JP4680361B2 - Separation / collection method - Google Patents

Description

(式中、Ｒ ¹ 、Ｒ ² 及びＲ ³ は同一もしくは異なる基であって、水素原子、炭素数１〜６のアルキル基又はヒドロキシアルキル基を示す。ｎは１〜４の整数である。)
【０００６】
【発明の実施の形態】
本発明に用いる分離材は、上記式(１)で表される基を少なくとも表面に有する分離材であって、その表面をＸ線光電子分光分析によって測定したスペクトルにおける、式(１)で表される基に由来するリン元素の量Ｐと、表面全体における炭素元素の量Ｃとの比(Ｐ／Ｃ)が０．００２〜０．３となる量の式(１)で表される基を有する分離材である。分離材の選択的分離能を向上させるために、上記Ｐ／Ｃの範囲は０．０１〜０．２が好ましい。
ここで、分離材の表面をＸ線光電子分光分析によって測定する方法としては、後述する実施例に基づいて測定することができる。
【０００７】
式(１)中のＲ¹、Ｒ²及びＲ³は、同一もしくは異なる基であって、水素原子、炭素数１〜６のアルキル基又はヒドロキシアルキル基を示す。
炭素数１〜６のアルキル基としては、例えば、メチル基、エチル基、プロピル基、ブチル基、ペンチル基、ヘキシル基、シクロヘキシル基、フェニル基等が挙げられる。
炭素数１〜６のヒドロキシアルキル基としては、例えば、ヒドロキシメチル基、２−ヒドロキシエチル基、３−ヒドロキシプロピル基、４−ヒドロキシブチル基、５−ヒドロキシペンチル基、６−ヒドロキシヘキシル基等が挙げられる。
【０００８】
式(１)で表される基としては、例えば、式(２)で表されるホスホリルコリン類似基含有単量体に由来する基、式(２)で表されるホスホリルコリン類似基含有単量体を含む単量体組成物を重合してなる重合体に由来する基等が挙げられる。
【化４】

式(２)中、Ｒ¹、Ｒ²及びＲ³は、式(１)と同じであり、Ｒ⁴は炭素数１〜６のアルキル基を示し、Ｒ⁵は水素原子又はメチル基を示す。ｎは１〜４の整数である。
【０００９】
式(２)で表されるホスホリルコリン類似基含有単量体としては、例えば、２−((メタ)アクリロイルオキシ)エチル−２'−(トリメチルアンモニオ)エチルホスフェート、３−((メタ)アクリロイルオキシ)プロピル−２'−(トリメチルアンモニオ)エチルホスフェート、４−((メタ)アクリロイルオキシ)ブチル−２'−(トリメチルアンモニオ)エチルホスフェート、５−((メタ)アクリロイルオキシ)ペンチル−２'−(トリメチルアンモニオ)エチルホスフェート、６−((メタ)アクリロイルオキシ)ヘキシル−２'−(トリメチルアンモニオ)エチルホスフェート、２−((メタ)アクリロイルオキシ)エチル−２'−(トリエチルアンモニオ)エチルホスフェート、２−((メタ)アクリロイルオキシ)エチル−２'−(トリプロピルアンモニオ)エチルホスフェート、２−((メタ)アクリロイルオキシ)エチル−２'−(トリブチルアンモニオ)エチルホスフェート、２−((メタ)アクリロイルオキシ)エチル−２'−(トリシクロヘキシルアンモニオ)エチルホスフェート、２−((メタ)アクリロイルオキシ)エチル−２'−(トリフェニルアンモニオ)エチルホスフェート、２−((メタ)アクリロイルオキシ)プロピル−２'−(トリメチルアンモニオ)エチルホスフェート、２−((メタ)アクリロイルオキシ)ブチル−２'−(トリメチルアンモニオ)エチルホスフェート、２−((メタ)アクリロイルオキシ)ペンチル−２'−(トリメチルアンモニオ)エチルホスフェート、２−((メタ)アクリロイルオキシ)ヘキシル−２'−(トリメチルアンモニオ)エチルホスフェート等が挙げられる。
中でも２−((メタ)アクリロイルオキシ)エチル−２'−(トリメチルアンモニオ)エチルホスフェートが好ましく、さらにＭＰＣが入手性等の点でより好ましい。
【００１０】
式(２)で表されるホスホリルコリン類似基含有単量体は、公知の方法で製造できる。例えば、特開昭５４−６３０２５号公報、特開昭５８−１５４５９１号公報等に示される公知の方法に準じて製造できる。
具体的には、環状リン化合物と２−ヒドロキシエチル(メタ)アクリレートとを、脱ハロゲン化水素剤のもとで反応させ、次いで、トリメチルアミンを反応させることにより、開環させて目的物を得る方法等が挙げられる。
【００１１】
式(２)で表されるホスホリルコリン類似基含有単量体を含む単量体組成物を重合してなる重合体としては、(Ａ)式(２)で表されるホスホリルコリン類似基含有単量体１０〜１００ｍｏｌ％、(Ｂ)疎水性単量体０〜９０ｍｏｌ％及び(Ｃ)親水性単量体０〜７０ｍｏｌ％からなる単量体組成物を重合してなる重合体が好ましく、特に、(Ａ)成分２０〜８０ｍｏｌ％、(Ｂ)成分２０〜６０ｍｏｌ％及び(Ｃ)成分０〜２０ｍｏｌ％からなる単量体組成物を重合してなる重合体が望ましい。(Ａ)成分が１０ｍｏｌ％未満の場合は、分離材としての性能を発現させることが困難であるので好ましくない。(Ｂ)成分を２０〜６０ｍｏｌ％の範囲で適宜配合することにより、分離材としての性能を安定して発現させることが容易となる。また、(Ｃ)成分を２０ｍｏｌ％以下配合することにより、得られる重合体の水溶液等に対する親和性を向上させることができる。
【００１２】
(Ｂ)成分としては、例えば、メチル(メタ)アクリレート、エチル(メタ)アクリレート、ブチル(メタ)アクリレート、２−エチルヘキシル(メタ)アクリレート、ラウリル(メタ)アクリレート、ステアリル(メタ)アクリレート等の直鎖又は分岐アルキル(メタ)アクリレート；シクロヘキシル(メタ)アクリレート等の環状アルキル(メタ)アクリレート；ベンジル(メタ)アクリレート、フェノキシエチル(メタ)アクリレート等の芳香族(メタ)アクリレート；ポリプロピレングリコール(メタ)アクリレート等の疎水性ポリアルキレングリコール(メタ)アクリレート；スチレン、メチルスチレン、クロロメチルスチレン等のスチレン系単量体；メチルビニルエーテル、ブチルビニルエーテル等のビニルエーテル系単量体；酢酸ビニル、プロピオン酸ビニル等のビニルエステル系単量体等が挙げられる。これらは単独若しくは混合物として使用できる。
【００１３】
(Ｃ)成分としては、例えば、ヒドロキシル基、カルボキシル基、スルホン酸基、アミド基、アミノ基、ジアルキルアミノ基、トリアルキルアミノ塩基、トリアルキルホスホニウム塩基及びポリオキシエチレン基からなる群より選ばれる親水性基を有する単量体等が挙げられる。具体的には、２−ヒドロキシエチル(メタ)アクリレート、２−ヒドロキシブチル(メタ)アクリレート、４−ヒドロキシブチル(メタ)アクリレート等の水酸基含有(メタ)アクリレート；アクリル酸、メタクリル酸等のカルボン酸；スチレンスルホン酸、(メタ)アクリロイルオキシホスホン酸、２−ヒドロキシ−３−(メタ)アクリルオキシプロピルトリメチルアンモニウムクロライド等のイオン性基含有単量体；(メタ)アクリルアミド、アミノエチルメタクリレート、ジメチルアミノエチル(メタ)アクリレート等の含窒素単量体；ポリエチレングリコール(メタ)アクリレート等が挙げられる。これらは単独若しくは混合物として使用できる。
【００１４】
前記ホスホリルコリン類似基含有単量体を含む単量体組成物を重合してなる重合体は、前記(Ａ)成分だけからなる単量体組成物、前記(Ａ)成分及び(Ｂ)成分からなる単量体組成物、前記(Ａ)成分及び(Ｃ)成分からなる単量体組成物、あるいは前記(Ａ)成分、(Ｂ)成分及び(Ｃ)成分からなる単量体組成物を重合したものであればよく、通常のラジカル(共)重合により製造することができる。
この重合体の分子量は、重量平均分子量で、５０００〜５００００００の範囲が好ましく、選択的分離性能を向上させる上で、また各種溶媒に対する重合体の耐溶出性を向上させる上で、１０００００〜２００００００の範囲が特に好ましい。
【００１５】
本発明に用いる分離材の調製は、上記(Ｐ／Ｃ)比が特定範囲となるような方法であれば特に限定されない。例えば、上記式(２)で表されるホスホリルコリン類似基含有単量体等の式(１)で表される基を有する単量体、若しくは上記式(２)で表されるホスホリルコリン類似基含有単量体を含む単量体組成物を重合してなる重合体を用いて、基材の少なくとも表面に化学的に修飾させるか、若しくは基材の表面にコートして乾燥固定させる方法等により得ることができる。
【００１６】
前記基材の材料としては、例えば、ポリエチレンテレフタレート、ポリスチレン、ポリメチルメタクリレート等の合成樹脂；磁性粉、シリカ、アルミナ、ガラス等の無機物が挙げられる。基材の形態は、特に限定されず、例えば、粉体、粒子、ビーズ、フレーク形状物、シート、フィルム、プレート、ゲル、繊維状物、織布、布織布等が挙げられる。
【００１７】
本発明に用いる分離材を調製するにあたり、前記(Ｐ／Ｃ)の制御は、後述する分離対象物質である特定成分によって、使用するホスホリルコリン類似基含有単量体、該単量体を含む単量体組成物を重合してなる重合体の種類や、基材の種類及び形状等を適宜選択して行うことができる。
分離対象物質が、赤血球、白血球、血小板の血球細胞の場合は、例えば、前記ホスホリルコリン類似基含有単量体を含む単量体組成物として、ＭＰＣと、ブチルメタクリレート(ＢＭＡ)、ステアリルメタクリレート(ＳＭＡ)等の炭素数４〜１８のアルキル(メタ)アクリレート、カチオン性基含有単量体とを用い、ＭＰＣ／(疎水性基含有単量体＋カチオン性基含有単量体)のモル比が１０／９０〜１００／０、特に２０／８０〜９０／１０となる単量体組成物を用いて重合体を調製し、該重合体を所定の(Ｐ／Ｃ)比となるように基材に固定することが好ましい。
【００１８】
分離対象物質が、リンパ球の分離の場合は、例えば、前記ホスホリルコリン類似基含有単量体を含む単量体組成物として、ＭＰＣと、ＢＭＡ等のアルキル(メタ)アクリレートとを用い、ＭＰＣ／疎水性基含有単量体のモル比が１０／９０〜１００／０、特に５０／５０〜９０／１０となる単量体組成物を用いて重合体を調製し、該重合体を所定の(Ｐ／Ｃ)比となるように基材に固定することが好ましい。
【００１９】
分離対象物質が、ＩｇＧ等の免疫グロブリンの場合は、例えば、前記ホスホリルコリン類似基含有単量体を含む単量体組成物として、ＭＰＣと、ＢＭＡ等のアルキル(メタ)アクリレートとを用い、ＭＰＣ／疎水性基含有単量体のモル比が１０／９０〜１００／０、特に３０／７０〜９０／１０となる単量体組成物を用いて重合体を調製し、該重合体を所定の(Ｐ／Ｃ)比となるように基材に固定することが好ましい。
【００２０】
分離対象物質が、株細胞及び初代培養細胞の場合は、例えば、前記ホスホリルコリン類似基含有単量体を含む単量体組成物として、ＭＰＣと、ＢＭＡ等のアルキル(メタ)アクリレートとを用い、ＭＰＣ／疎水性基含有単量体のモル比が１０／９０〜１００／０、特に４０／６０〜８５／１５となる単量体組成物を用いて重合体を調製し、該重合体を所定の(Ｐ／Ｃ)比となるように基材に固定することが好ましい。
【００２１】
分離対象物質が、ダイオキシン、その誘導体であるジベンゾフランの場合は、例えば、前記ホスホリルコリン類似基含有単量体を含む単量体組成物として、ＭＰＣと、ＢＭＡ等のアルキル(メタ)アクリレートとを用い、ＭＰＣ／疎水性基含有単量体のモル比が１０／９０〜１００／０、特に１５／８５〜９０／１０となる単量体組成物を用いて重合体を調製し、該重合体を所定の(Ｐ／Ｃ)比となるように基材に固定することが好ましい。
【００２２】
本発明の分離・回収方法は、上記分離材を、特定成分を含む溶液に接触させ、溶液中に含まれる１種類又は複数の特定成分を選択的に分離・回収する。特定成分を含む溶液と接触させる条件は、分離材表面に選択的に特定成分を付着させることができる条件を、分離対象物質である特定成分の種類等に応じて適宜選択することができる。更に特定成分を付着させた分離材は、洗浄又は溶剤で処理する方法等により、付着している特定成分を分離材表面から剥がして回収することができる。
【００２３】
特定成分を含む溶液としては、体液等が挙げられる。ここで、体液とは、動物体内の脈管又は組織・細胞の間を満たす全ての液体、及び体内から体外へ放出又は分泌される液体のことを指し、例えば、血液、血漿、血清、リンパ液、涙液、尿、脊髄液等が挙げられる。
体液以外の溶液としては、例えば、水溶液、有機溶媒を１種類あるいは複数種類含んだ溶液、水溶液と１種類あるいは複数種類の有機溶媒を混合した混合溶液等が挙げられる。具体的には、水、生理的食塩水、リン酸緩衝溶液、生理的リン酸緩衝溶液、培地、アルコール、アセトン、クロロホルム、エーテル、トルエン、アセトニトリル又はこれらの２種以上の混合溶液等が挙げられる。なお、前記溶液には、体液を希釈したものも含まれる。
【００２４】
本発明において、分離対象とする特定成分は、血球細胞、株細胞及び初代培養細胞からなる群より選択される１種又は２種以上の生体に由来する細胞、免疫グロブリン、又はダイオキシンもしくはその誘導体である。
【００２５】
前記血球細胞としては、赤血球、白血球、血小板又はこれらの混合物等が挙げられ、白血球としては、好中球、好酸球、好塩基球、リンパ球、単球が挙げられる。リンパ球としては、Ｔ細胞、Ｂ細胞等が挙げられる。血小板としては、血小板の活性化度合い(凝集反応の進行度合い)は特に限定されず、未活性化及び活性化した血小板等が挙げられる。これらの血球細胞の中で特に好ましくは、種々の疾患に際してその数が増減する白血球等が挙げられる。
前記株細胞及び初代培養細胞としては、由来の動物、由来の臓器及び形態は特に限定されず、繊維芽細胞、表皮細胞、内皮細胞、平滑筋細胞、神経細胞等が挙げられる。特に好ましくは、ティシュ・エンジニアリング(tissue engineering)等で利用可能な、血管内皮細胞、血管平滑筋細胞、肝細胞等が挙げられる。
【００２７】
前記免疫グロブリン(抗体)は、以下の(１)〜(７)が例示できる。
(１)Ｃ反応性蛋白質(ＣＲＰ)、リューマチ因子(ＲＦ)、トランスフェリン等の血漿蛋白質又はこれら血漿蛋白質に対する抗体、
(２)卵胞ホルモン、黄体ホルモン、男性ホルモン、インシュリン、グルカゴン、性腺刺激ホルモン、卵胞刺激ホルモン、黄体形成ホルモン、成長ホルモン、副腎皮質ホルモン、甲状腺ホルモン、副甲状腺ホルモン、プロラクチン、甲状腺刺激ホルモン、副腎皮質刺激ホルモン等、更に好ましくは、甲状腺刺激ホルモン(ＴＳＨ)、トリヨードサイロニン(Ｔ３)、サイロキシン(Ｔ４)、チロキシン結合蛋白質(ＴＢＧ)、サイログロブリン、インスリン、エストリオール(Ｅ３)、絨毛性ゴナドトロピン(ＨＣＧ)、ヒト胎盤性ラクトーゲン(ＨＰＬ)等のホルモンに対する抗体、
(３)癌胎児性抗原(ＣＥＡ)、β２−マイクログロブリン、α−フェトプロテイン(ＡＦＰ)等の腫瘍関連物質に対する抗体、
(４)ＨＢＳ抗体、ＨＢｅ抗体等のウィルス肝炎の抗体、
(５)ムンプス、ヘルペス、麻疹、風疹、サイトメガロ等のウィルス、抗エイズ抗体等の各種生体成分に対する抗体、
(６)フェノバルビタール、アセトアミノフェン、サリチル酸、シクロスポリン等の各種薬剤に対する抗体、
(７)酵素に対する抗体。
ただし、前記抗体はＦａｂフラグメント、Ｆ(ａｂ)'２フラグメント、又は還元型抗体であってもよい。前記(１)〜(７)のうち、特に好ましくは、種々の疾患に際してその量が増減し、診断あるいは治療にも利用することが可能である抗体が挙げられる。
【００２８】
【発明の効果】
本発明に用いる分離材は、少なくとも表面に、特定のホスホリルコリン類似構造を有する基を、リン元素と炭素元素との割合を特定とする範囲で含まれるので、広範囲に及ぶ特定成分を選択的に分離することができ、本発明の方法では、この分離材を用いるので、特定成分を選択的に分離・回収することが可能である。すなわち、目的とする特定成分を選択的に効率よく、他の成分から分離することで、目的成分以外の成分の混入を極力抑えることができ、さらに分離した成分を回収することで、回収した成分を別の用途に応用することが可能となる。
【００２９】
【実施例】
以下、本発明を実施例及び比較例に基づきさらに詳細に説明するが、本発明はこれらに限定されるものではない。なお、例中の分離材表面のＸ線光電子分光分析による測定は、以下の方法に基づいて行った。
＜分離材表面のＸ線光電子分光分析の測定方法＞
Ｘ線光電子分光分析機(商品名「ＥＳＣＡ−３３００」、島津製作所社製)を用いて、分離材表面に対して、Ｘ線の照射角が９０°のときの、各元素のスペクトルを測定し、リン元素及び炭素元素の各ピーク面積を求め、以下の式によりリン元素の量Ｐ／炭素元素の量Ｃ(Ｐ／Ｃ)を算出した。
Ｐ／Ｃ＝Ａｐ／Ａｃ
ただし、Ａｐ：リン元素のピーク面積、Ａｃ：炭素元素のピーク面積とする。
【００３０】
合成例１
ＭＰＣ ３５．７ｇ及びｎ−ブチルメタクリレート(以下、ＢＭＡと略記する)４．３ｇ(単量体組成モル比：ＭＰＣ／ＢＭＡ＝８０／２０)をエタノール１６０ｇに溶解して４つ口フラスコに入れ、３０分間窒素を吹き込んだ後、６０℃に昇温し、アゾビスイソブチロニトリル０．８２ｇを加えて８時間重合反応させた。得られた重合液を３リットルのジエチルエーテル中に攪拌しながら滴下し、析出した沈澱を濾過し、４８時間室温で真空乾燥を行って、粉末２９．６ｇを得た。得られた粉末をＧＰＣにより評価したところ、重量平均分子量は１５３０００であった。この粉末を共重合体(Ａ)とする。
【００３１】
合成例２〜４
単量体の種類、組成比及び溶媒種を表１に示すとおりに代えた以外は、合成例１と同様の操作により粉末を得、分子量を測定した。結果を表１に示す。なお、合成例２で調製した粉末を共重合体(Ｂ)、合成例３で調製した粉末を共重合体(Ｃ)、合成例４で調製した粉末を共重合体(Ｄ)とする。また、表１中のＳＭＡはステアリルメタクリレート、ＱＡは２−ヒドロキシ−３−メタクリルオキシプロピルトリメチルアンモニウムクロライドを示す。
【００３２】
【表１】

【００３３】
実施例１：白血球の分離
＜分離材の調製＞
合成例１により合成した共重合体(Ａ)０．５ｇを、エタノール１００ｍＬに溶解し、共重合体溶液を調製した。次いで、ポリエチレンテレフタレート(以下、ＰＥＴと略記する)製のシートをφ１３ｍｍの大きさの円形に切り抜いた円形シートを、上記共重合体溶液中に静かに浸漬させてから引き上げ、そのまま室温で１時間乾燥させた。さらに、乾燥器内で６０℃まで昇温し、そのままの温度で減圧下１６時間乾燥させることにより、ディスク形状の分離材を調製した。
得られた分離材表面のＸ線光電子分光分析の測定を行い、Ｐ／Ｃの値を求めた。結果を表２に示す。
【００３４】
＜選択的分離操作＞
ウサギから採取した新鮮血１０ｍＬにハンクス緩衝液１０ｍＬを加えて１／２に希釈することで、血球成分を選択的に分離するために用いる希釈血を調製した。
次に、上記で調製したディスク形状の分離材を、２４穴プレートに１枚／ウェルずつセットし、３ｍＬのろ過滅菌した蒸留水を加えて１時間湿潤させた。アスピレータで蒸留水を除去し、３ｍＬのハンクス緩衝液を加えて溶媒置換した後、これを完全に除去した。
次いで、上記で調製した希釈血を、この２４穴プレートに７００μＬ／ウェルずつ加えて、２５℃で６時間静置した。続いて、２４穴プレートから分離材を取り出し、３７℃に加温したDulbecco−リン酸緩衝生理食塩水を満たしたシャーレ中に浸してリンスした。この操作を２回繰り返した。その後、シャーレに満たした１０％中性緩衝ホルマリン液中で５分間、さらに、２．５％グルタルアルデヒド水溶液中で１時間固定化処理した。取り出した分離材は蒸留水で十分に洗った後、２５℃で１０時間風乾し、さらに減圧下で１６時間乾燥させた。
以上の操作を行った分離材の表面を走査型電子顕微鏡(ＳＥＭ)で観察し、単位面積あたりの表面に付着している各血球が占める面積の割合(占有率％)を計算した。結果を表２に示す。
【００３５】
比較例１
実施例１で調製したディスク形状を有する分離材の代わりに、何も処理していないφ１３ｍｍのＰＥＴ製円形シートを用いた以外は、実施例１と同様の方法で選択的分離を行い、分離材表面をＳＥＭで観察し、各血球が占める面積の割合(占有率％)を計算した。結果を表２に示す。
【００３６】
実施例２：赤血球の分離
実施例１において、共重合体(Ａ)の代わりに合成例２により合成した共重合体(Ｂ)を用いた以外は、実施例１と同様の方法で選択分離を行い、分離材表面をＳＥＭで観察し、各血球が占める面積の割合(占有率％)を計算した。結果を表２に示す。
【００３７】
実施例３：白血球の分離
実施例１において、共重合体(Ａ)の代わりに合成例３により合成した共重合体(Ｃ)を用いた以外は、実施例１と同様の方法で選択分離を行い、分離材表面をＳＥＭで観察し、各血球が占める面積の割合(占有率％)を計算した。結果を表２に示す。
【００３８】
【表２】

【００３９】
表２の結果より、実施例１及び実施例３で調製した分離材は、希釈血中に含まれる赤血球、白血球、血小板のうち、白血球だけを選択的に分離材表面の約１割程度の範囲に付着させうることが確認できた。また実施例２で調製した分離材は、希釈血中に含まれる赤血球、白血球、血小板のうち、赤血球だけを選択的に分離材表面の６割以上の範囲に付着させうることが確認できた。これに対して、比較例１では、赤血球、白血球、血小板全ての血球成分の付着が確認され、特に血小板については、基材表面の半分を覆うという結果になった。
【００４０】
実施例４：リンパ球の分離及び回収
＜分離材の調製＞
平均粒径０．２ｍｍのガラスビーズ(ｉｕｃｈｉ社製)を、アセトン及びエタノールで順に洗浄した後、さらに、蒸留水で十分すすぎ、乾燥器内で６０℃まで昇温させ、そのままの温度で１６時間減圧乾燥させて前処理ビーズを調製した。
一方、合成例１により合成した共重合体(Ａ)０．５ｇをエタノール１００ｍＬに溶解し、共重合体溶液を調製した。
次いで、上記前処理ビース５０ｇをポリプロピレン製の容器に入れ、上記共重合体溶液２０ｍＬを加えた後に密閉した。次に、３０分間容器ごと回転させることで内容物を攪拌した後、ロートを使ってビーズを濾別した。取り出したビーズをシャーレ上に広げてから乾燥器に入れ、徐々に５０℃まで温度を上げた後、そのままの温度で１０時間減圧下で乾燥することにより、ビーズ形状を有する分離材を調製した。
得られた分離材表面のＸ線光電子分光分析の測定を行い、Ｐ／Ｃの値を求めた。結果を表３に示す。
【００４１】
＜選択的分離操作＞
リンパ球懸濁液を以下の方法により調製した。市販のリンパ球分離試薬であるFicoll−Paque(ファルマシアバイオテク社製)を用い、手順書に従ってヒトから採取した血液からリンパ球を採取した。まず、Ficoll−Paqueを３ｍＬずつシリコン処理した試験管に取り分け、この上にハンクス溶液で１／２に希釈したヒト全血４ｍＬを静かに重層した。次いで、２０℃、１５５０ｒｐｍで４０分間遠心分離し、最上層部にある血漿を除去した。その後、ピペットでリンパ球層を丁寧に採取し、シリコン処理した試験管に移した。続いて、採取したリンパ球懸濁液の３倍量のハンクス溶液を加えて、ピペッティングにより静かに攪拌した。次いで２０℃、７００ｒｐｍで１０分間遠心分離し、上澄みを除去した。さらに、ハンクス溶液を加えて再懸濁させ、２０℃、７００ｒｐｍで１０分間遠心分離した。採取したリンパ球を、１０％血清を添加したＲＰＭＩ１６４０培地(ＧＩＢＣＯ社製)で再懸濁することでリンパ球懸濁液を調製し、血球計算盤を用いて培地１．０ｍＬ中に含まれるリンパ球の数を測定した。さらに、リンパ球を活性化させる目的で１ｇ／Ｌのリポ多糖類(以下、ＬＰＳと略記する。ＳＩＧＭＡ社製)を１／１００量加えたリンパ球懸濁液を調製した。すなわち、ＬＰＳを加えていないリンパ球懸濁液(以下、ＬＰＳ−と略記する)とＬＰＳを加えたリンパ球懸濁液(以下、ＬＰＳ＋と略記する)の２種類のリンパ球懸濁液を調製した。
上記ビーズ形状の分離材５．０ｇをシリコン処理した試験管に入れ、ＬＰＳ−又はＬＰＳ＋を５．０ｍＬ加えてから、室温で２時間インキュベートした。その後３０分間静置した後、上清を静かに除いた。次いで、生理的リン酸緩衝溶液(以下、ＰＢＳと略記する)を５．０ｍＬ加え、室温で１０分間インキュベートした。その後３０分間静置した後、上清を静かに除いた。この洗浄操作を３回繰り返した。
洗浄操作後、試験管内の分離材が流出しないように注意しながら、残っているＰＢＳを完全に除去した。その後、素早くＰＢＳ５．０ｍＬを加えてから、攪拌子を用いてスターラー上で緩やかに１分間かき混ぜ、ビーズ表面に付着していたリンパ球をＰＢＳ中に分散させた。その後、上清からＰＢＳを採取し、血球計算盤を用いてＰＢＳ１．０ｍＬ中に含まれるリンパ球の数を測定し、添加したリンパ球数(Ｃ₀)に対する回収したリンパ球数(Ｃ_R)の割合を、回収率として下記式により算出し、結果を表３に示す。
回収率(％)＝(Ｃ_R／Ｃ₀)×１００
【００４２】
比較例２
分離材として、実施例４で調製した前処理ビーズを用いた以外は、実施例４と同様の方法で分離を行い、リンパ球の回収率を求めた。結果を表３に示す。
【００４３】
【表３】

【００４４】
表３の結果より、実施例４で調製した分離材は、リンパ球を活性化させる目的でＬＰＳを加えたＬＰＳ＋と、加えていないＬＰＳ−のいずれの場合においても、約半分のリンパ球を分離した後、さらに回収可能であることが確認できた。一方、比較例２では、ＬＰＳ−の場合、約１割程度のリンパ球を分離・回収できたが、ＬＰＳ＋の場合では、一切の分離・回収ができなかった。すなわち、実施例４で使用した分離材は、リンパ球の活性化の程度に関わらず、選択的にリンパ球を分離・回収できる機能を有することが判った。
【００４５】
実施例５：免疫グロブリンの分離
＜分離材の調製＞
実施例４における分離材の調製において、共重合体(Ａ)の代わりに、合成例４により合成した共重合体(Ｄ)を使用した以外は、実施例４と同様の方法で分離材を調製した。
得られた分離材表面のＸ線光電子分光分析の測定を行い、Ｐ／Ｃの値を求めた。結果を表４に示す。
【００４６】
＜選択的分離操作＞
上記分離材５．０ｇをポリプロピレン製試験管に入れ、ＰＢＳを用いて１／２５６に希釈したウシ胎児血清(以下、ＦＣＳと略記する、ＧＩＢＣＯ社製)５．０ｍＬを加えて、室温で２時間インキュベートした。その後３０分間静置した後、上清を静かに除いた。次いで、ＰＢＳ５．０ｍＬを加え、室温で１０分間インキュベートした後、３０分間静置し、上清を静かに除いた。この洗浄操作を３回繰り返した。
洗浄操作後、２０倍濃度のＰＢＳ５．０ｍＬを加え、室温で２時間インキュベートすることで、分離材表面に付着した蛋白をＰＢＳ中に溶解させた。３０分間静置した後、上清を静かに分取し、これを検体とした。
次に、ポリスチレン製９６穴プレート(Ｎｕｎｃ社製)に、抗(ウシＩｇＧ)ウサギＩｇＧ((株)ヤガイ社製)、抗(ウシアルブミン)ウサギＩｇＧ((株)ヤガイ社製)又は抗(ウシファイブロネクチン)ウサギＩｇＧ(ＳＩＧＭＡ社製)を各々１００μＬ／ウェル注入し、室温で２時間インキュベートした。次いで、各抗体溶液を除去した後に、各ウェルを３００μＬのＰＢＳで４回洗浄した。
１／４に希釈したブロックエース溶液(雪印乳業(株)社製)２００μＬ／ウェルを加えて、室温で２時間インキュベートした。ウェル中にあるブロックエース溶液を除去した後、前記検体１００μＬ／ウェルを加えて室温で２時間インキュベートし、各ウェルを３００μＬのＰＢＳで４回洗浄した。
西洋ワサビ過酸化酵素(以下、ＨＲＰと略記する)を修飾した抗(ウサギＩｇＧ)ヤギＩｇＧ(ＳＩＧＭＡ社製)溶液１００μＬ／ウェルを注入し、室温で２時間インキュベートした。次いで、各抗体溶液を除去した後に、各ウェルを３００μＬのＰＢＳで４回洗浄した。
各ウェルに、ＨＲＰ発色溶液(ペルオキシダーゼ用発色キットＴ、住友ベークライト(株)社製)１００μＬ／ウェルを注入し、室温で２時間インキュベートした後、各ウェルを３００μＬのＰＢＳで４回洗浄した。次いで、ＨＲＰ発色溶液(ペルオキシダーゼ用発色キットＴ、住友ベークライト(株)社製)１００μＬ／ウェルを注入し、室温で１０分間インキュベートした。さらに、反応停止液(ペルオキシダーゼ用発色キットＴ、住友ベークライト(株)社製)１００μＬ／ウェルを注入し、反応を停止した。次いで、プレートリーダー(SPECTRA MAX250、モレキュラーデバイス社製)を用いて４５０ｎｍの吸光度を測定した。
予め作成しておいた検量線を使って、吸光度から検体中の各蛋白質量を求め、回収したアルブミン量に対する選択的に分離したＩｇＧ量の割合(Ｕｉ／Ｕａ)、及び回収したファイブロネクチン量に対する選択的に分離したＩｇＧ量の割合(Ｕｉ／Ｕｆ)をそれぞれ求めた。結果を表４に示す。
【００４７】
比較例３
分離材として、実施例４で調製した前処理ビーズを用いた以外は、実施例５と同様の方法で選択的分離を行い、その測定値からＵｉ／Ｕａ及びＵｉ／Ｕｆを求めた。結果を表４に示す。
【００４８】
【表４】

【００４９】
表４の結果より、実施例５で調製した分離材は、アルブミンに対するＩｇＧの割合(Ｕｉ／Ｕａ)では、比較例３の分離材より７０倍以上の分離性能を示し、ファイブロネクチンに対するＩｇＧの割合(Ｕｉ／Ｕｆ)では、比較例３の３０倍以上の分離性能を示すことが判った。すなわち、実施例５の分離材を用いることで、他のアルブミンやファイブロネクチンの数十倍、ＩｇＧを選択的分離によって濃縮できることを意味している。以上の結果から、実施例５で使用した分離材は、選択的にＩｇＧを分離・回収できる機能を有していることが確認できた。
【００５０】
実施例６：株細胞の分離
＜分離材の調製＞
合成例１により合成した共重合体(Ａ)０．５ｇを蒸留水１００ｍＬに溶解した共重合体溶液に、低蛍光セルデスク(住友ベークライト社製)を室温で５分間浸漬した。溶液からセルデスクを取り出し、室温で１時間乾燥させた。さらに、乾燥器内で６０℃まで昇温し、そのままの温度で４時間減圧乾燥させ、セルデスク形状の分離材を調製した。
得られた分離材表面のＸ線光電子分光分析の測定を行い、Ｐ／Ｃの値を求めた。結果を表５に示す。
【００５１】
＜選択的分離操作＞
φ９ｃｍのポリスチレンディッシュ中で、１０％ＦＣＳを含んだダルベッコ改変イーグルメディウム(ＤＭＥＭ、以下、培地と略記する)を用いて、ＮＩＨ３Ｔ３細胞(マウス胎児繊維芽細胞)及びＳＩＲＣ細胞(ウサギ角膜上皮細胞)をサブコンフルエント程度にまで培養した。これらの細胞を０．１％トリプシン溶液で処理して剥がし、遠心操作した後に培地にそれぞれ懸濁した。各懸濁液の細胞濃度を血球計算盤を用いて測定し、２０００個／ｍＬとなるように培地で希釈した。
２４穴プレートの各ウェルに、それぞれの細胞懸濁液０．５ｍＬを注入し、その中に上記セルデスク状の分離材を沈めた。次いで、２４時間炭酸ガスインキュベータ内で培養した後、分離材表面に接着した細胞の同定を行った。細胞の同定は、細胞内に発現している細胞骨格蛋白質であるビメンチンに対する特異抗体を用いる方法で行った。まず、細胞をメタノールで固定した後、抗ビメンチン抗体(コスモバイオ社製)を添加した。次に、洗浄処理を行い、ＦＩＴＣ標識抗(マウスＩｇＧ)抗体(ＳＩＧＭＡ社製)を用いてビメンチン陽性細胞を検出した。一方、全細胞数の測定はプロピジウムイオダイド(ＳＩＧＭＡ社製)を用いた核染色により行った。蛍光顕微鏡下で、ＦＩＴＣ観察用及びプロピジウムイオダイド観察用フィルターをそれぞれ用いて、同一画面を写真撮影し、全細胞数中に占めるＦＩＴＣ陽性細胞(ビメンチン陽性細胞：ＳＩＲＣ細胞)の割合から、ＮＩＨ３Ｔ３細胞及びＳＩＲＣ細胞の接着率(％)を計算した。結果を表５に示す。
【００５２】
比較例４
セルデスク形状の分離材の代わりに、何も処理していない低蛍光セルデスクを用いた以外は、実施例６と同様の方法でそれぞれの細胞の接着率を計算した。結果を表５に示す。
【００５３】
【表５】

【００５４】
表５の結果より、実施例６で調製した分離材は、表面にＮＩＨ３Ｔ３細胞を９６％選択的に接着していることが判った。これに対して、比較例４では、ＮＩＨ３Ｔ３細胞及びＳＩＲＣ細胞がほぼ同じ割合で表面に接着していることが判った。以上より、実施例６で使用した分離材は、選択的にＮＩＨ３Ｔ３細胞を分離できる機能を有していることが確認できた。
【００５５】
実施例７：初代培養細胞の分離
＜分離材の調製＞
２４穴プレート(Ｎｕｎｃ社製)の各ウェルに、合成例１により合成した共重合体(Ａ)０．５ｇをエタノール１００ｍＬに溶解した共重合体溶液１．０ｍＬずつを分注し、室温で３０分間インキュベートした。次いで、ウェル中の溶液を除去した後に、乾燥器内で６０℃まで昇温し、そのままの温度で４時間、減圧下で乾燥することにより、培養プレート形状の分離材を調製した。
得られた分離材表面のＸ線光電子分光分析の測定を行い、Ｐ／Ｃの値を求めた。結果を表６に示す。
【００５６】
＜選択的分離操作＞
生後２４時間以内のラット胎児から皮膚の切片を採取し、０．０５％のコラゲナーゼを含むＤＭＥＭで処理した。得られた溶液をメッシュで濾過した後、遠心操作を行い、回収した細胞を５％のＦＣＳを含むＤＭＥＭに再懸濁した。細胞濃度を血球計算盤を用いて測定し、５０００個／ｍＬとなるように培地で希釈した。
上記培養プレート形状の分離材のウェル中に、上記で調製した細胞懸濁液０．５ｍＬずつを分注し、１６時間炭酸ガスインキュベータ内で培養した。その後、ウェル内を生理緩衝食塩水で洗浄し、位相差顕微鏡を用いて写真撮影を行った。細胞の形態を観察することで、繊維芽細胞及び表皮細胞の識別を行い、分離材表面への各細胞の接着率を計算した。結果を表６に示す。
【００５７】
比較例５
培養プレート形状の分離材の代わりに、何も処理していない２４穴プレートを用いた以外は、実施例７と同様の方法でそれぞれの細胞の接着率を計算した。結果を表６に示す。
【００５８】
【表６】

【００５９】
表６の結果より、実施例７で調製した分離材は、生体組織から採取した繊維芽細胞と表皮細胞を含む懸濁液から、繊維芽細胞だけを９２％選択的に接着分離できることが判った。これに対して、比較例５では、繊維芽細胞及び表皮細胞がほぼ同じ割合で表面に接着していることが判った。以上より、実施例７で使用した分離材は、選択的に繊維芽細胞を分離できる機能を有していることが確認できた。
【００６０】
実施例８：初代培養細胞の回収
＜選択的分離操作＞
実施例７で調製した分離材を用いて、実施例７と同様に位相差顕微鏡を用いた写真撮影の前までの操作を行った。その後、写真撮影は行わず、分離材の各ウェルを０．１％トリプシン溶液で処理し、細胞を剥離した。次いで、回収した細胞を遠心処理した後、培地にそれぞれ懸濁した。懸濁液の細胞数を、血球計算盤を用いて測定し、２０００個／ｍＬとなるように培地で希釈し、細胞懸濁液とした。
得られた細胞懸濁液０．５ｍＬを２４穴プレートの各ウェルに分注し、炭酸ガスインキュベータ内で４８時間培養した。その後、位相差顕微鏡で細胞の形態を確認し、繊維芽細胞及び表皮細胞の識別を行った。結果を表７に示す。
【００６１】
比較例６
分離材として、比較例５と同様の２４穴プレートを用いた以外は、実施例８と同様の方法で細胞の識別を行った。結果を表７に示す。
【００６２】
【表７】

【００６３】
実施例８は、実施例７と同じ２種の細胞を含む懸濁液から１種類の初代培養細胞だけを選択的に分離した後、さらにその細胞を回収することを目的としている。ここでは、回収した細胞を培養し、その増殖の様子を観察することで、どちらの細胞が分離・回収できたかを評価している。表７より、分離材を用いて分離・回収したものは、繊維芽細胞のみ増殖していることが確認された。これに対して、比較例６では、繊維芽細胞及び表皮細胞のいずれの細胞も増殖していることが確認された。以上より、実施例８で使用した分離材は、選択的に繊維芽細胞を分離・回収できる機能を有していることが確認できた。
【００６４】
実施例９：情報伝達物質の分離
＜選択的分離操作＞
実施例５で調製したビーズ形状の分離材を使用し、この分離材５．０ｇをガラス製容器に入れ、ジベンゾフラン飽和水溶液２．５ｍＬとジベンゾ−ｐ−ジオキシン飽和水溶液２．５ｍＬとを加えてから容器を密閉した。６０分間容器ごと回転させることで内容物を攪拌した後、ロートを使って溶液を濾別した。回収した溶液は、吸光光度計を用いて、ジベンゾフランは２４９ｎｍ、ジベンゾ−ｐ−ジオキシンは２８９ｎｍの波長でそれぞれ吸光度を測定した。
予め作成しておいた検量線を使って、吸光度から溶液の濃度(Ｃ_n)を求め、分離材を入れないで上記と同様の操作をした各飽和水溶液の濃度(Ｃ₀)に対する変化の割合を、変化率として以下の式により算出した。結果を表８に示す。
変化率(％)＝((Ｃ₀−Ｃ_n)／Ｃ₀)×１００
【００６５】
比較例７
分離材として、比較例３と同様な前処理ビーズを用いた以外は、実施例９と同様の方法で選択的分離を行い、その測定値から変化率を計算した。結果を表８に示す。
【００６６】
【表８】

【００６７】
表８の結果より、実施例９の分離材では、情報伝達物質である、ジベンゾフラン及びジベンゾ−ｐ−ジオキシンを含む水溶液から、ジベンゾフランを選択的に分離できることが判った。すなわち、ジベンゾフランの濃度の変化率は、比較例７の３倍以上の値を示した。一方のジベンゾ−ｐ−ジオキシンは分離材を用いた場合も、比較例７の場合もほぼ同じ変化率であった。すなわち、実施例９で使用した分離材によって、溶液中のジベンゾフランを選択的に分離した結果、溶液中のジベンゾフランの濃度が減少したと考えられる。以上より、実施例９で使用した分離材は、選択的にジベンゾフランを分離できる機能を有していることが判った。
【００６８】
以上の実施例及び比較例の結果より次のことがわかる。すなわち、実施例１〜９で調製した分離材は、ホスホリルコリン基に由来するリン元素が含まれており、それぞれ異なったＰ／Ｃ値を有していることを確認した。実施例で使用した分離材は、ＭＰＣと他の単量体とを共重合させてできた重合体を、ＰＥＴ、ポリスチレン、ガラス等からなる種々の形状をした基材の上にコートしたものである。実施例１、４、６、７及び８で使用した分離材は、全て同じ共重合体(Ａ)を表面にコートして調製したにも関わらず、Ｐ／Ｃの値は、基材の種類や形状によっていずれも異なる値を示した。すなわち、分離材を調製する際の、ホスホリルコリン基を含む共重合体と、それをコートする基材との組み合わせによって、Ｐ／Ｃの値をある程度制御することが可能であり、分離材のＰ／Ｃ値をある特定の範囲内に制御することで、結果として、所望の特定成分を選択的に分離・回収する機能が発現されたと考えられる。[0001]
BACKGROUND OF THE INVENTION
  The present invention is a detailedCell etc.Separation material that can be separated with high selectivityUseThe present invention relates to a separation / recovery method that can selectively separate and recover a specific component from a solution such as a body fluid.
[0002]
[Prior art]
In recent years, in research on medical materials, polymers containing phosphorylcholine groups have characteristics such as blood compatibility, complement activity, and non-adsorption of biological substances due to phospholipid-like structures derived from biological membranes. As a result, the development of biomaterials using these functions has been actively conducted. For example, JP-A-54-36025 discloses that a method for producing 2-methacryloyloxyethyl phosphorylcholine (hereinafter abbreviated as MPC) and that the resulting polymer has excellent biocompatibility. JP-A-9-183819 discloses that a copolymer of MPC and methacrylic acid ester is useful as a medical material because it is difficult for platelets to adhere and aggregate and adhere to plasma proteins. In addition, a medical material using a copolymer having a phosphorylcholine-like group in the side chain is disclosed in JP-T-6-502200 (WO92 / 07885) and JP-A-7-502053 (WO93 / 01221). It has been disclosed that excellent biocompatibility can be obtained by coating a polymer surface having a phosphorylcholine-like group on a resin surface.
The conventional materials using these copolymers containing phosphorylcholine-like groups are all copolymers containing phosphorylcholine-like groups that prevent or suppress nonspecific adsorption of blood cells, plasma proteins, etc. to the material surface. The research is being conducted in the direction of minimizing the interaction between the components derived from the living body and the material surface.
By the way, no studies have been reported so far on the application of a polymer containing a phosphorylcholine-like group to a material for separating a specific component such as a living body. In addition, it is not known that an action capable of selectively separating a specific component such as a living body can be obtained by applying a polymer containing a phosphorylcholine-like group.
A component derived from a living body needs to be obtained with high purity while maintaining physiological activity depending on the use, and development of a simple separation method capable of achieving such an object is desired.
[0003]
[Problems to be solved by the invention]
  Object of the present inventionIs specialAn object of the present invention is to provide a separation / recovery method capable of selectively separating constant components and further recovering the components.
[0004]
[Means for Solving the Problems]
  As a result of intensive studies to solve the above-mentioned problems, the present inventors have controlled the ratio of the specific phosphorylcholine-like group in a material having a specific phosphorylcholine-like group at least on the surface and in a specific range, Cells derived from living bodiesSpecialIt has been found that it has an action of selectively separating constant components, and furthermore, it has been found that the separated specific components can be easily recovered, thereby completing the present invention.
[0005]
  That is, according to the present inventionFor example, at least the surface has a group represented by the formula (1)A separation material is brought into contact with a solution containing a specific component, and one or more specific components contained in the solution are selectively separated and recovered.A method,
  The ratio of the amount P of phosphorus element derived from the group represented by the formula (1) and the amount C of carbon element (P / C) in the spectrum of the separating material measured by X-ray photoelectron spectroscopy. ) Has a group represented by the formula (1) in an amount of 0.002 to 0.3,
  The specific component is a cell derived from one or more organisms selected from the group consisting of blood cells, cell lines and primary cultured cells, immunoglobulin, or dioxin or a derivative thereof.A separation / recovery method is provided.
[Chemical Formula 3]

(Where R ¹ , R ² And R ^Three Are the same or different groups, and each represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a hydroxyalkyl group. n is an integer of 1-4. )
[0006]
DETAILED DESCRIPTION OF THE INVENTION
  The present inventionUsed forThe separating material is a separating material having at least the group represented by the above formula (1) on the surface, and the surface is derived from the group represented by the formula (1) in the spectrum measured by X-ray photoelectron spectroscopy. A separation material having a group represented by the formula (1) in which the ratio (P / C) of the amount P of phosphorus element to the amount C of carbon element in the entire surface is 0.002 to 0.3 is there. In order to improve the selective separation ability of the separating material, the range of P / C is preferably 0.01 to 0.2.
  Here, as a method of measuring the surface of the separating material by X-ray photoelectron spectroscopy, it can be measured based on Examples described later.
[0007]
R in formula (1)¹, R²And R^ThreeAre the same or different groups, and each represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a hydroxyalkyl group.
Examples of the alkyl group having 1 to 6 carbon atoms include a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a cyclohexyl group, and a phenyl group.
Examples of the hydroxyalkyl group having 1 to 6 carbon atoms include hydroxymethyl group, 2-hydroxyethyl group, 3-hydroxypropyl group, 4-hydroxybutyl group, 5-hydroxypentyl group, 6-hydroxyhexyl group and the like. It is done.
[0008]
Examples of the group represented by the formula (1) include a group derived from a phosphorylcholine-like group-containing monomer represented by the formula (2) and a phosphorylcholine-like group-containing monomer represented by the formula (2). And a group derived from a polymer obtained by polymerizing the monomer composition.
[Formula 4]

In formula (2), R¹, R²And R^ThreeIs the same as in formula (1) and R^FourRepresents an alkyl group having 1 to 6 carbon atoms, R^FiveRepresents a hydrogen atom or a methyl group. n is an integer of 1-4.
[0009]
Examples of the phosphorylcholine-like group-containing monomer represented by the formula (2) include 2-((meth) acryloyloxy) ethyl-2 ′-(trimethylammonio) ethyl phosphate, 3-((meth) acryloyloxy ) Propyl-2 '-(trimethylammonio) ethyl phosphate, 4-((meth) acryloyloxy) butyl-2'-(trimethylammonio) ethyl phosphate, 5-((meth) acryloyloxy) pentyl-2'- (Trimethylammonio) ethyl phosphate, 6-((meth) acryloyloxy) hexyl-2 '-(trimethylammonio) ethyl phosphate, 2-((meth) acryloyloxy) ethyl-2'-(triethylammonio) ethyl Phosphate, 2-((meth) acryloyloxy) ethyl-2 ′-(tripropylammonio) ethyl phosphate, 2-((meth) acrylate Royloxy) ethyl-2 ′-(tributylammonio) ethyl phosphate, 2-((meth) acryloyloxy) ethyl-2 ′-(tricyclohexylammonio) ethyl phosphate, 2-((meth) acryloyloxy) ethyl-2 '-(Triphenylammonio) ethyl phosphate, 2-((meth) acryloyloxy) propyl-2'-(trimethylammonio) ethyl phosphate, 2-((meth) acryloyloxy) butyl-2 '-(trimethylammoni E) ethyl phosphate, 2-((meth) acryloyloxy) pentyl-2 ′-(trimethylammonio) ethyl phosphate, 2-((meth) acryloyloxy) hexyl-2 ′-(trimethylammonio) ethyl phosphate, etc. Can be mentioned.
Of these, 2-((meth) acryloyloxy) ethyl-2 ′-(trimethylammonio) ethyl phosphate is preferable, and MPC is more preferable in view of availability.
[0010]
The phosphorylcholine-like group-containing monomer represented by the formula (2) can be produced by a known method. For example, it can be produced according to known methods disclosed in JP-A-54-63025 and JP-A-58-154591.
Specifically, a method for obtaining a target product by ring-opening by reacting a cyclic phosphorus compound and 2-hydroxyethyl (meth) acrylate under a dehydrohalogenating agent and then reacting with trimethylamine. Etc.
[0011]
A polymer obtained by polymerizing a monomer composition containing a phosphorylcholine-like group-containing monomer represented by formula (2) includes (A) a phosphorylcholine-like group-containing monomer represented by formula (2) A polymer obtained by polymerizing a monomer composition consisting of 10 to 100 mol%, (B) a hydrophobic monomer 0 to 90 mol% and (C) a hydrophilic monomer 0 to 70 mol% is preferable. A polymer obtained by polymerizing a monomer composition comprising A) 20 to 80 mol%, (B) 20 to 60 mol%, and (C) 0 to 20 mol% is desirable. When the component (A) is less than 10 mol%, it is difficult to develop the performance as a separating material, which is not preferable. (B) By mix | blending a component suitably in 20-60 mol%, it becomes easy to make the performance as a separating material express stably. Moreover, the affinity with respect to the aqueous solution etc. of the polymer obtained can be improved by mix | blending 20 mol% or less of (C) component.
[0012]
Examples of the component (B) include linear chains such as methyl (meth) acrylate, ethyl (meth) acrylate, butyl (meth) acrylate, 2-ethylhexyl (meth) acrylate, lauryl (meth) acrylate, and stearyl (meth) acrylate. Or branched alkyl (meth) acrylate; cyclic alkyl (meth) acrylate such as cyclohexyl (meth) acrylate; aromatic (meth) acrylate such as benzyl (meth) acrylate and phenoxyethyl (meth) acrylate; polypropylene glycol (meth) acrylate, etc. Hydrophobic polyalkylene glycol (meth) acrylates; Styrene monomers such as styrene, methyl styrene and chloromethyl styrene; Vinyl ether monomers such as methyl vinyl ether and butyl vinyl ether; Vinyl such as vinyl acetate and vinyl propionate Ester-based monomer and the like. These can be used alone or as a mixture.
[0013]
As the component (C), for example, a hydrophilic group selected from the group consisting of hydroxyl group, carboxyl group, sulfonic acid group, amide group, amino group, dialkylamino group, trialkylamino base, trialkylphosphonium base and polyoxyethylene group And monomers having a functional group. Specifically, hydroxyl-containing (meth) acrylates such as 2-hydroxyethyl (meth) acrylate, 2-hydroxybutyl (meth) acrylate and 4-hydroxybutyl (meth) acrylate; carboxylic acids such as acrylic acid and methacrylic acid; Ionic group-containing monomers such as styrene sulfonic acid, (meth) acryloyloxyphosphonic acid, 2-hydroxy-3- (meth) acryloxypropyltrimethylammonium chloride; (meth) acrylamide, aminoethyl methacrylate, dimethylaminoethyl ( Nitrogen-containing monomers such as (meth) acrylate; polyethylene glycol (meth) acrylate and the like. These can be used alone or as a mixture.
[0014]
A polymer obtained by polymerizing a monomer composition containing the phosphorylcholine-like group-containing monomer comprises a monomer composition comprising only the component (A), the component (A), and the component (B). Monomer composition, monomer composition comprising component (A) and component (C), or monomer composition comprising component (A), component (B) and component (C) was polymerized. Any material can be used, and it can be produced by ordinary radical (co) polymerization.
The molecular weight of this polymer is preferably 5000 to 5000000 in terms of weight average molecular weight. For improving the selective separation performance and for improving the elution resistance of the polymer to various solvents, the molecular weight is 100,000 to 2,000,000. A range is particularly preferred.
[0015]
  The present inventionUsed forThe preparation of the separating material is not particularly limited as long as the (P / C) ratio is within a specific range. For example, a monomer having a group represented by the formula (1) such as a phosphorylcholine-like group-containing monomer represented by the above formula (2), or a phosphorylcholine-like group-containing monomer represented by the above formula (2) Using a polymer obtained by polymerizing a monomer composition containing a monomer, at least the surface of the substrate is chemically modified, or obtained by a method of coating the surface of the substrate and drying and fixing it. Can do.
[0016]
Examples of the material for the base material include synthetic resins such as polyethylene terephthalate, polystyrene, and polymethyl methacrylate; and inorganic substances such as magnetic powder, silica, alumina, and glass. The form of the substrate is not particularly limited, and examples thereof include powders, particles, beads, flake shaped materials, sheets, films, plates, gels, fibrous materials, woven fabrics, and woven fabrics.
[0017]
  The present inventionUsed forIn preparing the separating material, the control of (P / C) is carried out by using a phosphorylcholine-like group-containing monomer to be used, a monomer composition containing the monomer, depending on the specific component that is a separation target substance described later. It can carry out by selecting suitably the kind of polymer formed by superposition | polymerization, the kind and shape of a base material, etc.
  When the substance to be separated is blood cells of red blood cells, white blood cells, and platelets, for example, as a monomer composition containing the phosphorylcholine-like group-containing monomer, MPC, butyl methacrylate (BMA), stearyl methacrylate (SMA) And a molar ratio of MPC / (hydrophobic group-containing monomer + cationic group-containing monomer) is 10 / A polymer is prepared using a monomer composition of 90 to 100/0, particularly 20/80 to 90/10, and the polymer is fixed to a substrate so as to have a predetermined (P / C) ratio. It is preferable to do.
[0018]
When the substance to be separated is lymphocyte separation, for example, as a monomer composition containing the phosphorylcholine-like group-containing monomer, MPC and alkyl (meth) acrylate such as BMA are used. A polymer is prepared using a monomer composition in which the molar ratio of the functional group-containing monomer is 10/90 to 100/0, particularly 50/50 to 90/10. / C) It is preferable to fix to a base material so that it may become ratio.
[0019]
  The substance to be separated is IgG or the likeImmunoglobulinIn this case, for example, as a monomer composition containing the phosphorylcholine-like group-containing monomer, MPC and alkyl (meth) acrylate such as BMA are used, and the molar ratio of MPC / hydrophobic group-containing monomer is used. Is prepared using a monomer composition having a ratio of 10/90 to 100/0, particularly 30/70 to 90/10, and the polymer is prepared so as to have a predetermined (P / C) ratio. It is preferable to fix to the material.
[0020]
When the substance to be separated is a cell line or a primary cultured cell, for example, as a monomer composition containing the phosphorylcholine-like group-containing monomer, MPC and alkyl (meth) acrylate such as BMA are used. A polymer is prepared using a monomer composition having a molar ratio of the / hydrophobic group-containing monomer of 10/90 to 100/0, particularly 40/60 to 85/15. It is preferable to fix to a base material so that it may become (P / C) ratio.
[0021]
When the substance to be separated is dioxin or dibenzofuran which is a derivative thereof, for example, as a monomer composition containing the phosphorylcholine-like group-containing monomer, MPC and alkyl (meth) acrylate such as BMA are used. A polymer is prepared using a monomer composition having a molar ratio of MPC / hydrophobic group-containing monomer of 10/90 to 100/0, particularly 15/85 to 90/10. It is preferable to fix to a base material so that it may become (P / C) ratio.
[0022]
  The separation / recovery method of the present inventionReportThe release material is brought into contact with a solution containing a specific component, and one or more specific components contained in the solution are selectively separated and collected. The condition for bringing the specific component into contact with the solution containing the specific component can be appropriately selected according to the type of the specific component that is the substance to be separated, etc. Further, the separating material to which the specific component is adhered can be recovered by peeling the adhered specific component from the surface of the separating material by a method such as washing or treatment with a solvent.
[0023]
A body fluid etc. are mentioned as a solution containing a specific component. Here, the body fluid refers to all fluids that fill between the blood vessels or tissues / cells in the animal body, and fluids that are released or secreted from the body to the outside of the body, for example, blood, plasma, serum, lymph fluid, Examples include tears, urine, spinal fluid.
Examples of the solution other than the body fluid include an aqueous solution, a solution containing one or more kinds of organic solvents, and a mixed solution obtained by mixing an aqueous solution with one or more kinds of organic solvents. Specifically, water, physiological saline, phosphate buffer solution, physiological phosphate buffer solution, medium, alcohol, acetone, chloroform, ether, toluene, acetonitrile, or a mixed solution of two or more of these can be used. . The solution includes a diluted body fluid.
[0024]
  In the present invention, the specific component to be separatedMinutes,It is a cell derived from one or more organisms selected from the group consisting of blood cells, cell lines and primary cultured cells, immunoglobulin, or dioxin or a derivative thereof..
[0025]
  in frontThe hematopoietic cells include erythrocytes, leukocytes, platelets or a mixture thereof, and the leukocytes include neutrophils, eosinophils, basophils, lymphocytes and monocytes. Examples of lymphocytes include T cells and B cells. The platelet activation level (aggregation reaction progress level) is not particularly limited, and examples include platelets that have been inactivated and activated. Among these blood cells, particularly preferred are white blood cells whose number increases or decreases in various diseases.
  The cell line and the primary cultured cell are not particularly limited to the origin animal, the origin organ and the form, and examples thereof include fibroblasts, epidermis cells, endothelial cells, smooth muscle cells and nerve cells. Particularly preferred are vascular endothelial cells, vascular smooth muscle cells, hepatocytes and the like which can be used in tissue engineering and the like.
[0027]
  SaidImmunoglobulin (antibody)IsThe following (1) to (7) can be exemplified.
(1) Plasma proteins such as C-reactive protein (CRP), rheumatoid factor (RF), transferrin or antibodies to these plasma proteins,
(2) Follicular hormone, lutein hormone, male hormone, insulin, glucagon, gonadotropin, follicle stimulating hormone, luteinizing hormone, growth hormone, adrenal cortex hormone, thyroid hormone, parathyroid hormone, prolactin, thyroid stimulating hormone, adrenal cortex More preferably, thyroid stimulating hormone (TSH), triiodothyronine (T3), thyroxine (T4), thyroxine binding protein (TBG), thyroglobulin, insulin, estriol (E3), chorionic gonadotropin (HCG) ), Hormo such as human placental lactogen (HPL)ToAntibodies against,
(3) Tumor-related substances such as carcinoembryonic antigen (CEA), β2-microglobulin, α-fetoprotein (AFP)To qualityAntibodies against,
(4) HBS resistanceBody, HViral hepatitis such as Be antibodyAntibodies,
(5) Anti-virus against various biological components such as mumps, herpes, measles, rubella, cytomegalo and other anti-AIDS antibodiesbody,
(6) antibodies against various drugs such as phenobarbital, acetaminophen, salicylic acid, cyclosporine,
(7)EnzymeAntibody against.
  However, the antibody may be a Fab fragment, a F (ab) ′ 2 fragment, or a reduced antibody. Among the above (1) to (7), particularly preferred is an antibody whose amount increases or decreases in various diseases and can be used for diagnosis or treatment.
[0028]
【The invention's effect】
  The present inventionUsed forThe separation material includes a group having a specific phosphorylcholine-like structure at least on the surface in a range that specifies the ratio of phosphorus element and carbon element, so that a wide range of specific components can be selectively separated. In the method of the present invention, since this separation material is used, it is possible to selectively separate and recover a specific component. That is, the target specific component can be selectively and efficiently separated from other components to minimize the mixing of components other than the target component, and the separated component can be recovered to recover the recovered component. Can be applied to other uses.
[0029]
【Example】
EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example and a comparative example, this invention is not limited to these. In addition, the measurement by the X-ray photoelectron spectroscopic analysis of the separating material surface in an example was performed based on the following method.
<Measurement method of X-ray photoelectron spectroscopic analysis of separation material surface>
Using an X-ray photoelectron spectrometer (trade name “ESCA-3300”, manufactured by Shimadzu Corporation), the spectrum of each element was measured when the X-ray irradiation angle was 90 ° with respect to the surface of the separating material. The respective peak areas of phosphorus element and carbon element were determined, and the amount of phosphorus element P / the amount of carbon element C (P / C) was calculated by the following formula.
P / C = Ap / Ac
However, Ap is the peak area of the phosphorus element, and Ac is the peak area of the carbon element.
[0030]
Synthesis example 1
35.7 g of MPC and 4.3 g of n-butyl methacrylate (hereinafter abbreviated as BMA) (monomer composition molar ratio: MPC / BMA = 80/20) were dissolved in 160 g of ethanol and placed in a four-necked flask. After nitrogen was blown for 30 minutes, the temperature was raised to 60 ° C., 0.82 g of azobisisobutyronitrile was added, and a polymerization reaction was carried out for 8 hours. The obtained polymerization solution was added dropwise to 3 liters of diethyl ether with stirring, and the deposited precipitate was filtered and vacuum dried at room temperature for 48 hours to obtain 29.6 g of powder. When the obtained powder was evaluated by GPC, the weight average molecular weight was 153000. This powder is referred to as a copolymer (A).
[0031]
Synthesis Examples 2-4
A powder was obtained by the same operation as in Synthesis Example 1, except that the monomer type, composition ratio, and solvent type were changed as shown in Table 1, and the molecular weight was measured. The results are shown in Table 1. The powder prepared in Synthesis Example 2 is referred to as copolymer (B), the powder prepared in Synthesis Example 3 is referred to as copolymer (C), and the powder prepared in Synthesis Example 4 is referred to as copolymer (D). In Table 1, SMA represents stearyl methacrylate, and QA represents 2-hydroxy-3-methacryloxypropyltrimethylammonium chloride.
[0032]
[Table 1]

[0033]
Example 1: Leukocyte separation
<Preparation of separation material>
0.5 g of the copolymer (A) synthesized in Synthesis Example 1 was dissolved in 100 mL of ethanol to prepare a copolymer solution. Next, a circular sheet obtained by cutting a polyethylene terephthalate (hereinafter abbreviated as PET) sheet into a circular shape having a diameter of 13 mm is gently dipped in the copolymer solution, and then pulled up and dried at room temperature for 1 hour. I let you. Furthermore, the temperature was raised to 60 ° C. in a drier, and the disc-shaped separating material was prepared by drying at that temperature for 16 hours under reduced pressure.
The surface of the obtained separating material was measured by X-ray photoelectron spectroscopy, and the value of P / C was determined. The results are shown in Table 2.
[0034]
<Selective separation operation>
Diluted blood used for selectively separating blood cell components was prepared by adding 10 mL of Hank's buffer to 10 mL of fresh blood collected from rabbits and diluting to 1/2.
Next, the disc-shaped separating material prepared above was set on a 24-well plate one by one / well, and 3 mL of filter-sterilized distilled water was added and moistened for 1 hour. Distilled water was removed with an aspirator, 3 mL of Hanks buffer solution was added to replace the solvent, and this was completely removed.
Next, 700 μL / well of the diluted blood prepared above was added to this 24-well plate and allowed to stand at 25 ° C. for 6 hours. Subsequently, the separating material was taken out from the 24-well plate and rinsed by dipping in a petri dish filled with Dulbecco-phosphate buffered saline heated to 37 ° C. This operation was repeated twice. Thereafter, the mixture was fixed in a 10% neutral buffered formalin solution filled in a petri dish for 5 minutes, and further in a 2.5% glutaraldehyde aqueous solution for 1 hour. The separated separating material was thoroughly washed with distilled water, then air-dried at 25 ° C. for 10 hours, and further dried under reduced pressure for 16 hours.
The surface of the separation material subjected to the above operation was observed with a scanning electron microscope (SEM), and the proportion of the area occupied by each blood cell per unit area (occupancy rate%) was calculated. The results are shown in Table 2.
[0035]
Comparative Example 1
In place of the disc-shaped separation material prepared in Example 1, a PET 13 circular sheet with a diameter of 13 mm that was not treated at all was used to perform selective separation in the same manner as in Example 1, and the separation material The surface was observed with SEM, and the proportion of the area occupied by each blood cell (occupancy%) was calculated. The results are shown in Table 2.
[0036]
Example 2: Red blood cell separation
In Example 1, except that the copolymer (B) synthesized in Synthesis Example 2 was used instead of the copolymer (A), selective separation was performed in the same manner as in Example 1, and the surface of the separating material was subjected to SEM. The percentage of the area occupied by each blood cell (occupancy%) was calculated. The results are shown in Table 2.
[0037]
Example 3: Leukocyte separation
In Example 1, except that the copolymer (C) synthesized in Synthesis Example 3 was used instead of the copolymer (A), selective separation was performed in the same manner as in Example 1, and the surface of the separating material was subjected to SEM. The percentage of the area occupied by each blood cell (occupancy%) was calculated. The results are shown in Table 2.
[0038]
[Table 2]

[0039]
From the results of Table 2, the separation material prepared in Example 1 and Example 3 is a range of about 10% of the surface of the separation material by selectively selecting only white blood cells among red blood cells, white blood cells, and platelets contained in the diluted blood. It was confirmed that it can be adhered to the surface. In addition, it was confirmed that the separation material prepared in Example 2 can selectively adhere only red blood cells among the red blood cells, white blood cells, and platelets contained in the diluted blood to a range of 60% or more of the surface of the separation material. On the other hand, in Comparative Example 1, adhesion of blood cell components of all red blood cells, white blood cells, and platelets was confirmed, and in particular, for platelets, the result was that half of the substrate surface was covered.
[0040]
Example 4: Separation and collection of lymphocytes
<Preparation of separation material>
Glass beads having an average particle size of 0.2 mm (manufactured by Iuchi) were sequentially washed with acetone and ethanol, then rinsed thoroughly with distilled water, heated to 60 ° C. in a dryer, and kept at that temperature for 16 hours. Pretreated beads were prepared by drying under reduced pressure.
On the other hand, 0.5 g of the copolymer (A) synthesized in Synthesis Example 1 was dissolved in 100 mL of ethanol to prepare a copolymer solution.
Next, 50 g of the pretreatment beads were placed in a polypropylene container, and 20 mL of the copolymer solution was added, followed by sealing. Next, the contents were stirred by rotating the entire container for 30 minutes, and then the beads were filtered using a funnel. The taken-out beads were spread on a petri dish, placed in a drier, gradually raised in temperature to 50 ° C., and then dried under reduced pressure at the same temperature for 10 hours to prepare a separating material having a bead shape.
The surface of the obtained separating material was measured by X-ray photoelectron spectroscopy, and the value of P / C was determined. The results are shown in Table 3.
[0041]
<Selective separation operation>
A lymphocyte suspension was prepared by the following method. Lymphocytes were collected from blood collected from humans using Ficoll-Paque (Pharmacia Biotech), which is a commercially available lymphocyte separation reagent, according to the procedure manual. First, 3 mL of Ficoll-Paque was divided into silicon-treated test tubes, and 4 mL of human whole blood diluted 1/2 with Hanks' solution was gently overlaid thereon. Subsequently, it centrifuged at 20 degreeC and 1550 rpm for 40 minutes, and the plasma in the uppermost layer part was removed. Thereafter, the lymphocyte layer was carefully collected with a pipette and transferred to a test tube treated with silicon. Subsequently, 3 times the amount of Hanks solution of the collected lymphocyte suspension was added, and gently agitated by pipetting. Subsequently, it centrifuged at 20 degreeC and 700 rpm for 10 minutes, and the supernatant liquid was removed. Furthermore, Hanks solution was added and resuspended, and centrifuged at 20 ° C. and 700 rpm for 10 minutes. The collected lymphocytes were resuspended in RPMI 1640 medium (GIBCO) supplemented with 10% serum to prepare a lymphocyte suspension, and the lymphocytes contained in 1.0 mL of the medium using a hemocytometer. The number of spheres was measured. Furthermore, for the purpose of activating lymphocytes, a lymphocyte suspension was prepared by adding 1/100 amount of 1 g / L lipopolysaccharide (hereinafter abbreviated as LPS, manufactured by SIGMA). That is, two types of lymphocyte suspensions were prepared: a lymphocyte suspension not added with LPS (hereinafter abbreviated as LPS-) and a lymphocyte suspension added with LPS (hereinafter abbreviated as LPS +). did.
5.0 g of the above bead-shaped separation material was placed in a silicon-treated test tube, and 5.0 mL of LPS- or LPS + was added, followed by incubation at room temperature for 2 hours. Then, after standing for 30 minutes, the supernatant was gently removed. Next, 5.0 mL of physiological phosphate buffer solution (hereinafter abbreviated as PBS) was added and incubated at room temperature for 10 minutes. Then, after standing for 30 minutes, the supernatant was gently removed. This washing operation was repeated three times.
After the washing operation, the remaining PBS was completely removed while taking care not to allow the separating material in the test tube to flow out. Thereafter, 5.0 mL of PBS was quickly added, and then gently stirred for 1 minute on a stirrer using a stir bar to disperse lymphocytes adhering to the bead surface in PBS. Thereafter, PBS was collected from the supernatant, the number of lymphocytes contained in 1.0 mL of PBS was measured using a hemocytometer, and the number of added lymphocytes (C₀Lymphocyte count (C)_R) Was calculated as the recovery rate according to the following formula, and the results are shown in Table 3.
Recovery rate (%) = (C_R/ C₀) × 100
[0042]
Comparative Example 2
Separation was performed in the same manner as in Example 4 except that the pretreated beads prepared in Example 4 were used as the separation material, and the lymphocyte recovery rate was determined. The results are shown in Table 3.
[0043]
[Table 3]

[0044]
From the results of Table 3, the separation material prepared in Example 4 separated about half of the lymphocytes in both cases of LPS + with LPS + added for the purpose of activating lymphocytes and LPS− with no LPS added. After that, it was confirmed that further recovery was possible. On the other hand, in Comparative Example 2, about 10% of lymphocytes could be separated and collected in the case of LPS−, but no separation / collection was possible in the case of LPS +. That is, it was found that the separation material used in Example 4 has a function of selectively separating and collecting lymphocytes regardless of the degree of activation of lymphocytes.
[0045]
Example 5: Isolation of immunoglobulin
<Preparation of separation material>
In the preparation of the separation material in Example 4, the separation material was prepared in the same manner as in Example 4 except that the copolymer (D) synthesized in Synthesis Example 4 was used instead of the copolymer (A). did.
The surface of the obtained separating material was measured by X-ray photoelectron spectroscopy, and the value of P / C was determined. The results are shown in Table 4.
[0046]
<Selective separation operation>
5.0 g of the above separating material is put into a polypropylene test tube, and 5.0 mL of fetal bovine serum (hereinafter abbreviated as FCS, manufactured by GIBCO) diluted to 1/256 with PBS is added, and then at room temperature for 2 hours. Incubated. Then, after standing for 30 minutes, the supernatant was gently removed. Subsequently, 5.0 mL of PBS was added, and the mixture was incubated at room temperature for 10 minutes, then allowed to stand for 30 minutes, and the supernatant was gently removed. This washing operation was repeated three times.
After the washing operation, 5.0 mL of 20-fold PBS was added and incubated at room temperature for 2 hours to dissolve the protein adhering to the surface of the separating material in PBS. After leaving still for 30 minutes, the supernatant was gently separated and used as a specimen.
Next, an anti- (bovine IgG) rabbit IgG (manufactured by Yagai Co., Ltd.), anti- (bovine albumin) rabbit IgG (manufactured by Yagai Co., Ltd.) or anti- (bovine bovine IgG) is applied to a polystyrene 96-well plate (manufactured by Nunc). Fibronectin) rabbit IgG (manufactured by SIGMA) was injected at 100 μL / well and incubated at room temperature for 2 hours. Then, after removing each antibody solution, each well was washed 4 times with 300 μL of PBS.
200 μL / well of Block Ace solution diluted by 1/4 (manufactured by Snow Brand Milk Products Co., Ltd.) was added and incubated at room temperature for 2 hours. After removing the Block Ace solution in the well, 100 μL / well of the sample was added and incubated at room temperature for 2 hours, and each well was washed 4 times with 300 μL of PBS.
100 μL / well of an anti- (rabbit IgG) goat IgG (manufactured by SIGMA) solution modified with horseradish peroxidase (hereinafter abbreviated as HRP) was injected and incubated at room temperature for 2 hours. Then, after removing each antibody solution, each well was washed 4 times with 300 μL of PBS.
Each well was injected with 100 μL / well of HRP color development solution (color development kit T for peroxidase, manufactured by Sumitomo Bakelite Co., Ltd.) and incubated at room temperature for 2 hours, and then each well was washed 4 times with 300 μL of PBS. Next, 100 μL / well of HRP coloring solution (peroxidase coloring kit T, manufactured by Sumitomo Bakelite Co., Ltd.) was injected and incubated at room temperature for 10 minutes. Further, 100 μL / well of a reaction stop solution (color development kit T for peroxidase, manufactured by Sumitomo Bakelite Co., Ltd.) was injected to stop the reaction. Next, absorbance at 450 nm was measured using a plate reader (SPECTRA MAX250, manufactured by Molecular Devices).
Using a calibration curve prepared in advance, the amount of each protein in the sample was determined from the absorbance. The ratio of the selectively separated IgG amount to the recovered albumin amount (Ui / Ua) and the recovered fibronectin amount The ratio (Ui / Uf) of the amount of selectively separated IgG was determined. The results are shown in Table 4.
[0047]
Comparative Example 3
Except for using the pretreated beads prepared in Example 4 as the separation material, selective separation was performed in the same manner as in Example 5, and Ui / Ua and Ui / Uf were obtained from the measured values. The results are shown in Table 4.
[0048]
[Table 4]

[0049]
From the results in Table 4, the separation material prepared in Example 5 showed a separation performance 70 times or higher than the separation material of Comparative Example 3 in the ratio of IgG to albumin (Ui / Ua), and the ratio of IgG to fibronectin. In (Ui / Uf), it was found that the separation performance was 30 times or more that of Comparative Example 3. That is, using the separation material of Example 5 means that IgG can be concentrated by selective separation several tens of times that of other albumins and fibronectin. From the above results, it was confirmed that the separation material used in Example 5 had a function of selectively separating and collecting IgG.
[0050]
Example 6: Separation of cell lines
<Preparation of separation material>
A low fluorescent cell desk (manufactured by Sumitomo Bakelite Co., Ltd.) was immersed in a copolymer solution prepared by dissolving 0.5 g of the copolymer (A) synthesized in Synthesis Example 1 in 100 mL of distilled water at room temperature for 5 minutes. The cell desk was removed from the solution and dried at room temperature for 1 hour. Furthermore, the temperature was raised to 60 ° C. in a drier and dried under reduced pressure for 4 hours at the same temperature to prepare a cell desk-shaped separation material.
The surface of the obtained separating material was measured by X-ray photoelectron spectroscopy, and the value of P / C was determined. The results are shown in Table 5.
[0051]
<Selective separation operation>
NIH3T3 cells (mouse fetal fibroblasts) and SIRC cells (rabbit corneal epithelial cells) using Dulbecco's modified Egourdium (DMEM, hereinafter abbreviated as medium) containing 10% FCS in a 9 cm polystyrene dish The cells were cultured to subconfluence. These cells were treated with a 0.1% trypsin solution, detached, centrifuged, and then suspended in the medium. The cell concentration of each suspension was measured using a hemocytometer, and diluted with a medium so as to be 2000 cells / mL.
Into each well of the 24-well plate, 0.5 mL of each cell suspension was injected, and the cell desk-like separation material was submerged therein. Subsequently, after culturing in a carbon dioxide incubator for 24 hours, cells adhered to the surface of the separating material were identified. The cells were identified by a method using a specific antibody against vimentin, a cytoskeletal protein expressed in the cells. First, after fixing the cells with methanol, an anti-vimentin antibody (manufactured by Cosmo Bio) was added. Next, washing treatment was performed, and vimentin positive cells were detected using a FITC-labeled anti- (mouse IgG) antibody (manufactured by SIGMA). On the other hand, the total cell number was measured by nuclear staining using propidium iodide (manufactured by SIGMA). Under a fluorescence microscope, the same screen was photographed using a filter for FITC observation and a filter for propidium iodide observation, and NIH3T3 cells were calculated from the ratio of FITC positive cells (vimentin positive cells: SIRC cells) in the total number of cells. And the adhesion rate (%) of SIRC cells was calculated. The results are shown in Table 5.
[0052]
Comparative Example 4
The adhesion rate of each cell was calculated in the same manner as in Example 6 except that a low-fluorescence cell desk that had not been treated at all was used in place of the cell desk-shaped separation material. The results are shown in Table 5.
[0053]
[Table 5]

[0054]
From the results in Table 5, it was found that the separation material prepared in Example 6 had 96% NIH3T3 cells selectively adhered to the surface. In contrast, in Comparative Example 4, it was found that NIH3T3 cells and SIRC cells adhered to the surface at approximately the same rate. From the above, it was confirmed that the separation material used in Example 6 had a function of selectively separating NIH3T3 cells.
[0055]
Example 7: Isolation of primary cultured cells
<Preparation of separation material>
To each well of a 24-well plate (manufactured by Nunc), 1.0 mL of a copolymer solution prepared by dissolving 0.5 g of the copolymer (A) synthesized in Synthesis Example 1 in 100 mL of ethanol was dispensed at room temperature. Incubated for minutes. Next, after removing the solution in the well, the temperature was raised to 60 ° C. in a drier, and dried under reduced pressure for 4 hours at that temperature, thereby preparing a culture plate-shaped separating material.
The surface of the obtained separating material was measured by X-ray photoelectron spectroscopy, and the value of P / C was determined. The results are shown in Table 6.
[0056]
<Selective separation operation>
A section of skin was collected from a rat fetus within 24 hours of birth and treated with DMEM containing 0.05% collagenase. The obtained solution was filtered through a mesh and then centrifuged, and the collected cells were resuspended in DMEM containing 5% FCS. The cell concentration was measured using a hemocytometer, and diluted with a medium so as to be 5000 cells / mL.
0.5 mL of the cell suspension prepared above was dispensed into each well of the culture plate-shaped separating material and cultured in a carbon dioxide incubator for 16 hours. Thereafter, the inside of the well was washed with physiological buffered saline and photographed using a phase contrast microscope. By observing cell morphology, fibroblasts and epidermal cells were identified, and the adhesion rate of each cell to the surface of the separation material was calculated. The results are shown in Table 6.
[0057]
Comparative Example 5
The adhesion rate of each cell was calculated in the same manner as in Example 7 except that a 24-well plate that had not been treated at all was used instead of the culture plate-shaped separation material. The results are shown in Table 6.
[0058]
[Table 6]

[0059]
From the results in Table 6, it was found that the separation material prepared in Example 7 can selectively adhere and separate only fibroblasts from a suspension containing fibroblasts and epidermal cells collected from living tissue. . On the other hand, in Comparative Example 5, it was found that fibroblasts and epidermal cells adhered to the surface at substantially the same rate. From the above, it was confirmed that the separation material used in Example 7 had a function of selectively separating fibroblasts.
[0060]
Example 8: Recovery of primary cultured cells
<Selective separation operation>
Using the separating material prepared in Example 7, the same operation as in Example 7 was carried out until photography using a phase contrast microscope. Thereafter, no photography was performed, and each well of the separation material was treated with a 0.1% trypsin solution to detach the cells. Next, the collected cells were centrifuged and then suspended in the medium. The number of cells in the suspension was measured using a hemocytometer and diluted with a medium so as to be 2000 cells / mL to obtain a cell suspension.
0.5 mL of the obtained cell suspension was dispensed into each well of a 24-well plate and cultured in a carbon dioxide incubator for 48 hours. Thereafter, the morphology of the cells was confirmed with a phase contrast microscope, and fibroblasts and epidermal cells were identified. The results are shown in Table 7.
[0061]
Comparative Example 6
Cells were identified by the same method as in Example 8 except that the same 24-well plate as in Comparative Example 5 was used as the separation material. The results are shown in Table 7.
[0062]
[Table 7]

[0063]
The purpose of Example 8 is to selectively separate only one type of primary cultured cell from a suspension containing the same two types of cells as in Example 7, and then recover the cells. Here, the recovered cells are cultured, and the state of their proliferation is observed to evaluate which cells have been separated and recovered. From Table 7, it was confirmed that only the fibroblasts proliferated from the material separated and collected using the separating material. In contrast, in Comparative Example 6, it was confirmed that both fibroblasts and epidermal cells were proliferating. From the above, it was confirmed that the separation material used in Example 8 had a function of selectively separating and collecting fibroblasts.
[0064]
Example 9: Separation of information transfer material
<Selective separation operation>
Using the bead-shaped separating material prepared in Example 5, 5.0 g of this separating material was put in a glass container, and 2.5 mL of a saturated aqueous solution of dibenzofuran and 2.5 mL of a saturated aqueous solution of dibenzo-p-dioxin were added. The container was sealed. The contents were stirred by rotating the whole container for 60 minutes, and then the solution was filtered off using a funnel. The collected solution was measured for absorbance at a wavelength of 249 nm for dibenzofuran and 289 nm for dibenzo-p-dioxin using an absorptiometer.
Using a calibration curve prepared in advance, the concentration of the solution (C_n), And the concentration of each saturated aqueous solution (C₀) Was calculated by the following formula as the rate of change. The results are shown in Table 8.
Rate of change (%) = ((C₀-C_n) / C₀) × 100
[0065]
Comparative Example 7
Except for using the same pretreated beads as in Comparative Example 3 as the separating material, selective separation was performed in the same manner as in Example 9, and the rate of change was calculated from the measured values. The results are shown in Table 8.
[0066]
[Table 8]

[0067]
From the results of Table 8, it was found that the separation material of Example 9 can selectively separate dibenzofuran from an aqueous solution containing dibenzofuran and dibenzo-p-dioxin, which are information transmission substances. That is, the change rate of the concentration of dibenzofuran showed a value three times or more that of Comparative Example 7. One dibenzo-p-dioxin had almost the same rate of change both in the case of using the separating material and in the case of Comparative Example 7. That is, it is considered that the concentration of dibenzofuran in the solution decreased as a result of the selective separation of dibenzofuran in the solution by the separating material used in Example 9. From the above, it was found that the separating material used in Example 9 has a function of selectively separating dibenzofuran.
[0068]
The following can be seen from the results of the above Examples and Comparative Examples. That is, it was confirmed that the separation materials prepared in Examples 1 to 9 contained phosphorus elements derived from phosphorylcholine groups and had different P / C values. The separating material used in the examples is obtained by coating a polymer obtained by copolymerizing MPC and other monomers on a substrate having various shapes made of PET, polystyrene, glass and the like. is there. Although the separating materials used in Examples 1, 4, 6, 7, and 8 were all prepared by coating the same copolymer (A) on the surface, the value of P / C is the kind of the substrate. The values differed depending on the shape. That is, the value of P / C can be controlled to some extent by the combination of a copolymer containing a phosphorylcholine group and a base material on which it is coated when preparing the separation material. By controlling the C value within a specific range, it is considered that the function of selectively separating and recovering a desired specific component was developed as a result.

Claims (5)

In this method, a separation material having at least a group represented by formula (1) is brought into contact with a solution containing a specific component, and one or more specific components contained in the solution are selectively separated and recovered. And
The ratio of the amount P of phosphorus element derived from the group represented by the formula (1) and the amount C of carbon element (P / C) in the spectrum of the separating material measured by X-ray photoelectron spectroscopy. ) Has a group represented by the formula (1) in an amount of 0.002 to 0.3,
Separation characterized in that the specific component is a cell derived from one or more organisms selected from the group consisting of blood cells, cell lines and primary cultured cells, immunoglobulin, or dioxin or a derivative thereof.・ Recovery method.

(In the formula, R ¹ , R ² and R ³ are the same or different groups and represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a hydroxyalkyl group. N is an integer of 1 to 4) The separation / recovery method according to claim 1, wherein the group represented by the formula (1) is a group derived from a phosphorylcholine-like group-containing monomer represented by the formula (2).

Wherein R ¹ , R ² and R ³ are the same or different groups and represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a hydroxyalkyl group, and R ⁴ is an alkyl group having 1 to 6 carbon atoms. R ⁵ represents a hydrogen atom or a methyl group, and n is an integer of 1 to 4.) The group represented by the formula (1) is a group derived from a polymer obtained by polymerizing a monomer composition containing a phosphorylcholine-like group-containing monomer represented by the formula (2). The separation / recovery method according to claim 1 .

Wherein R ¹ , R ² and R ³ are the same or different groups and represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a hydroxyalkyl group, and R ⁴ is an alkyl group having 1 to 6 carbon atoms. R ⁵ represents a hydrogen atom or a methyl group, and n is an integer of 1 to 4.)   A polymer obtained by polymerizing a monomer composition containing a phosphorylcholine-like group-containing monomer is (A) 10-100 mol% of a phosphorylcholine-like group-containing monomer represented by formula (2), and (B) hydrophobic The separation according to claim 3, which is a polymer obtained by polymerizing a monomer composition comprising 0 to 90 mol% of a polymerizable monomer and 0 to 70 mol% of (C) a hydrophilic monomer.・ Recovery method.   The separation / recovery method according to any one of claims 1 to 4, wherein the blood cells are one or more selected from the group consisting of red blood cells, white blood cells, and platelets.