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WO2006036003A1 - Microparticule ayant, liee a celle-ci, une substance d’affinite biospecifique et utilisation de celle-ci - Google Patents

Microparticule ayant, liee a celle-ci, une substance d’affinite biospecifique et utilisation de celle-ci Download PDF

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Publication number
WO2006036003A1
WO2006036003A1 PCT/JP2005/018399 JP2005018399W WO2006036003A1 WO 2006036003 A1 WO2006036003 A1 WO 2006036003A1 JP 2005018399 W JP2005018399 W JP 2005018399W WO 2006036003 A1 WO2006036003 A1 WO 2006036003A1
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WIPO (PCT)
Prior art keywords
segment
polyethylene glycol
substance
fine particles
integer
Prior art date
Application number
PCT/JP2005/018399
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English (en)
Japanese (ja)
Inventor
Yukio Nagasaki
Tomoko Jomura
Yoshinori Katsuyama
Tadahito Takahashi
Kazunori Kataoka
Original Assignee
Yukio Nagasaki
Tomoko Jomura
Yoshinori Katsuyama
Tadahito Takahashi
Kazunori Kataoka
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Yukio Nagasaki, Tomoko Jomura, Yoshinori Katsuyama, Tadahito Takahashi, Kazunori Kataoka filed Critical Yukio Nagasaki
Priority to JP2006537856A priority Critical patent/JPWO2006036003A1/ja
Publication of WO2006036003A1 publication Critical patent/WO2006036003A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form

Definitions

  • Fine particles bound with a substance having biospecific affinity and use thereof Fine particles bound with a substance having biospecific affinity and use thereof
  • the present invention provides a pair of bio-specific affinities.
  • 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.
  • the present invention relates to use for detecting, separating and purifying members, and to the fine particles.
  • biological samples biological samples
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • impurities are often non-specifically adsorbed on the surface of the microsphere used as a solid phase carrier, resulting in poor separation efficiency.
  • Reference 4 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.
  • 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. .
  • BSA ushi serum albumin
  • 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.
  • biological samples other than the target that is, those of interest, proteins, lipids, inorganic salts, etc.
  • the particles may easily aggregate and settle. In some cases, the rate dropped.
  • 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.
  • 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.
  • 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
  • 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.
  • PE G polyethylene glycol
  • 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 is bound to the surface directly or via a spacer.
  • 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.
  • 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.
  • 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,
  • 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.
  • 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.
  • spacers may be, for example, ethylene dust as described in Table 1.
  • a member of a pair of substances having biospecific affinity is bound to a microparticle, in particular a covalent bond.
  • 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.
  • 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.
  • such fine particles have an average particle size of 0.02 ⁇ ! ⁇ 5 mm, preferably 0.0 5 ⁇ ! ⁇ 1 0 0 ⁇ .
  • 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.
  • 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.
  • 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.
  • 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):
  • n represents an integer of 1 ⁇ 1 0, n is 1: Represents an integer of L 0 0, and 1 E ⁇ Pi 1 2, independently of one another, also a hydrogen atom is properly carbon atoms Is an alkyl group of 1 to 5.
  • 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.
  • 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);
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • the polyamine segment has the following general formulas (1) and (2):
  • n represents an integer of 1 to 100
  • R 2 independently of each other, have a hydrogen atom or a carbon atom number of 1 to 5
  • 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.
  • 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.
  • a condensation dehydrating agent for example, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (ED C), N
  • 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.
  • 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.
  • the target substance can be isolated from the body.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • A PEG-modified magnetic particles
  • B BSA
  • C gelatin
  • Fig. 3 shows a comparison of recovery rates from cell extracts containing the same concentration of ⁇ -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.
  • APT ⁇ -fetoprotein
  • NX-PEG (1) Manufacture of an oligoamine-polyethylene dallic copolymer (hereinafter abbreviated as NX-PEG (1))
  • THF dehydrated tetrahydrofuran
  • an initiator Add lmmol (80.4 ⁇ L) 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.
  • 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.
  • EO distilled ethylene oxide
  • MeO-PEG-Methylbenzoate Abbreviated as MeO-PEG-Methylbenzoate.
  • 50 mL of Nasflask was charged with 6.2 mL (263 mmol) of the above MeO-PEG-Methylbenzoate lg (263 ⁇ mol) and pentaethylene hexamine (hereinafter abbreviated as PEHA) corresponding to 100 times the amount of PEG.
  • PEHA pentaethylene hexamine
  • 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).
  • IPA cooled isopropyl alcohol
  • N 5 -PEG MeO-PEG-Phenyl-N5 (hereinafter abbreviated as N 5 -PEG)) was obtained.
  • MeO-PEG-NH 2 manufactured by Nippon Oil & Fats Co., Ltd .: SUNBRIGHT MEPA-50H
  • lg 196 ⁇ mol
  • dimethyl succinate having two methinoesters in the molecule
  • 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.
  • 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.
  • MES 2-morpho Renoethane Norenophonic acid
  • 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, ⁇ ⁇ 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.
  • 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.
  • 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.

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Abstract

L’invention porte sur une microparticule ayant, lié à sa surface, un élément constitutif d’une substance possédant une paire d’affinités biospécifiques, où de plus un dérivé de polymère contenant un segment de glycol polyéthylène est directement lié à la surface. En outre, l’invention concerne l’utilisation de la microparticule dans la purification de substances bio-associées, etc. L’utilisation de la microparticule procure un procédé extrêmement efficace de purification de substances bio-associées, etc.
PCT/JP2005/018399 2004-09-28 2005-09-28 Microparticule ayant, liee a celle-ci, une substance d’affinite biospecifique et utilisation de celle-ci WO2006036003A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2006537856A JPWO2006036003A1 (ja) 2004-09-28 2005-09-28 生体特異的親和性を有する物質を結合した微粒子及びその使用

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JP2004329734 2004-09-28
JP2004-329734 2004-09-28

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Cited By (3)

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Publication number Priority date Publication date Assignee Title
US8962342B2 (en) 2007-06-06 2015-02-24 Beckton, Dickinson And Company Near-infrared dyes as surface enhanced raman scattering reporters
JP2016090570A (ja) * 2014-11-04 2016-05-23 三洋化成工業株式会社 磁性シリカ粒子を用いた対象物質の分離方法
CN112710826A (zh) * 2020-11-17 2021-04-27 北京九强生物技术股份有限公司 一种提高试剂稳定性的包被和封闭方法

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WO2002096977A1 (fr) * 2001-05-30 2002-12-05 Nanocarrier Co., Ltd. Procede de liaison d'une substance a incorporer a une terminaison polymere
JP2004177402A (ja) * 2002-11-12 2004-06-24 Matsushita Electric Ind Co Ltd 特異結合反応測定方法、それに用いる試薬キットおよび特異結合反応測定装置
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8962342B2 (en) 2007-06-06 2015-02-24 Beckton, Dickinson And Company Near-infrared dyes as surface enhanced raman scattering reporters
US9546957B2 (en) 2007-06-06 2017-01-17 Becton, Dickinson And Company Near-infrared dyes as surface enhanced raman scattering reporters
JP2016090570A (ja) * 2014-11-04 2016-05-23 三洋化成工業株式会社 磁性シリカ粒子を用いた対象物質の分離方法
CN112710826A (zh) * 2020-11-17 2021-04-27 北京九强生物技术股份有限公司 一种提高试剂稳定性的包被和封闭方法

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