JP2009171853A - Method and kit for collecting charged entity by magnetic fine particle with surface modified by temperature responsive polymer having higher critical solution temperature - Google Patents
Method and kit for collecting charged entity by magnetic fine particle with surface modified by temperature responsive polymer having higher critical solution temperature Download PDFInfo
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- JP2009171853A JP2009171853A JP2008010831A JP2008010831A JP2009171853A JP 2009171853 A JP2009171853 A JP 2009171853A JP 2008010831 A JP2008010831 A JP 2008010831A JP 2008010831 A JP2008010831 A JP 2008010831A JP 2009171853 A JP2009171853 A JP 2009171853A
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- temperature
- magnetic fine
- adsorbent
- fine particles
- responsive
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Images
Abstract
Description
本発明は、上限臨界溶液温度を有する温度応答性ポリマーで表面修飾された磁性微粒子による電荷物質の回収方法及びこれを用いた電荷物質を回収するためのキットに関する。 The present invention relates to a method for recovering a charged substance using magnetic fine particles surface-modified with a temperature-responsive polymer having an upper critical solution temperature, and a kit for recovering a charged substance using the same.
核酸等の電荷物質を、上限臨界溶液温度を有する温度応答性ポリマーで表面修飾された磁性微粒子を用いて回収を行うと、電荷物質が存在することから、磁性微粒子の凝集が阻害される。したがって、回収するのに時間がかかり、回収率が低く、改良が望まれている（例えば、特許文献１参照）。 When a charged substance such as a nucleic acid is recovered using magnetic fine particles whose surface is modified with a temperature-responsive polymer having an upper critical solution temperature, aggregation of the magnetic fine particles is inhibited due to the presence of the charged substance. Therefore, it takes a long time to recover, the recovery rate is low, and improvement is desired (for example, see Patent Document 1).
本発明の課題は、核酸等の電荷物質を、上限臨界溶液温度を有する温度応答性ポリマーで表面修飾された磁性微粒子を用いて回収する場合において、回収する時間を短縮し、回収率を高める方法を提供することである。 An object of the present invention is a method for shortening the collection time and increasing the collection rate when collecting charged substances such as nucleic acids using magnetic fine particles whose surface is modified with a temperature-responsive polymer having an upper critical solution temperature. Is to provide.
本発明者らは、上記課題を解決するために鋭意検討を重ねた。その結果、以下の構成を採用することにより、本発明の課題を解決することができることを見出し、この知見に基づいて本発明を完成させた。 The present inventors have made extensive studies to solve the above problems. As a result, it has been found that the problems of the present invention can be solved by adopting the following configuration, and the present invention has been completed based on this finding.
本発明は以下の構成を有する。
［１］検体中の電荷物質を回収する方法であって、
上限臨界溶液温度を有する温度応答性ポリマーで表面修飾された磁性微粒子と、電荷物質に対する親和性物質とが結合した吸着剤と、電荷物質を含む検体とを混合し、吸着剤に電荷物質を吸着させ、さらにポリアルキレングリコールと混合する工程、
前記工程で得られた混合物を上限臨界溶液温度未満にし、吸着剤を凝集させる工程、及び、
磁力により吸着剤を回収する工程、
を含む、検体中の電荷物質を回収する方法。
［２］前記上限臨界溶液温度を有する温度応答性ポリマーが、アクリロイルグリシンアミド、メタクリロイルグリシンアミド、アクリロイルアスパラギンアミド、メタクリロイルアスパラギンアミド、アクリロイルグルタミンアミド及びメタクリロイルグルタミンアミドからなる群から選ばれる少なくとも１種類のモノマーを重合して得られるポリマーである、［１］に記載の方法。
［３］前記上限臨界溶液温度を有する温度応答性ポリマーが、少なくとも２種類のモノマーを共重合して得られるコポリマーである、［２］に記載の方法。
［４］前記上限臨界溶液温度を有する温度応答性ポリマーが、アクリロイルグリシンアミドとアクリロイルアスパラギンアミドとを共重合して得られるコポリマーである、［３］に記載の方法。
［５］前記ポリアルキレングリコールが、ポリエチレングリコール、ポリプロピレングリコール、ポリエチレングリコールポリプロピレングリコールランダムコポリマー及びポリエチレングリコールポリプロピレングリコールブロックコポリマーからなる群から選ばれる少なくとも１種である、［１］〜［４］のいずれかに記載の方法。
［６］前記ポリアルキレングリコールが、ポリエチレングリコールである、［５］に記載の方法。
［７］前記電荷物質が、核酸である、［１］〜［６］のいずれかに記載の方法。
［８］前記核酸が、ＤＮＡ又はｍＲＮＡである、［７］に記載の方法。
［９］上限臨界溶液温度を有する温度応答性ポリマーで表面修飾された磁性微粒子と、電荷物質に対する親和性物質とが結合した吸着剤と、ポリアルキレングリコールを含む、検体中の電荷物質を回収するためのキット。
The present invention has the following configuration.
[1] A method for recovering charged substances in a specimen,
Adsorbing the charged substance to the adsorbent by mixing the adsorbent with the magnetic fine particle surface-modified with the temperature-responsive polymer having the upper critical solution temperature, the adsorbent combined with the affinity substance for the charged substance, and the specimen containing the charged substance And further mixing with polyalkylene glycol,
Bringing the mixture obtained in the previous step below the upper critical solution temperature and aggregating the adsorbent; and
Recovering the adsorbent by magnetic force,
A method for recovering a charged substance in a specimen, comprising:
[2] The temperature-responsive polymer having the upper critical solution temperature is at least one monomer selected from the group consisting of acryloyl glycinamide, methacryloyl glycinamide, acryloyl asparagine amide, methacryloyl asparagine amide, acryloyl glutamine amide, and methacryloyl glutamine amide. The method according to [1], which is a polymer obtained by polymerizing
[3] The method according to [2], wherein the temperature-responsive polymer having the upper critical solution temperature is a copolymer obtained by copolymerizing at least two types of monomers.
[4] The method according to [3], wherein the temperature-responsive polymer having the upper critical solution temperature is a copolymer obtained by copolymerizing acryloylglycinamide and acryloylasparagine amide.
[5] Any of [1] to [4], wherein the polyalkylene glycol is at least one selected from the group consisting of polyethylene glycol, polypropylene glycol, polyethylene glycol polypropylene glycol random copolymer, and polyethylene glycol polypropylene glycol block copolymer. The method described in 1.
[6] The method according to [5], wherein the polyalkylene glycol is polyethylene glycol.
[7] The method according to any one of [1] to [6], wherein the charged substance is a nucleic acid.
[8] The method according to [7], wherein the nucleic acid is DNA or mRNA.
[9] Collecting a charged substance in a specimen, including an adsorbent in which a magnetic fine particle surface-modified with a temperature-responsive polymer having an upper critical solution temperature, an affinity substance for the charged substance, and a polyalkylene glycol are bound. Kit for.
本発明の回収方法を用いることで、核酸等の電荷物質を、上限臨界溶液温度を有する温度応答性ポリマーで表面修飾された磁性微粒子を用いて回収する場合に、回収する時間を大幅に短縮することができ、また、回収率を高くすることができる。 By using the recovery method of the present invention, when recovering charged substances such as nucleic acids using magnetic fine particles whose surface is modified with a temperature-responsive polymer having an upper critical solution temperature, the recovery time is significantly shortened. In addition, the recovery rate can be increased.
以下、本発明について詳細に説明する。
１．電荷物質を回収する方法
本発明の方法は、検体中の電荷物質を回収する方法であって、上限臨界溶液温度を有する温度応答性ポリマーで表面修飾された磁性微粒子（以下、単に「温度応答性磁性微粒子」ということがある。）と電荷物質に対する親和性物質（以下、単に「リガンド」ということがある。）とが結合した吸着剤とを混合し、吸着剤に電荷物質を吸着させ、さらにポリアルキレングリコールと混合し、得られた混合物を温度応答性ポリマーが凝集する温度（上限臨界溶液温度）未満にすることにより、吸着剤を凝集させ、磁力により吸着剤を回収し、検体中の電荷物質を回収する方法である。
Hereinafter, the present invention will be described in detail.
1. Method of recovering charged substance The method of the present invention is a method of recovering a charged substance in a specimen, which is a magnetic fine particle surface-modified with a temperature-responsive polymer having an upper critical solution temperature (hereinafter simply referred to as “temperature responsiveness”). A magnetic fine particle ”) and an adsorbent in which an affinity substance for a charged substance (hereinafter sometimes simply referred to as“ ligand ”) is mixed to adsorb the charged substance to the adsorbent, and Mixing with polyalkylene glycol and lowering the resulting mixture below the temperature at which the temperature-responsive polymer aggregates (upper critical solution temperature) causes the adsorbent to aggregate, collect the adsorbent by magnetic force, and charge in the specimen This is a method for recovering a substance.
本発明の方法は、電荷物質を吸着させた吸着剤とポリアルキレングリコールとを混合する工程を含む。通常、電荷物質が存在すると、吸着剤の凝集が阻害されるが、吸着剤とポリアルキレングリコールとを混合することにより、吸着剤の凝集能が強化され、温度応答性ポリマーの上限臨界溶液温度が上昇することにより、電荷物質が存在しても吸着剤の凝集が可能又は容易となる。 The method of the present invention includes a step of mixing an adsorbent adsorbed with a charged substance and polyalkylene glycol. In general, the presence of a charged substance inhibits the aggregation of the adsorbent, but mixing the adsorbent and the polyalkylene glycol enhances the aggregating ability of the adsorbent and increases the upper critical solution temperature of the temperature-responsive polymer. By raising, the adsorbent can be aggregated or facilitated even in the presence of a charged substance.
２．温度応答性磁性微粒子
本発明に用いる温度応答性磁性微粒子は、磁性微粒子に上限臨界溶液温度を有する温度応答性ポリマーが表面修飾された粒子である。表面修飾とは、前記ポリマーが、磁性微粒子の表面に直接又は間接に化学的に固定されている状態や、磁性微粒子の表面に直接又は間接に絡まった状態も含む。ここで、間接とは、磁性微粒子と温度応答性ポリマーとが、他の物質を介していることをいう。温度応答性磁性微粒子の平均粒径は、１〜１０００ｎｍであることが好ましく、より好ましくは３〜２００ｎｍである。
2. Temperature-responsive magnetic fine particles The temperature-responsive magnetic fine particles used in the present invention are particles in which a temperature-responsive polymer having an upper critical solution temperature is surface-modified on the magnetic fine particles. The surface modification includes a state in which the polymer is chemically fixed directly or indirectly to the surface of the magnetic fine particles and a state in which the polymer is directly or indirectly entangled with the surface of the magnetic fine particles. Here, indirect means that the magnetic fine particles and the temperature-responsive polymer are via another substance. The average particle diameter of the temperature-responsive magnetic fine particles is preferably 1 to 1000 nm, more preferably 3 to 200 nm.
２−１．上限臨界溶液温度を有する温度応答性ポリマー
温度応答性ポリマーとは、溶液中で温度変化により可逆的に凝集状態と溶解状態とに変化するポリマーをいう。温度応答性ポリマーとしては、下限臨界溶液温度（以下、「ＬＣＳＴ」ということがある。）を有する温度応答性ポリマーと、上限臨界溶液温度（以下、「ＵＣＳＴ」ということがある。）を有する温度応答性ポリマーとが知られているが、本発明には、上限臨界溶液温度を有する温度応答性ポリマーを用いる。
2-1. Temperature-responsive polymer having an upper critical solution temperature A temperature-responsive polymer refers to a polymer that reversibly changes into an aggregated state and a dissolved state in solution due to temperature change. As the temperature-responsive polymer, a temperature-responsive polymer having a lower critical solution temperature (hereinafter sometimes referred to as “LCST”) and a temperature having an upper critical solution temperature (hereinafter sometimes referred to as “UCST”). Although a responsive polymer is known, a temperature responsive polymer having an upper critical solution temperature is used in the present invention.
本発明に用いる上限臨界溶液温度を有する温度応答性ポリマーは特に限定されないが、分散状態では回収困難な磁性微粒子に前記温度応答性ポリマーを表面修飾することで、ＵＣＳＴ未満に温度を低下させることにより、回収可能な大きさの塊に温度応答性磁性微粒子を凝集させる能力を有していることが必要である。逆に、ＵＣＳＴ以上に温度を上昇させることにより、凝集した温度応答性磁性微粒子を分散させることができる。 The temperature-responsive polymer having the upper critical solution temperature used in the present invention is not particularly limited, but by surface-modifying the temperature-responsive polymer to magnetic fine particles that are difficult to recover in a dispersed state, by reducing the temperature below UCST. It is necessary to have the ability to agglomerate temperature-responsive magnetic fine particles in a lump of a size that can be recovered. Conversely, the temperature-responsive magnetic fine particles can be dispersed by raising the temperature to more than UCST.
上限臨界溶液温度を有する温度応答性ポリマーとしては、アクリロイルグリシンアミド、メタクリロイルグリシンアミド、アクリロイルアスパラギンアミド、メタクリロイルアスパラギンアミド、アクリロイルニペコタミド、アクリロイルグルタミンアミド、メタクリロイルグルタミンアミド等から選ばれるモノマーを単独で重合して得られるホモポリマー又は少なくとも２種類のモノマーを共重合して得られるコポリマーを挙げることができる。また、上記モノマーとアクリルアミド、アセチルアクリルアミド、ビオチノールアクリレート、Ｎ−ビオチニル−Ｎ′−メタクロイルトリメチレンアミド、アクリロイルザルコシンアミド、メタクリロイルザルコシンアミド及びアクリロイルメチルウラシル等から選ばれる少なくとも１種類のモノマーとを共重合して得られるコポリマーを挙げることができる。同コポリマー中、温度応答性ポリマーを構成するモノマー含量は、通常、コポリマーを構成する全モノマー含有量の９５ｍｏｌ％以上である。 As a temperature-responsive polymer having an upper critical solution temperature, a monomer selected from acryloyl glycinamide, methacryloyl glycinamide, acryloyl asparagine amide, methacryloyl asparagine amide, acryloyl nipecotamide, acryloyl glutamine amide, methacryloyl glutamine amide alone is polymerized. And a copolymer obtained by copolymerizing at least two kinds of monomers. In addition, at least one monomer selected from the above monomers and acrylamide, acetylacrylamide, biotinol acrylate, N-biotinyl-N′-methacryloyl trimethylene amide, acryloyl sarcosine amide, methacryloyl sarcosine amide, acryloylmethyl uracil and the like. And a copolymer obtained by copolymerization of In the copolymer, the monomer content constituting the temperature-responsive polymer is usually 95 mol% or more of the total monomer content constituting the copolymer.
上限臨界溶液温度を有する温度応答性ポリマーのうち、ホモポリマーとしては、例えば、ポリ（アクリロイルグリシンアミド）、ポリ（アクリロイルアスパラギンアミド）、又はポリ（アクリロイルグルタミンアミド）等が好ましく利用できる。また、コポリマーとしては、例えば、アクリロイルグリシンアミドとアクリロイルアスパラギンアミドとのコポリマー等が好ましく利用できる。
上限臨界溶液温度を有する温度応答性ポリマーのＵＣＳＴは、このポリマーを構成するモノマーの種類及びモノマー成分の比率により様々に変化させることができる。
Among the temperature-responsive polymers having the upper critical solution temperature, as the homopolymer, for example, poly (acryloylglycinamide), poly (acryloylasparagineamide), poly (acryloylglutamineamide), or the like can be preferably used. As the copolymer, for example, a copolymer of acryloylglycinamide and acryloyl asparagine amide can be preferably used.
The UCST of a temperature-responsive polymer having an upper critical solution temperature can be variously changed depending on the type of monomer constituting the polymer and the ratio of monomer components.
本発明に好適に用いることができる温度応答性ポリマーの重合度は、通常５０〜１００００である。 The degree of polymerization of the temperature-responsive polymer that can be suitably used in the present invention is usually 50 to 10,000.
温度応答性ポリマーは、例えば、上記モノマーを有機溶媒又は水に溶解し、不活性ガスで系中を置換した後、重合温度まで昇温し、有機溶媒中であればアゾビスイソブチロニトリル等のアゾ系開始剤、過酸化ベンゾイル等の過酸化物、水系であれば過硫酸アンモニウム、過硫酸カリウム、２，２’−アゾビス（２−アミジノプロパン）二塩酸塩、４，４’−アゾビス（４−シアノ吉草酸）等の重合開始剤を添加し、攪拌下加熱を続けることにより得ることができる。その後、貧溶媒中で再沈殿を行い、析出したポリマーをろ取することで、製造したポリマーを精製することができる。 For example, the temperature-responsive polymer is obtained by dissolving the monomer in an organic solvent or water and replacing the system with an inert gas, then raising the temperature to the polymerization temperature, and azobisisobutyronitrile in the organic solvent. Azo initiators, peroxides such as benzoyl peroxide, ammonium persulfate, potassium persulfate, 2,2′-azobis (2-amidinopropane) dihydrochloride, 4,4′-azobis (4 It can be obtained by adding a polymerization initiator such as -cyanovaleric acid) and continuing heating with stirring. Thereafter, reprecipitation is performed in a poor solvent, and the produced polymer can be purified by filtering the precipitated polymer.
２−２．磁性微粒子
磁性微粒子は、多価アルコールとマグネタイトとから製造することができる。例えば、特開２００５−０８２５３８号公報に、デキストランを用いた磁性微粒子の製造方法が開示されており、この方法によって製造することができる。
2-2. Magnetic fine particles Magnetic fine particles can be produced from polyhydric alcohol and magnetite. For example, JP 2005-082538 A discloses a method for producing magnetic fine particles using dextran, which can be produced by this method.
磁性微粒子は特に制限はないが、平均粒径が、０．９ｎｍ以上、１０００ｎｍ未満であることが好ましい。この範囲であれば、温度応答性ポリマーで表面修飾を行った後で良好な分散性を有する。さらに、目的物である電荷物質の認識性を高め、親和性物質との吸着速度を高めるためには、平均粒径が、２．９ｎｍ以上、２００ｎｍ未満であることが特に好ましい。また、特に、吸着剤の回収において、磁集速度を速めるためには、３０ｎｍ以上が好ましく、４０ｎｍ以上がより好ましい。 The magnetic fine particles are not particularly limited, but the average particle diameter is preferably 0.9 nm or more and less than 1000 nm. If it is this range, it will have favorable dispersibility after surface modification with a temperature-responsive polymer. Furthermore, in order to increase the recognizability of the target charged substance and increase the adsorption rate with the affinity substance, the average particle size is particularly preferably 2.9 nm or more and less than 200 nm. In particular, in the recovery of the adsorbent, in order to increase the magnetic collection speed, 30 nm or more is preferable, and 40 nm or more is more preferable.
磁性微粒子の素材としては、例えば、マグネタイト、酸化ニッケル、フェライト、コバルト鉄酸化物、バリウムフェライト、炭素鋼、タングステン鋼、ＫＳ鋼、希土類コバルト磁石及びヘマタイト等の微粒子が挙げられる。 Examples of the magnetic fine particle material include fine particles such as magnetite, nickel oxide, ferrite, cobalt iron oxide, barium ferrite, carbon steel, tungsten steel, KS steel, rare earth cobalt magnet, and hematite.
２−３．温度応答性ポリマーと磁性微粒子との結合
磁性微粒子を温度応答性ポリマーの表面に固定するためには、例えば、多価アルコール
（以下、多価アルコール誘導体を含めて、「多価アルコール等」という。）等を介して行うことができる。磁性微粒子と、多価アルコール等との結合体は、当技術分野における周知の方法によって調製することができる。例えば、米国特許第４４５２７７３号に記載されているように、デキストラン５０重量％水溶液（１０ｍｌ）中に、塩化第二鉄・六水和物（１．５１ｇ）及び塩化第一鉄・四水和物（０．６４ｇ）混合水溶液（１０ｍｌ）を加えて攪拌し、７．４（Ｖ／Ｖ）％アンモニア水溶液をｐＨ１０〜１１程度になるように滴下しながら、６０〜６５℃に水浴中で加熱し、１５分反応させる方法で得ることができる。
2-3. Bonding of temperature-responsive polymer and magnetic fine particles In order to fix the magnetic fine particles on the surface of the temperature-responsive polymer, for example, polyhydric alcohol (hereinafter referred to as “polyhydric alcohol or the like including polyhydric alcohol derivatives”). ) And the like. A conjugate of magnetic fine particles and polyhydric alcohol can be prepared by a well-known method in this technical field. For example, as described in U.S. Pat. No. 4,452,773, ferric chloride hexahydrate (1.51 g) and ferrous chloride tetrahydrate in a 50% by weight aqueous solution (10 ml) of dextran. (0.64 g) Add a mixed aqueous solution (10 ml), stir, and heat in a water bath at 60-65 ° C. while dropping a 7.4 (V / V)% aqueous ammonia solution to a pH of about 10-11. For 15 minutes.
磁性微粒子において、磁性微粒子上の鉄イオンと、多価アルコール等の有する水酸基、カルボキシル、リン酸基等の基との結合により、磁性微粒子と多価アルコール等とが結合される。多価アルコール等の有する水酸基は、温度応答性ポリマーとの結合に利用されるだけでなく、電荷物質に対する親和性物質（リガンド）又はリガンドと結合した物質に対する親和性物質の有する反応性官能基との結合に利用され、更に磁性微粒子の水溶液中での分散性の向上に効果がある。そのため、本発明に用いる多価アルコールは、構成単位に水酸基を少なくとも２個有し、鉄イオンと結合可能なアルコール構造体であれば、特に制限なく使用することができる。例えば、デキストラン、ポリビニルアルコール、マンニトール、ソルビトール等が挙げられる。また、グリシジルメタクリレート重合体のようにエポキシを有し、開環後多価アルコール構造体を形成する化合物も使用できる。多価アルコール誘導体としては、修飾により、カルボキシル、アミノ、エポキシ、チオール、メタクリル又はアクリル等の反応性官能基や重合性基が導入された多価アルコールが使用できる。 In the magnetic fine particle, the magnetic fine particle and the polyhydric alcohol are bonded by the bond between the iron ion on the magnetic fine particle and a group such as a hydroxyl group, a carboxyl, and a phosphate group that the polyhydric alcohol has. Hydroxyl groups such as polyhydric alcohols are not only used for bonding with temperature-responsive polymers, but also have reactive functional groups possessed by affinity substances (ligands) for charged substances or affinity substances for substances bound to ligands. And is effective in improving the dispersibility of the magnetic fine particles in an aqueous solution. Therefore, the polyhydric alcohol used in the present invention can be used without particular limitation as long as it is an alcohol structure having at least two hydroxyl groups in the structural unit and capable of binding to iron ions. For example, dextran, polyvinyl alcohol, mannitol, sorbitol and the like can be mentioned. Moreover, the compound which has an epoxy like a glycidyl methacrylate polymer and forms a polyhydric alcohol structure after ring-opening can also be used. As the polyhydric alcohol derivative, a polyhydric alcohol into which a reactive functional group or a polymerizable group such as carboxyl, amino, epoxy, thiol, methacryl, or acrylic is introduced by modification can be used.
温度応答性磁性微粒子は、磁性微粒子存在下で温度応答性ポリマーを反応性の官能基を介して結合する方法、又は磁性微粒子中の多価アルコール上の活性水素と、又は多価アルコールに重合性不飽和結合を導入し、磁性微粒子にグラフト重合する方法等の当技術分野で周知の方法で得ることができる（ＡＤＶ．Ｐｏｌｙｍ．Ｓｃｉ．，Ｖｏｌ．４、ｐ１１１、１９６５や、Ｊ．Ｐｏｌｙｍｅｒ Ｓｃｉ．，Ｐａｒｔ−Ａ，３，ｐ１０３１，１９６５に記載されている）。 Temperature-responsive magnetic fine particles are a method of bonding a temperature-responsive polymer via a reactive functional group in the presence of magnetic fine particles, or active hydrogen on polyhydric alcohol in magnetic fine particles, or polymerizable to polyhydric alcohol. It can be obtained by a method known in the art such as a method of introducing an unsaturated bond and graft polymerization to magnetic fine particles (ADV. Polym. Sci., Vol. 4, p111, 1965, J. Polymer Sci. , Part-A, 3, p1031, 1965).
３．検体
本発明の方法に用いられる検体とは、本発明の方法の検出対象物質である電荷物質を含有するものであれば特に限定されない。例えば、微生物や細胞の培養破砕懸濁液、遺伝子増幅装置（例えば、ＰＣＲ（Polymerase chain reaction）等）で増幅されたＤＮＡを含有する溶液等が挙げられる。
3. Specimen The specimen used in the method of the present invention is not particularly limited as long as it contains a charged substance that is a substance to be detected by the method of the present invention. Examples thereof include a culture disruption suspension of microorganisms and cells, and a solution containing DNA amplified by a gene amplification apparatus (for example, PCR (Polymerase chain reaction)).
４．電荷物質
本発明の方法における検出対象物質である電荷物質としては、電荷を有する核酸、タンパク質等が挙げられ、好ましくは、核酸（ＤＮＡ、ｍＲＮＡ）等である。
4). Charged substance Examples of the charged substance that is a substance to be detected in the method of the present invention include charged nucleic acids and proteins, preferably nucleic acids (DNA, mRNA) and the like.
５．吸着剤
本発明の方法においては、電荷物質を回収するために、温度応答性磁性微粒子の表面又は温度応答性ポリマーに、電荷物質に対する親和性物質が結合されている吸着剤を用いる。例えば、電荷物質がｓｓＤＮＡの場合には、それの相補鎖となるアンチセンスＤＮＡが親和性物質であり、ｍＲＮＡの場合には、ｍＲＮＡのポリＡ配列と相補鎖となるオリゴｄＴが親和性物質である。アンチセンスＤＮＡやオリゴｄＴを温度応答性磁性微粒子の表面又は温度応答性ポリマーに固定することで、それらに対する特異的吸着作用を有するｓｓＤＮＡ又はｍＲＮＡを特異的に結合できる。電荷物質に対する親和性物質とは、上記に例示したような電荷物質と親和性を有する物質だけでなく、電荷物質に結合した他の物質と親和性を有する物質であってもよい。具体的には、ビオチン−アビジンの特異的な結合を
介して、親和性物質と温度応答性磁性微粒子とを結合することもできる。すなわち、ビオチン化した親和性物質を、ビオチンに対する親和性物質であるアビジンを介してビオチン化した温度応答性磁性微粒子と結合することができる。本発明では、市販されているアビジン化温度応答性磁性微粒子が利用でき、あるいはアビジン化、ビオチン化は、当技術分野で周知の方法に従えばよい。遺伝子増幅装置（例えば、ＰＣＲ等）で増幅されたＤＮＡの場合には、増幅の開始の起点となるプライマーにビオチンやヒスチジンタグ等のタグを結合させておき、ビオチンの親和性物質であるアビジンやヒスチジンタグの親和性物質であるイミノジ酢酸ニッケルを結合させた温度応答性磁性微粒子で回収する方法がある。
5. Adsorbent In the method of the present invention, an adsorbent in which an affinity substance for the charge substance is bound to the surface of the temperature-responsive magnetic fine particle or the temperature-responsive polymer is used to collect the charge substance. For example, when the charged substance is ssDNA, the antisense DNA that is complementary to it is an affinity substance, and in the case of mRNA, the oligo dT that is complementary to the poly A sequence of mRNA is the affinity substance. is there. By fixing the antisense DNA or oligo dT to the surface of the temperature-responsive magnetic fine particles or the temperature-responsive polymer, it is possible to specifically bind ssDNA or mRNA having a specific adsorption action on them. The affinity substance for the charged substance is not limited to a substance having an affinity for the charged substance as exemplified above, but may be a substance having an affinity for another substance bonded to the charged substance. Specifically, the affinity substance and the temperature-responsive magnetic fine particles can be bound through specific binding of biotin-avidin. That is, the biotinylated affinity substance can be combined with the biotinylated temperature-responsive magnetic fine particles via avidin, which is an affinity substance for biotin. In the present invention, commercially available avidinized temperature-responsive magnetic fine particles can be used, or avidinization and biotinylation may be performed according to methods well known in the art. In the case of DNA amplified by a gene amplification device (for example, PCR), a tag such as biotin or histidine tag is bound to a primer that is the starting point of amplification, and avidin, which is an affinity substance for biotin, There is a method of recovering with temperature-responsive magnetic fine particles to which nickel iminodiacetate, which is an affinity substance of histidine tag, is bound.
電荷物質がタンパク質の場合には、電荷物質に対する親和性物質として、ビオチン、アビジン、グルタチオン、レクチン及び抗体等を温度応答性磁性微粒子に表面修飾することで、それらに対する特異的吸着作用を有するタンパク質と特異的に結合できる。ビオチンの場合は、アビジンとの特異的な結合を介してビオチン化された検出対象のタンパク質と、またビオチン化された抗体を用いてそれらの抗原である種々のタンパク質と更に結合することが可能である。本発明では、市販されているアビジン、ビオチン化タンパク質が利用でき、ビオチン化は、当技術分野で周知の方法に従えばよい。グルタチオンの場合は、グルタチオン−Ｓ−トランスフェラーゼ（以下、「ＧＳＴ」という。）を含有するタンパク質と特異的に結合できる。このようなＧＳＴ含有タンパク質の調製は当技術分野で周知の方法に従えばよい。 When the charged substance is a protein, as a substance having an affinity for the charged substance, biotin, avidin, glutathione, lectin, an antibody and the like are surface-modified to temperature-responsive magnetic fine particles, thereby having a protein having a specific adsorption action on them. Can bind specifically. In the case of biotin, it is possible to further bind to the protein to be detected biotinylated through specific binding to avidin, and to various proteins that are antigens using biotinylated antibodies. is there. In the present invention, commercially available avidin and biotinylated protein can be used, and biotinylation may be performed according to a method well known in the art. In the case of glutathione, it can specifically bind to a protein containing glutathione-S-transferase (hereinafter referred to as “GST”). Such a GST-containing protein may be prepared by methods well known in the art.
温度応答性ポリマーとリガンドとの結合の例として、温度応答性ポリマーと抗体の結合方法を示す。国際公開第０１／００９１４１号パンフレットに記載されているように、ビオチンを、メタクリルやアクリル等の重合性官能基と結合させて付加重合性モノマーとし、他のモノマーと共重合することにより温度応答性ポリマーにビオチンを結合させることができる。一方、リガンドとしての抗体にアビジンを結合させ、ビオチン結合温度応答性ポリマーと混合することにより、アビジンとビオチンの結合を利用して、温度応答性ポリマーに抗体を結合させることができる。なお、ビオチンの代わりにグルタチオンを用いた場合は、アビジンではなく、グルタチオンＳトランスフェラーゼを用いればよい。また、ポリマーの製造時にカルボキシル、アミノ又はエポキシ等の官能基を持つモノマーを他のモノマーと共重合させ、当技術分野で周知の方法に従い、この官能基を介して、抗体親和性物質（例えば、メロンゲル、プロテインＡ、プロテインＧ等）をポリマー上に結合させる方法も利用できる。このようにして得られた抗体親和性物質にリガンドとしての抗体を結合させることにより、温度応答性ポリマーに抗体を結合させることができる。 As an example of the binding between the temperature-responsive polymer and the ligand, a method for binding the temperature-responsive polymer and the antibody will be described. As described in the pamphlet of WO 01/009141, biotin is combined with a polymerizable functional group such as methacryl or acryl to form an addition polymerizable monomer, and copolymerized with other monomers to react with temperature. Biotin can be attached to the polymer. On the other hand, by binding avidin to an antibody as a ligand and mixing it with a biotin-binding temperature-responsive polymer, the antibody can be bound to the temperature-responsive polymer using the bond between avidin and biotin. When glutathione is used instead of biotin, glutathione S transferase may be used instead of avidin. In addition, a monomer having a functional group such as carboxyl, amino or epoxy is copolymerized with another monomer during the production of the polymer, and an antibody affinity substance (for example, Melon gel, protein A, protein G, etc.) can also be used on the polymer. By binding an antibody as a ligand to the antibody affinity substance thus obtained, the antibody can be bound to the temperature-responsive polymer.
このようにして、リガンドと結合した温度応答性ポリマーを得ることができる。リガンドと結合した温度応答性ポリマーは、温度応答性ポリマーが凝集する条件にし、遠心分離、磁石での磁集等により、精製することができる。 In this way, a temperature-responsive polymer bonded with a ligand can be obtained. The temperature-responsive polymer bound to the ligand can be purified by conditions such that the temperature-responsive polymer aggregates, and centrifugation, magnetic collection with a magnet, or the like.
６．ポリアルキレングリコール（凝集促進剤）
本発明の方法において、ポリアルキレングリコールは凝集促進剤として機能し、温度応答性磁性微粒子又は吸着剤の凝集を促進する。本発明の方法では、温度応答性磁性微粒子又は吸着剤の回収において、温度応答性磁性微粒子又は吸着剤の凝集促進剤としてポリアルキレングリコールを添加することで、ＵＣＳＴを有する温度応答性ポリマーの上限臨界溶液温度を変化（上昇）させることにより、温度応答性磁性微粒子又は吸着剤を回収する時間を短縮し、また、回収率を高めることができる。ポリアルキレングリコールとしては、本発明の効果を発揮するものであれば特に限定されないが、例えば、ポリエチレングリコール、ポリプロピレングリコール、ポリエチレングリコールポリプロピレングリコールランダムコポリマー、ポリエチレングリコールポリプロピレングリコールブロックコポリマー等が挙げられる。これらのポリアルキレングリコールを単独で、又は２種以上を組み合わせて用いることができる。なかでも、少量で温度応答性磁性微粒子を凝集させること
ができる点で、好ましくはポリエチレングリコール、ポリプロピレングリコールであり、より好ましくは、ポリエチレングリコールである。ポリエチレングリコールは、実験により最適な分子量を持つものを選んで使用することができ、例えば、約２００〜２５，０００の範囲の数平均分子量を持つもの、好ましくは約３，０００〜２０，０００、より好ましくは約６，０００〜１５，０００の範囲の数平均分子量を持つものが挙げられる。これらは、例えば、シグマ社、和光純薬社等から入手できる。
6). Polyalkylene glycol (aggregation accelerator)
In the method of the present invention, the polyalkylene glycol functions as an aggregation promoter and promotes aggregation of the temperature-responsive magnetic fine particles or the adsorbent. In the method of the present invention, in the recovery of temperature-responsive magnetic fine particles or adsorbent, polyalkylene glycol is added as an aggregation accelerator for the temperature-responsive magnetic fine particles or adsorbent, so that the upper criticality of the temperature-responsive polymer having UCST is reached. By changing (raising) the solution temperature, it is possible to shorten the time for collecting the temperature-responsive magnetic fine particles or the adsorbent and to increase the recovery rate. The polyalkylene glycol is not particularly limited as long as it exhibits the effects of the present invention, and examples thereof include polyethylene glycol, polypropylene glycol, polyethylene glycol polypropylene glycol random copolymer, and polyethylene glycol polypropylene glycol block copolymer. These polyalkylene glycols can be used alone or in combination of two or more. Among these, polyethylene glycol and polypropylene glycol are preferable, and polyethylene glycol is more preferable because the temperature-responsive magnetic fine particles can be aggregated in a small amount. The polyethylene glycol having an optimum molecular weight can be selected and used by experiment, for example, having a number average molecular weight in the range of about 200 to 25,000, preferably about 3,000 to 20,000, More preferred are those having a number average molecular weight in the range of about 6,000 to 15,000. These can be obtained from Sigma, Wako Pure Chemicals, etc., for example.
本発明の方法においては、温度応答性磁性微粒子又は吸着剤が所望の温度で容易に凝集する程度まで、温度応答性磁性微粒子又は吸着剤の分散液中にポリアルキレングリコールを添加すればよい。 In the method of the present invention, polyalkylene glycol may be added to the dispersion liquid of temperature-responsive magnetic fine particles or adsorbent to such an extent that the temperature-responsive magnetic fine particles or adsorbent easily aggregates at a desired temperature.
例えば、ポリエチレングリコールによる温度応答性ポリマーのＵＣＳＴの上昇は、以下のとおりである。アクリロイルグリシンアミドであれば通常１〜１０℃であり、アクリロイルアスパラギンアミドであれば通常１〜１０℃である。
コポリマーのＵＣＳＴの変化は、コポリマーを構成するモノマーの種類及びモノマー成分の比率に応じて決定される。
For example, the increase in UCST of the temperature-responsive polymer by polyethylene glycol is as follows. If it is acryloyl glycinamide, it is 1-10 degreeC normally, and if it is acryloyl asparagine amide, it is 1-10 degreeC normally.
The change in the UCST of the copolymer is determined depending on the type of monomer constituting the copolymer and the ratio of the monomer components.
ポリアルキレングリコールは、粉末のまま使用することもできるが、水溶液として用いることが好ましい。この場合、ポリアルキレングリコールの濃度は、好ましくは３０重量％以下である。この濃度範囲内であれば、粘度が高くなり過ぎることがなく、扱い易く、特に少量を分取する際にも問題が生じることがない。 The polyalkylene glycol can be used as a powder, but is preferably used as an aqueous solution. In this case, the concentration of polyalkylene glycol is preferably 30% by weight or less. Within this concentration range, the viscosity does not become excessively high and is easy to handle, and no problem arises even when a small amount is taken.
ポリアルキレングリコールの添加量は、温度応答性磁性微粒子又は吸着剤の凝集を促進する効果を指標に当業者が適宜決定することができる。例えば、最終濃度として通常１０〜０．５重量％、好ましくは２〜０．５重量％とすることができる。また、温度応答性磁性微粒子又は吸着剤に使用される温度応答性ポリマーのＵＣＳＴの上昇として、通常１〜１０℃、好ましくは２〜５℃とすることができる。 The addition amount of the polyalkylene glycol can be appropriately determined by those skilled in the art based on the effect of promoting aggregation of the temperature-responsive magnetic fine particles or the adsorbent. For example, the final concentration is usually 10 to 0.5% by weight, preferably 2 to 0.5% by weight. Moreover, as raise of UCST of the temperature-responsive polymer used for a temperature-responsive magnetic fine particle or an adsorbent, it can be normally 1-10 degreeC, Preferably it can be 2-5 degreeC.
例えば、４ｍｇ／ｍｌの温度応答性磁性微粒子又は吸着剤、及び０．５ｍｇ／ｍｌの電荷物質を含む溶液は、ポリエチレングリコール等のポリアルキレングリコール溶液を添加することで、温度応答性磁性微粒子又は吸着剤濃度４ｍｇ／ｍｌ、ポリエチレングリコール濃度１重量％の条件下、容易に凝集状態にすることができる。 For example, a solution containing 4 mg / ml temperature-responsive magnetic fine particles or adsorbent and 0.5 mg / ml charged substance is added with a polyalkylene glycol solution such as polyethylene glycol, so that the temperature-responsive magnetic fine particles or adsorbent is added. It can be easily agglomerated under the conditions of an agent concentration of 4 mg / ml and a polyethylene glycol concentration of 1% by weight.
一方、凝集させた吸着剤を再び分散させるには、溶液を上昇後のＵＣＳＴ以上の温度にするか、精製水等でポリアルキレングリコール濃度を希釈すればよい。 On the other hand, in order to disperse the agglomerated adsorbent again, the solution may be brought to a temperature higher than the raised UCST, or the polyalkylene glycol concentration may be diluted with purified water or the like.
７．温度応答性磁性微粒子の製造方法
本発明の方法に用いる温度応答性磁性微粒子は、例えば、下記手順を有する１〜３のいずれかの製造方法を用いることで得ることができる。
7). Method for Producing Temperature Responsive Magnetic Fine Particles The temperature responsive magnetic fine particles used in the method of the present invention can be obtained, for example, by using any one of
７−１．温度応答性磁性微粒子の製造方法１
１）多価アルコール等を磁性微粒子の表面に固定するか、又は多価アルコール等の溶液中で磁性微粒子を調製する。
２）上限臨界溶液温度を有するポリマーが得られるモノマーと、前記多価アルコール等が固定された磁性微粒子とを混合し、ラジカル重合法で重合を行う。
３）重合未反応物を除去し、温度応答性磁性微粒子を分離する。
7-1.
1) Fix polyhydric alcohol or the like on the surface of magnetic fine particles, or prepare magnetic fine particles in a solution of polyhydric alcohol or the like.
2) A monomer from which a polymer having an upper critical solution temperature is obtained and magnetic fine particles to which the polyhydric alcohol or the like is fixed are mixed, and polymerization is performed by a radical polymerization method.
3) The polymerization unreacted material is removed, and the temperature-responsive magnetic fine particles are separated.
７−２．温度応答性磁性微粒子の製造方法２
１）多価アルコール等と、上限臨界溶液温度を有するポリマーが得られるモノマーとを混合し、ラジカル重合法でグラフト重合を行うか、又は上限臨界溶液温度を有するポリマー
上の官能基を介してグラフト重合法で結合を行う。
２）多価アルコール等を固定した温度応答性ポリマーの溶液中で磁性微粒子を調製する。３）多価アルコール等の未反応物を除去し、温度応答性磁性微粒子を分離する。
7-2.
1) A polyhydric alcohol and the like are mixed with a monomer capable of obtaining a polymer having an upper critical solution temperature, and graft polymerization is performed by a radical polymerization method, or grafting is performed via a functional group on the polymer having an upper critical solution temperature. Bonding is performed by a polymerization method.
2) Magnetic fine particles are prepared in a solution of a temperature-responsive polymer to which polyhydric alcohol or the like is fixed. 3) Unreacted substances such as polyhydric alcohol are removed, and temperature-responsive magnetic fine particles are separated.
７−３．温度応答性磁性微粒子の製造方法３
１）多価アルコール等を磁性微粒子の表面に固定するか、又は多価アルコール等の溶液中で磁性微粒子を調製する。
２）温度応答性ポリマーの末端又はポリマーの側鎖に反応性の官能基を導入したポリマーを調製、精製する。
３）磁性微粒子表面に官能基を介して温度応答性ポリマーを結合する。
４）未結合の温度応答性ポリマーを除去し、温度応答性磁性微粒子を分離する。
7-3.
1) Fix polyhydric alcohol or the like on the surface of magnetic fine particles, or prepare magnetic fine particles in a solution of polyhydric alcohol or the like.
2) A polymer having a reactive functional group introduced at the end of the temperature-responsive polymer or the side chain of the polymer is prepared and purified.
3) A temperature-responsive polymer is bonded to the surface of the magnetic fine particle through a functional group.
4) The unbonded temperature-responsive polymer is removed, and the temperature-responsive magnetic fine particles are separated.
８．吸着剤の製造方法
本発明の方法に用いる吸着剤は、温度応答性磁性微粒子にリガンドが固定された粒子である。温度応答性磁性微粒子へのリガンドの固定は、例えば、共有結合、水素結合、イオン結合又は疎水結合等から適切な結合手段を選択することによって行うことができる。また、温度応答性磁性微粒子とリガンドとの結合に、リガンドに対する親和性を有する他の物質を介させてもよい。例えば、ビオチン／アビジンの結合を介して温度応答性磁性微粒子とオリゴｄＴとを結合させることができる。この場合、温度応答性磁性微粒子−ビオチン／アビジン／ビオチン化されたオリゴｄＴの組み合わせが結合に介することになる。
8). Method for Producing Adsorbent The adsorbent used in the method of the present invention is a particle in which a ligand is fixed to temperature-responsive magnetic fine particles. The ligand can be fixed to the temperature-responsive magnetic fine particles by selecting an appropriate binding means from, for example, a covalent bond, a hydrogen bond, an ionic bond or a hydrophobic bond. Further, another substance having affinity for the ligand may be mediated through the binding between the temperature-responsive magnetic fine particles and the ligand. For example, temperature-responsive magnetic fine particles and oligo dT can be bound via a biotin / avidin bond. In this case, a combination of temperature-responsive magnetic fine particles-biotin / avidin / biotinylated oligo dT is mediated by the binding.
９．温度応答性磁性微粒子又は吸着剤の回収方法
本発明においては、温度応答性磁性微粒子又は吸着剤を含む溶液を、温度応答性ポリマーのＵＣＳＴ未満の温度にして、温度応答性磁性微粒子又は吸着剤を回収する。
この場合、ＵＣＳＴと冷却温度の差が大きければ大きいほど回収速度（操作性）及び回収率がよい。凝集促進剤を添加するとＵＣＳＴが上昇するため、一定の冷却温度を設定する場合において、ＵＣＳＴと冷却温度の差が大きくなり、温度応答性ポリマーの凝集が促進され、温度応答性磁性微粒子又は吸着剤の回収速度及び回収率が向上する。なお、本発明の方法においては、ＵＣＳＴが、溶液が凍結する温度を上回らなければならないため、ＵＣＳＴが溶液の凝固点に近く、冷却温度を下げられない場合において、凝集促進剤を添加するとＵＣＳＴが上昇し、温度応答性微粒子の回収が可能となる。
9. Method for recovering temperature-responsive magnetic fine particles or adsorbent In the present invention, a solution containing temperature-responsive magnetic fine particles or adsorbent is brought to a temperature lower than the UCST of the temperature-responsive polymer, and the temperature-responsive magnetic fine particles or adsorbent is used. to recover.
In this case, the larger the difference between the UCST and the cooling temperature, the better the recovery speed (operability) and the recovery rate. When the aggregation accelerator is added, the UCST rises. Therefore, when a constant cooling temperature is set, the difference between the UCST and the cooling temperature is increased, and the aggregation of the temperature-responsive polymer is promoted, and the temperature-responsive magnetic fine particles or the adsorbent The recovery speed and recovery rate of the are improved. In the method of the present invention, UCST must exceed the temperature at which the solution freezes. Therefore, when UCST is close to the freezing point of the solution and the cooling temperature cannot be lowered, the addition of the aggregation promoter increases the UCST. In addition, the temperature-responsive fine particles can be collected.
溶液（凝集促進剤添加後）中の温度応答性磁性微粒子又は吸着剤の濃度は、検出対象物質（電荷物質）、温度応答性磁性微粒子の種類等によって異なるが、操作性等の点から、通常１〜４ｍｇ／ｍｌが好ましい。 The concentration of the temperature-responsive magnetic fine particles or adsorbent in the solution (after the addition of the aggregation accelerator) varies depending on the substance to be detected (charged substance), the type of temperature-responsive magnetic fine particles, etc. 1-4 mg / ml is preferred.
温度応答性磁性微粒子又は吸着剤の回収に用いる磁石等の磁力は、用いる磁性微粒子の有する磁力の大きさによって異なる。磁力は、目的の磁性微粒子を磁集可能な程度の磁力を適宜使用できる。磁石の素材としては、例えば、上述した磁性微粒子の素材で構成されたものを使用することができる。例えば、マグナ社製ネオジ磁石等が利用できる。このように本発明では、磁石等の磁力によって、温度応答性磁性微粒子又は吸着剤を回収できるが、磁性微粒子の表面に温度応答性ポリマーが固定されていることで、分散状態では回収困難なナノサイズの磁性微粒子を意図的に凝集させて、回収率を高めることが可能になる。なお、本発明は、このような温度応答性磁性微粒子又は吸着剤を、溶液中で凝集促進剤濃度を変化させることにより凝集又は分散させることが可能である。したがって、短時間で、効率よく、核酸等の電荷物質を回収することが可能である。また、ＵＣＳＴが溶液の凝固点に近く、冷却温度を下げられない場合において、電荷物質の回収を可能にすることができる。 The magnetic force of the magnet or the like used for collecting the temperature-responsive magnetic fine particles or the adsorbent varies depending on the magnitude of the magnetic force of the magnetic fine particles used. As the magnetic force, a magnetic force that can collect magnetic particles of interest can be used as appropriate. As the material for the magnet, for example, a material composed of the magnetic fine particle material described above can be used. For example, a neodymium magnet manufactured by Magna can be used. As described above, in the present invention, the temperature-responsive magnetic fine particles or the adsorbent can be recovered by the magnetic force of a magnet or the like. However, since the temperature-responsive polymer is fixed on the surface of the magnetic fine particles, it is difficult to recover in a dispersed state. It is possible to intentionally agglomerate magnetic particles of a size and increase the recovery rate. In the present invention, such temperature-responsive magnetic fine particles or adsorbents can be aggregated or dispersed in the solution by changing the aggregation accelerator concentration. Therefore, it is possible to efficiently collect charged substances such as nucleic acids in a short time. Further, when the UCST is close to the freezing point of the solution and the cooling temperature cannot be lowered, it is possible to collect the charged substance.
本発明の方法による電荷物質の回収方法の具体例を示す。
アビジン固定温度応答性磁性微粒子とビオチン化されたオリゴｄＴの複合物を用いて、ｍＲＮＡを濃縮する方法は下記のとおりである。
１）目的ｍＲＮＡを含有する細胞破砕懸濁液（検体）を調製する。
２）ビオチン化されたオリゴｄＴを温度応答性磁性微粒子に結合させる。
３）オリゴｄＴを結合した温度応答性磁性微粒子（吸着剤）とｍＲＮＡを含む細胞破砕懸濁液を混ぜて、ハイブリダイズさせ、ｍＲＮＡ／オリゴｄＴ−温度応答性磁性微粒子の結合体を生成させる。
４）前記混合液に凝集促進剤を加え、冷却後、攪拌もしくはピペッティングを行い、ｍＲＮＡ／オリゴｄＴ−温度応答性磁性微粒子／凝集促進剤の複合体を凝集させる。
５）磁気分離装置を用いて、凝集物を回収し、上澄をピペット等により注意深く除去する。
６）ｍＲＮＡはオリゴｄＴ−温度応答性磁性微粒子に結合したままでＲＴ−ＰＣＲを行ってもよいし、目的によっては、オリゴｄＴ−温度応答性磁性微粒子からｍＲＮＡを分離した後、ＲＴ−ＰＣＲを行ってもよい。ＲＴ−ＰＣＲ後にアガロース電気泳動にてｃＤＮＡのバンドを確認する。
The specific example of the collection method of the charge substance by the method of this invention is shown.
A method for concentrating mRNA using a complex of avidin-fixed temperature-responsive magnetic fine particles and biotinylated oligo dT is as follows.
1) A cell disruption suspension (specimen) containing the target mRNA is prepared.
2) The biotinylated oligo dT is bound to the temperature-responsive magnetic fine particles.
3) The temperature-responsive magnetic fine particles (adsorbent) to which oligo dT is bound and the cell disruption suspension containing mRNA are mixed and hybridized to produce a conjugate of mRNA / oligo dT-temperature-responsive magnetic fine particles.
4) An aggregation promoter is added to the mixed solution, and after cooling, stirring or pipetting is performed to aggregate the mRNA / oligo dT-temperature-responsive magnetic fine particle / aggregation promoter complex.
5) Collect aggregates using a magnetic separator and carefully remove the supernatant with a pipette or the like.
6) RT-PCR may be performed while mRNA is bound to oligo dT-temperature responsive magnetic microparticles. Depending on the purpose, mRNA may be separated from oligo dT-temperature responsive magnetic microparticles and then RT-PCR may be performed. You may go. After RT-PCR, the cDNA band is confirmed by agarose electrophoresis.
本発明の検体中の電荷物質を回収するためのキットは、例えば、以下の試薬から構成される。
試薬Ａ：吸着剤（温度応答性磁性微粒子にリガンド又はリガンドに対する親和性物質が結合した粒子）の分散液
試薬Ｂ：凝集促進剤溶液
試薬Ｃ：希釈用バッファー（上記試薬Ａ及びＢの希釈、並びに検体の希釈に使用可能な緩衝液である。一例として、トリス塩酸緩衝液、リン酸緩衝液等が挙げられる。）
試薬Ｄ：検出対象物質（電荷物質）の標準品（一例として、精製したＤＮＡが挙げられる。）。
The kit for recovering the charged substance in the specimen of the present invention is composed of, for example, the following reagents.
Reagent A: Dispersion liquid of adsorbent (particles in which ligand or affinity substance for ligand is bound to temperature-responsive magnetic fine particles) Reagent B: Aggregation accelerator solution Reagent C: Dilution buffer (dilution of the above-mentioned reagents A and B, and (It is a buffer solution that can be used for diluting a sample. Examples thereof include Tris-HCl buffer solution and phosphate buffer solution.)
Reagent D: Standard product of detection target substance (charged substance) (an example is purified DNA).
以下、実施例によって本発明を具体的に説明するが、本発明はこれらによって何ら限定されることはない。また、実施例及び比較例中における物性の測定方法、用いた吸着剤（ストレプトアビジン固定化温度応答性磁性微粒子）の製造方法は以下の通りである。 EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited at all by these. Moreover, the measuring method of the physical property in an Example and a comparative example, and the manufacturing method of the used adsorption agent (streptavidin fixed temperature-responsive magnetic fine particle) are as follows.
［物性の測定方法１］ＳＤＳポリアクリルアミドゲル電気泳動(ＳＤＳ−ＰＡＧＥ)
アクリルアミド、ビスアクリルアミドから作られた１２．５重量％のポリアクリルアミドゲルを備えた電気泳動装置を用いて、２０ｍＡの電流で電気泳動を行った。なお、マーカーは、アトー社製タンパク質電気泳動用有色分子量マーカーを使用した。電気泳動を行った後、ポリアクリルアミドゲルを０．５重量％のクーマシーブリリアントブルー溶液に浸すことによって、該ゲル中のタンパク質の染色を行った。
[Physical property measurement method 1] SDS polyacrylamide gel electrophoresis (SDS-PAGE)
Electrophoresis was performed at an electric current of 20 mA using an electrophoresis apparatus equipped with a 12.5 wt% polyacrylamide gel made from acrylamide and bisacrylamide. The marker used was a colored molecular weight marker for protein electrophoresis manufactured by Ato. After electrophoresis, the protein in the gel was stained by immersing the polyacrylamide gel in a 0.5% by weight Coomassie brilliant blue solution.
［物性の測定方法２］温度応答性磁性微粒子の平均粒径の測定
大塚電子社製ＥＬＳ−８０００ＳＤ型「光散乱光度計」によって測定した。
測定数は、Ｎ＝１００である。
[
The number of measurements is N = 100.
［物性の測定方法３］アガロース電気泳動
２重量％のアガロースから作られたゲルを備えたサブマリン電気泳動装置を用いて、１２５ｍＡの電流で電気泳動を行った。なお、マーカーはタカラバイオ社製ＤＮＡ電気泳動用分子量マーカーを使用した。電気泳動を行った後、アガロースゲルを０．０５重量％のエチジウムブロマイド溶液に浸すことによって、該ゲル中のＤＮＡの染色を行った。
[Physical Property Measuring Method 3] Agarose Electrophoresis Electrophoresis was performed at a current of 125 mA using a submarine electrophoresis apparatus equipped with a gel made from 2% by weight of agarose. As a marker, a molecular weight marker for DNA electrophoresis manufactured by Takara Bio Inc. was used. After electrophoresis, DNA in the gel was stained by immersing the agarose gel in a 0.05 wt% ethidium bromide solution.
［製造例１］磁性微粒子の調製
磁性微粒子（平均粒径４０ｎｍ）は、以下の方法で製造した。
１００ｍｌのフラスコに、塩化第二鉄・六水和物（１．０ｍｏｌ）及び塩化第一鉄・四水和物（０．５ｍｏｌ）混合水溶液を３ｍｌ、多価アルコールであるデキストラン（和光純薬社製、分子量３２０００〜４００００）の１０重量％水溶液６０ｍｌを入れ、メカニカルスターラーで攪拌し、この混合溶液を５０℃に昇温した後、これに２５重量％アンモニア水溶液５．０ｍｌを滴下し、１時間程度攪拌した。この操作で、デキストランが固定された、平均粒径が約４０ｎｍの磁性微粒子が得られた。
[Production Example 1] Preparation of magnetic fine particles Magnetic fine particles (average particle size 40 nm) were produced by the following method.
In a 100 ml flask, 3 ml of a mixed aqueous solution of ferric chloride / hexahydrate (1.0 mol) and ferrous chloride / tetrahydrate (0.5 mol), dextran, a polyhydric alcohol (Wako Pure Chemical Industries, Ltd.) 60 ml of a 10 wt% aqueous solution having a molecular weight of 32,000 to 40,000) was added and stirred with a mechanical stirrer. After the temperature of the mixed solution was raised to 50 ° C, 5.0 ml of a 25 wt% aqueous ammonia solution was added dropwise thereto for 1 hour. Stir to a certain degree. By this operation, magnetic fine particles having an average particle diameter of about 40 nm and having fixed dextran were obtained.
［製造例２］ビオチンモノマー〔Ｎ−ビオチニル−Ｎ′−メタクロイルトリメチレンアミド〕の調製
ビオチンモノマーは、以下の方法で製造した。
Ｎ−（３−アミノプロピル）メタクリルアミド塩酸塩１８ｇ、ビオチン２４ｇ及びトリエチルアミン３０ｇを３００ｍｌのＮ，Ｎ−ジメチルホルムアミド（ＤＭＦ）に溶解し、０℃に冷却し、ジフェニルホスフォニルアジド２８ｇを５０ｍｌのＤＭＦに溶解させた溶液を１時間かけて、この混合物中に滴下した。滴下終了後、０℃で３時間攪拌し、更に室温で１２時間攪拌した。この後、減圧下で、溶媒を留去し、展開溶媒としてクロロホルム−メタノール混合溶媒を用いて、カラムクロマトグラフィーに残留物をかけることで目的物であるＮ−ビオチニル−Ｎ′−メタクロイルトリメチレンアミドを得た。
[Production Example 2] Preparation of biotin monomer [N-biotinyl-N'-methacryloyl trimethylene amide] A biotin monomer was produced by the following method.
18 g of N- (3-aminopropyl) methacrylamide hydrochloride, 24 g of biotin and 30 g of triethylamine are dissolved in 300 ml of N, N-dimethylformamide (DMF), cooled to 0 ° C., and 28 g of diphenylphosphonlazide is added to 50 ml of A solution dissolved in DMF was added dropwise to the mixture over 1 hour. After completion of dropping, the mixture was stirred at 0 ° C. for 3 hours, and further stirred at room temperature for 12 hours. Thereafter, the solvent was distilled off under reduced pressure, and the residue, N-biotinyl-N′-methacryloyl trimethylene, was obtained by subjecting the residue to column chromatography using a chloroform-methanol mixed solvent as a developing solvent. The amide was obtained.
［製造例３］ビオチン固定化温度応答性磁性微粒子の調製
温度応答性磁性微粒子は、以下の方法で製造した。
１０ｍＭ 炭酸ナトリウム溶液１５ｍｌに前記方法で調製した磁性微粒子（平均粒径４０ｎｍ）１ｍｌを添加し、２時間攪拌後、グリシジルメタクリレート１００ｍｇを添加し、７２時間反応させた。透析後に濃縮し、メタクリル化磁性微粒子を得た。５０ｍｌの三口フラスコに、Ｎ−アクリロイルグリシンアミド２００ｍｇ、前記メタクリル化磁性微粒子２ｍｇ及びビオチンモノマー３ｍｇを入れ、蒸留水で２０ｍｌに調整した。この溶液を窒素置換した後、過硫酸アンモニウム３０ｍｇを添加し、５０℃で２時間反応させることで、温度応答性磁性微粒子が得られた。この温度応答性磁性微粒子の平均粒径は、約１００ｎｍであった（光散乱光度計による測定）。また、この温度応答性磁性微粒子はＵＣＳＴを１０℃に有し、ＵＣＳＴ未満の水溶液中では凝集し、磁石で容易に回収できる。溶液をＵＣＳＴ以上にすると直ちに分散し、磁石では容易に回収できなかった。
[Production Example 3] Preparation of biotin-immobilized temperature-responsive magnetic fine particles Temperature-responsive magnetic fine particles were produced by the following method.
1 ml of magnetic fine particles (average particle size 40 nm) prepared by the above method was added to 15 ml of 10 mM sodium carbonate solution, and after stirring for 2 hours, 100 mg of glycidyl methacrylate was added and reacted for 72 hours. Concentration after dialysis gave methacrylic magnetic fine particles. A 50 ml three-necked flask was charged with 200 mg of N-acryloylglycinamide, 2 mg of the methacrylic magnetic particles and 3 mg of biotin monomer, and adjusted to 20 ml with distilled water. After replacing this solution with nitrogen, 30 mg of ammonium persulfate was added and reacted at 50 ° C. for 2 hours to obtain temperature-responsive magnetic fine particles. The average particle size of the temperature-responsive magnetic fine particles was about 100 nm (measured with a light scattering photometer). The temperature-responsive magnetic fine particles have UCST at 10 ° C., and aggregate in an aqueous solution lower than UCST and can be easily recovered with a magnet. When the solution was more than UCST, it was immediately dispersed and could not be easily recovered with a magnet.
［製造例４］ストレプトアビジン固定化温度応答性磁性微粒子の調製
ストレプトアビジン固定化温度応答性磁性微粒子は、以下の方法で製造した。
前記ビオチン固定化温度応答性磁性微粒子溶液の０．１重量％水溶液５０μｌを１．５ｍｌエッペンドルフチューブにとり、さらに１ｇ／ｌのストレプトアビジン溶液５０μｌをチューブに加え、室温で、２０分混合した。その後、チューブを２℃に冷却し、磁性微粒子を凝集させ、磁石で回収し、上清部分と分離した。凝集部分にバッファー（２０ｍＭ
Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５），１５０ｍＭ ＮａＣｌ，０．０５％ＴＷＥＥＮ２０）２００μｌを加え、磁性微粒子を加温して分散させ、再度冷却して凝集させ、凝集塊に混入した成分を洗浄し取り除いた。この操作を４回繰り返した。各操作で凝集後に回収した磁性微粒子にそれぞれ２×ＳＤＳ サンプルバッファー（２０重量％グリセリン、２５０ｍＭ Ｔｒｉｓ−ＨＣｌ、４重量％ＳＤＳ、１０重量％メルカプトエタノール、８Ｍ 尿素、０．００４重量％ブロモフェノールブルー、ｐＨ６．８）を２０μｌ加え、９５℃で、１０分間加熱し、磁性微粒子に結合したタンパク質を抽出し、この抽出液を用いてＳＤＳ−ＰＡＧＥを行い、ＳＤＳポリアクリルアミドゲルをクーマシーブリリアントブルー染色液で染色、脱色した後、バンドの濃度を確認し、ストレプトアビジンの固定化を確認した。
[Production Example 4] Preparation of streptavidin-immobilized temperature-responsive magnetic fine particles Streptavidin-immobilized temperature-responsive magnetic fine particles were produced by the following method.
50 μl of a 0.1 wt% aqueous solution of the biotin-immobilized temperature-responsive magnetic fine particle solution was placed in a 1.5 ml Eppendorf tube, and 50 μl of a 1 g / l streptavidin solution was added to the tube and mixed at room temperature for 20 minutes. Thereafter, the tube was cooled to 2 ° C., the magnetic fine particles were aggregated, collected with a magnet, and separated from the supernatant portion. Buffer (20 mM) in the aggregation part
200 μl of Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% TWEEN 20) was added, and the magnetic fine particles were heated and dispersed, cooled again and aggregated, and the components mixed in the aggregate were washed and removed. . This operation was repeated 4 times. 2 × SDS sample buffer (20 wt% glycerin, 250 mM Tris-HCl, 4 wt% SDS, 10 wt% mercaptoethanol, 8M urea, 0.004 wt% bromophenol blue, 20 μl of pH 6.8) was added and heated at 95 ° C. for 10 minutes to extract the protein bound to the magnetic fine particles, SDS-PAGE was performed using this extract, and SDS polyacrylamide gel was subjected to Coomassie Brilliant Blue staining solution. After staining and decoloring, the concentration of the band was confirmed, and the immobilization of streptavidin was confirmed.
なお、上記製造例で製造した温度応答性磁性微粒子以外に、Ｔｈｅｒｍａ−Ｍａｘ（登録商標）ＵＳＡ Ｓｔｒｅｐｔａｖｉｄｉｎ（以下、「ＴＭ−ＵＳＡ」と略す。）として
、マグナビート社から温度応答性磁性微粒子を入手することができる。実施例ではマグナビートから入手したＴＭ−ＵＳＡを使用した。このＴＭ−ＵＳＡは、約２℃で十分に凝集が完了する。このＴＭ−ＵＳＡはミリポア製マイレスクＧＶ（孔径０．２２μｍ）で濾過滅菌した後に実験に使用し、Ｔｏｔａｌ−ＲＮＡからのｍＲＮＡの分離操作に関してはクリーンベンチ内で行い、使用する試薬も滅菌したものを使用した。
In addition to the temperature-responsive magnetic fine particles produced in the above production example, temperature-responsive magnetic fine particles were obtained from Magnabeat as Thermo-Max (registered trademark) USA Streptavidin (hereinafter abbreviated as “TM-USA”). can do. In the examples, TM-USA obtained from Magna Beat was used. This TM-USA is fully agglomerated at about 2 ° C. This TM-USA was used for experiments after sterilization by filtration with Millipore Mylesque GV (pore size 0.22 μm), and the mRNA separation from Total-RNA was performed in a clean bench, and the reagents used were also sterilized. used.
［実施例１］
ポリエチレングリコール（ＰＥＧ）によるＴＭ−ＵＳＡの凝集促進効果の確認
ＴＭ−ＵＳＡ（４ｍｇ／ｍｌ） ５０μｌと１×Ｂｉｎｄｉｎｇ ｂｕｆｆｅｒ（２０ｍＭ Ｔｒｉｓ−ＨＣｌ、２ｍＭ ＥＤＴＡ、１Ｍ ＬｉＣｌ、ｐＨ８．０） ４０μｌと、重量均分子量６０００のポリエチレングリコール１０％（ｗ／ｖ）水溶液 １０μｌを添加し、混合したサンプルをサンプル２とした。
[Example 1]
Confirmation of aggregation promotion effect of TM-USA by polyethylene glycol (PEG) 50 μl of TM-USA (4 mg / ml) and 40 μl of 1 × Binding buffer (20 mM Tris-HCl, 2 mM EDTA, 1M LiCl, pH 8.0), weight
［比較例１］
ＴＭ−ＵＳＡ（４ｍｇ／ｍｌ） ５０μｌと１×Ｂｉｎｄｉｎｇ ｂｕｆｆｅｒ ４０μｌと超純水（ミリポア社製Ｄｉｒｅｃｔ−Ｑで精製した水） １０μｌを添加し、混合したサンプルをサンプル１とした。
[Comparative Example 1]
TM-USA (4 mg / ml) 50 μl, 1 × Binding buffer 40 μl, and ultrapure water (water purified by Direct-Q manufactured by Millipore) 10 μl were added, and the mixed sample was used as
調製したサンプル１及び２を、氷水中で１分間冷却し、凝集させ、磁石で１分間回収した後、上清を回収し、上清の透過率（５５０ｎｍ）を測定した。コントロールとして、磁気回収を行っていないサンプルの透過率（吸光度）を測定した。測定した結果を表１に示す。
The
比較例１はコントロールの透過率（吸光度）とほとんど数値が変わらず、ＴＭ−ＵＳＡを回収できていないことが確認できた。実施例１はコントロールの透過率（吸光度）と比べ、数値が大きく減少していることからＴＭ-ＵＳＡを回収できていることが確認できた。 In Comparative Example 1, the numerical value of the transmittance (absorbance) of the control hardly changed, and it was confirmed that TM-USA could not be recovered. In Example 1, compared with the transmittance (absorbance) of the control, it was confirmed that TM-USA could be recovered since the value was greatly reduced.
［実施例２］
ＴＭ−ＵＳＡを用いたＴｏｔａｌ−ＲＮＡからのｍＲＮＡの分離
[Example 2]
Separation of mRNA from Total-RNA using TM-USA
（ＴＭ−ＵＳＡへのビオチン−オリゴｄＴの固定）
ＴＭ−ＵＳＡ（１６ｍｇ／ｍｌ）２５μｌとＴＥ Ｂｕｆｆｅｒ（１０ｍＭ Ｔｒｉｓ−ＨＣｌ、１ｍＭ ＥＤＴＡ、ｐＨ８．０）７５μｌを１．５ｍｌエッペンドルフチューブに採取し、よく混合し、氷水中で冷却し、凝集させ、磁石で回収し、上清部分を取り除いた。１×Ｂｉｎｄｉｎｇ ｂｕｆｆｅｒ（２０ｍＭ Ｔｒｉｓ−ＨＣｌ、２ｍＭ ＥＤＴＡ、１Ｍ ＬｉＣｌ、ｐＨ８．０）５０μｌ、重量平均分子量６０００のポリエチレングリコール１０％（ｗ／ｖ）水溶液１０μｌを加え、分散させた。再度チューブを氷水中で冷却し、凝集させ、磁石で回収し、上清部分を取り除いた。凝集部分に１×Ｂｉｎｄｉｎｇ ｂｕｆｆｅｒ ２４μｌ、ビオチン化オリゴｄＴ（０．７５ ｐｍｏｌ／μｌ）１
μｌを加え、分散させ、室温で５００ｒｐｍ、５分間、振とうし、反応させた。
このようにして、ビオチン化オリゴｄＴ固定化ＴＭ−ＵＳＡを調製した。
(Immobilization of biotin-oligo dT to TM-USA)
Take 25 μl of TM-USA (16 mg / ml) and 75 μl of TE Buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) in a 1.5 ml Eppendorf tube, mix well, cool in ice water, aggregate, magnet And the supernatant was removed. 50 μl of 1 × Binding buffer (20 mM Tris-HCl, 2 mM EDTA, 1M LiCl, pH 8.0) and 10 μl of a 10% (w / v) aqueous solution of polyethylene glycol having a weight average molecular weight of 6000 were added and dispersed. The tube was cooled again in ice water, aggregated, collected with a magnet, and the supernatant was removed. 1 × Binding buffer 24 μl in the aggregation part, biotinylated oligo dT (0.75 pmol / μl) 1
μl was added and dispersed, and the mixture was reacted by shaking at 500 rpm for 5 minutes at room temperature.
In this way, biotinylated oligo dT-immobilized TM-USA was prepared.
（ビオチン化オリゴｄＴ固定化ＴＭ−ＵＳＡによるＴｏｔａｌ ＲＮＡからのｍＲＮＡの分離）
Ｈｕｍａｎ Ｔｉｓｓｕｅ Ｔｏｔａｌ ＲＮＡ ５μｌと、Ｎｕｃｌｅａｓｅ ｆｒｅｅ ｗａｔｅｒ ３５μｌとを１．５ｍｌエッペンドルフチューブに取り、混合し、７０℃で１５分加熱し、氷上に２分静置後に５×Ｂｉｎｄｉｎｇ ｂｕｆｆｅｒ（１００ｍＭ Ｔｒｉｓ−ＨＣｌ、１０ｍＭ ＥＤＴＡ、５Ｍ ＬｉＣｌ、ｐＨ８．０）１０μｌを加え、調製したビオチン化オリゴｄＴ固定化ＴＭ−ＵＳＡ（４ｍｇ／ｍｌ）１００μｌと混合し、室温で５００ｒｐｍ、１０分間、振とうし、反応させた。その後、１０重量％ＰＥＧ（ＭＷ．６０００） １０μｌを加えて氷水中で冷却し、凝集させ、磁石で回収し、上清部分を取り除いた。凝集部分にＷａｓｈｉｎｇ ｂｕｆｆｅｒ（１０ｍＭ Ｔｒｉｓ−ＨＣｌ、０．１５Ｍ ＥＤＴＡ、０．１５Ｍ ＬｉＣｌ、ｐＨ７．５）５０μｌと１０重量％ＰＥＧ（ＭＷ．６０００） １０μｌを加え、溶液中に分散させた。再度チューブを氷水中で冷却し、凝集させ、磁石で回収し、上清部分を取り除いた。この洗浄の操作を２回繰り返した。洗浄後、凝集部分にＮｕｃｌｅａｓｅ ｆｒｅｅ ｗａｔｅｒ ５０μｌを加え、分散させ、１．５ｍｌエッペンドルフチューブに２０μｌ分注し（１）、残りの溶液を３７℃で５分間加熱後、氷水中で２分冷却し、凝集させ、磁石で回収し、上清部分を１．５ｍｌエッペンドルフチューブに移し（２）、Ｒｅｖｅｒｓｅ ｔｒａｎｓｃｒｉｐｔｉｏｎ ｐｏｌｙｍｅｒａｓｅ ｃｈａｉｎ ｒｅａｃｔｉｏｎ（以下、「ＲＴ−ＰＣＲ」と略す。）を行った。
(Separation of mRNA from total RNA by biotinylated oligo dT immobilized TM-USA)
5 μl of Human Tissue Total RNA and 35 μl of Nuclease free water were taken in a 1.5 ml Eppendorf tube, mixed, heated at 70 ° C. for 15 minutes, and left on ice for 2 minutes, then 5 × Binding buffer (100 mM Tris-HCl, 10 mM) 10 μl of EDTA, 5M LiCl, pH 8.0) was added, mixed with 100 μl of the prepared biotinylated oligo dT-immobilized TM-USA (4 mg / ml), shaken at room temperature for 10 minutes, and reacted. Thereafter, 10 μl of 10 wt% PEG (MW.6000) was added, cooled in ice water, aggregated, collected with a magnet, and the supernatant portion was removed. To the aggregated portion, 50 μl of Washing buffer (10 mM Tris-HCl, 0.15 M EDTA, 0.15 M LiCl, pH 7.5) and 10 μl of 10 wt% PEG (MW.6000) were added and dispersed in the solution. The tube was cooled again in ice water, aggregated, collected with a magnet, and the supernatant was removed. This washing operation was repeated twice. After washing, add 50 μl of Nuclease free water to the aggregated portion, disperse, dispense 20 μl into a 1.5 ml Eppendorf tube (1), heat the remaining solution at 37 ° C. for 5 minutes, cool in ice water for 2 minutes, Aggregate, collect with a magnet, transfer the supernatant to a 1.5 ml Eppendorf tube (2), and perform reverse transcription polymer chain reaction (hereinafter abbreviated as “RT-PCR”).
（ｍＲＮＡを用いたＲＴ−ＰＣＲによるｃｏｍｐｌｅｍｅｎｔａｒｙ ＤＮＡ（以下、「ｃＤＮＡ」と略す。）の増幅の確認）
ビオチン化オリゴｄＴ固定化ＴＭ−ＵＳＡを用いて分離したｍＲＮＡ（上記（１）,（２））をＯｎｅ ｓｔｅｐ ＲＴ−ＰＣＲ ｋｉｔ（ＱＩＡＧＥＮ社製）、β−Ａｃｔｉｎ ｐｒｉｍｅｒ ｐａｉｒ（Ｐｒｏｍｅｇａ社製）を用いてＲＴ−ＰＣＲを行った（反応液組成及び反応条件については、表２を参照）。ＰＣＲ産物５μｌとＴａＫａＲａ ６×Ｌｏａｄｉｎｇ ｂｕｆｆｅｒ（３０重量％Ｇｌｙｃｅｒｏｌ、３０ｍＭ ＥＤＴＡ、０．０３重量％Ｂｒｏｍｏｐｈｅｎｏｌ Ｂｌｕｅ、０．０３重量％Ｘｙｌｅｎｅ Ｃｙａｎｏｌ）１μｌを混合し、アガロースゲル電気泳動を行った。エチジウムブロマイド染色液で染色し、脱色後、ＰＣＲ産物のバンド（２８５ｂｐ）を確認した。
(Confirmation of amplification of complementary DNA (hereinafter abbreviated as “cDNA”) by RT-PCR using mRNA)
MRNA (above (1) and (2)) separated using biotinylated oligo-dT-immobilized TM-USA was used using One step RT-PCR kit (QIAGEN) and β-Actin primer pair (Promega). RT-PCR was performed (see Table 2 for the reaction solution composition and reaction conditions). 5 μl of PCR product and 1 μl of TaKaRa 6 × Loading buffer (30 wt% Glycerol, 30 mM EDTA, 0.03 wt% Bromophenol Blue, 0.03 wt% Xylene Cyanol) were mixed and subjected to agarose gel electrophoresis. After staining with ethidium bromide staining solution and decolorization, a band (285 bp) of the PCR product was confirmed.
ビオチン化オリゴｄＴ固定化ＴＭ−ＵＳＡにｍＲＮＡを結合させた後にＰＥＧを添加し、冷却磁気回収したｍＲＮＡに基づいて、ＲＴ−ＰＣＲで増幅したｃＤＮＡの目的産物のバンドを図１のレーン１、２に示す。レーン１は、ＴＭ−ＵＳＡとｍＲＮＡが結合した状態でＲＴ−ＰＣＲにかけ、増幅したＰＣＲ産物をアガロース電気泳動で確認した結果である。レーン２は、ＴＭ−ＵＳＡとｍＲＮＡの結合を外し、ＴＭ−ＵＳＡを除去した後にサンプルをＲＴ−ＰＣＲにかけ、増幅したＰＣＲ産物をアガロース電気泳動で確認した結果である。レーン３は、対象例として、ｍＲＮＡ分離キット（mRNAdembeads Purification Mini kit、ademtech社製）を用いて分離したｍＲＮＡからｃＤＮＡを増幅させたＰＣＲ産物をアガロース電気泳動で確認した結果である。レーン４は、１００ｂｐ ＬａｄｄｅｒのＤＮＡ分子量マーカーである。
After binding mRNA to biotinylated oligo dT-immobilized TM-USA, PEG was added, and the target product bands of cDNA amplified by RT-PCR based on the cooled magnetically recovered mRNA were shown in
ＴＭ−ＵＳＡと結合したｍＲＮＡについてＰＣＲを行った場合、及びＴＭ−ＵＳＡから分離したｍＲＮＡについてＰＣＲを行った場合ともに、ｃＤＮＡが増幅されており、検体からｍＲＮＡが回収されたことが確認できた。 Both when PCR was performed on mRNA bound to TM-USA and when PCR was performed on mRNA isolated from TM-USA, it was confirmed that cDNA was amplified and mRNA was recovered from the specimen.
ＲＴ−ＰＣＲの条件を以下に示す。 The conditions for RT-PCR are shown below.
Claims (9)
上限臨界溶液温度を有する温度応答性ポリマーで表面修飾された磁性微粒子と、電荷物質に対する親和性物質とが結合した吸着剤と、電荷物質を含む検体とを混合し、吸着剤に電荷物質を吸着させ、さらにポリアルキレングリコールと混合する工程、
前記工程で得られた混合物を上限臨界溶液温度未満にし、吸着剤を凝集させる工程、及び、
磁力により吸着剤を回収する工程、
を含む、検体中の電荷物質を回収する方法。 A method for recovering charged substances in a specimen,
Adsorbing the charged substance to the adsorbent by mixing the adsorbent with the magnetic fine particle surface-modified with the temperature-responsive polymer having the upper critical solution temperature, the adsorbent combined with the affinity substance for the charged substance, and the specimen containing the charged substance And further mixing with polyalkylene glycol,
Bringing the mixture obtained in the previous step below the upper critical solution temperature and aggregating the adsorbent; and
Recovering the adsorbent by magnetic force,
A method for recovering a charged substance in a specimen, comprising:
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| JP2015036624A (en) * | 2013-08-12 | 2015-02-23 | オーソ・クリニカル・ダイアグノスティックス株式会社 | Kit and method for detecting and quantifying detection object |
| WO2016148090A1 (en) * | 2015-03-13 | 2016-09-22 | 静岡県公立大学法人 | Gene examination method using stimulation-responsive magnetic nanoparticles |
| JP2016168020A (en) * | 2015-03-13 | 2016-09-23 | 静岡県公立大学法人 | Genetic test method using stimulus-responsive magnetic nanoparticles |
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