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WO2012111686A1 - Procédé de fabrication de particules magnétiques liées à la streptavidine - Google Patents

Procédé de fabrication de particules magnétiques liées à la streptavidine Download PDF

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Publication number
WO2012111686A1
WO2012111686A1 PCT/JP2012/053464 JP2012053464W WO2012111686A1 WO 2012111686 A1 WO2012111686 A1 WO 2012111686A1 JP 2012053464 W JP2012053464 W JP 2012053464W WO 2012111686 A1 WO2012111686 A1 WO 2012111686A1
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Prior art keywords
streptavidin
magnetic particles
antibody
cross
linked
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English (en)
Japanese (ja)
Inventor
荒井　信之
泰弘 松岡
豪 永井
和樹 守田
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Minaris Medical Co Ltd
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Kyowa Medex Co Ltd
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Priority to JP2012557981A priority Critical patent/JPWO2012111686A1/ja
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    • 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
    • G01N2446/00—Magnetic particle immunoreagent carriers

Definitions

  • the present invention relates to a method for producing streptavidin-coupled magnetic particles and a method for producing protein-coupled magnetic particles.
  • magnetic particles are often used as a solid phase carrier to detect substances to be examined such as hormones, cancer markers, and infectious disease markers.
  • an antibody or an antigen (primary probe) or the like is bound on a magnetic particle, bound to a measurement target substance in a sample, and further labeled with a fluorescent substance, a chemiluminescent substrate, an enzyme, or the like. By binding to the next probe, the substance to be measured is detected qualitatively or quantitatively.
  • a method in which a primary probe and a secondary probe are reacted in a liquid phase and then bonded onto the magnetic particles is often used.
  • a biotin-labeled primary probe in which biotin is bound to a primary probe is reacted with a measurement target component in a sample and a secondary probe to form a biotin-labeled primary probe-measurement target component-secondary probe.
  • a complex is formed, then avidin-bound magnetic particles are allowed to act, and the complex is bound onto the magnetic particles by avidin-biotin interaction.
  • streptavidin-bonded magnetic particles using streptavidin having the same properties as avidin are more useful.
  • streptavidin binds very strongly to biotin and has the property of being more resistant to denaturation than avidin.
  • the isoelectric point is known to have less non-specific binding to other proteins because avidin is basic, whereas streptavidin is weakly acidic or neutral. Streptavidin-coupled magnetic particles using this streptavidin are used in many applications.
  • Patent Document 1 discloses a method for separating a substance to be detected in a specimen, which uses magnetic particles whose surface is modified with a temperature-responsive polymer, and has high temperature responsiveness even for magnetic particles having an average particle diameter of 50 to 1,000 nm.
  • a method is described in which magnetic particles are recovered from an aqueous solution by particle aggregation with molecules. While such particles have merit in the reaction due to the smaller magnetic particles, there are non-specific adsorption due to the particle surface being covered with temperature-responsive polymer, and under special conditions to aggregate There was a process to change.
  • Patent Document 2 describes a method of chemically forming a porous layer on the outer layer of magnetic particles. In this method, an immune reaction between an antigen and an antibody, or between DNAs or between DNA and RNA. In hybridization, the binding ability per surface area was improved, but the reaction efficiency in the pores was poor, and it was difficult to obtain the expected performance.
  • the present inventors prepared a cross-linked product of streptavidin by reacting glutaraldehyde and streptavidin, and obtained cross-linked product of streptavidin and magnetic particles.
  • the present inventors have found that streptavidin-coupled magnetic particles having a high biotin-binding ability can be produced by reacting with. That is, the present invention relates to the following [1] to [4].
  • a method for producing streptavidin-coupled magnetic particles comprising the following steps. (1) reacting glutaraldehyde and streptavidin to prepare a cross-linked product of streptavidin; and (2) A step of reacting the cross-linked streptavidin prepared in step (1) with magnetic particles. [2] The method for producing streptavidin-coupled magnetic particles according to [1], further comprising the following steps. (3) A step of reacting the streptavidin-bound magnetic particles prepared in step (2) with a reducing agent. [3] The method for producing streptavidin-coupled magnetic particles according to [1] or [2], wherein the streptavidin-coupled magnetic particles have a structure in which streptavidin is crosslinked on the magnetic particles. [4] A method for producing protein-bound magnetic particles, comprising reacting streptavidin-bound magnetic particles produced by the production method according to any one of [1] to [3] with a biotinylated protein.
  • the present invention provides a method for producing streptavidin-coupled magnetic particles having a high biotin-binding ability and a method for producing protein-coupled magnetic particles using the streptavidin-coupled magnetic particles.
  • the streptavidin-coupled magnetic particles and protein-coupled magnetic particles produced by the production method of the present invention are useful for clinical diagnosis.
  • FIG. 2 is a graph showing the change over time in the particle size of a cross-linked streptavidin after addition of a glutaraldehyde solution in Example 1.
  • FIG. The horizontal axis represents the reaction time (hr), and the vertical axis represents the particle size (nm) of the cross-linked streptavidin.
  • ⁇ shows the change over time when using a 0.005% solution of glutaraldehyde
  • ⁇ shows the change over time when using a 0.0075% solution of glutaraldehyde
  • ⁇ shows the change over time when using a 0.010% solution of glutaraldehyde
  • Lane 2 is an SDS-PAGE electrophoretic image showing the structure of streptavidin on a magnetic particle in a streptavidin crosslinked product and a streptavidin-coupled magnetic particle produced by the production method of the present invention.
  • Lane 1 is molecular weight marker
  • lane 2 is streptavidin
  • lane 3 is streptavidin crosslinked with a particle size of 13.6 nm
  • lane 4 is streptavidin crosslinked with a particle size of 29.2 nm
  • lane 5 is 63.9 nm in particle size streptavidin crosslinked
  • lane 8 biotin It represents streptavidin-bound magnetic particles with a binding capacity of 5.16 pmol / mm 2 .
  • Band A represents a monomer
  • band B represents a dim
  • the method for producing streptavidin-coupled magnetic particles of the present invention comprises a step of preparing a cross-linked product of streptavidin by reacting glutaraldehyde and streptavidin (primary reaction step), and A step (secondary reaction step) of reacting the cross-linked streptavidin prepared in the primary reaction step with magnetic particles.
  • the streptavidin-coupled magnetic particles produced by the production method of the present invention have a structure in which streptavidin is cross-linked on the magnetic particles. Streptavidin has a tetrameric structure, and monomers are linked by non-covalent bonds.
  • streptavidin-coupled magnetic particles obtained by the method for producing streptavidin-coupled magnetic particles of the present invention streptavidin having this tetrameric structure is covalently bonded via glutaraldehyde on the magnetic particles to form a crosslinked structure. ing. Streptavidin is bound to the amino group of the magnetic particle via glutaraldehyde.
  • a part of the tetrameric streptavidin is bound to the amino group of the magnetic particle via glutaraldehyde.
  • This streptavidin cross-linked structure is obtained by, for example, replacing streptavidin-bound magnetic particles with 1% SDS solution and treating them at 60 ° C. for 1 hour, thereby binding the streptavidin between subunits bound on the magnetic particles. It can be dissociated and confirmed by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), gel filtration HPLC or the like. SDS-PAGE is a method for separating proteins by electrophoresis depending on the size.
  • the SDS-PAGE is not particularly limited as long as it is a method capable of confirming the streptavidin cross-linked structure, and includes, for example, the method described in Bio-Experiment Illustrated 5 (Separate volume of Cell Engineering, Shujunsha).
  • the tetrameric structure of streptavidin is unwound by denaturation treatment in the presence of SDS. If streptavidin on the magnetic particles does not have a cross-linked structure, the degradation product obtained by the modification treatment in the presence of SDS is only a monomer derived from streptavidin. On the other hand, if streptavidin on the magnetic particles has a cross-linked structure, a dimer related to the streptavidin cross-linked structure in addition to the streptavidin-derived monomer by modification in the presence of SDS. , Trimers and higher order multimers will be obtained. Therefore, when a band derived from streptavidin-derived monomers, dimers, trimers, and higher-order multimers is observed by SDS-PAGE, the streptavidin cross-links on the magnetic particles. A structure is formed.
  • the streptavidin-coupled magnetic particles produced by the production method of the present invention has a structure in which streptavidin is cross-linked on the magnetic particles, and thus has a high biotin-binding ability.
  • Streptavidin coupled magnetic particles produced by the production method of the present invention biotin-binding capacity is normally 0.5 ⁇ 18 pmol / mm 2, preferably 1 ⁇ 17 pmol / mm 2, 2 ⁇ 16 pmol / mm 2 is Particularly preferred.
  • the streptavidin-coupled magnetic particles produced by the production method of the present invention also have an excellent property that the dispersibility of the streptavidin-coupled magnetic particles is good.
  • the dispersibility of the streptavidin-bonded magnetic particles can be evaluated by, for example, visually checking the state in the cuvette after the streptavidin-bonded magnetic particles stored in a cuvette are mixed by inversion. it can.
  • the biotin-binding ability per particle in the streptavidin-coupled magnetic particles of the present invention can be measured by any method that can measure biotin-binding ability. For example, a certain amount of fluorescently labeled biotin can be measured. After reacting with a certain amount of streptavidin-bound magnetic particles and collecting the streptavidin-bound magnetic particles with a magnet, a certain amount of supernatant is collected, the fluorescence of the collected supernatant is measured, and the measured value obtained is It can be calculated by comparing with a calibration curve prepared in advance showing the relationship between the fluorescence intensity and the biotin concentration.
  • streptavidin may be naturally derived or genetically modified, but is preferably genetically modified.
  • the magnetic particles to which the streptavidin is fixed are not particularly limited as long as they are magnetic particles that enable the production of the streptavidin-coupled magnetic particles of the present invention.
  • Examples include magnetic particles having a core / shell structure made of an organic polymer, magnetic particles having a structure in which a magnetic material is not uniformly dispersed in an organic polymer without including an outer layer, and clustered magnetic particles made of only a magnetic material.
  • Magnetic particles having functional groups such as amino group, carboxyl group, epoxy group, and tosyl group on the surface can also be used.
  • the magnetic substance contained in the magnetic particles is preferably a superparamagnetic fine magnetic particle with little residual magnetization, for example, triiron tetroxide (Fe 3 O 4 ), ⁇ -heavy sesquioxide ( ⁇ -Fe 2 O 3 ). And various metals such as ferrite, iron, manganese, cobalt, and chromium, or alloys of these metals.
  • the content of the magnetic substance in the magnetic particles composed of the organic polymer and the magnetic substance is preferably 10% by weight or more, more preferably 30 to 60% by weight, based on the total weight of the magnetic particles.
  • the shape of the magnetic particle examples include a spherical shape and a needle shape, and a spherical shape is preferable.
  • the particle diameter of the magnetic particles is, for example, 0.1 to 5 ⁇ m, and preferably 0.5 to 3 ⁇ m.
  • Specific examples of magnetic particles include amino group type Estapor magnetic particles (Merck), hydrophobic type Estapor magnetic particles (Merck), and the like.
  • the primary reaction step is a step in which glutaraldehyde and streptavidin are reacted to prepare a streptavidin cross-linked product.
  • streptavidin has a tetramer structure, and monomers are linked by non-covalent bonds.
  • the streptavidin cross-linked body is a structure in which streptavidin having a tetrameric structure is covalently bonded to each other via glutaraldehyde.
  • the reaction between glutaraldehyde and streptavidin can be used under any conditions that can prepare a cross-linked product of streptavidin.
  • streptavidin is obtained by dissolving streptavidin in a dispersion or the like in an aqueous solution of streptavidin.
  • a cross-linked streptavidin can be prepared by adding glutaraldehyde and reacting streptavidin with glutaraldehyde.
  • the dispersion include an aqueous solution containing a surfactant.
  • the pH of the dispersion is usually pH 4.5-7, preferably pH 5-6.
  • Examples of the aqueous medium used for the aqueous solution include distilled water, purified water, and a buffer solution.
  • a buffering agent corresponding to the set pH.
  • the buffer used in the buffer include an acetate buffer, a citrate buffer, a succinate buffer, a phosphate buffer, and Good's buffer.
  • Good buffering agents include, for example, 2-morpholinoethanesulfonic acid (MES), bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris), piperazine-N, N′-bis (2-ethanesulfone) Acid) (PIPES), N- (2-acetamido) -2-aminoethanesulfonic acid (ACES), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO), N, N-bis (2-hydroxyethyl)- 2-aminoethanesulfonic acid (BES), 3-morpholinopropanesulfonic acid (MOPS), N- [tris (hydroxymethyl) methyl] -2-aminoethanesulfonic acid (TES), 2- [4- (2-hydroxy Ethyl) -1-piperazinyl] ethanesulfonic acid (HEPES), 3- [N, N-bis (2-hydroxyethyl) amino] -2
  • the surfactant is not particularly limited as long as it can disperse magnetic particles, and examples thereof include an anionic surfactant, a cationic surfactant, an amphoteric surfactant, and a nonionic surfactant.
  • the concentration of the surfactant in the dispersion is not particularly limited as long as it is a concentration capable of dispersing the magnetic particles, and is, for example, 0.01 to 5.0%.
  • the reaction temperature in the reaction of glutaraldehyde and streptavidin is usually 0 to 40 ° C, preferably 20.0 to 37.5 ° C, particularly preferably 25 ° C.
  • the reaction time is usually 0.5 to 72 hours, preferably 6 to 48 hours, and particularly preferably 18 to 36 hours.
  • the amount of glutaraldehyde is not particularly limited as long as it can prepare a cross-linked streptavidin by reaction with streptavidin.
  • the molar concentration ratio is 0.5 to 5, and 1 to 3 is particularly preferable.
  • the concentration of glutaraldehyde is usually 0.001 to 0.05%, preferably 0.003 to 0.02%, particularly preferably 0.005 to 0.015%.
  • the concentration of streptavidin is usually 2 to 40 mg / mL, preferably 5 to 25 mg / mL, and particularly preferably 10 to 15 mg / mL.
  • the degree of formation of the streptavidin crosslinked product can be monitored by measuring the particle size of the streptavidin crosslinked product.
  • the particle size of the streptavidin crosslinked product can be measured, for example, with a particle size distribution meter.
  • the reaction solution is sampled at regular intervals, and the particle size of the streptavidin crosslinked product can be measured.
  • the secondary reaction step may be performed before the particle size of the streptavidin cross-linked product becomes constant in the primary reaction, or after it becomes constant or at a stage where the degree of change in particle size becomes moderate.
  • the particle size of the cross-linked streptavidin is usually 10 to 300 nm, preferably 20 to 250 nm, particularly preferably 50 to 200 nm.
  • Streptavidin may be naturally derived or genetically modified, preferably genetically modified.
  • the secondary reaction step is a step of binding the cross-linked streptavidin obtained in the primary reaction step to magnetic particles by covalent bonding or physical adsorption.
  • the streptavidin cross-linked product used for the reaction with the magnetic particles may be the reaction mixture itself in the primary reaction step or may be isolated from the reaction mixture in the primary reaction step. The reaction mixture itself in the next reaction step is preferred.
  • a streptavidin cross-linked product is reacted with a magnetic particle having a functional group on the surface in the above-mentioned dispersion to thereby react the streptavidin.
  • Avidin crosslinked can be bonded to magnetic particles.
  • a cross-linked streptavidin and magnetic particles having a functional group on the surface may be reacted in the above dispersion.
  • the functional group include the functional groups described above.
  • the magnetic particles having a functional group include amino group type Estapor magnetic particles (manufactured by Merck).
  • the linker is not particularly limited as long as it is bonded to both the streptavidin cross-linked product and the magnetic particle having a functional group on the surface, and the streptavidin cross-linked product is bonded to the magnetic particle.
  • glutaraldehyde examples thereof include bifunctional crosslinking agents such as imide esters, N-hydroxysuccinimide esters, and compounds having a maleimide group and an N-hydroxysuccinimide active ester in the molecule.
  • the reaction between the streptavidin cross-linked product and the magnetic particles having a functional group on the surface may be performed under any reaction conditions as long as the streptavidin cross-linked product can bind to the magnetic particles.
  • Streptavidin used in the primary reaction step is usually 10-30 mg and preferably 50-100 mg for the magnetic particles.
  • the linker is usually 1 to 20 times mol and preferably 5 to 10 mol (w / w) times mol of the functional group on the magnetic particle surface.
  • the streptavidin cross-linked product is bonded to the magnetic particles by physical adsorption
  • the streptavidin cross-linked product is reacted with the magnetic particles in, for example, the above-mentioned dispersion to convert the streptavidin cross-linked product to the magnetic particles.
  • the magnetic particles used in this reaction include the above-described magnetic particles, and hydrophobic type Estapor magnetic particles (manufactured by Merck) can be used.
  • the reaction between the cross-linked streptavidin and the magnetic particles may be under any conditions as long as the cross-linked streptavidin is capable of binding to the magnetic particles.
  • the reaction temperature for the reaction between the streptavidin crosslinked product and the magnetic particles is usually 15 to 50 ° C, preferably 20 to 40 ° C, and particularly preferably 35 ° C.
  • the reaction time is usually 30 minutes to 6 hours, preferably 1 to 2 hours.
  • the reaction mixture itself obtained in the secondary reaction step can also be used as streptavidin-coupled magnetic particles, but the magnetic particles in the reaction mixture obtained in the secondary reaction step are collected by a magnet, and other than the magnetic particles Magnetic particles obtained by removing the solution and then washing with a washing solution can also be used as streptavidin-coupled magnetic particles.
  • the cleaning liquid is not particularly limited as long as it is a cleaning liquid capable of cleaning substances other than the streptavidin-coupled magnetic particles obtained according to the present invention, and examples thereof include the aforementioned aqueous medium.
  • An aqueous medium containing protein and preservative can also be used as a cleaning liquid.
  • the protein include bovine serum albumin (BSA).
  • Examples of the preservative include sodium azide.
  • the washed magnetic particles can be suspended and stored in a storage solution.
  • the storage solution is not particularly limited as long as it can stably store the streptavidin-bound magnetic particles obtained by the present invention.
  • BSA bovine serum albumin
  • the method for producing streptavidin-coupled magnetic particles of the present invention may further include a reduction reaction step after the secondary reaction step.
  • the reduction reaction step is a reaction step between the streptavidin-coupled magnetic particles generated in the secondary reaction step and the reducing agent.
  • streptavidin having a cross-linked structure is formed on the magnetic particles. Since the streptavidin having the cross-linked structure contains a Schiff base (imine), the Schiff base (imine) is reduced by a reducing agent. By doing so, a more stable crosslinked structure can be obtained.
  • the reaction mixture itself in the secondary reaction step or the washed magnetic particles may be used.
  • the solvent used for the reaction between the streptavidin-bonded magnetic particles and the reducing agent is not particularly limited as long as it is a solvent capable of allowing the reduction reaction to proceed, and examples thereof include the above-described dispersion liquid.
  • a dispersion containing an organic solvent can also be used as a solvent in the reduction reaction.
  • the organic solvent is not particularly limited as long as it is soluble in water and can cause a reduction reaction, and examples thereof include methanol, ethanol, and tetrahydrofuran.
  • the reducing agent is not particularly limited as long as it is a reducing agent that can reduce the Schiff base (imine) and maintain a crosslinked structure, and examples thereof include a borane-based reducing agent.
  • examples of the borane reducing agent include 2-picoline borane and sodium borohydride.
  • the amount of the reducing agent added is usually 0.0001 to 0.1 (w / w)% of the magnetic particles, preferably 0.0005 to 0.05 (w / w)%, and particularly preferably 0.001 (w / w)%.
  • the reaction temperature of the reduction reaction is usually 30 to 50 ° C, preferably 35 to 45 ° C, particularly preferably 40 ° C.
  • the reaction time for the reduction reaction is usually 2 days to 10 days, preferably 5 days to 8 days, and particularly preferably 6 days.
  • the magnetic particles can be separated from solution components other than the magnetic particles by a magnet.
  • the separated magnetic particles are washed with a washing solution, and the washed magnetic particles can be stored in a state of being suspended in a storage solution.
  • the cleaning liquid is not particularly limited as long as it is a cleaning liquid capable of cleaning the solution components in the separated magnetic particles, and examples thereof include the above-described cleaning liquid.
  • the storage solution is not particularly limited as long as it is a solution that can stably store the obtained streptavidin-coupled magnetic particles, and examples thereof include the storage solution described above.
  • Protein-bound magnetic particles can be produced by reacting the streptavidin-coupled magnetic particles produced by the production method of the present invention with a biotinylated protein.
  • the protein binds to the magnetic particle by the interaction between streptavidin on the magnetic particle and biotin that binds to the protein.
  • the reaction between the streptavidin-bound magnetic particles and the biotinylated protein may be performed under any conditions as long as the protein binds on the magnetic particles.
  • the reaction temperature is usually 25 to 50 ° C, preferably 30 to 40 ° C.
  • the reaction time is usually 30 minutes to 24 hours, preferably 2 to 18 hours.
  • Examples of the protein include an antibody that binds to the measurement target component, and a competitive substance that competes with the measurement target component in the antigen-antibody reaction.
  • Examples of the competitive substance include a measurement target component and a substance containing an epitope recognized by an antibody that binds to the measurement target component.
  • proteins include IgG, anti-IgG antibody, IgM, anti-IgM antibody, IgA, anti-IgA antibody, IgE, anti-IgE antibody, apoprotein AI, anti-apoprotein AI antibody, apoprotein AII, anti-apoprotein AII antibody Apoprotein B, anti-apoprotein B antibody, apoprotein E, anti-apoprotein E antibody, rheumatoid factor, anti-rheumatic factor antibody, D-dimer, anti-D-dimer antibody, oxidized LDL, antioxidant LDL antibody, glycated LDL, Anti-glycated LDL antibody, glycoalbumin, anti-glycoalbumin antibody, triiodothyronine (T3), anti-T3 antibody, total thyroxine (T4), anti-T4 antibody, drug (anti-tencan drug, etc.), antibody binding to drug, C -Reactive protein (CRP), anti-CRP antibody, cytokines, antibodies that bind to cytok
  • biotinylated hydrocarbon compounds and biotinylated nucleic acids can also be used.
  • Hydrocarbon compound-bonded magnetic particles can be produced by reacting the streptavidin-bonded magnetic particles of the present invention with a biotinylated hydrocarbon compound.
  • nucleic acid-binding magnetic particles can be produced by reacting the streptavidin-binding magnetic particles of the present invention with biotinylated nucleic acids.
  • hydrocarbon compounds in biotinylated hydrocarbon compounds include mold toxins [deoxynivalenol (DON), nivalenol (NIV), T-2 toxin (T2), etc.], endocrine disruptors [bisphenol A, nonylphenol, Dibutyl phthalate, polychlorinated biphenyls (PCBs), dioxins, p, p'-dichlorodiphenyltrichloroethane, tributyltin, etc.], steroid hormones (aldosterone, testosterone, etc.) and the like.
  • mold toxins deoxynivalenol (DON), nivalenol (NIV), T-2 toxin (T2), etc.
  • endocrine disruptors bisphenol A, nonylphenol, Dibutyl phthalate, polychlorinated biphenyls (PCBs), dioxins, p, p'-dichlorodiphenylt
  • nucleic acid in the biotinylated nucleic acid examples include DNA, RNA, aptamer, and derivatives thereof.
  • the component to be measured in a sample can be measured using the streptavidin-coupled magnetic particles and protein-coupled magnetic particles obtained by the production method of the present invention. Furthermore, the measurement target component in the sample can be measured using the streptavidin-binding magnetic particles obtained by the production method of the present invention and the biotinylated protein.
  • an immunological measurement method using ordinary magnetic particles can be used, and examples thereof include a sandwich method and a competition method.
  • the sample is not particularly limited as long as it enables a method for measuring a component to be measured using streptavidin-coupled magnetic particles and protein-coupled magnetic particles obtained by the production method of the present invention.
  • whole blood, plasma, Serum, cerebrospinal fluid, saliva, amniotic fluid, urine, sweat, pancreatic juice and the like can be mentioned, and plasma, serum and the like are preferable.
  • the component to be measured is not particularly limited as long as it can be measured by a measurement method using streptavidin-coupled magnetic particles and protein-coupled magnetic particles obtained by the production method of the present invention, and examples thereof include the following substances. . IgG, IgM, IgA, IgE, apoprotein AI, apoprotein AII, apoprotein B, apoprotein E, rheumatoid factor, D-dimer, oxidized LDL, glycated LDL, glycoalbumin, triiodothyronine (T3), total thyroxine (T4), drugs (anti-tencan, etc.), C-reactive protein (CRP), cytokines, ⁇ -fetoprotein (AFP), carcinoembryonic antigen (CEA), CA19-9, CA15-3, CA-125 , PIVKA-II, parathyroid hormone (PTH), human chorionic gonadotropin (hCG), thyroid stimulating hormone (TSH), insulin, C-peptide,
  • streptavidin-bound magnetic particles obtained by the production method of the present invention and biotinylated hydrocarbon-based compounds can be used to measure components to be measured in a sample.
  • components to be measured include a hydrocarbon compound constituting a biotinylated hydrocarbon compound, an antibody that binds to the hydrocarbon compound, and the like.
  • hydrocarbon compound include the aforementioned hydrocarbon compounds.
  • the measurement of a measurement target component in a sample using a biotinylated hydrocarbon compound can be performed using a normal immunological measurement method such as a sandwich method or a competitive method.
  • nucleic acid-bound magnetic particles produced using streptavidin-bound magnetic particles obtained by the production method of the present invention and biotinylated nucleic acid or in place of protein-bound magnetic particles
  • the component to be measured in the sample can be measured using the streptavidin-coupled magnetic particles and biotinylated nucleic acid obtained by the production method of the present invention.
  • components to be measured include nucleic acids and proteins that bind to nucleic acids constituting biotin nucleic acids.
  • the protein include the aforementioned proteins.
  • Measurement of a measurement target component in a sample using a biotinylated nucleic acid can be performed using a normal nucleic acid measurement method or a normal immunological measurement method.
  • the prepared solution was transferred to a plastic disposable and dispersed in 0.1% BSA / PBS to a concentration of 0.05 mg / mL.
  • a streptavidin crosslinked product is formed by the reaction of streptavidin and glutaraldehyde, and the particle size of the crosslinked product increases.
  • the particle size of the formed streptavidin crosslinked product was measured under a constant temperature condition of 25 ° C. using a particle size distribution analyzer (manufactured by Sysmex Corporation, ZETASIZERSIZENano-ZS), and the change in particle size with time was followed. The result is shown in FIG.
  • the median particle diameters after adding glutaraldehyde for 22 hours were 190 nm, 82 nm, 37 nm, and 15 nm, respectively. As is apparent from FIG. 1, it was found that the particle diameter of the streptavidin crosslinked body increases with time, particularly when glutaraldehyde 0.0075%, 0.01%, and 0.0125% is used.
  • dispersion A 10 mmol / L acetate buffer
  • the magnetic particles have a core-shell structure, have a particle size of 1.62 ⁇ m, the inner core portion contains a magnetic material with a total mass ratio of 41.2%, and the shell portion made of polystyrene has 97 ⁇ eq of amino groups chemically. Particles modified with / g.
  • washing a series of operations of dispersion of magnetic particles, collection of magnetic particles, and suction removal was referred to as “washing”. (Omitted). The washing was continued 4 times.
  • dispersion A 1.5 ⁇ mL was added to sufficiently disperse the magnetic particles, and then 3.5 ⁇ mL of 25% glutaraldehyde aqueous solution (Nacalai Tesque) was added and shaken incubator (AS-One, SI-300C). Incubated for 2 hours at 37 ° C. with shaking at 1,500 rpm.
  • washing operation was performed 10 times using pH 5.5, 10 mmol / L acetate buffer (hereinafter referred to as dispersion B) containing 0.1% Trimethylstearylammonium Chloride (manufactured by Tokyo Chemical Industry Co., Ltd.) (hereinafter obtained) Magnetic particles are abbreviated as “activated particles”).
  • the activated particles (5 mg) were transferred to 150 ⁇ L of each reaction solution 22 hours after addition of glutaraldehyde and quickly dispersed. This was incubated for 16 hours at 40 ° C. with shaking at 1,500 rpm in a shaking incubator.
  • Streptavidin-bound magnetic particles are dispersed in 0.1% BSA / PBS [PBS: 10 mmol / L ⁇ phosphate buffer (pH 7.2) containing 0.15 mol / L sodium chloride] at 1 mg / mL, and double dilution method is used. Dilute to 6 levels (64-fold dilution) of 0.0156 mg / mL. Each of these 6 samples and blank (0.1% BSA / PBS) was dispensed into a 96-well black plate by 50 ⁇ L.
  • Biotin-Fluorescein® (manufactured by Thermo® Scientific) was diluted to 1 ⁇ g / mL with 0.1% BSA / PBS, and 50 ⁇ L was dispensed into each well into which the sample was dispensed.
  • the plate into which the sample was dispensed was incubated at 37 ° C. for 10 minutes with shaking in a shaker incubator (Amalite), and the fluorescence intensity in the state where particles were dispersed was measured with a fluorescence plate reader “Plate Chameleon V” (manufactured by HIDEX). It was measured.
  • the fluorescently labeled biotin when bound to streptavidin on the magnetic particles, the fluorescently labeled biotin bound to streptavidin exists in close proximity, and fluorescence quenching occurs. Fluorescence quenching increases as the amount of fluorescently labeled biotin bound to streptavidin bound to magnetic particles per unit area increases.
  • the biotin-binding ability of the streptavidin-coupled magnetic particles of the present invention is calculated by preliminarily evaluating the fluorescence reduction rate of this streptavidin using a commercially available magnetic particle with a known biotin-binding ability as a reference. did.
  • the concentration of streptavidin-bound magnetic particles when the fluorescence intensity decreased by 50% was calculated by linear approximation, and compared with the reference streptavidin, biotin binding capacity ( pmol / mm 2 ) was calculated.
  • the streptavidin-coupled magnetic particles obtained in (1) above are compared with commercially available streptavidin-coupled magnetic particles (Dynal Dynabeads T1 and Merck BE-M08 / 10). Thus, it was found to have a high biotin binding ability.
  • streptavidin has a crosslinked structure in the streptavidin-coupled magnetic particles produced by the production method of the present invention.
  • the present invention provides a method for producing streptavidin-coupled magnetic particles having a high biotin-binding ability and a method for producing protein-coupled magnetic particles using the streptavidin-coupled magnetic particles.
  • the streptavidin-coupled magnetic particles and protein-coupled magnetic particles produced by the production method of the present invention are useful for clinical diagnosis.

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Abstract

La présente invention concerne un procédé de fabrication de particules magnétiques liées à la streptavidine qui présentent une forte capacité de liaison à la biotine. L'invention concerne également un procédé de fabrication de particules magnétiques liées à la streptavidine, caractérisé en ce qu'il comprend les étapes suivantes. (1) Une étape de préparation de streptavidine réticulée, par réaction de glutaraldéhyde et de streptavidine ; (2) une étape de réaction des particules magnétiques avec la streptavidine réticulée, préparée à l'étape (1). Les particules magnétiques liées à la streptavidine, fabriquées au moyen de ce procédé de fabrication, peuvent être utilisées pour le diagnostic clinique.
PCT/JP2012/053464 2011-02-15 2012-02-15 Procédé de fabrication de particules magnétiques liées à la streptavidine Ceased WO2012111686A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10481158B2 (en) 2015-06-01 2019-11-19 California Institute Of Technology Compositions and methods for screening T cells with antigens for specific populations
CN115356477A (zh) * 2022-10-20 2022-11-18 苏州纳微生命科技有限公司 一种链霉亲和素磁珠及其制备方法和应用
US20230341388A1 (en) * 2019-12-26 2023-10-26 Precision Biosensor Inc. Method for manufacturing test strip for multiplex immunoassay analysis, and test strip manufactured using same
US12258613B2 (en) 2017-03-08 2025-03-25 California Institute Of Technology Pairing antigen specificity of a T cell with T cell receptor sequences

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JPH07209166A (ja) * 1993-12-13 1995-08-11 Hewlett Packard Co <Hp> 基体表面への被検体の結合方法、低分子量被検体の定量方法、及びビオチン−被検体複合体
JP2000146965A (ja) * 1998-11-06 2000-05-26 Iatron Lab Inc 免疫学的分析用試薬、免疫学的分析方法及び免疫学的分析用キット
JP2005523027A (ja) * 2002-04-22 2005-08-04 ユニヴァーシティ オヴ フロリダ 機能化されたナノ粒子及びその使用方法
JP2006042654A (ja) * 2004-08-03 2006-02-16 Onchip Cellomics Consortium 細胞分離回収方法および細胞分離チップおよび細胞分離装置

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JPH07209166A (ja) * 1993-12-13 1995-08-11 Hewlett Packard Co <Hp> 基体表面への被検体の結合方法、低分子量被検体の定量方法、及びビオチン−被検体複合体
JP2000146965A (ja) * 1998-11-06 2000-05-26 Iatron Lab Inc 免疫学的分析用試薬、免疫学的分析方法及び免疫学的分析用キット
JP2005523027A (ja) * 2002-04-22 2005-08-04 ユニヴァーシティ オヴ フロリダ 機能化されたナノ粒子及びその使用方法
JP2006042654A (ja) * 2004-08-03 2006-02-16 Onchip Cellomics Consortium 細胞分離回収方法および細胞分離チップおよび細胞分離装置

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10481158B2 (en) 2015-06-01 2019-11-19 California Institute Of Technology Compositions and methods for screening T cells with antigens for specific populations
US12258613B2 (en) 2017-03-08 2025-03-25 California Institute Of Technology Pairing antigen specificity of a T cell with T cell receptor sequences
US20230341388A1 (en) * 2019-12-26 2023-10-26 Precision Biosensor Inc. Method for manufacturing test strip for multiplex immunoassay analysis, and test strip manufactured using same
CN115356477A (zh) * 2022-10-20 2022-11-18 苏州纳微生命科技有限公司 一种链霉亲和素磁珠及其制备方法和应用
CN115356477B (zh) * 2022-10-20 2023-02-10 苏州纳微生命科技有限公司 一种链霉亲和素磁珠及其制备方法和应用

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