Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
  • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
  • C07F9/06—Phosphorus compounds without P—C bonds
  • C07F9/08—Esters of oxyacids of phosphorus
  • C07F9/09—Esters of phosphoric acids
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 and B01D15/30 - B01D15/36, e.g. affinity, ligand exchange or chiral chromatography
  • B01D15/3804—Affinity chromatography
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
  • B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
  • B01J20/28004—Sorbent size or size distribution, e.g. particle size
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/30—Processes for preparing, regenerating, or reactivating
  • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
  • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
  • B01J20/3204—Inorganic carriers, supports or substrates
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/30—Processes for preparing, regenerating, or reactivating
  • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
  • B01J20/3214—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating
  • B01J20/3217—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond
  • B01J20/3219—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond involving a particular spacer or linking group, e.g. for attaching an active group
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/30—Processes for preparing, regenerating, or reactivating
  • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
  • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
  • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/30—Processes for preparing, regenerating, or reactivating
  • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
  • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
  • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
  • B01J20/3244—Non-macromolecular compounds
  • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
  • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/30—Processes for preparing, regenerating, or reactivating
  • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
  • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
  • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
  • B01J20/3244—Non-macromolecular compounds
  • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
  • B01J20/3257—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one of the heteroatoms nitrogen, oxygen or sulfur together with at least one silicon atom, these atoms not being part of the carrier as such
  • B01J20/3259—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one of the heteroatoms nitrogen, oxygen or sulfur together with at least one silicon atom, these atoms not being part of the carrier as such comprising at least two different types of heteroatoms selected from nitrogen, oxygen or sulfur with at least one silicon atom
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/30—Processes for preparing, regenerating, or reactivating
  • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
  • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
  • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
  • B01J20/3268—Macromolecular compounds
  • B01J20/3272—Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
  • B01J20/3274—Proteins, nucleic acids, polysaccharides, antibodies or antigens
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/30—Processes for preparing, regenerating, or reactivating
  • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
  • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
  • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
  • B01J20/3285—Coating or impregnation layers comprising different type of functional groups or interactions, e.g. different ligands in various parts of the sorbent, mixed mode, dual zone, bimodal, multimodal, ionic or hydrophobic, cationic or anionic, hydrophilic or hydrophobic
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
  • C07F9/06—Phosphorus compounds without P—C bonds
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
  • C07F9/06—Phosphorus compounds without P—C bonds
  • C07F9/08—Esters of oxyacids of phosphorus
  • C07F9/09—Esters of phosphoric acids
  • C07F9/091—Esters of phosphoric acids with hydroxyalkyl compounds with further substituents on alkyl
• • 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
  • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2220/00—Aspects relating to sorbent materials
  • B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
  • B01J2220/58—Use in a single column
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T428/00—Stock material or miscellaneous articles
  • Y10T428/29—Coated or structually defined flake, particle, cell, strand, strand portion, rod, filament, macroscopic fiber or mass thereof
  • Y10T428/2982—Particulate matter [e.g., sphere, flake, etc.]

Definitions

• the present invention relates to an affinity particle and an affinity separation method.
• the present invention also relates to an affinity particle utilizing inorganic particles and an affinity separation method capable of easily and accurately separating an objective substance. It is also very useful for various separation, purification, and detection methods such as immunoprecipitation and latex agglutination, which can detect target substances with high sensitivity and ease.
• affinity column particles carrying a ligand are used for separation and purification of a target substance (Patent Documents 1 and 2).
• the desired target substance is not selectively separated. That is, in addition to the target substance captured by the ligand, an undesired target substance is also adsorbed on the column.
• Non-Patent Document 1 agarose or the like is used as an affinity separation method for dispersing and separating affinity particles in a liquid sample (Non-Patent Document 1), but has the following problems.
• the desired target substance is not selectively separated. That is, in addition to the target substance captured by the ligand, an undesired target substance is also adsorbed on the affinity particles.
• Patent Document 1 Japanese Patent Publication No. Hei 8-26076
• Patent Document 2 Japanese Patent Publication No. 2002-511141
• Non-patent document l Bioconjugate Chem .; 2002; 13 (2); 163-166
• the present invention provides an epoch-making affinity separation method that can easily and accurately separate a target substance of interest from affinity particles using inexpensive inorganic particles. It is also very useful for various separation, purification and testing methods, including immunoprecipitation and latex agglutination, which can detect the target substance with high sensitivity and ease.
• the present invention provides an affinity particle characterized by having a phosphorylcholine group represented by the following formula (1) on the surface of an inorganic particle by a covalent bond.
• the present invention provides a reactive group or an adsorptive group that has a phosphorylcholine group represented by the following formula (1) in a covalent bond on the surface of an inorganic particle and can bind to a ligand having a specific affinity for a target substance Is provided on the surface of the inorganic particle by covalent bond or adsorption.
• the present invention provides a phosphorylcholine group represented by the following formula (1) in a covalent bond on the surface of an inorganic particle, and a ligand having a specific affinity for a target substance is provided on the surface of the inorganic particle.
• An object of the present invention is to provide an affinity particle having a covalent bond or an adsorption.
• the present invention provides one or two kinds of inorganic particles in which a group force including silica, titanium oxide, zinc white, alumina, oxidized iron, talc, myc, sericite, and gold colloid is also selected.
• the present invention provides the above-mentioned affinity particles, wherein the above-mentioned inorganic particles have an average particle diameter of 20 nm to 500 m and a specific gravity of 1. OgZcm 2 or more.
• the present invention provides the above-mentioned, wherein the ligand is an antibody, an antigen, an enzyme, a substrate, a receptor, a lectin, a peptide, a DNA, an RNA, an aptamer, a protein A, a protein G, avidin, a biotin, a chelating conjugate.
• the present invention provides the above-mentioned affinity particle, wherein the affinity particle is one or more selected ligands.
• the present invention provides (1) a first step of binding an arbitrary ligand to the affinity particle according to claim 1 or 2, and (2) a method of converting the affinity particle produced in the first step into an arbitrary ligand.
• Inorganic particles comprising: a second step of dispersing in a liquid sample containing a target substance selectively captured by the apparatus; and (3) a third step of recovering the target substance captured by the affinity particles.
• the present invention provides (1) a first step of dispersing the affinity particles according to claim 3 in a liquid sample containing a target substance selectively captured by an arbitrary ligand, (2) an affinity It is intended to provide a method for separating an objective substance by affinity using inorganic particles, which comprises a second step of recovering the objective substance captured by a particle.
• the affinity particles of the present invention are used for detecting an antibody or a protein by an immunoprecipitation method or a latex agglutination method, the recovery step (2) is unnecessary, and the dispersion state of the particles is changed by visual observation. Can be easily checked.
• the affinity particle of the present invention has a very high selectivity for separation because it captures only a certain target substance (a target substance desired to be separated) with a ligand and suppresses the adsorption of other substances to the particles. . And, because of its excellent dispersibility and extremely easy separation from a liquid sample, it is possible to easily and accurately separate a target substance by affinity particles using inexpensive inorganic powder particles.
• the method for separating a target substance of the present invention can efficiently and simply separate a target substance to be separated in a short time.
• substances since substances have the property of adsorbing to foreign substances, it is difficult to efficiently separate only the target substance with conventional affinity particles. Non-specific adsorption of this substance to affinity particles can be prevented very efficiently, and purification efficiency can be increased.
• the phosphorylcholine group has extremely high hydrophilicity, and also has a function of improving the dispersibility of the affinity particles in a liquid sample containing water.
• the affinity particles used in the present invention are composed of inorganic particles, they can be easily recovered by static or light centrifugation with a high specific gravity. Further, the particles can be packed in a column as a carrier, and used as an affinity column for recovering a target substance.
• FIG. 1 shows the capture of protein by affinity particles of the present invention and conventional affinity particles. It is a schematic diagram which shows the difference of selectivity !.
• FIG. 2 is a structural formula and an NMR spectrum of the compound obtained in Synthesis Example 1.
• FIG. 3 is a structural formula and an NMR spectrum of the compound prepared in Synthesis Example 2.
• FIG. 5 is a 31P-CPMAS spectrum of PC particles (A) produced in Reference Example 1.
• FIG. 6 is an FT-IR ⁇ vector of the PC particle (A) produced in Reference Example 1.
• FIG. 7 is a 13 C-CPMAS spectrum of PC particles (C) produced in Reference Example 3.
• FIG. 8 Evaluation of non-specific protein adsorption of PC particles (A), (B), and (C) produced in Reference Examples 1, 2, and 3.
• FIG. 9 is an evaluation of selectivity for anti-esialbumin antibody and anti-human hemoglobin using Af particles (A) performed in Example 1.
• FIG. 10 shows the evaluation of selectivity for anti-esialbumin antibody and anti-human hemoglobin using Af particles (B) performed in Example 2.
• FIG. 11 is an evaluation of selectivity for anti-esialbumin antibody and anti-human hemoglobin using Af particles (C) performed in Example 3.
• FIG. 12 shows the evaluation of selectivity for goat antiserum using Af particles (A) and (C) performed in Examples 1 and 3.
• FIG. 13 shows the evaluation of selectivity for anti-human hemoglobin in goat serum using Af particles (A) and (C) performed in Examples 1 and 3.
• FIG. 14 is an evaluation of selectivity for anti-diasialbumin antibody and anti-human hemoglobin using amino particles performed in Comparative Example 1.
• the inorganic particles constituting the affinity particles are not particularly limited.
• Inorganic particles generally mean 20 ⁇ ! It means an inorganic object of about 500 m.
• Specific particles include, for example, talc, kaolin, mica, sericite (sericite), muscovite, gold Mica, synthetic mica, mica, biotite, permikulite, magnesium carbonate, calcium carbonate, aluminum silicate, barium silicate, calcium silicate, magnesium silicate, strontium silicate, metal tungstate, magnesium , Silica, zeolite, barium sulfate, calcined calcium sulfate (baked gypsum), calcium phosphate, fluoroapatite, hydroxyapatite, ceramic powder, metal stone (for example, zinc myristate, calcium palmitate, aluminum stearate), boron nitride And inorganic particles such as cerium oxide, gold colloid and the like.
• Particularly preferred particles are silica, titanium oxide, zinc white, alumina, iron oxide, talc, myric, sericite, gold colloid, and the like. Further, nonporous inorganic particles are preferable to porous inorganic particles.
• the phosphorylcholine group of the above formula (1) and a reactive group or an adsorptive group capable of binding to a ligand are introduced into the particle surface by a covalent bond, particles having an amino group on the surface are preferable.
• affinity particles having an average particle diameter of the inorganic particles of 20 nm to 500 ⁇ m and a specific gravity of 1.OgZcm 2 or more are preferable.
• silica titanium oxide, zinc white, alumina, iron oxide, talc, myriki, sericite, gold colloid, and the like.
• the ligand can bind.
• amide, ester, urethane, ether, secondary amamine, urea bond, disulfide bond and the like are preferable.
• an amino group, a hydroxyl group, a carboxyl group, a thiol group, an aldehyde group, and the like, which are preferred by a reactive group capable of forming a ligand in these covalent bond forms are preferred.
• the adsorption form is preferably avidin-biotin, metal-chelate conjugate, or the like. Accordingly, avidin, biotin, chelate conjugates, and the like, in which an adsorbing group in which a ligand can be in these adsorbing forms are preferable.
• a ligand is a substance that specifically binds to a certain target substance, and includes various antibodies, antigens, enzymes, substrates, receptors, peptides, DNA, RNA, aptamers, proteins, and the like.
• K protein g avidin, biotin, chelate conjugate, various metal ions, and the like.
• various antibodies are IgG, IgM, IgA, IgD, IgE, IgY
• antigens are proteins, polysaccharides
• enzymes are daltathione S-transferases
• substrates are daltathione
• receptors are hormone receptors, cytokinin receptors, and chelating enzymes.
• the product is tri-triacetate, and the various metal ions are Ni 2+ , Co 2+ , Cu 2+ , Zn 2+ , and Fe 3+ .
• the phosphorylcholine group represented by the formula (1) has a covalent bond on the surface of the inorganic particle, and a reactive group or an adsorptive group capable of binding to a ligand having a specific affinity for a target substance is provided on the surface of the inorganic particle. Since it is the essence of the present invention that the inorganic particles exist directly on the surface of the inorganic particles by covalent bonding or adsorption, the production method is not limited, and the particles may be bonded by any method.
• this does not include an embodiment in which a polymer having a reactive group or an adsorbing group to which a phosphorylcholine group and a ligand can bind in advance and the particle surface is simply coated without chemical bonding is used. This is because the coated polymer may peel off or may be affected by the coated polymer.
• the affinity particles of the present invention can be produced by the following method and the like.
• Step 1 A reactive group or an adsorptive group capable of binding a phosphorylcholine group represented by the following formula (1) and a ligand is introduced into the particles.
• the reactive group or the adsorptive group is not limited, but includes an amino group, a hydroxyl group, a carboxyl group, and an aldehyde group.
• Step 2 The phosphorylcholine group represented by the formula (1) and the ligand are bound to the reactive group or the adsorptive group introduced into the particles.
• the chemical structure (spacer) existing between the phosphorylcholine group or the ligand and the reactive group or the adsorptive group is arbitrary.
• an arbitrary spacer may be an methylene chain, an oxyethylene chain, or an alkylene chain containing one or more amino groups.
• Step 1 Introduce an amino group to any particle by a known method or a method developed in the future. Amino groups are introduced directly on the particle surface. The amino group is a primary amine or a secondary amine.
• Step 2 An aldehyde or hydrate obtained by the oxidative cleavage reaction of glycerol phosphorylcholine is applied to a particle having an amino group to form a phosphorylcholine group directly on the particle surface by a reductive amination reaction. Is added.
• the phosphorylcholine group is directly added to the particle surface of the particles having an amino group by the amidation reaction of the carboxyl compound obtained by the oxidative cleavage reaction of glycerol phosphorylcholine.
• Known methods for introducing an amino group into the particles include the following. 1. Introduction of amino group by surface reaction in plasma treatment
• Amino groups are introduced into the particle surface by low-temperature plasma in a nitrogen gas atmosphere.
• the particles are contained in a plasma reaction vessel, the inside of the reaction vessel is evacuated by a vacuum pump, and nitrogen gas is introduced. Subsequently, an amino group can be introduced into the particle surface by glow discharge. It is also possible to mechanically granulate the plasma-treated inorganic material.
• the literature on plasma processing is shown below.
• Plasma aminofunctionalisation of PVDr microfiltration membranes comparison of the in plasma modifications with a grafting method using ESCA and an
• the surface of inorganic particles such as silanol-containing particles is treated with a surface modifier such as alkoxysilane, chlorosilane, and silazane having an amino group.
• silica particles are treated with 3-aminopropyltrimethoxysilane having a primary amino group to introduce an amino group.
• the silica is immersed in a water-2-propanol mixed solution, 3-aminopropyltrimethoxysilane is added, and the mixture is heated to 100 ° C and reacted for 6 hours. After cooling to room temperature, the silica is washed with methanol and dried to obtain particles having amino groups introduced directly to the silica surface.
• Preferred particles to be treated include particles of glass, alumina, talc, clay, myriki, asbestos, titanium oxide, zinc oxide, iron oxide and the like, in addition to silica.
• the particle surface is first treated with 1.3.5.7-tetramethylcyclotetrasiloxane, and the aminated surface is obtained by reacting the Si-H group introduced on the surface with a monomer having an amino group.
• a monomer having an amino group For example, put My power and 1.3.5.7-tetramethylcyclotetrasiloxane in a desiccator and degas with an aspirator. After reacting at 80 ° C for 16 hours, remove my strength and dry at 120 ° C. The obtained force is dispersed in ethanol, and allyluamine is added, followed by addition of an ethanol solution of chloroplatinic acid, and the mixture is stirred at 60 ° C. for 2 hours.
• aminated amyloid After completion of the reaction, filtration, washing with ethanol, and drying under reduced pressure are performed to obtain aminated amyloid.
• Various inorganic particles my strength, talc, kaolin, alumina, titanium oxide, zinc oxide, iron oxide, various inorganic pigments are preferably treated.
• an amine-based monomer can be used as the monomer used in this method.
• the amine-based monomer is not limited to arylamine, but may be any as long as it has an amino group and a polymerizable reactive site such as butyl or acrylic.
• the amino group may be protected with a butoxycarbol group, a benzyloxycarbol group, or the like.
• a monomer having a functional group into which an amino group can be more easily introduced may be used.
• Step 2 a method for introducing a phosphorylcholine group on the surface of the aminated particles will be described below.
• the particles are immersed in methanol, phosphatidyl glyceraldehyde is added, and left at room temperature for 6 hours. Then, sodium cyanoboronate is added at 0 ° C, and the mixture is heated and stirred overnight to add a phosphorylcholine group to the amino group. The particles are washed with methanol and dried to obtain particles having phosphorylcholine groups directly on the surface.
• a protic solvent such as water, ethanol, or 2-propanol other than methanol can be used, and the introduction rate when using methanol is high.
• particles having an amino group are prepared, and reductive amination with an aldehyde compound or a hydrate compound obtained by an oxidative cleavage reaction of glycerol phosphorylcholine is performed. Particles having phosphorylcholine groups directly added to the particle surface by the reaction can be produced.
• This method has a great advantage that the introduction ratio of phosphorylcholine groups is high and that the surface of various inorganic particles can be modified.
• the above-mentioned aldehyde-containing compound is a very simple step, which oxidatively cleaves a known glycerol phosphorylcholine by a known method.
• the bond is cleaved by oxidizing 1,2-diol using an oxidizing agent such as periodate, periodate, or bismuth triacid, thereby obtaining an aldehyde compound.
• the reaction is usually performed in water or an organic solvent containing water, and the reaction temperature is from 0 ° C. to room temperature.
• the aldehyde form may form a hydrate through an equilibrium reaction in water, but does not affect the subsequent reaction with amine.
• An example of a scheme for preparing a monofunctional aldehyde containing a phosphorylcholine group is shown below.
• the reductive amination reaction in which an aldehyde compound (or a hydrate compound) obtained by an oxidative cleavage reaction of glycerol phosphorylcholine is bonded to an amino group of particles is easily performed by stirring both components in a solvent. Can be performed. In this reaction, both are dissolved or dispersed in water or alcohol (the organic solvent of the third component may be mixed) to form an imine, which is then reduced with a reducing agent to obtain a secondary amine. It is.
• a mild reducing agent such as sodium cyanoboronate is preferable, but other reducing agents can be used as long as phosphorylcholine is stable.
• the reaction is usually carried out at 0 ° C to room temperature.
• amino group may be reacted with an arbitrary amount of the compound represented by the formula (2) by a conventional method, and the remaining amino group may be used as a reactive group to which a ligand can be bound or an adsorptive group.
• ⁇ integer from 1 to 12
• a compound of the formula (2) is reacted with chlorodithionium chloride in ⁇ , N'-dimethylformamide to form an acid chloride, and in a ⁇ , N'-dimethylformamide in amino.
• a phosphorylcholine group represented by the formula (1) can be introduced through an amide bond.
• the compound of the formula (2) can be synthesized according to the following scheme.
• the phosphorylcholine group is not bonded to all amino groups (the amount of reaction is adjusted), and the remaining amino group becomes a reactive group or an adsorbable group to which a ligand can be bonded.
• These particles are the affinity particles according to claim 2, and are particles in which a phosphorylcholine group represented by the formula (1) and a reactive group or an adsorptive group capable of binding a ligand are directly present on the surface of the inorganic particles. Then, the remaining amino groups are bonded with a ligand to form the affinity particles according to claim 3, wherein the phosphorylcholine group represented by the formula (1) and the ligand are directly present on the surface of the inorganic particles. Become.
• the affinity particle according to claim 2 is a product form in which a user can bind an arbitrary ligand according to a substance (target substance) to be captured.
• the affinity particle according to claim 3 is a product embodiment in which a ligand is bound in advance.
• the affinity particle according to claim 1 is an affinity particle in which at least the phosphorylcholine group of the formula (1) is present on the particle surface, and is not limited to the presence or absence of a ligand—a reactive group or an adsorptive group capable of binding the ligand. This is a product mode in which a user can bind an arbitrary ligand according to a substance (target substance) to be captured.
• the phosphorylcholine group of the formula (1) includes any aspect of the affinity particle, and includes, for example, the aspects of claims 2 and 3.
• the above reaction in order to leave an amino group as a reactive group or an adsorptive group to which a ligand can bind, 3-aminopropyltrimethoxysilane and 3-aminopropyltrimethoxysilane into which phosphorylcholine group has been introduced. It can be carried out by a method of competing with methoxysilane or by adjusting the reaction amount.
• the amino group may be reacted with a compound having an arbitrary functional group, and the functional group may be a reactive group or an adsorptive group to which a ligand can bind.
• a compound having an arbitrary functional group and the functional group may be a reactive group or an adsorptive group to which a ligand can bind.
• daltaraldehyde, alkyl diimidate, acyl azides, isocyanates and the like can be considered.
• the reaction amount of the surface modifier was adjusted to adjust the hydroxyl groups (OH) existing on the particle surface.
• OH hydroxyl groups
• the amino group on the inorganic particles is reacted with one aldehyde group of dartartaldehyde, and the other aldehyde group is reacted with the amino group in the protein, thereby binding the protein.
• hydroxyl groups existing on the particle surface do not require the introduction of a reactive or adsorbable group capable of binding to a ligand such as the above-mentioned amino group.
• a reactive or adsorbable group capable of binding to a ligand such as the above-mentioned amino group.
• Is used as it is to introduce a phosphorylcholine group and a ligand or a reactive group or an adsorptive group to which the ligand can bind.
• the affinity particles of the present invention are preferably produced by this method.
• a chemical bond is formed by dehydration from the hydroxyl group on the particle surface and Si—OMe of the compound of the following formula (3) or (4).
• This chemical reaction proceeds quantitatively very easily in most organic solvents by heating and refluxing.
• This dehydration reaction is preferable because a chemically and physically extremely stable phosphorylcholine group can be introduced.
• the phosphorylcholine group-containing compound represented by the following formula (3) or (4) is a novel compound.
• OMe may be OEt, CI.
• OEt CI bonded to Si, up to two may be a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, or an isobutyl group.
• a phosphorylcholine derivative represented by the following formula (5) is dissolved in distilled water.
• the phosphorylcholine derivative of the following formula (5) is a known compound and can be obtained as a commercial product.
• the above procedure can be performed in exactly the same manner even if m and n in the compound shown in the formula (3) or (4) are changed.
• the reaction solvent is not particularly limited, and water, an alcohol such as ethanol, propanol or butanol, or an aprotic solvent such as DMF or DMSO can be used in addition to the above-mentioned methanol.
• dehydrated methanol is preferred, although a dehydrated solvent is preferred.
• a phosphorylcholine derivative represented by the following formula (5) is dissolved in a mixture of distilled water and acetonitrile.
• the phosphorylcholine derivative of the following formula (5) is a known compound, and a commercially available product can be obtained.
• the above procedure can be performed in exactly the same manner even if m and n in the compound shown in the formula (4) are changed.
• the reaction solvent is not particularly limited, and aprotic solvents such as acetonitrile, tetrahydrofuran, and dimethyl sulfoxide can be used in addition to N, N, and 1-dimethylformamide described above. However, a dehydrated solvent is preferred in order to prevent polymerization during the reaction.
• the remaining hydroxyl group becomes a reactive group or an adsorbable group to which a ligand can be bound without binding a phosphorylcholine group to all hydroxyl groups (adjusting the reaction amount).
• These particles are the affinity particles according to claim 2, and are particles in which a phosphorylcholine group represented by the formula (1) and a reactive group or an adsorptive group capable of binding a ligand are directly present on the surface of the inorganic particles.
• the remaining hydroxyl group bonded with a ligand becomes the affinity particle according to claim 3, wherein the phosphorylcholine group represented by the formula (1) and the ligand are directly present on the surface of the inorganic particle. Become.
• the affinity particles according to claim 2 are used depending on the substance (target substance) to be captured.
• This is a product embodiment that allows a person to bind an arbitrary ligand.
• the affinity particle according to claim 3 is a product embodiment in which a ligand is bound in advance.
• the affinity particle according to claim 1 is an affinity particle in which at least the phosphorylcholine group of the formula (1) is present on the particle surface, and the presence or absence of a ligand-ligand-binding reactive group or an adsorption group. Not limited to this, it is a product form that allows the user to bind an arbitrary ligand depending on the protein (a certain target substance) to be captured. Further, as long as at least the phosphorylcholine group of the formula (1) is present on the particle surface, this also includes the affinity particles of! / And such embodiments, and includes, for example, the embodiments of claims 2 and 3. It is a thing.
• the hydroxyl groups on the particles are activated using cyanogen bromide.
• the amino group in the protein is reacted to bind the protein.
• the hydroxyl group may be reacted with a compound having an arbitrary functional group, and the functional group may be a reactive group to which a ligand can be bonded or an adsorptive group.
• Step 1 Carboxyl groups are introduced into any particle by a known method or a method developed in the future. Carboxyl groups are introduced directly on the particle surface.
• Step 2 A phosphorylcholine-containing compound represented by the following formula (2) is reacted with a carboxyl group-containing particle by an ordinary method to form an acid amide bond between the phosphorylcholine group and a reaction capable of binding the remaining carboxyl group to a ligand. It may be a group or an adsorptive group.
• Step 1 "How to introduce carboxyl groups on the particle surface”
• Known methods (Step 1) for introducing a carboxyl group into particles include the following.
• the surface of inorganic particles such as silanol-containing particles is treated with a surface modifier such as alkoxysilane, chlorosilane, and silazane having a carboxyl group.
• a surface modifier such as alkoxysilane, chlorosilane, and silazane having a carboxyl group.
• silica particles are treated with triethoxysilylpropyl succinic anhydride to introduce carboxyl groups.
• triethoxysilylpropyl succinic anhydride is dissolved in N, N-dimethylformamide, distilled water and 4-dimethylaminopyridine are added thereto, and the mixture is stirred at room temperature for 16 hours.
• a silane coupling agent having the indicated carboxylic acid is obtained.
• This reaction is a hydrolysis reaction of succinic anhydride with 4-dimethylaminopyridine.
• Silica particles are treated with a silane coupling agent having a carboxylic acid to introduce a carboxyl group.
• silica is immersed in a water-2-propanol mixture, a silane coupling agent having carboxylic acid is added, and the mixture is heated to 100 ° C. and reacted for 6 hours. After cooling to room temperature, the silica is washed with methanol and dried to obtain particles having carboxyl groups introduced directly onto the silica surface.
• Preferred particles to be treated include particles of glass, alumina, talc, clay, myriki, asbestos, titanium oxide, zinc oxide, iron oxide and the like, in addition to silica.
• the surface of the particles is first treated with 1.3.5.7-tetramethylcyclotetrasiloxane, and the Si-H group introduced on the surface is reacted with a monomer having a carboxyl group to obtain a carboxylated surface.
• Various inorganic particles my strength, talc, kaolin, alumina, titanium oxide, zinc oxide, iron oxide, various inorganic pigments are preferably treated.
• a carboxyl-based monomer can be used as the monomer used in the present method.
• the carboxyl-based monomer only needs to have a carboxyl group and a reactive site such as polymerizable butyl or acrylic.
• Step 2 “Method for Introducing Phosphorylcholine Group into Particles Having Carboxyl Group” Next, a method for introducing a phosphorylcholine group onto the surface of the carboxylated particles (Step 2) will be described below.
• This particle is an affinity particle according to claim 2, wherein the phosphorylcholine group represented by the formula (1) and a reactive group or an adsorptive group capable of binding a ligand are directly present on the surface of the inorganic particle. Become. Then, the ligand bound to a reactive group or an adsorptive group capable of binding to the ligand becomes the affinity particle according to claim 3, wherein the phosphorylcholine group represented by the formula (1) and the ligand are directly attached to the surface of the inorganic particle. It becomes particles that exist.
• the affinity particle according to claim 2 is a product form in which a user can bind an arbitrary ligand according to a substance (target substance) to be captured.
• the affinity particle according to claim 3 is a product embodiment in which a ligand is bound in advance.
• the affinity particle according to claim 1 is an affinity particle in which at least the phosphorylcholine group of the formula (1) is present on the particle surface, and the presence or absence of a ligand-ligand-binding reactive group or an adsorption group. Not limited to this, it is a product form that allows the user to bind any ligand according to the substance (target substance) to be captured. Further, as long as at least the phosphorylcholine group of the formula (1) is present on the particle surface, it also includes the affinity particles of such an embodiment, and for example, also includes the embodiments of claims 2 and 3.
• the carboxyl group may be left as a reactive group or an adsorbing group to which a ligand can be bound by adjusting the reaction amount of a silane coupling agent having a carboxylic acid into which a phosphorylcholine group is introduced. You can do it.
• the carboxyl group is reacted with a compound having an arbitrary functional group
• the functional group may be a reactive group or an adsorptive group to which a ligand can bind.
• the carboxyl groups on the inorganic particles are immersed in a solution of N-hydroxysuccinimide (NHS), 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide to activate the particle surface Esterify.
• NHS N-hydroxysuccinimide
• the amino group in the protein is reacted to bind the protein.
• the hydroxyl group may be reacted with a compound having an arbitrary functional group, and the functional group may be a reactive group to which a protein can be bound or an adsorptive group.
• the affinity separation method of the target substance of the present invention is performed.
• the method of the present invention is an epoch-making method for separating and purifying a target substance in that high-precision separation is easily performed using inorganic particles.
• the method of the present invention includes the following three steps.
• the first step has already been performed, and thus is omitted.
• a phosphorylcholine group represented by the formula (1) and a reactive group or an adsorbable group to which a ligand can be bonded are attached to the surface of the inorganic particle.
• the second step of dispersing the affinity particles produced in the first step in a liquid sample containing a target substance selectively captured by an arbitrary ligand For example, the affinity particles produced in the first step are dispersed in a liquid sample containing a target substance selectively captured by an arbitrary ligand, and gently shaken at 4 ° C for 30 minutes. Centrifuge at 500 Orpm for 5 minutes and discard the supernatant. For washing, mix 1 ml of PBS solution, shake gently, centrifuge at 5,000 rpm for 5 minutes, and discard the supernatant. This washing operation is repeated three times. 3. Separated affinity particle force The third step of recovering the captured target substance.
• FIG. 1 is a schematic diagram showing the difference in selectivity of capturing the target substance between the affinity particles of the present invention and the conventional affinity particles.
• the phosphorylcholine group introduced on the particle surface can be confirmed and quantified by FT-IR and elemental analysis.
• 1-a-Glyceline phosphorylcholine (6.29 g) was dissolved in 210 ml of distilled water and cooled in an ice-water bath. Sodium periodate (10.23 g) was added and stirred for 5 hours. The reaction solution was concentrated under reduced pressure and dried under reduced pressure, and the desired product was extracted with methanol. The structure is shown in the following compound (6).
• FIG. 2 shows the 1H NMR spectrum of the compound of the formula (6) in heavy water. Since the compound of formula (6) is in equilibrium with water in formula (9), the actual spectrum reflects both formula (6) and formula (9).
• FIG. 3 shows the 1H NMR spectrum of the compound of the formula (7) in heavy water.
• FIG. 5 shows the 13C-CPMAS spectrum and 13C-PSTMAS spectrum of the PC particles (A) of Reference Example 1.
• the PSTMAS spectrum is a technique for selectively obtaining the staples of free-moving molecular chains, and is widely used for analyzing modified chains on the particle surface.
• a spectrum at 54.2 ppm due to the carbon of the phosphorylcholine group is observed.
• phosphorylcholine groups could be introduced onto the surface of the carrier silica gel. From FIG. 5, a spectrum derived from carbon of the propyl group which is a spacer is observed at around 9 ppm and 23 ppm, and a spectrum derived from ethyl in phosphorylcholine is observed at around 60 ppm and 69 ppm. From the above, it can be seen that the structures of formulas (10) and (11) can be introduced into silica gel without being destroyed.
• FIG. 7 shows the FT-IR ⁇ vector of the PC particle (C) of Reference Example 3. An absorption characteristic of an amide bond could be observed around 1650 cm- 1 .
• the phosphorylcholine group used in Reference Example 1 was introduced, and the untreated silica gel particles (abbreviated as untreated particles) and the PC particles produced in Reference Examples 1, 2, and 3 (A), (B), (C ) Were added in an amount of 25 mg each, and 1 ml of distilled water was added, followed by sonication for 1 minute. The distilled water was removed by centrifugation, and albumin (100 ⁇ gZml) or lysozyme (100 ⁇ gZml) was added to lml and reacted at room temperature for 1 hour.
• Fig. 8 shows the results. It was a component of PC particles (A) treated with phosphorylcholine groups that adsorption of both albumin and lysozyme was significantly suppressed compared to untreated particles. PC particles (B) and (C) further suppressed the adsorption of both albumin and lysozyme compared to untreated particles or PC particles (A).
• FIG. 4 shows the P quantitative measurement of the Af particles (B) treated with the surface modifier of Synthesis Example 4 by the above procedure.
• the obtained amount of introduced PC was 3.3 molZg particles, and it was confirmed that the PC group was guided to the particle surface.
• Fig. 4 shows the quantitative determination of P of Af particles (C) treated with the surface modifier of Synthesis Example 4 in the above procedure. From this, the amount of PC introduced was 6.3 ⁇ molZg particles, and it was confirmed that PC groups were introduced on the particle surface.
• perialbumin 1 mg Zml
• human hemoglobin 1 mg Zml
• sodium trihydroborate 1 mg
• This perialbumin or human hemoglobin is the ligand.
• the affinity separation method according to claim 7 is described.
• 1 ml of ethanolamine hydrochloride (0.5 M, pH 7.1) and 10 mg of sodium trihydroborate were added, reacted at room temperature for 1 hour, and centrifuged with PBS. Purification (5000 g) was performed four times to obtain the affinity particles of claim 3.
• a selectivity test was performed using goat antiserum against human hemoglobin.
• dartaraldehyde is a reactive group or an adsorptive group to which a ligand can bind.
• a selectivity test was performed using goat antiserum mixed with anti-human hemoglobin.
• 25 ml of the Af particles (A) and (C) prepared in Example 3 were mixed with 1 ml of distilled water and subjected to ultrasonic treatment for 1 minute. Remove the distilled water by centrifugation, add lml of daltaraldehyde solution (8%) and lOmg of sodium cyanotrihydroborate for stabilization of Schiff base, react at room temperature for 5 hours, and centrifuge with MQ water (5000g) Washed 5 times.
• lml of human hemoglobin (lmgZml) and lOmg of sodium trihydroborate were mixed, reacted at room temperature for 1 day, and centrifuged and purified (5,000g) four times with PBS.
• This human hemoglobin is the ligand.
• This is the affinity separation method described in claim 7.
• lml of ethanolamine hydrochloride (0.5M, pH 7.1) and lOmg of sodium trihydroborate were added, reacted for 1 hour at room temperature, and centrifuged and purified with PBS. 5000g) was performed 4 times to obtain the affinity particles of claim 3.
• the supernatant fraction and the eluted fraction were flowed by SDS-PAGE, and the result of silver staining was shown in FIG. Af particles In both (A) and (C), the eluting fraction shows a heavy antibody heavy chain band and only a faint band in the others. I know that It was. Also the density of the band, antibody capture volume was about 10 to 20 8.
• Af particles (A) had an activity of 13.1 g and Af particles (C) had an activity of about 10.1 ⁇ g.
• the affinity particles of the present invention have very high selectivity because they capture only the target protein desired to be separated. It has excellent dispersibility, and it is extremely easy to separate the liquid sample force. Affinity particles using inexpensive inorganic particles make it easy and highly accurate. Since it is possible to separate the target substance every time, it is useful for biological related industries that require high precision separation of the target substance.

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