WO2012111686A1

WO2012111686A1 - Manufacturing method for streptavidin-bonded magnetic particles

Info

Publication number: WO2012111686A1
Application number: PCT/JP2012/053464
Authority: WO
Inventors: 荒井　信之; 泰弘松岡; 豪永井; 和樹守田
Original assignee: 協和メデックス株式会社
Priority date: 2011-02-15
Filing date: 2012-02-15
Publication date: 2012-08-23
Also published as: JPWO2012111686A1

Abstract

Provided is a manufacturing method for streptavidin-bonded magnetic particles having a high biotin binding capability. A manufacturing method for streptavidin-bonded magnetic particles, said method being characterised by including the following steps. (1) A step for preparing cross-linked streptavidin by reacting glutaraldehyde and streptavidin; (2) a step for reacting magnetic particles with the cross-linked streptavidin prepared in step (1). The streptavidin-bonded magnetic particles manufactured by means of this manufacturing method can be used in clinical diagnosis.

Description

Method for producing streptavidin-coupled magnetic particles

The present invention relates to a method for producing streptavidin-coupled magnetic particles and a method for producing protein-coupled magnetic particles.

In diagnostic agents, 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. In such a measurement system, 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.

In recent years, there has been a demand for higher sensitivity of examinations by early detection of diseases, higher precision of examinations, high sensitivity, and support for trace markers. Furthermore, rapid output of examination results for the purpose of patient services and the large amount of processing associated with the establishment of an examination center have led to demand for rapid examination.

Therefore, as a means to achieve high sensitivity and speed in a measurement system using such magnetic particles, 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. Used. In a typical example, 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. There is a method in which 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.

In such avidin-bonded magnetic particles, streptavidin-bonded magnetic particles using streptavidin having the same properties as avidin are more useful. Like avidin, streptavidin binds very strongly to biotin and has the property of being more resistant to denaturation than avidin. In addition, 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.

However, the amount of streptavidin that can be bound on the magnetic particles is limited, and a large amount of streptavidin-bound magnetic particles must be used to obtain the biotin binding capacity required for the reagent per test. There are problems such as high manufacturing costs. Furthermore, there are the following problems in the measurement using streptavidin-coupled magnetic particles.
(1) Since magnetic particles precipitate under standing conditions, it is necessary to disperse them during use. However, if the amount of particles is large, it takes time and labor to disperse.
(2) When the amount of particles is large, the volume increases when the magnet is moved to one side of the container, and the efficiency of cleaning the reaction liquid confined inside during B / F separation and cleaning decreases.
(3) If the amount of magnetic particles is large when detecting the component to be measured, the turbidity due to coloring of the magnetic particles themselves increases, and for example, detection by chemiluminescence or fluorescence decreases the sensitivity due to optical shielding.

In addition, when the amount of streptavidin-coupled magnetic particles is reduced and the biotin-binding ability is kept to a minimum, the reaction in the measurement is inhibited by competing with biotin (vitamin H) present in the sample, and an accurate test is performed. There is a possibility that the value cannot be obtained. Biotin has been taken as a supplement and administered as a drug, and such problems are often pointed out.

On the other hand, several methods have been proposed as means for solving this problem. One method is to reduce the particle size of the particles in order to increase the surface area per magnetic particle weight. However, when the particle size is reduced, there are problems such that the time for collecting the particles with a magnet is significantly increased, and that the particles are easily flown by the discharge / suction operation of the cleaning liquid in the cleaning process. 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.

In addition, a method of making it porous to increase the surface area of the insoluble carrier has been proposed. For example, 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.

JP 2009-28711 A JP 2006-307126 A

An object of the present invention is to provide a method for producing streptavidin-coupled magnetic particles having a high biotin-binding ability. Another object of the present invention is to provide a method for producing protein-bound magnetic particles using streptavidin-bound magnetic particles having a high biotin-binding ability.

As a result of intensive studies to solve the problem, 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].

[1] 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.

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, It represents the change with time when a glutaraldehyde 0.0125% solution was used.

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, lanes 6 streptavidin-conjugated magnetic particles biotin binding capacity 1.96 pmol / mm ^2, lanes 7 streptavidin coupled magnetic particles biotin binding capacity 3.68 pmol / mm ^2, 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 dimer, band C represents a trimer, band D represents a tetramer, and band E represents a higher-order cross-linked body.

It is a photograph which shows the dispersibility of the streptavidin coupling | bonding magnetic particle manufactured by the manufacturing method of this invention. The top shows the streptavidin-coupled magnetic particles in a stationary state, and the bottom shows the dispersed state of the streptavidin-coupled magnetic particles after 5 times of inversion mixing.

1. Method for Producing Streptavidin-Coupled Magnetic Particles 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. In the 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. 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 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. 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. 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).

In SDS-PAGE, 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.

In the streptavidin-coupled magnetic particles of the present invention, streptavidin may be naturally derived or genetically modified, but is preferably genetically modified.

In the present invention, 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 ), γ-heavy sesquioxide (γ-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 particle 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 μm, and preferably 0.5 to 3 μm.
Specific examples of magnetic particles (commercially available) include amino group type Estapor magnetic particles (Merck), hydrophobic type Estapor magnetic particles (Merck), and the like.

(1) Primary reaction step The primary reaction step is a step in which glutaraldehyde and streptavidin are reacted to prepare a streptavidin cross-linked product. As described above, 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.For example, 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. Examples of 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. When a buffer solution is used as the aqueous medium, it is desirable to use a buffering agent corresponding to the set pH. Examples of 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-hydroxypropanesulfonic acid (DIPSO), N- [ Squirrel (hydroxymethyl) methyl] -2-hydroxy-3-amino propane sulfonic acid (TAPSO), and the like.

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. For example, 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.

In the next reaction step, 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. After adding glutaraldehyde to the aqueous solution of streptavidin, 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. Although it is good, it is preferable to carry out 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.

(2) Secondary reaction step 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. In the secondary reaction step, 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.

When a cross-linked streptavidin is bound to a magnetic particle by a covalent bond, for example, 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. In the presence of a linker, a cross-linked streptavidin and magnetic particles having a functional group on the surface may be reacted in the above dispersion. Examples of the functional group include the functional groups described above. Examples of 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. For example, 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. Examples of commercially available divalent cross-linking agents include, for example, the site of Thermo Fisher Scientific (http://www.piercenet.com/products/browse.cfm?fldID=43238248-EE54-4070-A6DF-F037FBFCABCA), Dojin Corporation Examples include the compounds described in the 27th edition general catalog of the Chemical Research Laboratory.

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.

When 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. Can be combined. Examples of 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. Examples of 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.For example, in a neutral to weakly acidic buffer, bovine serum albumin (BSA An aqueous solution containing a protein such as
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. In the secondary reaction step, 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.

As the streptavidin-coupled magnetic particles in the reduction reaction step, 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.

After the reduction reaction, 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.

2. Method for Producing Protein-Binding Magnetic Particles 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. Specific examples of 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 cytokines, α-fetoprotein (AFP), anti-AFP antibody, carcinoembryonic antigen (CEA), anti-CEA antibody, CA19-9, anti CA19-9 antibody, CA15-3, anti-CA15-3 antibody, CA-125, anti-CA-125 antibody, PIVKA-II, anti-PIVKA-II anti , Parathyroid hormone (PTH), anti-PTH antibody, human chorionic gonadotropin (hCG), anti-hCG antibody, thyroid stimulating hormone (TSH), anti-TSH antibody, insulin, anti-insulin antibody, C-peptide, anti-C-peptide antibody , Estrogen, anti-estrogen antibody, fibroblast growth factor-23 (FGF-23), anti-FGF-23 antibody, glutamate decarboxylase (GAD), anti-GAD antibody, pepsinogen, anti-pepsinogen antibody, hepatitis B virus (HBV ) Antigen, anti-HBV antibody, hepatitis C virus (HCV) antigen, anti-HCV antibody, adult T-cell leukemia virus type 1 (HTLV-I) antigen, anti-HTLV-I antibody, human immunodeficiency virus (HIV) antigen, Anti-HIV antibody, influenza virus antigen, anti-influenza virus antibody, tuberculosis antigen (TBGL), anti-tuberculosis antibody, mycoplasma antigen, anti-mycoplasma antibody, hemoglobin A1c, anti-hemoglobin A1c antibody, atrial natriuretic peptide Tide (ANP), anti-ANP antibody, brain natriuretic peptide (BNP), anti-BNP antibody, troponin T, anti-troponin T antibody, troponin I, anti-troponin I antibody, creatinine kinase-MB (CK-MB), anti-CK- MB antibody, myoglobin, anti-myoglobin antibody, L-FABP, anti-L-FABP antibody, H-FABP, anti-H-FABP antibody, CCP antigen, anti-CCP antibody, SP-D, anti-SP-D antibody, mold venom [ Antibodies that bind to 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.], antibodies that bind to steroid hormones (aldosterone, testosterone, etc.), fungi such as E. coli, antibodies that bind to fungi, food alleles It allergens over material mites such as antiallergic agent antibodies, and the like.

In addition to biotinylated proteins, 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. In addition, nucleic acid-binding magnetic particles can be produced by reacting the streptavidin-binding magnetic particles of the present invention with biotinylated nucleic acids.

Examples of 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.

Examples of the nucleic acid in the biotinylated nucleic acid 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. As the measurement method, 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. For example, 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, α-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, estrogen, fibroblast growth factor-23 (FGF-23), anti-glutamic acid Decarboxylase (GAD) antibody, pepsinogen, hepatitis B virus (HBV) antigen, anti-HBV antibody, hepatitis C virus (HCV) antigen, anti-HCV antibody, adult T-cell leukemia virus type 1 (HTLV-I) antigen Anti-HTLV-I antibody, human immunodeficiency virus (HIV) antibody, influenza virus antigen, anti-in Luenza virus antibody, anti-tuberculosis antibody, tuberculosis antigen (TBGL) mycoplasma antibody, hemoglobin A1c, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), troponin T, troponin I, creatinine kinase-MB (CK -MB), myoglobin, L-FABP, H-FABP, anti-CCP antibody, SP-D, 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.), fungi such as Escherichia coli And allergic substances such as food allergen mites, anti-allergic substance antibodies and the like.

Further, instead of protein-bound magnetic particles, using hydrocarbon compound-bound magnetic particles produced using streptavidin-bound magnetic particles obtained by the production method of the present invention and biotinylated hydrocarbon-based compounds, or Instead of protein-bound magnetic particles, streptavidin-bound magnetic particles obtained by the production method of the present invention and biotinylated hydrocarbon compounds can be used to measure components to be measured in a sample. Examples of components to be measured include a hydrocarbon compound constituting a biotinylated hydrocarbon compound, an antibody that binds to the hydrocarbon compound, and the like. Examples of the 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.

Further, in place of protein-bound magnetic particles, 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 In addition, 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. Examples of components to be measured include nucleic acids and proteins that bind to nucleic acids constituting biotin nucleic acids. Examples of 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.

Hereinafter, the present invention will be described in more detail with reference to examples, but these do not limit the scope of the present invention.

(1) Production of Streptavidin-Binding Magnetic Particles A genetically modified streptavidin (manufactured by Roche) is dissolved in pH 5.5, 10 mmol / L acetic acid buffer solution to 24.5 mg / mL, and ice-cooled to 1 Let stand for more than an hour. To 75 μL of this streptavidin solution, a 25% glutaraldehyde aqueous solution (manufactured by Nacalai Tesque) was diluted with pH 5.5, 10 mmol / L acetic acid buffer solution, and 75 μL of a glutaraldehyde solution adjusted to 0.0125% was added. Similarly, a solution to which 0.010%, 0.0075%, and 0.005% glutaraldehyde solution was added was prepared.

The prepared solution was transferred to a plastic disposable and dispersed in 0.1% BSA / PBS to a concentration of 0.05 mg / mL. With the passage of time, 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.

Next, as magnetic particles, amino group type Estapor magnetic particles EM2-100 / 40 (made by Merck & Co., Inc.) are used, 20 mg of the magnetic particles are added, and pH 5.5 containing 1.0% Trimethylstearylammonium Chloride (Tokyo Chemical Co., Ltd.) Then, it was dispersed in 4 mL of 10 mmol / L acetate buffer (hereinafter referred to as dispersion A). The magnetic particles have a core-shell structure, have a particle size of 1.62 μm, 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 μeq of amino groups chemically. Particles modified with / g. Subsequently, magnetic particles were collected by arranging a strong magnet beside the container, and the dispersion A was removed by suction (hereinafter, 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.

Next, 1.5 μmL of dispersion A was added to sufficiently disperse the magnetic particles, and then 3.5 μmL 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.

Furthermore, the 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 μL 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.

Next, a 0.53 mg / mL methanol solution (200 μL) of 2-picoline borane (manufactured by Junsei Chemical Co., Ltd.) was added, and the mixture was incubated at 40 ° C. for 6 days while shaking at 1,500 rpm in a shaking incubator. The obtained reaction mixture was separated into magnetic particles and other solution components by a magnet, and the separated magnetic particles were separated into 50 μmmmol / L MES buffer containing 1.0% BSA and 0.09% sodium azide ( Washed 10 times with pH 6.5) to obtain streptavidin-bound magnetic particles.

(2) Measurement of biotin-binding ability of streptavidin-coupled magnetic particles Biotin-binding ability of the streptavidin-coupled magnetic particles obtained in (1) and commercially available streptavidin-coupled magnetic particles was measured by the following method. .

Streptavidin-bound magnetic particles are dispersed in 0.1% BSA / PBS [PBS: 10 mmol / L 緩衝 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 μL. Next, Biotin-Fluorescein® (manufactured by Thermo® Scientific) was diluted to 1 μg / mL with 0.1% BSA / PBS, and 50 μL 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.

Here, when the fluorescently labeled biotin is 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. Using this property, 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. In this example, from the diluted sample of streptavidin-bound magnetic particles, 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 measurement results are shown in Table 1.

As is clear from Table 1, 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.

Analysis of streptavidin cross-linked structure on magnetic particles Streptavidin binding obtained by the same method as in Example 1 with biotin binding capacities of 1.96 pmol / mm ² , 3.68 pmol / mm ² and 5.16 pmol / mm ² For the magnetic particles, each streptavidin-bound magnetic particle was washed 10 times with 4 mL of PBS, replaced with 1% SDS / PBS, and then incubated at 60 ° C. for 1 hour. Next, the magnetic particles were collected with a magnet, and the protein solution in the supernatant was collected and analyzed by SDS-PAGE. The same operation was performed on commercially available streptavidin-coupled magnetic particles. The results of SDS-PAGE are shown in FIG.

As is clear from FIG. 2, for streptavidin, only the monomer constituting streptavidin was observed, whereas in the streptavidin-coupled magnetic particles produced by the production method of the present invention, In addition to the body, dimer, trimer, tetramer and higher order bands were observed (see lanes 6-8). In addition to the monomer, dimer, trimer, tetramer and higher-order bands were also observed in the cross-linked streptavidin before binding to the magnetic particles (see lanes 3-5). ). Thus, it was found that streptavidin has a crosslinked structure in the streptavidin-coupled magnetic particles produced by the production method of the present invention.

[Test Example 1] Evaluation of dispersibility of streptavidin-coupled magnetic particles About streptavidin-coupled magnetic particles having a biotin-binding ability of 5.22 pmol / mm ² prepared by the same method as in Example 1, the streptavidin-coupled magnetic particles Was dispersed in PBS containing 0.1% BSA at a concentration of 0.1 mg / mL and allowed to stand for 24 hours in a cool dark place. This was mixed by inversion and stirred 5 times, and the degree of dispersion was compared. The result is shown in FIG. The top of FIG. 3 represents the streptavidin-coupled magnetic particles in a stationary state, and the bottom represents the dispersed state of the streptavidin-coupled magnetic particles after 5 times of inversion mixing.

As apparent from FIG. 3, it was found that the streptavidin-bonded magnetic particles produced by the production method of the present invention have very good dispersibility.

Claims

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.
Furthermore, the manufacturing method of the streptavidin coupling | bonding magnetic particle of Claim 1 including the following processes.
(3) A step of reacting the streptavidin-bound magnetic particles prepared in step (2) with a reducing agent.
The method for producing streptavidin-coupled magnetic particles according to claim 1 or 2, wherein the streptavidin-coupled magnetic particles have a structure in which streptavidin is cross-linked on the magnetic particles.
A method for producing protein-bound magnetic particles, comprising reacting streptavidin-bound magnetic particles produced by the production method according to any one of claims 1 to 3 and biotinylated protein.