JP3441496B2 - Affinity separation material - Google Patents
Affinity separation materialInfo
- Publication number
- JP3441496B2 JP3441496B2 JP28854493A JP28854493A JP3441496B2 JP 3441496 B2 JP3441496 B2 JP 3441496B2 JP 28854493 A JP28854493 A JP 28854493A JP 28854493 A JP28854493 A JP 28854493A JP 3441496 B2 JP3441496 B2 JP 3441496B2
- Authority
- JP
- Japan
- Prior art keywords
- substance
- separation
- separation material
- target
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
- A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
- A61M1/3687—Chemical treatment
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/50—Conditioning of the sorbent material or stationary liquid
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/52—Physical parameters
- G01N30/54—Temperature
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- External Artificial Organs (AREA)
Description
【０００１】[0001]
【産業上の利用分野】本発明は、標的物質に対して特異
的親和性を有する物質と刺激応答性高分子を利用した新
規な物質分離材料及び分離システムに関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel substance separating material and a separating system using a substance having a specific affinity for a target substance and a stimuli-responsive polymer.
【０００２】[0002]
【従来の技術】近年、細胞工学や遺伝子工学等の発展に
伴い、細胞や遺伝子を利用した細胞治療や遺伝子治療の
研究が盛んとなっており、それらの生体物質を損傷させ
ることなく分離精製する技術が望まれている。また、バ
イオテクノジーの発展により、生物工学的手法によるペ
プチド、蛋白質、糖蛋白質類といった生理活性分子の生
産が行われるようになってきており、細胞やバイオプロ
ダクツの簡便で損傷の少ない分離精製技術が望まれてい
る。2. Description of the Related Art In recent years, with the development of cell engineering and genetic engineering, research on cell therapy and gene therapy using cells and genes has become active, and separation and purification of these biological substances without damaging them. Technology is desired. With the development of biotechnology, the production of bioactive molecules such as peptides, proteins and glycoproteins has been started by biotechnological methods, and a simple and less damaging separation and purification technique for cells and bioproducts has been developed. Is desired.
【０００３】従来より化学工業分野で使用されている分
離・精製の単位操作として、吸着・分配・蒸留・析出が
知られているが、これらの方法は、熱や有機溶媒の添加
などにより、被精製物質に対して、大幅な環境変化を強
いるプロセスである。従って、前述の細胞やバイオプロ
ダクツの分離には適していない。Adsorption, distribution, distillation and precipitation have been known as unit operations for separation and purification which have been conventionally used in the chemical industry field. However, in these methods, heat, an addition of an organic solvent, etc. This is a process that imposes drastic environmental changes on purified substances. Therefore, it is not suitable for the separation of the cells and bioproducts mentioned above.
【０００４】細胞やバイオプロダクツの分離方法とし
て、体積（分子量）や密度による方法（沈降速度法、密
度勾配遠心法、ゲル濾過法など）、電場中での移動度の
差による方法（電気泳動など）、等電点による方法（焦
点電気泳動など）、２液相間への分配による方法（２層
分配法、分配クロマトクラフィー）、固相への吸着性の
差による方法（吸着クロマトグラフィー、アフィニティ
ークロマトグラフィー）などが知られている。As a method for separating cells and bioproducts, a method based on volume (molecular weight) or density (sedimentation velocity method, density gradient centrifugation method, gel filtration method, etc.), a method based on difference in mobility in an electric field (electrophoresis, etc.) ), A method by isoelectric point (focusing electrophoresis, etc.), a method by partitioning between two liquid phases (two-layer partitioning method, partition chromatography), a method by difference in adsorptivity to solid phase (adsorption chromatography, Affinity chromatography) and the like are known.
【０００５】これらの分離方法の多くは、物理化学的性
状が大きく異なる細胞成分の分離には適用できるもの
の、物理化学的性状が良く似た成分や細胞、例えばリン
パ球亜集団の分離などには適用が困難であった。この中
で標的物質に対する選択性の高い方法は、アフィニティ
ークロマトグラフィーであり、近年広く利用されるよう
になってきている。細胞を対象とするアフィニティーク
ロマトグラフィーとしては、標的細胞の表層に存在する
膜蛋白等に対するモノクローナル抗体を結合したビーズ
やシャーレを用いた分離方法が報告されており、本方法
による各種リンパ球の亜集団分離も報告されている（例
えば、ジャーナル・オブ・イミュノロジカル・メソッ
ド、第５４号、２５１ページ、１９８３年に記載されて
いるブラウンらの研究報告）。この抗体を用いた方法
は、特異性が極めて高いことで利点であるが、欠点とし
て、吸着した細胞の脱着が困難なこと、抗体が細胞表層
の抗原に結合するための時間（接触時間）を長くする必
要があること、その結果、非特異的な吸着が増加するこ
となどがあった。Although many of these separation methods can be applied to the separation of cell components having greatly different physicochemical properties, they are applicable to the separation of components or cells having similar physicochemical properties, such as lymphocyte subpopulations. It was difficult to apply. Among them, the method having high selectivity for the target substance is affinity chromatography, which has been widely used in recent years. As an affinity chromatography targeting cells, a separation method using beads or a petri dish to which a monoclonal antibody against a membrane protein existing in the surface layer of a target cell is bound has been reported, and a subpopulation of various lymphocytes by this method. Separation has also been reported (eg, Brown et al., Research Report, Journal of Immunological Methods, 54, 251 page, 1983). The method using this antibody is advantageous in that it has extremely high specificity, but has the disadvantage that desorption of adsorbed cells is difficult, and the time (contact time) for the antibody to bind to the antigen on the cell surface is It was necessary to increase the length, and as a result, nonspecific adsorption was increased.
【０００６】前述の欠点を改良した方法としては、アビ
ジン−ビオチンのような親和性の高い結合を利用して、
短時間で分離材料に吸着させる方法が国際出願（ＷＯ９
１／１６１１６）で提案されている。すなわち、ビオチ
ンで標識した抗体を予め時間をかけて標的細胞に結合さ
せた後、アビジンを結合した分離材料に吸着させること
により、短時間で効率良く標的細胞を分離できることと
なる。しかしながら、この方法では、標的細胞の回収
が、物理的振動によりアビジン−ビオチン結合を解離す
ることにより行われているため、ビーズ同士の衝突によ
る細胞の損傷や機能低下が免れない。As a method for improving the above-mentioned drawbacks, a high affinity bond such as avidin-biotin is used,
International application for a method of adsorbing to a separation material in a short time (WO9
1/16116). That is, the target cells can be efficiently separated in a short time by binding the biotin-labeled antibody to the target cells in advance over time and then adsorbing the antibody to the separation material to which avidin is bound. However, in this method, since the target cells are collected by dissociating the avidin-biotin bond by physical vibration, cell damage and functional deterioration due to collision of beads are unavoidable.
【０００７】細胞機能を損なわないように回収する方法
としては、特開平２−２１１８６５号に記載された水に
対する上限または下限臨界溶解温度が０〜８０℃にある
ポリマーもしくはコポリマーで表面を被覆した細胞培養
基材が報告されている。この方法は、温度により疎水性
−親水性と相転移する温度応答性高分子を利用したもの
であり、温度応答性高分子が疎水性で収縮した状態の時
に細胞を吸着させた後、温度を変化させ、親水性となっ
て膨潤するときに吸着した細胞を脱着させる方法であ
る。この方法の欠点は、細胞に対する特異性が低いた
め、種々の細胞が存在する液体から特定の細胞を回収す
ることができないことである。特に、マクロファージ、
白血球、リンパ球などの多くは、曲率の小さい表面に吸
着することが知られており、フィルターや不織布形状に
加工した分離材料を用いて、特定のリンパ球などを選択
的に回収することは困難であった。As a method of recovering the cell function without impairing it, cells described in JP-A-2-211865, whose surface is coated with a polymer or copolymer having an upper or lower critical dissolution temperature in water of 0 to 80 ° C. Culture substrates have been reported. This method utilizes a temperature-responsive polymer that undergoes a phase transition from hydrophobic to hydrophilic depending on the temperature.When the temperature-responsive polymer is hydrophobic and contracts, cells are adsorbed and then the temperature is changed. This is a method of desorbing adsorbed cells when they are changed and become hydrophilic and swell. The disadvantage of this method is that it is not possible to recover specific cells from a liquid in which various cells are present due to their low specificity for cells. Especially macrophages,
Many white blood cells, lymphocytes, etc. are known to be adsorbed on the surface with a small curvature, and it is difficult to selectively collect specific lymphocytes etc. using a separation material processed into a filter or nonwoven fabric shape. Met.
【０００８】[0008]
【発明が解決しようとする課題】従って、本発明は、標
的物質に対する高い特異性を有し標的物質を簡便に回収
できる分離材料及び分離システムを提供することを目的
とする。SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide a separation material and a separation system which have high specificity for a target substance and can easily recover the target substance.
【０００９】[0009]
【問題点を解決するための手段】上記発明の目的は以下
のアフィニティー分離材料並びに分離方法によって達成
される。
（１）標的物質に対して特異的親和性を有する物質Ａ
を、互いに結合能のある少なくとも２種の化合物（物質
Ｂと物質Ｃ）の結合体からなるスペーサーを介して、刺
激応答性高分子とともに表面に担持させたことを特徴と
するアフィニティー分離材料。The above objects of the invention can be achieved by the following affinity separation material and separation method. (1) Substance A having a specific affinity for a target substance
The affinity separation material is characterized in that is supported on the surface together with the stimuli-responsive polymer via a spacer composed of a conjugate of at least two compounds (substance B and substance C) capable of binding to each other.
【００１０】（２）標的物質に対して特異的親和性を有
する物質Ａを、互いに結合能のある少なくとも２種の化
合物（物質Ｂと物質Ｃ）の結合体を介して、刺激応答性
高分子とともに表面に担持させたことを特徴とするアフ
ィニティー分離材料を用いて、標的物質を該分離材料に
結合させた後、刺激応答性高分子の高次構造を変化させ
て、標的物質を該分離材料より脱離させることを特徴と
する分離方法。(2) The stimuli-responsive polymer is prepared by interposing a substance A having a specific affinity for a target substance through a conjugate of at least two compounds (substance B and substance C) capable of binding each other. The target substance is bound to the separation material by using an affinity separation material characterized in that the target substance is bound to the separation material, and then the higher-order structure of the stimuli-responsive polymer is changed. A separation method characterized by further desorbing.
【００１１】（３）互いに結合能を有する物質Ｂと物質
Ｃのいずれか一方が結合した標的物質に対して特異的親
和性を有する物質Ａと、互いに結合能を有する物質Ｂと
物質Ｃの残った方を刺激応答性高分子とともに表面に担
持させた分離材料とからなる分離システムにおいて、物
質Ａと標的物質を接触させると同時もしくは接触させた
後に、さらに該分離材料と接触させて、該分離材料に標
的物質を結合させた後、刺激応答性高分子の高次構造を
変化させて、標的物質を脱離させることを特徴とする分
離方法。(3) Remaining substance A having a specific affinity for a target substance to which either one of substance B or substance C having a binding ability is bound, and substance B and substance C having a binding ability to each other In a separation system comprising a separation material supported on the surface together with a stimuli-responsive polymer, the substance A and the target substance are brought into contact with each other at the same time or after they are brought into contact with each other, and then the separation material is further brought into contact with the separation material. A method for separation, comprising binding a target substance to a material and then changing the higher-order structure of the stimuli-responsive polymer to release the target substance.
【００１２】本発明において、標的物質は特に限定され
ないが、機能維持が重要であり分離が困難な細胞、例え
ば、上皮系細胞、肝実質細胞、膵ラ島細胞、マクロファ
ージ、単核球、ＮＫ細胞（ＣＤ５６+）、血液幹細胞な
どの未分化細胞（ＣＤ３４+）、Ｂリンパ球、Ｔリンパ
球、及びそのサブセット（ＣＤ４+、ＣＤ８+、ＣＤ１９
+、ＣＤ７１+、ＩＬ２Ｒ+など）、腫瘍細胞などを好適
に例示できる。標的物質に対して特異的親和性を有する
物質とは、抗原−抗体、酵素−基質（阻害剤）、細胞表
層のレセプターとの反応などの生体の制御機構で見られ
る特異的親和性を例示できる。[0012] In the present invention, the target substance is not particularly limited, but cells whose function maintenance is important and difficult to separate, such as epithelial cells, hepatocytes, pancreatic islet cells, macrophages, mononuclear cells, NK cells (CD56 + ), undifferentiated cells such as blood stem cells (CD34 + ), B lymphocytes, T lymphocytes, and subsets thereof (CD4 + , CD8 + , CD19)
+ , CD71 + , IL2R +, etc.), tumor cells and the like can be preferably exemplified. The substance having a specific affinity for the target substance can be exemplified by a specific affinity found in a biological control mechanism such as an antigen-antibody, an enzyme-substrate (inhibitor), a reaction with a receptor on the cell surface. .
【００１３】互いに結合能のある少なくとも２種の化合
物（物質Ｂと物質Ｃ）の結合体とは、物質Ｂと物質Ｃと
が結合能を有していればよく生体由来物質であっても合
成化合物であっても、また、第３成分が結合していても
かまわないが、生理的条件で高い親和性を有するビオチ
ン−アビジン、ビオチン−ストレプトアビジン、リボフ
ラビン−リボフラビン結合蛋白などの組み合わせを好適
に例示できる。ビオチン−アビジンの組み合わせは、ビ
オチン標識抗体などが市販されており容易に入手できる
ため、応用性が高く好ましい。The conjugate of at least two compounds (substance B and substance C) capable of binding to each other may be any substance as long as the substances B and C have the ability to bind to each other, even if it is a biological substance. Although it may be a compound or the third component may be bound, a combination of biotin-avidin, biotin-streptavidin, riboflavin-riboflavin binding protein, etc., which has high affinity under physiological conditions, is preferably used. It can be illustrated. The biotin-avidin combination is preferable because it has high applicability since biotin-labeled antibodies and the like are commercially available and can be easily obtained.
【００１４】刺激応答性高分子とは、熱、ｐＨ、電位、
光などにより高次構造が変化して、水溶液中で膨潤した
り収縮する高分子であればよい。例えば、水に対する上
限臨界温度または下限臨界温度を有し、温度変化に応答
して、膨潤−収縮する高分子を好適に例示できる。その
ような高分子としては、Ｎ−イソプロピルアクリルアミ
ドやＮ、Ｎ−ジエチルアクリルアミド、Ｎ−イソプロピ
ルメタアクリルアミドなどのアクリルアミドやメタアク
リルアミドの誘導体類をはじめ、ビニルメチルエーテル
などのビニルエーテル類などのポリマーやコポリマーが
知られている。The stimuli-responsive polymer means heat, pH, electric potential,
Any polymer may be used as long as it is a polymer whose high-order structure is changed by light or the like and swells or contracts in an aqueous solution. For example, a polymer having an upper critical temperature or a lower critical temperature for water and swelling / contracting in response to a temperature change can be preferably exemplified. Examples of such a polymer include acrylamide and methacrylamide derivatives such as N-isopropylacrylamide, N, N-diethylacrylamide, and N-isopropylmethacrylamide, and polymers and copolymers such as vinyl ethers such as vinyl methyl ether. Are known.
【００１５】光応答性の場合は、例えば、アゾベンゼン
基を有する吸水性高分子のように光異性化をおこす高分
子、トリフェニルメタンロイコハイドロオキシドのビニ
ル誘導体とアクリルアミド系単量体との共重合体のよう
に光イオン解離する感応基を有する吸水性高分子、スピ
ロベンゾピランを含むＮ−イソプロピルアクリルアミド
ゲルのように疎水性相互作用が光変化する高分子などを
用いることができる。In the case of photoresponsiveness, for example, a polymer that undergoes photoisomerization such as a water-absorbing polymer having an azobenzene group, a copolymer of vinyl derivative of triphenylmethane leuco hydroxide and an acrylamide monomer is used. It is possible to use a water-absorbing polymer having a sensitive group that undergoes photoion dissociation such as coalescence, or a polymer having a hydrophobic interaction such as N-isopropylacrylamide gel containing spirobenzopyran that undergoes a photochange in hydrophobic interaction.
【００１６】物質Ａ、Ｂ、Ｃを材料表面に担持させる方
法は特に限定されず、公知の方法を組み合わせることに
よって目的の分離材料を作製することができる。例え
ば、官能基を有する単量体を共重合した刺激応答性高分
子を合成した後、該官能基を利用して物質Ｂの結合と基
材表面への結合を行なったり、電子線、オゾン、γ線、
プラズマなどを利用したグラフト法により官能基を有す
る単量体を共重合した刺激応答性グラフト鎖を形成させ
た後、物質Ｂを結合させたりすることができる。The method of supporting the substances A, B and C on the material surface is not particularly limited, and the desired separation material can be prepared by combining known methods. For example, after synthesizing a stimuli-responsive polymer in which a monomer having a functional group is copolymerized, the functional group is used to bond the substance B and the surface of the base material, or electron beam, ozone, gamma rays,
After forming a stimuli-responsive graft chain by copolymerizing a monomer having a functional group by a graft method using plasma or the like, the substance B can be bound thereto.
【００１７】一方、標的物質への特異的親和性を有する
物質Ａに対しては、縮合剤や架橋剤を用いて物質Ｃ結合
させることができる。さらに、これらを混和することに
より物質Ｂと物質Ｃとを結合させて、物質Ａが、物質Ｂ
と物質Ｃの結合体を介して、刺激応答性高分子とともに
表面に担持された分離材料を作製することができる。On the other hand, the substance C having a specific affinity for the target substance can be bound to the substance C using a condensing agent or a crosslinking agent. Further, by mixing these, the substance B and the substance C are bound to each other so that the substance A becomes the substance B.
A separation material supported on the surface together with the stimuli-responsive polymer can be produced through the conjugate of the substance C and the substance.
【００１８】分離材料の形態は、特に限定されず、プレ
ート状、シャーレ状、繊維状、不織布状、多孔質膜、フ
ィルター状、ビーズ状などを例示でき、それぞれの形態
にあったカラムなりモジュ−ルに収納されて使用されて
もかまわない。また、その基材となる材質についても水
に対して非溶解性であれば特に限定されず、ポリオレフ
ィン、ハロゲン化ポリオレフィン、ポリウレタン、ポリ
アミド、ポリエステル、綿、ポリスチレン、及びそれら
の変性物や共重合体など、既存の材料を例示することが
できる。The form of the separating material is not particularly limited, and examples thereof include plate, petri dish, fibrous, non-woven fabric, porous membrane, filter, and bead forms. It may be stored in a package and used. Also, the material for the base material is not particularly limited as long as it is insoluble in water, and includes polyolefins, halogenated polyolefins, polyurethanes, polyamides, polyesters, cotton, polystyrene, and modified products and copolymers thereof. For example, existing materials can be exemplified.
【００１９】標的物質の分離方法は、前記の分離材料に
標的物質を接触させてもかまわないし、予め物質Ｂと物
質Ｃのいずれかを結合した物質Ａを標的物質と接触させ
た後、物質Ｂと物質Ｃの残った方を結合させた分離材料
と接触させることにより、分離材料に吸着する方法でも
よい。In the method of separating the target substance, the target substance may be brought into contact with the above-mentioned separation material, or the substance A in which either the substance B or the substance C is bound in advance is brought into contact with the target substance and then the substance B is contacted. Alternatively, a method may be used in which the remaining one of the substance C and the remaining substance C is brought into contact with the bound separation material to adsorb to the separation material.
【００２０】標的物質の回収は、温度等の刺激により刺
激応答性高分子の高次構造を急速に変化せしめることに
より、物質Ｂと物質Ｃの結合を弱めることにより行う
が、回収率の向上などを目的として、必要に応じて物理
的な方法や化学的な方法を併用してもかまわない。細胞
やタンパク質などの非特異的吸着を抑制するため、刺激
応答性高分子が緩衝液、培養液、血液、血漿などで膨潤
した状態で標的物質を特異的に吸着させた後、温度等を
変化させて収縮させるこにより、物質Ｂと物質Ｃの結合
を解離させることが好ましい。また、このとき物質Ａと
標的物質との特異的結合が解離することによって、標的
物質が脱離してもかまわない。The target substance is recovered by rapidly changing the higher-order structure of the stimulus-responsive polymer by stimulating with temperature or the like to weaken the bond between the substance B and the substance C, but the recovery rate is improved. For the purpose, a physical method or a chemical method may be used together if necessary. In order to suppress non-specific adsorption of cells and proteins, the stimuli-responsive polymer specifically adsorbs the target substance when swollen in buffer, culture medium, blood, plasma, etc., and then changes the temperature etc. It is preferable to dissociate the bond between the substance B and the substance C by causing the substance to contract. Further, at this time, the target substance may be desorbed by dissociating the specific bond between the substance A and the target substance.
【００２１】本発明の分離材料は、標的物質に対する特
異的親和性を有するリガンドと刺激応答性高分子とを有
しているため、標的物質を選択的に吸着し、刺激応答性
高分子の高次構造変化により、該標的物質を簡便に脱離
できる分離材料や分離システムとなる。Since the separation material of the present invention has a ligand having a specific affinity for a target substance and a stimulus-responsive polymer, the target substance is selectively adsorbed, and the high level of the stimulus-responsive polymer. Due to the change in the secondary structure, a separation material or a separation system can be easily desorbed from the target substance.
【００２２】[0022]
（実施例１）標的物質としてＣＤ４+細胞を設定し、標
的物質に対して特異的親和性を有する物質としてＣＤ４
に対する抗体、物質Ｂと物質Ｃの結合体としてアビジン
−ビオチン結合、刺激応答性高分子としてポリ（Ｎ−イ
ソプロピルアクリルアミド）を選定して、アフィニティ
分離材料を作製し、ＣＤ４+細胞の分離を行った。(Example 1) CD4 + cells were set as a target substance, and CD4 + was used as a substance having a specific affinity for the target substance.
Antibody, avidin-biotin bond as a conjugate of substance B and substance C, and poly (N-isopropylacrylamide) as a stimuli-responsive polymer, an affinity separation material was prepared, and CD4 + cells were separated. .
【００２３】分子内に複数のパ−オキサイド基を有する
ポリブチルメタクリレートを重合開始剤として、Ｎ−イ
ソプロピルアクリルアミドとメタクリル酸とを重合さ
せ、ブチルメタクリレートを疎水性ドメインとしＮ−イ
ソプロピルアクリルアミドとメタクリル酸との共重合体
（モル比２２：１）を温度応答性ドメインとするブロッ
ク共重合体を合成した。本ブロック共重合体を、クロロ
ホルム−エタノール（９：１）の混合溶媒に溶解し、プ
ラズマ放電処理した厚さ１００μのポリエステル不織布
にコーティングした。Polymerization of N-isopropylacrylamide and methacrylic acid with polybutylmethacrylate having a plurality of peroxide groups in the molecule as a polymerization initiator, butylmethacrylate as a hydrophobic domain and N-isopropylacrylamide and methacrylic acid. A block copolymer having the temperature-responsive domain of the copolymer (22: 1 in molar ratio) was synthesized. This block copolymer was dissolved in a mixed solvent of chloroform-ethanol (9: 1) and coated on a polyester non-woven fabric having a thickness of 100 μm and subjected to plasma discharge treatment.
【００２４】本不織布をφ２５に打ち抜いて容器に入
れ、５ｍｇ／ｍｌの１−エチル−３−（ジメチルアミノ
プロピル）カルボジイミド（シグマ社製）溶液を２０ｍ
ｌ（ｐＨ５．５）シャーレに注入し、５分間室温で放置
した。続いて、アビジンの１０ｍｇ／ｍｌ溶液を１ｍｌ
添加し、室温で１時間時々撹拌しながら放置した。さら
に、グリシンを最終濃度で０．２モルとなるように添加
して１時間放置した後、リン酸バッファー（ＰＢＳ）で
リンスした。このアビジン固定化不織布は、ＰＢＳ中で
４℃で保存した。This non-woven fabric was punched out to a diameter of 25 and placed in a container. A 5 mg / ml solution of 1-ethyl-3- (dimethylaminopropyl) carbodiimide (manufactured by Sigma) was added to 20 m of the solution.
It was poured into a petri dish (pH 5.5) and left for 5 minutes at room temperature. Then, add 1 ml of a 10 mg / ml solution of avidin.
Added and left at room temperature for 1 hour with occasional stirring. Further, glycine was added to a final concentration of 0.2 mol, left for 1 hour, and then rinsed with a phosphate buffer (PBS). This avidin-immobilized nonwoven fabric was stored in PBS at 4 ° C.
【００２５】ＣＤ４+細胞とアビジンに特異的に吸着す
る物質として、ビオチン標識したＣＤ４抗体を購入（イ
ムノテック）し、アビジン固定化不織布にＰＢＳ中でイ
ンキュベートすることにより結合させて分離材料を得
た。この分離用不織布を５枚積層してφ２５ｍｍのフィ
ルターホルダーにセットして、人新鮮血のバフィーコー
トより採取し１％アルブミンを添加したＰＢＳで洗浄し
て調整した白血球液（１×１０5 ／ｍｌ）を２５℃でゆ
っくりと１０ｍｌ流すことにより分離用不織布に吸着さ
せた。吸着した細胞の溶出は、温度を３５℃に上昇させ
て、１％アルブミンを添加したＰＢＳを流すことで行っ
た。その結果、純度９４％、回収率５３％でＣＤ４+細
胞を回収することができた。As a substance that specifically adsorbs to CD4 + cells and avidin, a biotin-labeled CD4 antibody was purchased (immunotech) and allowed to bind to an avidin-immobilized nonwoven fabric by incubating in PBS to obtain a separation material. . Five layers of this non-woven fabric for separation were stacked and set on a φ25 mm filter holder, and leukocyte fluid (1 × 10 5 / ml) was prepared by collecting human fresh blood from a buffy coat and washing it with PBS containing 1% albumin. ) Was adsorbed on the separating nonwoven fabric by slowly flowing 10 ml at 25 ° C. Elution of the adsorbed cells was performed by raising the temperature to 35 ° C. and flowing PBS containing 1% albumin. As a result, CD4 + cells could be recovered with a purity of 94% and a recovery rate of 53%.
【００２６】（実施例２）実施例１と同様の方法で作製
したアビジン固定化不織布を、φ２５ｍｍのフィルター
ホルダーに５枚積層することにより分離モジュ−ルを作
製した。骨髄から得たバフィーコートより１％アルブミ
ンを添加したＰＢＳで洗浄して調整した白血球液（１×
１０6 ／ｍｌ）に、ビオチン標識したＣＤ３４抗体を添
加した後、前述の分離モジュールに定量ポンプで０．５
ｍｌ／ｍｉｎで流した。吸着した細胞の溶出は、温度を
３５℃に上昇させて１％アルブミンを添加したＰＢＳを
流すことで行った。その結果、純度９５％、回収率４８
％でＣＤ３４+細胞を回収することができた。(Example 2) A separation module was produced by laminating five avidin-immobilized nonwoven fabrics produced by the same method as in Example 1 on a filter holder having a diameter of 25 mm. White blood cell liquid prepared by washing with PBS containing 1% albumin from a buffy coat obtained from bone marrow (1 x
Biotin-labeled CD34 antibody was added to 10 6 / ml), and then 0.5 μm was added to the aforementioned separation module with a metering pump.
Flowed at ml / min. Elution of the adsorbed cells was performed by raising the temperature to 35 ° C. and flowing PBS containing 1% albumin. As a result, the purity was 95% and the recovery rate was 48.
It was possible to recover CD34 + cells in%.
【００２７】[0027]
【発明の効果】本発明の分離材料や分離方法は、標的物
質を選択的に吸着する物質Ａを使用して標的物質を吸着
し、刺激応答性高分子の高次構造変化により解離する物
質Ｂと物質Ｃの結合により分離材料に保持させているた
め、標的物質への高い選択性が得られ、かつ標的物質の
変性や機能変化が少ない状態で、外部刺激（環境変化）
により簡便に脱離させて回収することが可能となる。ま
た、従来困難であった血球系細胞や機能細胞の詳細な分
離が容易にできるため、特殊な細胞を分離して、必要に
より増殖・機能変換させて再び生体内に戻す細胞治療や
遺伝子治療に有効な分離技術等として効果を発揮するこ
ととなる。また、本分離材料や分離方法は、標的物質を
各種のバイオプロダクツ、生体関連物質や化学物質とす
ることで、バイオ産業、医薬品産業、化学工業から診断
・治療などの医療分野に至るまで、広範に利用できる技
術となる。Industrial Applicability The separation material and the separation method of the present invention use the substance A that selectively adsorbs the target substance to adsorb the target substance, and dissociate the substance B by dissociation due to the conformational change of the stimuli-responsive polymer. Since it is held in the separation material by the binding of the substance C and the substance C, high selectivity to the target substance is obtained, and external stimulus (environmental change) is obtained in a state where the target substance is not denatured or its function is changed little.
By this, it becomes possible to easily desorb and collect. In addition, since it is easy to perform detailed separation of blood cells and functional cells, which was difficult in the past, it is suitable for cell therapy and gene therapy in which special cells are separated, and if necessary, undergo proliferation / function conversion and then return to the living body. It will be effective as effective separation technology. In addition, this separation material and separation method can be applied to a wide range of fields from the bio industry, the pharmaceutical industry, the chemical industry to the medical field such as diagnosis and treatment by using various bio products, bio-related substances and chemical substances as target substances. Technology that can be used for.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.7 識別記号 ＦＩ Ｃ１２Ｎ 1/02 Ｃ１２Ｎ 1/02 (56)参考文献 特開 昭64−72054（ＪＰ，Ａ) 特開 平１−157000（ＪＰ，Ａ) 特開 平５−133947（ＪＰ，Ａ) 特開 昭63−159756（ＪＰ，Ａ) 特開 平７−136508（ＪＰ，Ａ) 特開 平５−220389（ＪＰ，Ａ) (58)調査した分野(Int.Cl.7，ＤＢ名) B01J 20/26 A61M 1/36 545 B01D 15/00 G01N 30/48 A61K 35/12 C12N 1/02 ─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. 7 Identification code FI C12N 1/02 C12N 1/02 (56) Reference JP-A 64-72054 (JP, A) JP-A 1-157000 (JP , A) JP-A-5-133947 (JP, A) JP-A-63-159756 (JP, A) JP-A-7-136508 (JP, A) JP-A-5-220389 (JP, A) (58) Fields investigated (Int.Cl. 7 , DB name) B01J 20/26 A61M 1/36 545 B01D 15/00 G01N 30/48 A61K 35/12 C12N 1/02
Claims (3)
度制御のみによって回収させられる、特異的親和性を有
する物質を、互いに結合能のある少なくとも２種の化合
物の結合体からなるスペーサーを介して、温度応答性高
分子とともに表面に担持させたことを特徴とするアフィ
ニティ分離材料。1. A target substance is specifically adsorbed and further heated.
The substance having a specific affinity, which can be recovered only by controlling the temperature , is supported on the surface together with the temperature- responsive polymer through a spacer composed of a conjugate of at least two compounds capable of binding to each other. Characterized affinity separation material.
物質Ａを、互いに結合能のある少なくとも２種の化合物
（物質Ｂと物質Ｃ）の結合体を介して、温度応答性高分
子とともに表面に担持させたことを特徴とするアフィニ
ティ分離材料を用いて、標的物質を該分離材料に結合さ
せた後、温度応答性高分子の高次構造を変化させて、標
的物質を該分離材料より脱離させることを特徴とする分
離方法。 2. It has a specific affinity for a target substance.
At least two compounds capable of binding substance A to each other
Through the combination of (substance B and substance C), high temperature responsiveness
Affinity characterized by being carried on the surface together with the child
The target substance is bound to the separation material using the separation material.
Then, the higher-order structure of the temperature-responsive polymer is changed to
Of separating a specific substance from the separation material
Separation method.
いずれか一方が結合した標的物質に対して特異的親和性
を有する物質Ａと、互いに結合能を有する物質Ｂと物質
Ｃの残った方を温度応答性高分子とともに表面に担持さ
せた分離材料とからなる分離システムにおいて、物質Ａ
と標的物質を接触させると同時もしくは接触させた後
に、さらに該分離材料を接触させて、該分離材料に標的
物質を結合させた後、温度応答性高分子の高次構造を変
化させて、標的物質を脱離させることを特徴とする分離
方法。 3. A substance B and a substance C capable of binding to each other
Specific affinity for target substance bound by either one
A having a substance, and a substance B and a substance capable of binding to each other
The remaining C is supported on the surface together with the temperature-responsive polymer.
In the separation system consisting of the separated material, the substance A
At the same time as or after contacting the target substance with
And further contact the separation material to target the separation material.
After binding substances, the higher-order structure of the temperature-responsive polymer is changed.
Separation characterized by releasing the target substance
Method.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP28854493A JP3441496B2 (en) | 1993-11-17 | 1993-11-17 | Affinity separation material |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP28854493A JP3441496B2 (en) | 1993-11-17 | 1993-11-17 | Affinity separation material |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH07136505A JPH07136505A (en) | 1995-05-30 |
| JP3441496B2 true JP3441496B2 (en) | 2003-09-02 |
Family
ID=17731621
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP28854493A Expired - Fee Related JP3441496B2 (en) | 1993-11-17 | 1993-11-17 | Affinity separation material |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3441496B2 (en) |
Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2069695A (en) * | 1995-03-13 | 1996-10-02 | Ao Forschungsinstitut Davos | An extracorporeal blood treatment apparatus and method for removal of free circulating infectious agents |
| AU9224698A (en) * | 1997-09-08 | 1999-03-29 | Fleximer, Llc | Smart polymer-coupled bioactive entities and uses thereof |
| EP0922715B8 (en) | 1997-12-09 | 2008-05-21 | National Institute of Advanced Industrial Science and Technology | Stimuli-responsive polymer utilizing keto-enol tautomerization |
| WO2000067901A1 (en) * | 1999-05-11 | 2000-11-16 | Japan Chemical Innovation Institute | Affinity-controlling material with the use of stimulus-responsive polymer and separation/purification method with the use of the material |
| JP4536879B2 (en) * | 1999-07-29 | 2010-09-01 | 学校法人 関西大学 | Method and apparatus for removing organic substance in liquid |
| JP5109003B2 (en) * | 2000-10-13 | 2012-12-26 | 岡野 光夫 | Separation material such as stimulus-responsive affinity chromatography material and separation purification method |
| JP2006158305A (en) * | 2004-12-08 | 2006-06-22 | National Institute Of Advanced Industrial & Technology | Adsorbent for separating cell, adsorbent module for separating cell and method for separating cell |
| WO2007055178A1 (en) * | 2005-11-08 | 2007-05-18 | Japan Health Sciences Foundation | Method of cell fractionation and substrate to be used for the method |
| JP6426503B2 (en) * | 2015-03-09 | 2018-11-21 | 株式会社日立製作所 | Adsorbent, separation and purification apparatus using the same, and separation and purification method |
| JP6778380B2 (en) | 2016-08-23 | 2020-11-04 | 昭和電工マテリアルズ株式会社 | Adsorbent |
| WO2023276937A1 (en) * | 2021-06-30 | 2023-01-05 | 日油株式会社 | Cell dissociating agent and cell separation method |
-
1993
- 1993-11-17 JP JP28854493A patent/JP3441496B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPH07136505A (en) | 1995-05-30 |
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