fine particles are latex particles in a form in which a magnetic substance is embedded or in the form of a surface layer, or the magnetic substance is covered with silica or metal alkoxide. Inorganic particles in a covered form, preferably latex particles embedded in a magnetic material or in a form having a surface layer, and having a carboxyl group on the surface of the lattice. On the other hand, the polymer derivative is a copolymer having a polyethylene glycol segment and a polyamine segment, and the polyethylene glycol segment has a degree of polymerization of 2 to 100 0 0 0 And the polyamine segment has the following general formulas (1) and (2):

(1)

In the formula, m represents an integer of 1 to 10; n represents an integer of 1 to 100; and R ₂ , independently of each other, have a hydrogen atom or a carbon atom number of 1 to 5 An alkyl group of

(2)

In the formula, x + y is an integer from 1 to 30 and Ri to R4 each independently represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms. It is preferable to use a polyamine represented by the general formula (2). The step of bringing the fine particles into contact with the polymer derivative and the step of bonding the polymer derivative to the surface of the fine particles adversely affect the binding characteristics of one member to the other member in an aqueous medium. For example, the polymer derivative is merely subjected to physical bonds such as ionic bonds, hydrogen bonds, and hydrophobic bonds, or a combination of two or more of these at the temperature below room temperature. Or when a functional group such as a carboxyl group is present on the surface of the fine particles, a condensation dehydrating agent (for example, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (ED C), N

— Conditions for forming an amide bond between the functional group and, for example, an amino group present in a polymer derivative under mild conditions in the presence of hydroxysuccinimide (NHS, etc.) Can be done below.

The PEG-modified microparticles have specific affinity with one member on the microparticle, and include a specific partner or another member that forms a complex, and Samples suspected of having non-specifically binding substances to the microparticles, especially biological samples such as blood, urine, body fluids, cell lysate-containing buffers, cell extracts, etc. themselves, Alternatively, when they are contacted in a diluted or buffered aqueous medium, the one member on the microparticle can selectively form a bond with the other member, so in the sample. It can be used in a method of forming a specific binding pair with one member of a substance having a biospecific affinity of the pair. Such use can detect the other member present in the sample, It can be used for purification or to provide a method for screening substances that form the bond.

For example, in the case of purifying the substance from a sample containing the other member substance, after forming the above specific binding pair, the bound substance is centrifuged and the particles to be used are If it is a magnetic particle, it is collected by applying a magnetic field, washed with water or a suitable buffer solution, and non-specifically adsorbed impurities are washed away, and then the bound substance is ionic strength. A complex that forms the specific binding pair by treatment with a buffer solution with a high pH, a surfactant solution, or a buffer solution with a high or low pH, for example, incubation in the solution. The target substance can be isolated from the body.

In addition, for example, when screening for a drug that specifically binds to a specific receptor, receptor proteins obtained from animal cells, microbial cells, and their fragments expressing the receptor of interest bind. The microparticles are prepared and incubated with the microparticles and the compound or substance to be screened in a suitable buffer solution. Next, when a specifically bound complex is obtained by washing, the specifically bound compound or substance is identified from the complex. The compounds or substances thus identified are subjected to in vitro or in vivo tests using cells bearing the receptor or animals having the cells, which are originally ligands for the receptor. It is only necessary to evaluate the strength of the anti-agonist or antagonist of the target and determine whether it is the target compound or not. Thus, according to the present invention, a method for detecting or separating or purifying the other member present in the sample using PEG-modified microparticles or a substance that forms the bond is used. A screening method is provided.

In the sample, a method for forming a specific binding pair with one member of the pair of biospecific affinity substances bound on the microparticle is not limited. However, it can be carried out by incubating the PEG-modified microparticles added to the sample at room temperature for an appropriate time, usually 1 to 24 hours.

As described above, when the purification method or the like according to the present invention is carried out, a fine particle having a specific ligand bound to its surface, preferably, a magnetic particle is provided with a substance having the biospecific affinity from a sample. An operation of separating the target substance from the magnetic particles may be performed by removing the non-specifically bound impurities by performing selective separation and magnetic separation and washing. The PE G-modified fine particles have excellent dispersibility under physiological conditions, high ionic strength, and the property that they are difficult to settle even under contaminants such as proteins. The binding efficiency with molecules having affinity is extremely high, and the yield and purity of purification can be improved. The magnetic substance that imparts magnetism to the fine particles can be used of any kind or origin as long as it meets the purpose of the present invention, but is limited to a metal oxide having magnetic responsiveness. However, examples include metal oxides such as iron, cobalt, and nickel, and ferromagnetic iron oxides such as magnetite, maghemite, and zinc manganate ferrite are particularly preferred. It can be cited as a thing.

The PEG-modified microparticles consist of a PEG solution for modifying magnetic particles and an unmodified microparticle, an extraction buffer solution, a washing buffer solution, and an elution buffer solution. It can also be used.

Brief Description of Drawings

FIG. 1 is a photograph showing a comparison of dispersibility after cell extract treatment when PEG-modified magnetic particles (A), BSA (B), and gelatin (C) of the present invention are used.

FIG. 2 shows the results of immunoprecipitation performed using magnetic particles coated with PEG-modified magnetic particles (A), BSA (B), and gelatin (C) of the present invention. Each is a photograph showing the results of silver staining on the left and the Western plot on the right.

Fig. 3 shows a comparison of recovery rates from cell extracts containing the same concentration of α-fetoprotein (APT) when the magnetic particles of the present invention and BSA-coated magnetic particles are used. It is a photograph showing the result of the stamp mouth.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described more specifically based on specific examples. However, the present invention is not limited to these examples.

Production Example 1: (Manufacture of an oligoamine-polyethylene dallic copolymer (hereinafter abbreviated as NX-PEG (1)))

In an eggplant flask under an argon atmosphere, 20 mL of dehydrated tetrahydrofuran (hereinafter abbreviated as THF) as a solvent, as an initiator Add lmmol (80.4 μL) of 2-methoxyethanol, add 1 mmol of potassium naphthalene (2.86 mL of 0.35 mol / L THF solution), stir for 10 minutes with a magnetic stirrer, and alkoxide. To the initiator, 114 mmol (about 5.7 mL) of distilled ethylene oxide (hereinafter abbreviated as EO) was added with a cooled glass syringe and stirred at room temperature for 24 hours to perform ring opening polymerization of E0. After 5 hours, 5 mmol of methyl 4- (bromomethyl) benzoate (1.15 mg of lmol / L THF solution) was added to the reaction system as a terminator, and the polymerization reaction was stopped by stirring at room temperature for 2 hours. . The reaction product was dropped into 20 times the amount of jetyl ether, and unreacted materials and oligomers were removed by a reprecipitation method. Next, the precipitate was dissolved in an appropriate amount of chloroform and then extracted with saturated saline to remove salts in the system. After recovering the chromatoform phase and removing the solvent under reduced pressure, it is redissolved in benzene and then freeze-dried to give a polymer having a methyl ester at the end

Abbreviated as MeO-PEG-Methylbenzoate). Next, 50 mL of Nasflask was charged with 6.2 mL (263 mmol) of the above MeO-PEG-Methylbenzoate lg (263 μmol) and pentaethylene hexamine (hereinafter abbreviated as PEHA) corresponding to 100 times the amount of PEG. The mixture was stirred for 24 hours at 120 ° C in a hot water bath to introduce oligoamine at the PEG end. After dissolving the reaction solution in a small amount of methanol, it is dropped into cooled isopropyl alcohol (hereinafter abbreviated as IPA). Unreacted PEHA is removed by IPA reprecipitation method, and further unreacted material. Centrifuge the precipitate (below 4 ° C, (5,00 rpm, 20 minutes), redissolved in a small amount of methanol, and purified by IPA reprecipitation a total of 3 times. Finally, the polymer derivative is the target product by freeze-drying benzene.

(MeO-PEG-Phenyl-N5 (hereinafter abbreviated as N 5 -PEG)) was obtained.

Production Example 2: (Manufacturing of NX-PEG (2))

MeO-PEG-NH ₂ (manufactured by Nippon Oil & Fats Co., Ltd .: SUNBRIGHT MEPA-50H) lg (196 μmol) having a primary amine at one end and dimethyl succinate having two methinoesters in the molecule Add 12.8 mL (equivalent to 500 times the molar amount of PEG) to 50 mL Naslasco, stir in a hot water bath at 120 ° C for 24 hours, dissolve the reaction solution in a small amount of methanol, IPA reprecipitation was performed. The precipitate is collected by centrifugation (4 ° C or less, 5, 00 rpm, 20 minutes), dissolved in a small amount of methanol again, and purified by IPA reprecipitation three times in total, which is unreacted. Dimethyl succinate was completely removed. The precipitate was recovered by benzene freeze-drying to obtain a PEG derivative (MeO-PEG-NHC OCH ₂ CH ₂ COOCHs) at one end.

Add 500 mg (102 mol) of the PEG derivative and 11.9 mL of PEHA (500 times the molar amount of PEG) to 50 mL Nasflask containing dehydrated black mouth form, stir in a hot water bath at 120 ° C for 24 hours, Gomin was introduced. After a period of time, the reaction solution was dissolved in a small amount of methanol, IPA was reprecipitated, and the precipitate was collected by centrifugation (4 ° C or less, 5,000 rpm, 20 minutes). Re-dissolve in a small amount of methanol and repeat the purification by IPA reprecipitation a total of 3 times, The corresponding PEHA was completely removed. The precipitate was recovered by benzene lyophilization to obtain the desired product (hereinafter abbreviated as MeO-PEG-N5) in which an oligoamine at the PEG end was introduced.

(Method of PEG coating of magnetic particles and evaluation of dispersibility) Method for supporting antibodies on magnetic particles:

Magnetic particles with carboxyl groups on the surface (Magnetic material is coated with a resin based on polystyrene: solid concentration 10% / PBS dispersion; manufactured by JSR, Tokyo, Japan) 150 1 50 mM 2-morpho Renoethane Norenophonic acid (hereinafter abbreviated as MES) Noffer (pH 5.0) After dispersion in 1.0 ml, the magnetic particles were collected with a magnet and the supernatant was removed. This operation was repeated once more to wash the magnetic particles. Add 50 ml of 50 mM MES buffer (pH 5.0) to disperse the magnetic particles, let stand for 15 minutes at room temperature, and then remove the supernatant, and then add 50 mM MES buffer.ー 360 1 was added and the magnetic particles were dispersed again. Add 50 mg / ml N-hydroxysulfosuccinimid (S-NHS) 7.5 1 and stir, and then add 50 mg / ml 1-ethyl-3- (3-dimethylamino) carbodiimide 7.5 μ 1 was added and stirred. Shake it for 20 minutes, remove the supernatant, disperse by adding 714 μl of lOOmM MES buffer (pH 6.0), and add it to the anti-fetoprotein (AFP) mouse monochromator. The final antibody solution (36 μl) was added and stirred. The mixture was reacted at room temperature for 2 hours while rotating. After completion of the reaction, the magnetic particles on which the antibody was immobilized were washed twice with 1 ml of lOmM Phosphate Buffer (l50r M NaCl pH 7.5) (PBS) and dispersed in 1500 μl of PBS. PEG coating method:

After immobilization of the antibody, blocking of the extra active u-ruboxyl active site was performed using N5-PEG. Take 200 1 minutes of the magnetic particle dispersion (1%) with the antibody immobilized on it, remove the supernatant, and then use 200 μl of 1% N5-PEG in PBS! After stirring, the mixture was shaken at room temperature for 1 hour, and allowed to stand at 4 ° C. After washing twice with PBS to remove unbound polymer, 100 μl of PBS was added to disperse the magnetic particles.

Comparison of dispersibility:

In accordance with the PEG coating method, coating was performed using BSA and gelatin. Each of these magnetic particles coated with PEG® BSA and gelatin was compared in terms of dispersibility when treated with cell extracts. The magnetic particle dispersion 50 1 was placed in an Eppendorf tube, and after removing the supernatant, 細胞 μΙ of cell extract (NIH 3T3) was added, stirred, and shaken for 1 hour at room temperature to react. After removing the supernatant, the dispersibility after washing once with 20 mM Tris-HCl 150 mM NaCl (pH 8.0) and 0.05% Tween20 (TBST) was compared.

As shown in Fig. 1, it is clear that the dispersion stability in the aqueous medium is remarkably improved in the PEG-modified fine particles of the present invention as compared with the conventional treatment system using BSA or gelatin. It is.

(Immunoprecipitation of specific binding protein using biospecific affinity magnetic particles)

The following operations were performed using magnetic particles coated with PEG after antibody binding. For comparison, BSA and Gelatin-coated magnetic particles were used. Magnetic particle dispersion

Place 50 μl in an Eppendorf tube, remove the supernatant, add 100 μl of M IH3T3 cell extract supplemented with AFP to 100 IU / ml, disperse, and shake at room temperature. I did. After removing the supernatant, wash 3 times with TBST200 M 1 SDS'PA GE sample buffer

(Laemmli) 25 μl was added. After hand 3 minutes treatment in boiling water, the supernatant was separated by SDS-PAGE (poly Accession Riruami de 10 ^o / o gel). Further, the protein band was confirmed by Western staining with silver staining, and the activity of AFP was confirmed. The silver staining method was carried out by a conventional method after the silver staining kit purchased from Amersham. The Western plot method uses the TRANS-BLOT SD SEMI DRY

Using TRANSFER CELL, the protein was transferred from a gel after SDS-PAGE electrophoresis to a polyvinylidene difluoride (PVDF) membrane. Transcription was performed at a constant current of 1 mA per cm ^{2 for} 2 hours. Thereafter, the mixture was blocked in a 1% BSA / PBS solution for 30 minutes, and further anti-AFP rabbit antibody (1% BSA / PBS solution) diluted 5000 times was shaken at room temperature for 1 hour. After washing 3 times with TBST, Al force phosphatase-labeled anti-rabbit antibody (goat) diluted 5000 times with ¹⁰ BSA / PBS solution was added, and the mixture was allowed to stand at 4 ° C for 1 hour. After washing 3 times with TBST, 5-bromo-4-chloro "3-mdolyl-phosphate / Nitro blue

The reaction was carried out in a terazolium (B CIP / NBT) substrate solution.

As shown in FIG. 2, by using the PE 0-modified magnetic fine particles of the present invention, non-specific contamination compared to conventional BSA and gelatin. At the same time as the amount of product decreased, the protein recovery from the same concentration of cell extract was significantly improved, as shown in Figure 3.

Industrial applicability

As described above, for example, PEG-modified magnetic fine particles with fixed ligands do not contain non-specific contaminants, and have a high yield and a high yield compared to conventional magnetic particles. It can be used for separation and purification of high purity biological materials. Therefore, the present invention can be used in the medical industry.

Claims

The scope of the claims

1. Fine particles in which one member of a substance having a pair of bio-specific affinity is bound to the surface either directly or via a spacer. Furthermore, a polymer derivative containing a poly (ethylene glycol) segment is directly combined, and the polyethylene glycol chain in the derivative freely moves in an aqueous medium. A sample that contains the other member of the substance having a specific affinity for the substance and is suspected of the presence of a substance that can non-specifically bind to the fine particle. Contacting in a medium, and

Selectively bonding one member on the microparticle to the other member.

A method of forming a specific binding pair with a member of a pair of substances having a biospecific affinity and a member of the other.

2. Fine particles in which one member of a substance having a bio-specific affinity is bound to the surface directly or via a spacer, In addition, a polymer derivative containing a polyethylene glycol segment is directly bonded to the surface, and the polyethylene glycol chain in the derivative is in a form that can move in an aqueous medium. The other of the microparticles in the sample containing the other member of the biospecific affinity substance and suspected of the presence of a substance that can bind nonspecifically to the microparticle. Use for a carrier to form a selective bond between a member of said and said one member on a microparticle.

3. The average particle size of the fine particles is 0.0 2 μ! The method of claim 1, which is ˜5 mm.

4. The average particle size of the fine particles is 0.0 2 μη! Use according to claim 2, which is ~ 5 mm.

5. Selective formation of a bond can be accomplished by detecting, purifying, or purifying the other member of a pair of substances having a biospecific affinity in a sample. The method according to claim 1 or 3, wherein the method is intended for screening.

6. Selectively form a bond by detecting the other member of a pair of bio-specific substances in the sample, purifying, or forming the bond 5. Use according to claim 2 or 4, which is intended for screening.

7. The method according to any one of claims 1, 3 and 5, wherein the polyethylene glycol segment of the polyethylene glycol derivative has a degree of polymerization of 2 to 100.

8. The method according to any one of claims 2, 4 and 6, wherein the polyethylene glycol segment of the polyethylene glycol derivative has a degree of polymerization of 2 to 100.

9. The method according to any one of claims 1, 3, 5 and 7, wherein the poly (ethylene glycol) derivative is a block copolymer having a polyamine segment.

The use according to any one of claims 2, 4, 6 and 8, wherein the 10 · polyethylene glycol derivative is a block copolymer having a polyamine segment.

11. The method according to claim 9, wherein the polyamine segment is represented by the following general formulas (1) and (2):

(l)

In the formula, m represents an integer of 1 to: L 0, and n represents:! Represents an integer of ˜100, and R ₂ , independently of one another, is a hydrogen atom or an alkyl group having 1 to 5 carbon atoms;

(2)

In the formula, x + y is an integer from 1 to 30 and Ri to R4 each independently represents a hydrogen atom or an anolequinole group having 1 to 5 carbon atoms.

1 2. Use according to claim 10, wherein the polyamine segment is represented by the following general formulas (1) and (2):

(1) In the formula, m represents an integer of 1 to 10; n represents an integer of 1 to 100; and R ₂ , independently of each other, have a hydrogen atom or a carbon atom number of 1 to 5 An alkyl group of

(CH ₂ CH ₂ NR ¹ ) _X -R ²

(CH ₂ CH ₂ NR ³ ) _y -R ⁴

(2)

In the formula, x + y is an integer from 1 to 30 and Ri ~! 4 each independently represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms.

1 3. The method according to any one of claims 1, 3, 5, 7, 9 and 11 wherein the fine particles comprise a magnetic material.

1 4. Use according to any one of claims 2, 4, 6, 8, 10 and 12 wherein the microparticles comprise a magnetic material.

1 5. The fine particles are latex particles embedded in a magnetic material or in a surface layer, or inorganic particles in a magnetic material coated with silica or metal alkoxide. Term

The method according to any one of 1, 3, 5, 7, 9, 11 and 13.

1 6. The fine particles are latex particles in a form in which a magnetic substance is embedded or in a surface layer, or inorganic particles in a form in which the magnetic substance is coated with silica or metal alkoxide.

2, 4, 6, 8,

10. Use according to any one of 0, 12 and 14.

1 7. A fine particle in which one member of a pair of substances having biospecific affinity dispersed in an aqueous medium is bound to the surface directly or via a spacer. Contacting with a polymer derivative containing polyethylene glycol segment at

Binding the polymer derivative to the surface of the microparticles;

A method for modifying the surface of a microparticle in which one member of a pair of substances having a biospecific affinity is bound to the surface directly or via a spacer.

18. The fine particles are latex particles embedded in a magnetic material or in a surface layer, or inorganic particles in a magnetic material coated with silica or metal alkoxide, The polymer derivative is a copolymer having a polyethylene glycol segment and a polyamine segment, and the polyethylene glycol segment has a degree of polymerization of 2 to 100 0 0 force, and is a polymer. The amine segment has the following general formulas (1) and (2):

(1)

Where m represents an integer from 1 to 10 and n is:! Represents an integer of ˜100, and D and ₁₂ are each independently a hydrogen atom or an alkyl group having 1 to 5 carbon atoms;

(2)

In the formula, x + y is an integer from 1 to 30 and Ri to R 4 each independently represents a hydrogen atom or an alkylene quinole group having 1 to 5 carbon atoms. The reforming method according to claim 15, which is any one of the following.

19 9. The fine particles are latex particles embedded in a magnetic material or in a form having a surface layer, and a force lupoxyl group is present on the latex surface. The polymer derivative is a copolymer having a polyethylene glycol segment and a pore segment, wherein the polyethylene glycol segment has a degree of polymerization of 2 to; The reforming method according to claim 18, wherein the polyamin segment is represented by the general formula (2):

20 One member of a pair of biospecific affinities is bonded to the surface directly or via a spacer, and the polyethylene glycol chain moves in an aqueous medium. A fine particle in which a polymer derivative containing a polyethylene glycol segment in such a form is directly bonded to the surface,

The fine particles are latex particles embedded in a magnetic substance or in a form having a surface layer, a carboxyl group is present on the latex surface, and a polymer derivative is a polymer. A copolymer having a polyethylene glycol segment and a polyamine segment, wherein the polyethylene glycol segment has a degree of polymerization of 2 to 100 and a polyamin segment. Is the general formula (2); (CHsCHsNR ¹ ) ^ ²

N ヽ (CH ₂ CH ₂ NR ³ ) _y —R ⁴

(2)

Where X + y is:! Fine particles which are integers up to 30 and each of R ¹ to R 4 independently represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms and represented by: