WO2012111687A1 - 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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WO2012111687A1
WO2012111687A1 PCT/JP2012/053465 JP2012053465W WO2012111687A1 WO 2012111687 A1 WO2012111687 A1 WO 2012111687A1 JP 2012053465 W JP2012053465 W JP 2012053465W WO 2012111687 A1 WO2012111687 A1 WO 2012111687A1
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magnetic particles
streptavidin
antibody
coupled
glutaraldehyde
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PCT/JP2012/053465
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English (en)
Japanese (ja)
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荒井　信之
泰弘 松岡
耕治 鵜澤
豪 永井
和樹 守田
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協和メデックス株式会社
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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 streptavidin-coupled magnetic particles, a method for producing the same, 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 reacted magnetic particles having amino groups on the surface with glutaraldehyde, and then obtained magnetic particles were subjected to streptogenesis in the presence of free glutaraldehyde.
  • the inventors have found that a streptavidin-binding magnetic particle having a high biotin-binding ability can be produced by reacting with avidin, and the present invention has been completed. 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 magnetic particles having amino groups on the surface with glutaraldehyde; and (2) A step of reacting the magnetic particles obtained in step (1) with streptavidin in the presence of free glutaraldehyde. [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 an SDS-PAGE migration image showing the structure of streptavidin on magnetic particles in streptavidin-coupled magnetic particles produced by the production method of the present invention and commercially available streptavidin-coupled magnetic particles.
  • Lane 1 is molecular weight marker
  • lane 2 is streptavidin
  • lane 3 is streptavidin-coupled magnetic particle of Example 1
  • lane 4 is commercially available streptavidin-coupled magnetic particle Dynabeads T1 (manufactured by Dynal)
  • lane 5 is It represents commercially available streptavidin-coupled magnetic particles BE-M08 / 10 (manufactured by Merck).
  • Band A represents a monomer
  • band B represents a dimer
  • band C represents a trimer
  • band D represents a tetramer
  • band E represents a higher-order cross-linked body.
  • the method for producing streptavidin-coupled magnetic particles of the present invention comprises a step of reacting magnetic particles having amino groups on the surface with glutaraldehyde (primary reaction step) and a primary reaction step.
  • a step (secondary reaction step) of reacting the obtained magnetic particles with streptavidin in the presence of free glutaraldehyde is included.
  • the streptavidin-coupled magnetic particles produced by the production method of the present invention have a structure in which streptavidin is crosslinked on the magnetic particles. Streptavidin has a tetrameric structure, and monomers are linked by non-covalent bonds.
  • streptavidin-bonded magnetic particles produced by the production method of the present invention streptavidin having a tetrameric structure is covalently bonded via glutaraldehyde on the magnetic particles to form a crosslinked structure.
  • Streptavidin is bonded to the amino group of the magnetic particle through glutaraldehyde. More specifically, 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 the streptavidin between subunits bound on the magnetic particles by treating at 60 ° C. for 1 hour. 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. After denaturing a sample with SDS, the denatured protein is subjected to polyacrylamide gel electrophoresis, and the obtained electrophoresis image is used. This is a method for separating and identifying proteins.
  • SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • 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 crosslinked 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 even higher order multimers are 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.
  • the biotin binding capacity per particle of the streptavidin-bound magnetic particles produced by the production method of the present invention is usually 0.5 to 3 pmol / mm 2 , preferably 1 to 2.5 pmol / mm 2 .
  • 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-coupled magnetic particles of the present invention 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 substance is not uniformly dispersed in an organic polymer without including an outer layer, and cluster-like magnetic particles made only of a magnetic substance. Specific examples (commercially available) of magnetic particles having an amino group on the surface include amino group type Estapor magnetic particles (manufactured by Merck).
  • 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.
  • Examples of the shape of the magnetic particles 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.
  • the primary reaction step is a step of reacting magnetic particles having amino groups on the surface with glutaraldehyde.
  • the magnetic particles having an amino group on the surface used in the primary reaction step are not particularly limited as long as they have an amino group on the surface and can produce the streptavidin-bonded magnetic particles of the present invention.
  • the above-mentioned magnetic particle etc. are mentioned.
  • magnetic particles having amino groups on the surface are used by being dispersed in a dispersion.
  • the dispersion include an aqueous solution containing a surfactant.
  • the pH of the dispersion is usually pH 4.5-7, preferably pH 5-6.
  • the aqueous medium used for the aqueous solution include distilled water, purified water, and a buffer solution.
  • a buffer solution is used as the aqueous medium, it is desirable to use 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 magnetic particles having amino groups on the surface used in the primary reaction step are fixed to the container, or when aggregation of magnetic particles is formed, the magnetic particles are easily dispersed by ultrasonic treatment. be able to. The presence or absence of aggregation of the magnetic particles can be confirmed by measuring the particle size of the magnetic particles with a particle size distribution meter.
  • the reaction conditions in the primary reaction step are not particularly limited as long as the magnetic particles having amino groups on the surface and glutaraldehyde can react with each other.
  • the reaction temperature in the primary reaction step 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.
  • Secondary reaction step is a step in which the magnetic particles obtained in the primary reaction step are reacted with streptavidin in the presence of free glutaraldehyde.
  • the free glutaraldehyde may be unreacted glutaraldehyde in the primary reaction or newly added glutaraldehyde.
  • the unreacted glutaraldehyde in the primary reaction is used as free glutaraldehyde
  • the unreacted glutaraldehyde can be used as it is in the secondary reaction step, but glutaraldehyde is added to the reaction solution after the primary reaction.
  • the magnetic particles are preferably washed to such an extent that glutaraldehyde remains on the magnetic particles.
  • the washing conditions for the magnetic particles are not particularly limited as long as glutaraldehyde remains on the washed magnetic particles.
  • the cleaning liquid is not particularly limited as long as it is a cleaning liquid capable of cleaning and removing excess glutaraldehyde, and examples thereof include an aqueous solution containing the aforementioned surfactant.
  • the number of washings is not particularly limited as long as glutaraldehyde remains in the magnetic particles after washing away excessive glutaraldehyde, and is usually 5 to 15 times, and preferably 8 to 12 times.
  • the temperature of the washing solution is not particularly limited as long as glutaraldehyde remains in the magnetic particles after washing and removing excessive glutaraldehyde, and is usually 20 to 30 ° C., preferably 22.5 to 27.5 ° C., 25 ° C is particularly preferred.
  • the washed magnetic particles can be stored until they are reacted with streptavidin.
  • the storage time until the reaction with streptavidin is not particularly limited as long as it can produce the streptavidin-coupled magnetic particles of the present invention, and is usually 2 to 6 hours, preferably 3 to 5 hours, and preferably 4 hours. Particularly preferred.
  • the amount of glutaraldehyde added is not particularly limited as long as it is an amount capable of producing the streptavidin-coupled magnetic particles of the present invention, and is usually 2 to 20 mg per 1 mg of magnetic particles. An amount of ⁇ 10 mg is preferred.
  • the reaction temperature is usually 25 to 40 ° C, preferably 27.5 to 37.5 ° C, particularly preferably 35 ° C.
  • the reaction time is usually 4 to 24 hours, preferably 8 to 20 hours, and particularly preferably 18 hours.
  • the amount of streptavidin is not particularly limited as long as it is an amount capable of producing the streptavidin-coupled magnetic particles of the present invention, and is usually 15 to 45 (w / w)% with respect to the magnetic particles, and 20 to 30 ( w / w)% is preferred, and 25 (w / w)% is particularly preferred.
  • the reaction mixture itself obtained in the secondary reaction step can also be used as the streptavidin-coupled magnetic particles produced by the production method of the present invention, but the magnetic particles in the reaction mixture obtained in the secondary reaction step are used.
  • the magnetic particles obtained by collecting with a magnet and removing the solution other than the magnetic particles and then washing with a cleaning liquid can also be used as the streptavidin-coupled magnetic particles produced by the production method of the present invention.
  • 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 of the present invention, and examples thereof include the above-described aqueous medium.
  • An aqueous medium containing protein and preservative can also be used as a cleaning liquid.
  • the protein examples include bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • the preservative examples include sodium azide.
  • the washed magnetic particles can be stored in a storage solution.
  • the storage solution is not particularly limited as long as it is a solution that can stably store the streptavidin-coupled magnetic particles of the present invention, for example, a protein such as bovine serum albumin (BSA) in a neutral to weakly acidic buffer solution.
  • An aqueous solution containing BSA bovine serum albumin
  • the method for producing streptavidin-coupled magnetic particles of the present invention may 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 proceeding the reduction reaction, 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 can be washed with, for example, the above-mentioned dispersion or diluted storage solution, and further suspended and stored in the storage solution.
  • the storage solution is not particularly limited as long as it is a solution that can stably store the streptavidin-coupled magnetic particles of the present invention, and examples thereof include the storage solution described above.
  • Protein-bound magnetic particles can be produced by reacting 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 measurement target component in the 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.
  • dispersion A pH 5.5, 10 mmol / L acetate buffer
  • Trimethylstearylammonium Chloride manufactured by Tokyo Chemical Industry Co., Ltd.
  • 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.
  • dispersion B pH 5.5, 10 mmol / L acetate buffer
  • Dispersion B was preliminarily incubated in a constant temperature bath at 25 ° C., and the work was performed in an environment at 25 ° C.
  • washing was performed 6 times in succession, 3 times after 3 hours and 30 minutes from the start of washing, and once after 4 hours (hereinafter, the obtained particles are abbreviated as “activated particles”).
  • Recombinant Streptavidin (Roche), dissolved in pH 5.5, 10 mmol / L acetate buffer to a concentration of 24.5 mg / mL, prepare a streptavidin solution, ice-cooled for 1 hour It was left above. After removing the dispersion B from the activated particles, 1.4 mL of a streptavidin solution was added and quickly dispersed. This was incubated for 16 hours at 40 ° C. with shaking at 1,500 rpm in a shaking incubator. Next, 0.53 mg / mL methanol solution (200 ⁇ L) of 2-picoline borane (manufactured by Junsei) was added and incubated at 40 ° C.
  • reaction mixture was separated into magnetic particles and other solution components by a magnet, and the separated magnetic particles were separated into 50 ⁇ mmol / L MES buffer solution containing 1.0% BSA and 0.09% sodium azide ( Washed 10 times with pH 6.5) to obtain streptavidin-bound magnetic particles.
  • 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 of dispersed particles was measured with a fluorescence plate reader “Plate Chameleon V” (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-coupled magnetic particles obtained in Example 1 the streptavidin crosslinked structure on the magnetic particles was confirmed by the following method. 20 mg of streptavidin-bound magnetic particles were washed 10 times with 5 mL of PBS and adjusted to 20 mg / mL with 1% SDS / PBS. Next, it was incubated in a 60 ° C. incubator for 1 hour, and the supernatant from which particles were removed was analyzed by SDS-PAGE.
  • Fig. 1 shows a migration image of the streptavidin-coupled magnetic particles obtained by this method and the structure of streptavidin in commercially available streptavidin-coupled magnetic particles confirmed by SDS-PAGE.
  • streptavidin and commercially available streptavidin-coupled magnetic particles only the monomer constituting streptavidin was observed, whereas it was produced by the production method of the present invention.
  • streptavidin-coupled magnetic particles dimers, trimers, tetramers and higher order bands were observed in addition to the monomers.
  • streptavidin-coupled magnetic particles does not have a crosslinked structure
  • streptavidin-coupled magnetic particles obtained by the production method of the present invention streptavidin has a crosslinked structure. Turned out to be taking.
  • 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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  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
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  • Peptides Or Proteins (AREA)

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 réaction des particules magnétiques, possédant des groupes aminés sur leur surface, avec du glutaraldéhyde ; et (2) une étape de réaction des particules magnétiques obtenues à l'étape (1) avec de la streptavidine, en présence de glutaraldéhyde libre. Les particules magnétiques liées à la streptavidine, fabriquées au moyen de ce procédé de fabrication, peuvent être utilisées pour le diagnostic clinique.
PCT/JP2012/053465 2011-02-15 2012-02-15 Procédé de fabrication de particules magnétiques liées à la streptavidine WO2012111687A1 (fr)

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WO2018124185A1 (fr) 2016-12-28 2018-07-05 Jsr株式会社 Dispersion de particules magnétiques
US10481158B2 (en) 2015-06-01 2019-11-19 California Institute Of Technology Compositions and methods for screening T cells with antigens for specific populations
US10858646B2 (en) 2015-10-08 2020-12-08 Toppan Printing Co., Ltd. Method and kit for concentrating target double-stranded nucleic acid molecules using a pyrrole-imidazole-containing polyamide

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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
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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
US10858646B2 (en) 2015-10-08 2020-12-08 Toppan Printing Co., Ltd. Method and kit for concentrating target double-stranded nucleic acid molecules using a pyrrole-imidazole-containing polyamide
US11479764B2 (en) 2015-10-08 2022-10-25 Toppan Printing Co., Ltd. Method and kit for concentrating target double-stranded nucleic acid molecules using a pyrrole-imidazole-containing polyamide
WO2018124185A1 (fr) 2016-12-28 2018-07-05 Jsr株式会社 Dispersion de particules magnétiques
US11320430B2 (en) 2016-12-28 2022-05-03 Jsr Corporation Magnetic particle dispersion

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