JPWO2012111687A1 - Method for producing streptavidin-coupled magnetic particles - Google Patents
Method for producing streptavidin-coupled magnetic particles Download PDFInfo
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- JPWO2012111687A1 JPWO2012111687A1 JP2012557982A JP2012557982A JPWO2012111687A1 JP WO2012111687 A1 JPWO2012111687 A1 JP WO2012111687A1 JP 2012557982 A JP2012557982 A JP 2012557982A JP 2012557982 A JP2012557982 A JP 2012557982A JP WO2012111687 A1 JPWO2012111687 A1 JP WO2012111687A1
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- magnetic particles
- streptavidin
- antibody
- coupled
- bound
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Abstract
ビオチン結合能の高いストレプトアビジン結合磁性粒子の製造方法を提供する。以下の工程を含むことを特徴とする、ストレプトアビジン結合磁性粒子の製造方法。（１）表面にアミノ基を有する磁性粒子をグルタルアルデヒドと反応させる工程；及び、（２）工程（１）で得られた磁性粒子を、遊離のグルタルアルデヒド存在下、ストレプトアビジンと反応させる工程。本発明の製造方法により製造されるストレプトアビジン結合磁性粒子は、臨床診断上、有用である。Provided is a method for producing streptavidin-binding magnetic particles having high biotin-binding ability. A method for producing streptavidin-coupled magnetic particles, comprising the following steps. (1) a step of reacting magnetic particles having an amino group 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. The streptavidin-bound magnetic particles produced by the production method of the present invention are useful for clinical diagnosis.
Description
本発明は、ストレプトアビジン結合磁性粒子及びその製造方法、及び、蛋白質結合磁性粒子の製造方法に関する。 The present invention relates to streptavidin-coupled magnetic particles, a method for producing the same, and a method for producing protein-coupled magnetic particles.
診断薬では、ホルモン・癌マーカー・感染症マーカー等の検査対象物質を検出するために固相担体として磁性粒子がしばしば用いられる。このような測定系においては、抗体または抗原（一次プローブ）等が磁性粒子上に結合され、検体中の測定対象物質と結合した後、さらに蛍光物質や化学発光基質、酵素などで標識された二次プローブと結合することにより、定性または定量的に測定対象物質が検出される。 In diagnostic agents, magnetic particles are often used as solid phase carriers to detect substances to be examined such as hormones, cancer markers, infection markers, and the like. 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.
また、近年においては、疾病の早期発見、検査の高精度化、高感度・微量マーカーへの対応等により検査の高感度化が求められている。さらに患者サービスを目的とした検査結果の迅速出力、検査センター化に伴う大量処理化などにより、検査の迅速性も求められるようになってきている。 In recent years, there has been a demand for higher sensitivity of testing by early detection of diseases, high accuracy of testing, 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.
そこで、このような磁性粒子を用いた測定系における高感度化・迅速化を実現する手段として、一次プローブおよび二次プローブを液相中で反応させ、次に磁性粒子上に結合させる方法がよく用いられる。代表的な例では一次プローブにビオチンを結合させたビオチン標識一次プローブを、試料中の測定対象成分、及び、二次プローブと反応させて、ビオチン標識一次プローブ−測定対象成分−二次プローブからなる複合体を形成させ、次いで、アビジン結合磁性粒子を作用させて、アビジン−ビオチン相互作用により、磁性粒子上に当該複合体を結合させる方法がある。 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.
このようなアビジン結合磁性粒子においては、アビジンと同じ性質を持つストレプトアビジンを用いたストレプトアビジン結合磁性粒子の方がより有用である。ストレプトアビジンはアビジンと同様にビオチンと非常に強く結合し、アビジンよりも変性に強いという特性がある。また、等電点はアビジンが塩基性であるのに対して、ストレプトアビジンは弱酸性または中性であるため、他の蛋白質との非特異的結合が少ないという利点が知られている。このストレプトアビジンを利用したストレプトアビジン結合磁性粒子は多くの用途で利用されている。 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.
しかしながら、磁性粒子上に結合できるストレプトアビジンの量は限られており、１テストあたりの試薬に必要とされるビオチン結合能を得るためには大量のストレプトアビジン結合磁性粒子を用いらなければならず、製造コストがかかる等の問題点があった。さらに、ストレプトアビジン結合磁性粒子を用いる測定においても以下のような問題点があった。
(1)磁性粒子は静置条件下で沈殿してしまうため使用時には分散させる必要があるが、粒子量が多いと分散させるのに時間と労力がかかる
(2)粒子量が多いと磁石で容器の片側に寄せた際にその体積が大きくなり、B/F分離及び洗浄時に内部に閉じ込められた反応液を洗浄する効率が低下する
(3)測定対象成分を検出する際に磁性粒子量が多いと磁性粒子そのものの着色による濁度が増加し、例えば化学発光や蛍光による検出では光学的な遮蔽によりその感度が低下する。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 solution trapped 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.
また、ストレプトアビジン結合磁性粒子の量を抑え、ビオチン結合能を必要最小限とした状態においては、検体中に存在するビオチン（ビタミンH）と競合することにより測定における反応が阻害され、正確な検査値を得ることができない可能性が生じる。ビオチンはサプリメントとしての服用、薬剤としての投与があり、しばしばこのような問題が指摘されている。 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.
一方で、この解決手段としていくつかの方法が提案されてきた。一つは、磁性粒子重量あたりの表面積を大きくするため、粒子の粒径を小さくする方法がある。しかしながら、粒径を小さくすると、磁石で粒子を回収する時間が大幅に長くなったり、洗浄工程における洗浄液の吐出・吸引操作で粒子が流れやすくなったりする等の問題がある。特許文献１には検体中の検出対象物質を分離する方法において、温度応答性高分子により表面が修飾された磁性粒子を用いて、平均粒径50〜1,000 nmの磁性粒子においても温度応答性高分子による粒子凝集により、水溶液中から磁性粒子を回収する方法が記載されている。このような粒子は磁性粒子が小さくなることによる反応におけるメリットがある一方、粒子表面が温度応答性高分子により覆われていることによる非特異吸着があり、また凝集させるために特殊な条件下におきかえる工程があった。
On the other hand, several methods have been proposed as this solution. 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.
また、不溶性担体の表面積を大きくするため多孔質化する方法も提案されている。例えば、特許文献２には磁性粒子の外層に多孔質の層を化学的に形成させる方法が記載されているが、この方法では、抗原と抗体との免疫反応やDNA同士またはDNAとRNAとのハイブリダイゼーションにおいて、表面積あたりの結合能は向上するものの、孔内の反応効率が悪く、期待された性能を得ることは難しかった。
In addition, a method of making it porous has been proposed in order to increase the surface area of the insoluble carrier. For example,
本発明の目的は、ビオチン結合能の高いストレプトアビジン結合磁性粒子の製造方法を提供することにある。また、本発明の別の目的は、ビオチン結合能の高いストレプトアビジン結合磁性粒子を用いる、蛋白質結合磁性粒子の製造方法を提供することにある。 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.
本発明者らは本課題を解決すべく鋭意検討を重ねた結果、表面にアミノ基を有する磁性粒子をグルタルアルデヒドと反応させた後、得られた磁性粒子を、遊離のグルタルアルデヒド存在下、ストレプトアビジンと反応させることにより、ビオチン結合能の高いストレプトアビジン結合磁性粒子を製造し得る、という知見を見出し、本発明を完成させた。すなわち、本発明は、以下の［１］〜［４］に関する。 As a result of intensive studies to solve this problem, 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].
［１］ 以下の工程を含むことを特徴とする、ストレプトアビジン結合磁性粒子の製造方法。
（１）表面にアミノ基を有する磁性粒子をグルタルアルデヒドと反応させる工程；及び、
（２）工程（１）で得られた磁性粒子を、遊離のグルタルアルデヒド存在下、ストレプトアビジンと反応させる工程。
［２］ さらに、以下の工程を含む［１］記載のストレプトアビジン結合磁性粒子の製造方法。
（３）工程（２）で調製したストレプトアビジン結合磁性粒子と還元剤とを反応させる工程。
［３］ ストレプトアビジン結合磁性粒子が、磁性粒子上に、ストレプトアビジンどうしが架橋された構造を有する、［１］または［２］記載のストレプトアビジン結合磁性粒子の製造方法。
［４］ ［１］〜［３］のいずれかに記載の製造方法により製造されるストレプトアビジン結合磁性粒子と、ビオチン化蛋白質とを反応させることを特徴とする、蛋白質結合磁性粒子の製造方法。[1] 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.
本発明により、ビオチン結合能の高いストレプトアビジン結合磁性粒子の製造方法、及び、当該ストレプトアビジン結合磁性粒子を用いる、蛋白質結合磁性粒子の製造方法が提供される。本発明の製造方法により製造されるストレプトアビジン結合磁性粒子、及び、蛋白質結合磁性粒子は、臨床診断上、有用である。 The present invention provides a method for producing streptavidin-coupled magnetic particles having a high biotin-binding ability, and a method for producing protein-bound 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.
１．ストレプトアビジン結合磁性粒子の製造方法
本発明のストレプトアビジン結合磁性粒子の製造方法は、表面にアミノ基を有する磁性粒子をグルタルアルデヒドと反応させる工程（１次反応工程）、及び、１次反応工程で得られた磁性粒子を、遊離のグルタルアルデヒドの存在下、ストレプトアビジンと反応させる工程（２次反応工程）を含む。1. Method for Producing Streptavidin-Coupled Magnetic Particles 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 of reacting the obtained magnetic particles with streptavidin in the presence of free glutaraldehyde (secondary reaction step) is included.
本発明の製造方法で製造されるストレプトアビジン結合磁性粒子は、磁性粒子上で、ストレプトアビジンが架橋された構造を有する。ストレプトアビジンは四量体構造を取っており、単量体どうしは非共有結合により結合している。本発明の製造方法により製造されるストレプトアビジン結合磁性粒子においては、磁性粒子上で、この四量体構造のストレプトアビジンどうしがグルタルアルデヒドを介して共有結合し、架橋構造を取っている。また、ストレプトアビジンは、グルタルアルデヒドを介して磁性粒子のアミノ基と結合している。より詳細には、四量体構造のストレプトアビジンのうちの一部が、グルタルアルデヒドを介して磁性粒子のアミノ基と結合している。このストレプトアビジンの架橋構造は、例えばストレプトアビジン結合磁性粒子を1%のSDS溶液に置換し、60℃で1時間処理することによって、磁性粒子上に結合していたストレプトアビジンのサブユニット間結合を解離させ、SDS-PAGE（ドデシル硫酸ナトリウム−ポリアクリルアミドゲル電気泳動）、ゲル濾過HPLC等により確認することができる。SDS-PAGEは、電気泳動により蛋白質を大きさに依存して分離する方法であり、試料をSDSで変性させた後、変性した蛋白質をポリアクリルアミドゲル電気泳動に供して、得られた泳動像から、蛋白質の分離、同定を行う方法である。SDS-PAGEとしては、ストレプトアビジンの架橋構造を確認できる方法であれば特に制限はなく、例えばバイオ実験イラストレイテッド５（細胞工学別冊 秀潤社）に記載されている方法等が挙げられる。 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. In the 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. 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 (cell engineering separate volume Shujunsha).
SDS-PAGEにおいて、四量体構造のストレプトアビジンは、SDS存在下での変性処理により、その四量体構造がほどかれる。もしも磁性粒子上のストレプトアビジンが架橋構造を取っていないのであれば、SDS存在下での変性処理により得られる分解物は、ストレプトアビジン由来の単量体のみとなる。一方、磁性粒子上のストレプトアビジンが架橋構造を取っているのであれば、SDS存在下での変性処理により、ストレプトアビジン由来の単量体に加え、ストレプトアビジンの架橋構造に関わった二量体、三量体、さらには高次の多量体が得られることになる。従って、SDS-PAGEにより、ストレプトアビジン由来の単量体、二量体、三量体、及び、高次の多量体に起因するバンドが見られる場合には、磁性粒子上に、ストレプトアビジンの架橋構造が形成されていることになる。 In SDS-PAGE, tetrameric streptavidin is unfolded 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 involved in the cross-linked structure of streptavidin in addition to the monomer derived from streptavidin by modification treatment 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.
本発明の製造方法で製造されるストレプトアビジン結合磁性粒子は、磁性粒子上にストレプトアビジンどうしが架橋された構造を有しているため、ビオチン結合能が高い。本発明の製造方法で製造されるストレプトアビジン結合磁性粒子の、粒子あたりのビオチン結合能は、通常、0.5〜3 pmol/mm2であり、1〜2.5 pmol/mm2が好ましい。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 ability per particle of the streptavidin-coupled magnetic particles produced by the production method of the present invention is usually 0.5 to 3 pmol / mm 2 and preferably 1 to 2.5 pmol / mm 2 .
本発明のストレプトアビジン結合磁性粒子における、粒子あたりのビオチン結合能は、ビオチン結合能を測定し得る方法であれば、いかなる方法によっても測定することができ、例えば一定量の蛍光標識されたビオチンを一定量のストレプトアビジン結合磁性粒子と反応させ、磁石でストレプトアビジン結合磁性粒子を収集した後に、一定量の上清を採取し、採取された上清の蛍光を測定し、得られた測定値を、予め作成された蛍光強度とビオチン濃度との関係を示す検量線に照らし合わせることにより算出することができる。 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.
本発明のストレプトアビジン結合磁性粒子において、ストレプトアビジンは、天然由来のものでも、遺伝子組み換え体でもよいが、遺伝子組み換え体が好ましい。
本発明において、ストレプトアビジンが固定される磁性粒子は、本発明のストレプトアビジン結合磁性粒子の製造を可能とする磁性粒子であれば特に制限はなく、例えば粒子の内部に磁性体を含有し、外層が有機ポリマーなどから成るコア・シェル構造の磁性粒子、外層を含まず磁性体が有機ポリマーに不均一に分散した構造の磁性粒子、磁性体のみから成るクラスター状の磁性粒子等が挙げられる。表面にアミノ基を有する磁性粒子の具体例（市販品）としては、例えばアミノ基タイプEstapor磁性粒子（メルク社製）等が挙げられる。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. Specific examples (commercially available) of magnetic particles having amino groups on the surface include amino group-type Estapor magnetic particles (manufactured by Merck).
磁性粒子中に含まれる磁性体は、残留磁化が少なく、超常磁性の磁性体微粒子が好ましく、例えば四三酸化鉄(Fe3O4)、γ−重三二酸化鉄(γ-Fe2O3)等の各種フェライト、鉄、マンガン、コバルト、クロムなどの金属またはこれら金属の合金等が用いられる。
有機ポリマーと磁性体から成る磁性粒子中の磁性体の含有量は、磁性粒子全体の重量に占める割合が10重量%以上であるのが好ましく、30〜60重量%であることがより好ましい。The magnetic substance contained in the magnetic particles has a small residual magnetization and is preferably a superparamagnetic magnetic fine particle, for example, iron 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.
磁性粒子の形状としては、例えば球状、針状等が挙げられ、球状が好ましい。磁性粒子の粒径は、例えば0.1〜5μm等が挙げられ、好ましくは0.5〜3μmである。 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.
（１）１次反応工程
１次反応工程は、表面にアミノ基を有する磁性粒子をグルタルアルデヒドと反応させる工程である。１次反応工程で使用される、表面にアミノ基を有する磁性粒子としては、表面にアミノ基を有し、かつ、本発明のストレプトアビジン結合磁性粒子を製造し得る磁性粒子であれば特に制限はなく、例えば前述の磁性粒子等が挙げられる。(1) Primary reaction step The primary reaction step is a step of reacting magnetic particles having amino groups on the surface with glutaraldehyde. The magnetic particle having an amino group on the surface used in the primary reaction step is not particularly limited as long as it is a magnetic particle having an amino group on the surface and capable of producing the streptavidin-bonded magnetic particle of the present invention. For example, the above-mentioned magnetic particle etc. are mentioned.
１次反応工程において、表面にアミノ基を有する磁性粒子は、分散液中に分散されて使用される。分散液としては、例えば界面活性剤を含む水溶液等が挙げられる。分散液のpHとしては、通常pH4.5〜7であり、pH5〜6が好ましい。水溶液に使用される水性媒体としては、例えば蒸留水、精製水、緩衝液等が挙げられる。水性媒体として緩衝液を用いる場合には、設定するpHに応じた緩衝剤を用いることが望ましい。緩衝液に用いる緩衝剤としては、例えば、酢酸緩衝剤、クエン酸緩衝剤、コハク酸緩衝剤、リン酸緩衝剤、グッドの緩衝剤等が挙げられる。 In the primary reaction step, magnetic particles having amino groups on the surface are used by being dispersed in a dispersion. 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.
グッドの緩衝剤としては、例えば２−モルホリノエタンスルホン酸(MES)、ビス（２−ヒドロキシエチル）イミノトリス（ヒドロキシメチル）メタン(Bis-Tris)、ピペラジン−Ｎ，Ｎ’−ビス（２−エタンスルホン酸）(PIPES)、Ｎ−（２−アセトアミド）−２−アミノエタンスルホン酸(ACES)、３−モルホリノ−２−ヒドロキシプロパンスルホン酸(MOPSO)、Ｎ，Ｎ−ビス（２−ヒドロキシエチル）−２−アミノエタンスルホン酸(BES)、３−モルホリノプロパンスルホン酸(MOPS)、Ｎ−〔トリス（ヒドロキシメチル）メチル〕−２−アミノエタンスルホン酸(TES)、２−〔４−（２−ヒドロキシエチル）−１−ピペラジニル〕エタンスルホン酸(HEPES)、３−〔Ｎ，Ｎ−ビス（２−ヒドロキシエチル）アミノ〕−２−ヒドロキシプロパンスルホン酸(DIPSO)、Ｎ−〔トリス（ヒドロキシメチル）メチル〕−２−ヒドロキシ−３−アミノプロパンスルホン酸(TAPSO)等が挙げられる。 Good buffers 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- [to Scan (hydroxymethyl) methyl] -2-hydroxy-3-amino propane sulfonic acid (TAPSO), and the like.
界面活性剤は、磁性粒子を分散し得るものであれば特に制限はなく、例えば陰イオン性界面活性剤、陽イオン性界面活性剤、両性界面活性剤、非イオン性界面活性剤等が挙げられる。界面活性剤の分散液中の濃度は、磁性粒子を分散し得る濃度であれば特に制限はなく、例えば0.01〜5.0%である。 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%.
また、１次反応工程に使用される、表面にアミノ基を有する磁性粒子が容器に固着した場合、又は、磁性粒子の凝集が形成された場合は、超音波処理により容易に磁性粒子を分散させることができる。磁性粒子の凝集の有無は、粒度分布計により磁性粒子の粒径を測定することにより確認することができる。 In addition, when 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.
１次反応工程の反応条件は、表面にアミノ基を有する磁性粒子とグルタルアルデヒドとが反応し得る条件であれば、特に制限はない。１次反応工程における反応温度は、通常15〜50℃であり、20〜40℃が好ましく、35℃が特に好ましい。反応時間は、通常30分間〜6時間であり、1〜2時間が好ましい。 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, particularly preferably 35 ° C. The reaction time is usually 30 minutes to 6 hours, preferably 1 to 2 hours.
（２）２次反応工程
２次反応工程は、１次反応工程で得られた磁性粒子を、遊離のグルタルアルデヒドの存在下、ストレプトアビジンと反応させる工程である。(2) Secondary reaction step The 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.
遊離のグルタルアルデヒドは、１次反応における未反応のグルタルアルデヒドであっても、新たに添加されるグルタルアルデヒドであってもよい。 The free glutaraldehyde may be unreacted glutaraldehyde in the primary reaction or newly added glutaraldehyde.
遊離のグルタルアルデヒドとして、１次反応における未反応のグルタルアルデヒドを用いる場合は、該未反応のグルタルアルデヒドをそのまま２次反応工程に用いることができるが、１次反応後の反応液にグルタルアルデヒドが過剰に存在する場合は、磁性粒子上にグルタルアルデヒドが残存する程度に磁性粒子を洗浄するのが好ましい。磁性粒子の洗浄条件としては、洗浄後の磁性粒子上にグルタルアルデヒドが残存する条件であれば特に制限はない。洗浄液としては、過剰に存在するグルタルアルデヒドを洗浄除去できる洗浄液であれば特に制限はなく、例えば前述の界面活性剤を含む水溶液等が挙げられる。洗浄回数は、過剰に存在するグルタルアルデヒドを洗浄除去した後の磁性粒子にグルタルアルデヒドが残存する回数であれば特に制限はなく、通常5〜15回であり、8〜12回が好ましい。洗浄液の温度は、過剰に存在するグルタルアルデヒドを洗浄除去した後の磁性粒子にグルタルアルデヒドが残存する温度であれば特に制限はなく、通常20〜30℃であり、22.5〜27.5℃が好ましく、25℃が特に好ましい。また、洗浄された磁性粒子は、ストレプトアビジンと反応させるまで保存することができる。ストレプトアビジンとの反応までの保存時間は、本発明のストレプトアビジン結合磁性粒子を製造し得る時間であれば特に制限はなく、通常2〜6時間であり、3〜5時間が好ましく、4時間が特に好ましい。 When 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. When it is present in excess, 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 and removing excessive glutaraldehyde, and is usually 5 to 15 times, and preferably 8 to 12 times. The temperature of the washing liquid is not particularly limited as long as glutaraldehyde remains in the magnetic particles after washing and removing excess 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 is a time capable of producing the streptavidin-coupled magnetic particles of the present invention, and is usually 2 to 6 hours, preferably 3 to 5 hours, and 4 hours Particularly preferred.
遊離のグルタルアルデヒドとして、グルタルアルデヒドを新たに添加する場合、１次反応の反応混合物から未反応のグルタルアルデヒドを除去し、新たにグルタルアルデヒドを添加する。なお、この場合、未反応のグルタルアルデヒドは完全に除去されていなくてもよい。添加されるグルタルアルデヒドの量は、本発明のストレプトアビジン結合磁性粒子を製造し得る量であれば特に制限はなく、通常、磁性粒子1 mgに対して2〜20 mgとなる量であり、5〜10 mgとなる量が好ましい。 When glutaraldehyde is newly added as free glutaraldehyde, unreacted glutaraldehyde is removed from the reaction mixture of the primary reaction, and glutaraldehyde is newly added. In this case, unreacted glutaraldehyde may not be completely removed. The amount of glutaraldehyde to be 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 an amount of 2 to 20 mg with respect to 1 mg of the magnetic particles. An amount of ~ 10 mg is preferred.
遊離のグルタルアルデヒドの存在下でのストレプトアビジンとの反応は、本発明のストレプトアビジン結合磁性粒子を製造し得る条件であれば如何なる条件も用いることができる。反応温度は、通常25〜40℃で、27.5〜37.5℃が好ましく、35℃が特に好ましい。反応時間は、通常4〜24時間であり、8〜20時間が好ましく、18時間が特に好ましい。ストレプトアビジンの量は、本発明のストレプトアビジン結合磁性粒子を製造し得る量であれば特に制限はなく、通常、磁性粒子に対して15〜45(w/w)%であり、20〜30(w/w)%が好ましく、25(w/w)%が特に好ましい。 For the reaction with streptavidin in the presence of free glutaraldehyde, any conditions can be used as long as the streptavidin-coupled magnetic particles of the present invention can be produced. 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.
２次反応工程で得られた反応混合物そのものも、本発明の製造方法によって製造されるストレプトアビジン結合磁性粒子として使用することができるが、２次反応工程で得られた反応混合物中の磁性粒子を磁石により収集し、磁性粒子以外の溶液を除去した後、洗浄液により洗浄して得られる磁性粒子も、本発明の製造方法によって製造されるストレプトアビジン結合磁性粒子として使用することができる。洗浄液としては、本発明のストレプトアビジン結合磁性粒子以外の物質を洗浄し得る洗浄液であれば特に制限はなく、例えば前述の水性媒体等が挙げられる。また、蛋白質や防腐剤を含む水性媒体も洗浄液として使用することができる。蛋白質としては、例えば牛血清アルブミン(BSA)等が挙げられる。防腐剤としては、例えばアジ化ナトリウム等が挙げられる。また、洗浄された磁性粒子は、保存用溶液に懸濁されて保存され得る。保存用溶液としては、本発明のストレプトアビジン結合磁性粒子を安定に保存し得る溶液であれば特に制限はなく、例えば中性から弱酸性の緩衝液中に、牛血清アルブミン(BSA)等の蛋白質を含む水溶液等が挙げられる。 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. Examples of the protein include bovine serum albumin (BSA). Examples of the preservative include sodium azide. In addition, 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
本発明のストレプトアビジン結合磁性粒子の製造方法は、２次反応工程後に、還元反応工程を含んでもよい。還元反応工程は、２次反応工程で生成したストレプトアビジン結合磁性粒子と還元剤との反応の工程である。２次反応工程により、磁性粒子上に架橋構造のストレプトアビジンが形成されるが、形成された架橋構造のストレプトアビジンはシッフ塩基（イミン）を含むため、このシッフ塩基（イミン）を還元剤により還元することにより、より安定な架橋構造とすることができる。 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. 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.
還元反応工程におけるストレプトアビジン結合磁性粒子として、２次反応工程の反応混合物そのものを用いても、洗浄された磁性粒子を用いてもよい。ストレプトアビジン結合磁性粒子と還元剤との反応に用いられる溶媒は、還元反応を進行させ得る溶媒であれば特に制限はなく、例えば前述の分散液等が挙げられる。また、有機溶媒を含む分散液も還元反応における溶媒として用いることができる。有機溶媒としては、水に可溶性で、還元反応を進行させ得る有機溶媒であれば特に制限はなく、例えばメタノール、エタノール、テトラヒドロフラン等が挙げられる。還元剤としては、シッフ塩基（イミン）を還元し、架橋構造を保持し得る還元剤であれば特に制限はなく、例えばボラン系の還元剤等が挙げられる。ボラン系の還元剤としては、例えば２−ピコリンボラン、水素化ホウ素ナトリウム等が挙げられる。還元剤の添加量は、通常磁性粒子の0.0001〜0.1(w/w)%であり、0.0005〜0.05(w/w)%が好ましく、0.001(w/w)%が特に好ましい。 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 a Schiff base (imine) and retain a crosslinked structure, and examples thereof include a borane-based reducing agent. Examples of the borane-based reducing agent include 2-picoline borane and sodium borohydride. The addition amount of the reducing agent is usually 0.0001 to 0.1 (w / w)%, preferably 0.0005 to 0.05 (w / w)%, particularly preferably 0.001 (w / w)% of the magnetic particles.
還元反応の反応温度は、通常30〜50℃であり、35〜45℃が好ましく、40℃が特に好ましい。還元反応の反応時間は、通常2日間〜10日間であり、5日間〜8日間が好ましく、6日間が特に好ましい。 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 of the reduction reaction is usually 2 days to 10 days, preferably 5 days to 8 days, and particularly preferably 6 days.
還元反応後、磁性粒子は磁石により、磁性粒子以外の溶液成分と分離することができる。分離された磁性粒子は、例えば前述の分散液又は希釈した保存用溶液により洗浄され、さらに、保存用溶液中に懸濁されて保存することができる。保存用溶液としては、本発明のストレプトアビジン結合磁性粒子を安定に保存し得る溶液であれば特に制限はなく、例えば前述の保存用溶液等が挙げられる。 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 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.
２．蛋白質結合磁性粒子の製造方法
本発明の製造方法で製造されるストレプトアビジン結合磁性粒子と、ビオチン化蛋白質とを反応させることにより蛋白質結合磁性粒子を製造することができる。磁性粒子上のストレプトアビジンと蛋白質に結合するビオチンとの相互作用により、蛋白質が磁性粒子上に結合する。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.
ストレプトアビジン結合磁性粒子と、ビオチン化蛋白質との反応は、蛋白質が磁性粒子上に結合する条件であれば如何なる条件でもよい。反応温度は、通常25〜50℃であり、30〜40℃が好ましい。反応時間は、通常30分間〜24時間であり、2〜18時間が好ましい。 The reaction between the streptavidin-bound magnetic particles and the biotinylated protein may be performed under any conditions as long as the protein is bound to 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.
蛋白質としては、測定対象成分に結合する抗体、抗原抗体反応において測定対象成分と競合する競合物質等が挙げられる。競合物質としては、例えば測定対象成分や、測定対象成分に結合する抗体が認識するエピトープを含む物質等が挙げられる。蛋白質の具体例としては、IgG、抗IgG抗体、IgM、抗IgM抗体、IgA、抗IgA抗体、IgE、抗IgE抗体、アポ蛋白AI、抗アポ蛋白AI抗体、アポ蛋白AII、抗アポ蛋白AII抗体、アポ蛋白B、抗アポ蛋白B抗体、アポ蛋白E、抗アポ蛋白E抗体、リウマチファクター、抗リウマチファクター抗体、D-ダイマー、抗D-ダイマー抗体、酸化LDL、抗酸化LDL抗体、糖化LDL、抗糖化LDL抗体、グリコアルブミン、抗グリコアルブミン抗体、トリヨードサイロニン(T3)、抗T3抗体、総サイロキシン(T4)、抗T4抗体、薬剤（抗テンカン剤等）、薬剤に結合する抗体、C-反応性蛋白(CRP)、抗CRP抗体、サイトカイン類、サイトカイン類に結合する抗体、α−フェトプロテイン(AFP)、抗AFP抗体、癌胎児性抗原(CEA)、抗CEA抗体、CA19-9、抗CA19-9抗体、CA15-3、抗CA15-3抗体、CA-125、抗CA-125抗体、PIVKA-II、抗PIVKA-II抗体、副甲状腺ホルモン(PTH)、抗PTH抗体、ヒト絨毛性ゴナドトロピン(hCG)、抗hCG抗体、甲状腺刺激ホルモン(TSH)、抗TSH抗体、インスリン、抗インスリン抗体、C-ペプタイド、抗C-ペプタイド抗体、エストロゲン、抗エストロゲン抗体、線維芽細胞増殖因子-23(FGF-23)、抗FGF-23抗体、グルタミン酸脱炭酸酵素(GAD)、抗GAD抗体、ペプシノーゲン、抗ペプシノーゲン抗体、B型肝炎ウイルス(HBV)抗原、抗HBV抗体、C型肝炎ウイルス(HCV)抗原、抗HCV抗体、成人T細胞性白血病ウイルス1型(HTLV-I)抗原、抗HTLV-I抗体、ヒト免疫不全ウイルス(HIV)抗原、抗HIV抗体、インフルエンザウイルス抗原、抗インフルエンザウイルス抗体、結核菌抗原(TBGL)、抗結核菌抗体、マイコプラズマ抗原、抗マイコプラズマ抗体、ヘモグロビンA1c、抗ヘモグロビンA1c抗体、心房性ナトリウム利尿ペプチド(ANP)、抗ANP抗体、脳性ナトリウム利尿ペプチド(BNP)、抗BNP抗体、トロポニンT、抗トロポニンT抗体、トロポニンI、抗トロポニンI抗体、クレアチニンキナーゼ-MB(CK-MB)、抗CK-MB抗体、ミオグロビン、抗ミオグロビン抗体、L-FABP、抗L-FABP抗体、H-FABP、抗H-FABP抗体、CCP抗原、抗CCP抗体、SP-D、抗SP-D抗体、カビ毒類［デオキシニバレノール(DON)、ニバレノール(NIV)、T-2トキシン(T2)等］に結合する抗体、内分泌撹乱物質類［ビスフェノールA、ノニルフェノール、フタル酸ジブチル、ポリ塩素化ビフェニル(PCB)類、ダイオキシン類、ｐ，ｐ’−ジクロロジフェニルトリクロロエタン、トリブチルスズ等］に結合する抗体、ステロイドホルモン類（アルドステロン、テストステロン等）に結合する抗体、大腸菌等の菌類、菌類に結合する抗体、食物アレルギー物質ダニ類等のアレルギー物質、抗アレルギー物質抗体等が挙げられる。 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.
また、ビオチン化蛋白質の他に、ビオチン化炭化水素系化合物やビオチン化核酸も用いることができる。本発明のストレプトアビジン結合磁性粒子と、ビオチン化炭化水素系化合物とを反応させることにより、炭化水素系化合物結合磁性粒子を製造することができる。また、本発明のストレプトアビジン結合磁性粒子と、ビオチン化核酸とを反応させることにより、核酸結合磁性粒子を製造することができる。 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.
ビオチン化炭化水素系化合物における炭化水素系化合物としては、例えばカビ毒類［デオキシニバレノール(DON)、ニバレノール(NIV)、T-2トキシン(T2)等］、内分泌撹乱物質類［ビスフェノールA、ノニルフェノール、フタル酸ジブチル、ポリ塩素化ビフェニル(PCB)類、ダイオキシン類、ｐ，ｐ’−ジクロロジフェニルトリクロロエタン、トリブチルスズ等］、ステロイドホルモン類（アルドステロン、テストステロン等）等が挙げられる。 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.
ビオチン化核酸における核酸としては、例えばDNA、RNA、アプタマー、これらの誘導体等が挙げられる。 Examples of the nucleic acid in the biotinylated nucleic acid include DNA, RNA, aptamer, and derivatives thereof.
本発明の製造方法により得られるストレプトアビジン結合磁性粒子及び蛋白質結合磁性粒子を用いて、試料中の測定対象成分を測定方法することができる。さらに、本発明の製造方法により得られるストレプトアビジン結合磁性粒子と、ビオチン化蛋白質とを用いて、試料中の測定対象成分を測定することもできる。当該測定方法は、通常の磁性粒子を用いる免疫学的測定方法を用いることができ、サンドイッチ法、競合法等が挙げられる。試料としては、本発明の製造方法により得られるストレプトアビジン結合磁性粒子及び蛋白質結合磁性粒子を用いた測定対象成分の測定方法を可能とする試料であれば特に制限はなく、例えば全血、血漿、血清、髄液、唾液、羊水、尿、汗、膵液等が挙げられるが、血漿、血清等が好ましい。 Using the streptavidin-coupled magnetic particles and protein-coupled magnetic particles obtained by the production method of the present invention, a measurement target component in a sample can be measured. 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.
測定対象成分としては、本発明の製造方法により得られるストレプトアビジン結合磁性粒子及び蛋白質結合磁性粒子を用いた測定方法により測定され得るものであれば特に制限はなく、例えば以下の物質等が挙げられる。IgG、IgM、IgA、IgE、アポ蛋白AI、アポ蛋白AII、アポ蛋白B、アポ蛋白E、リウマチファクター、D-ダイマー、酸化LDL、糖化LDL、グリコアルブミン、トリヨードサイロニン(T3)、総サイロキシン(T4)、薬剤（抗テンカン剤等）、C-反応性蛋白(CRP)、サイトカイン類、α−フェトプロテイン(AFP)、癌胎児性抗原(CEA)、CA19-9、CA15-3、CA-125、PIVKA-II、副甲状腺ホルモン(PTH)、ヒト絨毛性ゴナドトロピン(hCG)、甲状腺刺激ホルモン(TSH)、インスリン、C-ペプタイド、エストロゲン、線維芽細胞増殖因子-23(FGF-23)、抗グルタミン酸脱炭酸酵素(GAD)抗体、ペプシノーゲン、B型肝炎ウイルス(HBV)抗原、抗HBV抗体、C型肝炎ウイルス(HCV)抗原、抗HCV抗体、成人T細胞性白血病ウイルス1型(HTLV-I)抗原、抗HTLV-I抗体、ヒト免疫不全ウイルス(HIV)抗体、インフルエンザウイルス抗原、抗インフルエンザウイルス抗体、抗結核菌抗体、結核菌抗原(TBGL)マイコプラズマ抗体、ヘモグロビンA1c、心房性ナトリウム利尿ペプチド(ANP)、脳性ナトリウム利尿ペプチド(BNP)、トロポニンT、トロポニンI、クレアチニンキナーゼ-MB(CK-MB)、ミオグロビン、L-FABP、H-FABP、抗CCP抗体、SP-D、カビ毒類［デオキシニバレノール(DON)、ニバレノール(NIV)、T-2トキシン(T2)等］、内分泌撹乱物質類［ビスフェノールA、ノニルフェノール、フタル酸ジブチル、ポリ塩素化ビフェニル(PCB)類、ダイオキシン類、ｐ，ｐ’−ジクロロジフェニルトリクロロエタン、トリブチルスズ等］、ステロイドホルモン類（アルドステロン、テストステロン等）、大腸菌等の菌類、食物アレルギー物質ダニ類等のアレルギー物質、抗アレルギー物質抗体等が挙げられる。 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.
また、蛋白質結合磁性粒子の代わりに、本発明の製造方法により得られるストレプトアビジン結合磁性粒子とビオチン化炭化水素系化合物とを用いて製造される炭化水素系化合物結合磁性粒子を用いて、又は、蛋白質結合磁性粒子の代わりに、本発明の製造方法により得られるストレプトアビジン結合磁性粒子とビオチン化炭化水素系化合物とを用いて、試料中の測定対象成分を測定することができる。測定対象成分としては、例えばビオチン化炭化水素系化合物を構成する炭化水素系化合物や当該炭化水素系化合物に結合する抗体等が挙げられる。炭化水素系化合物としては、例えば前述の炭化水素系化合物等が挙げられる。ビオチン化炭化水素系化合物を用いた試料中の測定対象成分の測定は、例えばサンドイッチ法や競合法等の通常の免疫学的測定方法を用いて行うことができる。 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.
さらに、蛋白質結合磁性粒子の代わりに、本発明の製造方法により得られるストレプトアビジン結合磁性粒子とビオチン化核酸とを用いて製造される核酸結合磁性粒子を用いて、又は、蛋白質結合磁性粒子の代わりに、本発明の製造方法により得られるストレプトアビジン結合磁性粒子とビオチン化核酸とを用いて、試料中の測定対象成分を測定することができる。測定対象成分としては、例えばビオチン核酸を構成する核酸に結合する核酸や蛋白質等が挙げられる。蛋白質としては、例えば前述の蛋白質等が挙げられる。ビオチン化核酸を用いた試料中の測定対象成分の測定は、通常の核酸測定方法や、通常の免疫学的測定方法を用いて行うことができる。 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.
以下、実施例により本発明をより詳細に説明するが、これらは本発明の範囲を何ら限定するものではない。 EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, these do not limit the scope of the present invention at all.
（１）ストレプトアビジン結合磁性粒子の製造
磁性粒子にはアミノ基タイプEstapor磁性粒子 EM2-100/40（メルク社製）を用いた。当該磁性粒子はコア・シェル構造から成り、粒径が1.62μmであり、内部コア部分には全質量比41.2%の磁性体を含み、ポリスチレンからなるシェル部分には化学的にアミノ基が97μeq/gで修飾されている粒子である。(1) Production of Streptavidin-Coupled Magnetic Particles Amino group type Estapor magnetic particles EM2-100 / 40 (Merck) were used as magnetic particles. 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 / amino group chemically. Particles modified with g.
当該磁性粒子を100 mg採取し、1.0%のTrimethylstearylammonium Chloride（東京化成社製）を含むpH5.5、10 mmol/L酢酸緩衝液（以下、分散液Aという）を4 mL加え、分散させた。続いて容器横に強力磁石を配することで磁性粒子を収集し、分散液Aを吸引除去した（以下、分散液の添加、磁性粒子の収集、吸引除去の一連の操作を「洗浄」と略す）。該洗浄を続けて4回行った。 100 mg of the magnetic particles were collected, and 4 mL of pH 5.5, 10 mmol / L acetic acid buffer (hereinafter referred to as dispersion A) containing 1.0% Trimethylstearylammonium Chloride (manufactured by Tokyo Chemical Industry Co., Ltd.) was added and dispersed. 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 adding the dispersion, collecting the magnetic particles, and removing the suction was abbreviated as “washing”. ). The washing was continued 4 times.
次に、分散液Aを1.5 mL加え、磁性粒子を十分に分散させた後、25%グルタルアルデヒド水溶液（ナカライテスク社製）を3.5 mL加え、シェーキングインキュベーター（アズワン社製、SI-300C）により1,500 rpmで振盪しながら37℃で2時間インキュベートした。 Next, 1.5 mL of dispersion A was added to sufficiently disperse the magnetic particles, and then 3.5 mL of 25% glutaraldehyde aqueous solution (manufactured by Nacalai Tesque) was added and shaken incubator (manufactured by ASONE, SI-300C). Incubate for 2 hours at 37 ° C. with shaking at 1,500 rpm.
次に0.1%のTrimethylstearylammonium Chloride（東京化成社製）を含むpH5.5、10 mmol/L酢酸緩衝液（以下、分散液Bという）を用いて、洗浄操作を10回行った。分散液Bはあらかじめ25℃の恒温槽でインキュベートしておき、25℃の環境下で作業を行った。また洗浄は最初に連続して6回行い、洗浄開始から3時間30分後に3回、4時間後に1回行った（以後、得られた粒子を「活性化粒子」と略す）。 Next, the washing operation was performed 10 times using a pH 5.5, 10 mmol / L acetate buffer solution (hereinafter referred to as dispersion B) containing 0.1% Trimethylstearylammonium Chloride (manufactured by Tokyo Chemical Industry Co., Ltd.). Dispersion B was preliminarily incubated in a constant temperature bath at 25 ° C., and the work was performed in an environment at 25 ° C. In addition, washing was performed 6 times in succession, 3 times after 3 hours 30 minutes and once after 4 hours (hereinafter, the obtained particles are abbreviated as “activated particles”).
遺伝子組み換え体のストレプトアビジン（ロシュ社製）を、pH5.5、10 mmol/L酢酸緩衝液に24.5 mg/mLの濃度となるように溶解し、ストレプトアビジン溶液を調製し、氷冷で1時間以上静置した。活性化粒子から分散液Bを除去した後、ストレプトアビジン溶液1.4 mLを添加し、素早く分散させた。このままシェーキングインキュベーターにより1,500 rpmで振盪しながら40℃で16時間インキュベートした。次に、２−ピコリンボラン（純正化学社製）の0.53 mg/mLメタノール溶液(200μL)を添加し、シェーキングインキュベーターにより1,500 rpmで振盪しながら40℃で6日間インキュベートした。得られた反応混合物から、磁石により磁性粒子とそれ以外の溶液成分とに分離し、分離された磁性粒子を1.0%のBSA、0.09%のアジ化ナトリウムを含む50 mmol/LのMES緩衝液(pH6.5)で10回洗浄し、ストレプトアビジン結合磁性粒子を得た。 Recombinant Streptavidin (Roche) is dissolved in pH 5.5, 10 mmol / L acetate buffer to a concentration of 24.5 mg / mL, and a streptavidin solution is prepared. It was left above. After removing dispersion B from the activated particles, 1.4 mL of a streptavidin solution was added and dispersed quickly. 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 incubated at 40 ° C. for 6 days with 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 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.
（２）ストレプトアビジン結合磁性粒子のビオチン結合能の測定
上記（１）で得られたストレプトアビジン結合磁性粒子、及び、市販のストレプトアビジン結合磁性粒子について、以下の方法により、ビオチン結合能を測定した。(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. .
ストレプトアビジン結合磁性粒子を0.1%BSA・PBS［PBS：0.15 mol/L 塩化ナトリウムを含有する10 mmol/L リン酸緩衝液(pH7.2)］に1 mg/mLで分散させ、倍々希釈法で0.0156 mg/mLの6段階（64倍希釈）まで希釈した。この6サンプルとブランク(0.1%BSA・PBS)それぞれを96穴ブラックプレートに50μLずつ分注した。次にBiotin-Fluorescein (Thermo Scientific社製)を0.1%BSA・PBSで1μg/mLに希釈し、サンプルを分注したウエルに50μLずつ分注した。サンプルを分注したプレートはシェーカーインキュベーター(アマライト社製)で振盪しながら37℃で10分間インキュベートし、粒子が分散した状態での蛍光強度を蛍光プレートリーダー“プレートカメレオンＶ”（HIDEX社製）で測定した。 Streptavidin-binding magnetic particles are dispersed at 1 mg / mL in 0.1% BSA / PBS [PBS: 10 mmol / L phosphate buffer (pH 7.2) containing 0.15 mol / L sodium chloride] It diluted to 6 steps (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.
ここで磁性粒子上のストレプトアビジンに蛍光標識ビオチンが結合すると、ストレプトアビジンに結合した蛍光標識ビオチンどうしが近接して存在することになり、蛍光の消光が起こる。蛍光の消光は単位面積当たりの磁性粒子に結合したストレプトアビジンへの蛍光標識ビオチンの結合量が大きくなるほど高くなる。この性質を利用し、ビオチン結合能が既知の市販の磁性粒子をレファレンスとして、このストレプトアビジンの蛍光減少率をあらかじめ評価しておくことにより、本発明のストレプトアビジン結合磁性粒子のビオチン結合能を算出した。本実施例ではストレプトアビジン結合磁性粒子の希釈サンプルから、蛍光強度が５０％減少した時のストレプトアビジン結合磁性粒子濃度を直線近似で算出し、レファレンスのストレプトアビジンと比較することで、ビオチン結合能(pmol/mm2)を算出した。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.
測定結果を第１表に示す。 The measurement results are shown in Table 1.
第１表から明らかな様に、上記（１）で得られたストレプトアビジン結合磁性粒子は、市販のストレプトアビジン結合磁性粒子（ダイナル社製 DynabeadsT1、及びメルク社製 BE-M08/10）に比較して、高いビオチン結合能を有することが分かった。 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.
磁性粒子上のストレプトアビジンの架橋構造の分析
実施例１で得られたストレプトアビジン結合磁性粒子について、磁性粒子上のストレプトアビジンの架橋構造は以下の方法で確認した。ストレプトアビジン結合磁性粒子20 mgを5 mLのPBSで10回洗浄し、1%SDS/PBSで20 mg/mLとした。次に60℃のインキュベーターで1時間インキュベートし、粒子を除いた上清をSDS-PAGEで分析した。Analysis of Streptavidin Crosslinked Structure on Magnetic Particles For the 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.
本法によるストレプトアビジン結合磁性粒子、及び、市販のストレプトアビジン結合磁性粒子におけるストレプトアビジンの構造をSDS-PAGEで確認した泳動像を図１に示す。 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.
図１から明らかな様に、ストレプトアビジン、及び、市販のストレプトアビジン結合磁性粒子においては、ストレプトアビジンを構成する単量体のみが認められたのに対して、本発明の製造方法により製造されるストレプトアビジン結合磁性粒子においては、単量体に加え、二量体、三量体、四量体及びさらに高次のバンドが認められた。これにより、市販のストレプトアビジン結合磁性粒子においては、ストレプトアビジンが架橋構造を取っていないのに対して、本発明の製造方法で得られたストレプトアビジン結合磁性粒子においては、ストレプトアビジンが架橋構造を取っていることが判明した。 As is clear from FIG. 1, in 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. In streptavidin-coupled magnetic particles, dimers, trimers, tetramers and higher order bands were observed in addition to the monomers. Thus, in the commercially available streptavidin-coupled magnetic particles, streptavidin does not have a crosslinked structure, whereas in the streptavidin-coupled magnetic particles obtained by the production method of the present invention, streptavidin has a crosslinked structure. Turned out to be taking.
本発明により、ビオチン結合能の高いストレプトアビジン結合磁性粒子の製造方法、及び、当該ストレプトアビジン結合磁性粒子を用いる、蛋白質結合磁性粒子の製造方法が提供される。本発明の製造方法により製造されるストレプトアビジン結合磁性粒子、及び、蛋白質結合磁性粒子は、臨床診断上、有用である。 The present invention provides a method for producing streptavidin-coupled magnetic particles having a high biotin-binding ability, and a method for producing protein-bound 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.
Claims (4)
（１）表面にアミノ基を有する磁性粒子をグルタルアルデヒドと反応させる工程；及び、
（２）工程（１）で得られた磁性粒子を、遊離のグルタルアルデヒド存在下、ストレプトアビジンと反応させる工程。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.
（３）工程（２）で調製したストレプトアビジン結合磁性粒子と還元剤とを反応させる工程。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.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2011029332 | 2011-02-15 | ||
| JP2011029332 | 2011-02-15 | ||
| PCT/JP2012/053465 WO2012111687A1 (en) | 2011-02-15 | 2012-02-15 | Manufacturing method for streptavidin-bonded magnetic particles |
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| JP6920683B2 (en) | 2015-10-08 | 2021-08-18 | 凸版印刷株式会社 | Method and kit for concentrating target double-stranded nucleic acid molecules using pyrrole imidazole-containing polyamide |
| JP6218916B1 (en) | 2016-12-28 | 2017-10-25 | Jsr株式会社 | Magnetic particle dispersion |
| WO2018165475A1 (en) | 2017-03-08 | 2018-09-13 | California Institute Of Technology | Pairing antigen specificity of a t cell with t cell receptor sequences |
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| JPH07209166A (en) * | 1993-12-13 | 1995-08-11 | Hewlett Packard Co <Hp> | Bonding of specimen to surface of base body, determination of specimen having low molecular weight and biotin-specimen composite |
| JP2000146965A (en) * | 1998-11-06 | 2000-05-26 | Iatron Lab Inc | Immunological analysis reagent, immunological analysis method and immunological analysis kit |
| JP2005523027A (en) * | 2002-04-22 | 2005-08-04 | ユニヴァーシティ オヴ フロリダ | Functionalized nanoparticles and methods of use |
| JP2005526226A (en) * | 2001-08-10 | 2005-09-02 | エフ．ホフマン−ラ ロシュ アーゲー | Method for producing fine particles loaded with protein |
| JP2006042654A (en) * | 2004-08-03 | 2006-02-16 | Onchip Cellomics Consortium | Method for separating and collecting cell, cell separation chip and cell separator |
| JP2008501122A (en) * | 2004-05-28 | 2008-01-17 | ナノゲン・インコーポレイテッド | Nanoscale electronic detection system and manufacturing method thereof |
| US20100267166A1 (en) * | 2007-03-01 | 2010-10-21 | Church & Dwight Co., Inc. | Diagnostic detection device |
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- 2012-02-15 WO PCT/JP2012/053465 patent/WO2012111687A1/en active Application Filing
- 2012-02-15 JP JP2012557982A patent/JPWO2012111687A1/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07209166A (en) * | 1993-12-13 | 1995-08-11 | Hewlett Packard Co <Hp> | Bonding of specimen to surface of base body, determination of specimen having low molecular weight and biotin-specimen composite |
| JP2000146965A (en) * | 1998-11-06 | 2000-05-26 | Iatron Lab Inc | Immunological analysis reagent, immunological analysis method and immunological analysis kit |
| JP2005526226A (en) * | 2001-08-10 | 2005-09-02 | エフ．ホフマン−ラ ロシュ アーゲー | Method for producing fine particles loaded with protein |
| JP2005523027A (en) * | 2002-04-22 | 2005-08-04 | ユニヴァーシティ オヴ フロリダ | Functionalized nanoparticles and methods of use |
| JP2008501122A (en) * | 2004-05-28 | 2008-01-17 | ナノゲン・インコーポレイテッド | Nanoscale electronic detection system and manufacturing method thereof |
| JP2006042654A (en) * | 2004-08-03 | 2006-02-16 | Onchip Cellomics Consortium | Method for separating and collecting cell, cell separation chip and cell separator |
| US20100267166A1 (en) * | 2007-03-01 | 2010-10-21 | Church & Dwight Co., Inc. | Diagnostic detection device |
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