JP4261369B2 - 高密度リポタンパク質アッセイデバイスおよび方法 - Google Patents
高密度リポタンパク質アッセイデバイスおよび方法 Download PDFInfo
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/60—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving cholesterol
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Description
本発明は、血液流体サンプル中の高密度リポタンパク質(HDL)関連コレステロールの濃度を決定する方法およびこの方法を行うための診断アッセイデバイスに関する。
血中に存在するコレステロールの量は、冠状動脈疾患の危険性に関連することが公知である。コレステロールは、主にタンパク質結合形態で血中を循環する。コレステロールを輸送するタンパク質は、リポタンパク質であり、このリポタンパク質は、それらの密度に基づいて、3つのクラスに下位分類される。超低密度リポタンパク質(VLDL)は、ヒトにおいて、肝臓において合成され、最終的には、低密度リポタンパク質(LDL)に変換され、血漿コレステロールの大部分を輸送する、トリグリセリドリッチリポタンパク質である。高密度リポタンパク質(HDL)は、トリグリセリドリッチリポタンパク質の異化、ならびに末梢組織からのコレステロールの除去および肝臓への輸送に関与する、リポタンパク質である。血清HDLレベルと冠状動脈疾患の危険性との間の逆の関係が確立された。特に、HDLと関連した血清コレステロールの割合が、低い場合、冠状動脈疾患の危険性は、増大する。
一局面において、本発明は、低密度リポタンパク質(LDL)および/または超低密度リポタンパク質(VLDL)もまた含む血液流体サンプル中の高密度リポタンパク質(HDL)と関連する血清コレステロールを測定するアッセイデバイスを提供する。このデバイスは、以下を備える:
(i)複数の多孔性要素(これを通って、このような血液流体サンプルは、順番に、毛細管作用および/または重力によって流動し得る)であって、
ここでこの複数の要素は、以下を備える:サンプル分配マトリクス;このマトリクスの下流にあるHDL試験パッドであって、この試験HDLパッドにおいてHDL濃度がアッセイされ得る、HDL試験パッド;およびHDL試験パッドの上流にある、流体サンプルに由来する非HDLリポタンパク質を選択的に結合して、除去するために有効な固定化試薬を含む要素。
(a)このようなサンプルと、吸収性サンプル分配マトリクスとを接触させる工程であって、ここでこのサンプル分配マトリクスは、アッセイデバイス内に含まれる複数の多孔性要素のうちの1つであり、この要素を通って、このサンプルは、毛細管作用および/または重力によって順番に流れ得、
ここでこの複数の要素は、このサンプル分配マトリクスの下流にあるHDL試験パッドであって、このHDL試験パッドにおいて、HDL濃度がアッセイされ得;そして
HDL試験パッドの上流に、流体サンプル由来の非HDLリポタンパク質を選択的に結合して除去するに有効な固定化試薬を含む要素、
をさらに備える、工程;
(b)サンプルと、固定化試薬を含む要素とを接触させる工程;
(c)マトリクスを、HDL試験パッドと流体連絡させ、それにより、サンプルを、要素からHDL試験パッドへと移す工程;ならびに
(d)血液流体サンプル中のHDLリポタンパク質の含有量を決定する工程。
(I.定義)
以下の用語は、別段示されなければ、以下の意味を有する。
図1〜4は、本発明に従って構築されたマルチ分析物アッセイデバイス14の種々の実施形態を示す。図4は、分解組み立て様式で示される。このデバイスは、少量の血液サンプル、代表的には、10〜50μlの間の血液を用いて、特にHDLと関連する血清コレステロール(HDL関連コレステロールまたは単にHDLコレステロールともいわれる)を決定するために設計される。他のアッセイ(例えば、総コレステロールまたはトリグリセリドレベル)は、同じサンプルから同時に決定され得る。HDL関連コレステロールの決定はまた、単に、HDLの決定またはHDLアッセイといわれ得る。
操作時に、血液サンプルは、ウェル16に配置され、篩分けパッド22を通して吸収され、ここで大きな粒子(赤血球を含む)が除去され、そこからサンプル分配マトリクス26へと入る。これらの工程は、デバイスが「サンプル分配」ステージにある間に起こり、その結果、サンプル分配マトリクスは、試験パッドと流体連絡しておらず、特定の実施形態(例えば、図1〜2)においては、試薬パッド74とも流体連絡していない。
5〜20mg/mlの硫酸デキストラン(500,000MW)および(必要に応じて)12.5mM Mg(OAc)2を含む水溶液は、上記のように、約125μmの厚みを有するカチオン膜(例えば、表面四級アンモニウム基を有するナイロン膜)に分配される。この試薬溶液は、約16μl/インチの速度で分配され、次いで、その膜は、連続ロールプロセスにおいて50℃で20分間乾燥される。例えば、100フィートの長さは、このように調製され得、アッセイデバイスに適合するように切断され得る。
HDL反応膜を調製するために、ポリスルホン膜は、以下の水溶性処方物に浸漬される:コレステロールオキシダーゼ36.5ユニット/ml、コレステロールエステラーゼ215ユニット/ml、ペルオキシダーゼ200ユニット/ml、4−アミノアンチピリン1.88mg/ml、およびTOOS(3−[エチル(3−メチルフェニル)アミノ]−2−ヒドロキシプロパンスルホン酸)12.05mg/ml。この試薬は、16.6μl/インチの速度で分配され、その膜は、連続ロールプロセスにおいて50℃で20分間乾燥される。例えば、100フィートの長さは、このように調製され得、アッセイデバイスに適合するように切断され得る。
代表的アッセイは、本質的に実施例1〜2に記載されるように調製された試薬パッドおよびHDL試験パッドを使用して、LDX(登録商標)分析器において行われる。図1に示されるようなアッセイデバイス構成について、サンプル(35μlの血清または全血)は、サンプルウェルに付与され、サンプル分配マトリクスを通して2分間分配させる。その反応バーは、次いで、マトリクスと3秒間(試薬パッドおよび試験パッド(約1.5μlの合わせた収容能)を満たすに十分な血清を移動させるに十分な時間)接触させ、その後、そのバーを、その元の位置に戻す。反射率の読み取りを、3秒ごとに150秒間、HDL試験パッドの上部表面から行って、HDLアッセイ反応の進行をモニターする。得られた最小の反射率値を、次いで、予め確立した較正曲線に従って、HDLコレステロールのmg/dLに変換する。
Claims (32)
- 低密度リポタンパク質(LDL)または超低密度リポタンパク質(VLDL)もまた含む血液流体サンプル中の高密度リポタンパク質(HDL)と関連する血清コレステロールを測定するためのアッセイデバイスであって、
該デバイスは、以下:
(i)複数の多孔性要素であって、該要素を通って、該血液流体サンプルが毛細管作用および/または重力によって順番に流れ得る、複数の多孔性要素であって、
該複数の要素は、以下:
サンプル分配マトリクス;
該マトリクスの下流にあるHDL試験パッドであって、ここでHDL濃度がアッセイされ得る、HDL試験パッド;および
該HDL試験パッドの上流に、該流体サンプルから非HDLリポタンパク質を選択的に結合して除去するに有効な、該流体サンプルに不溶性である固定化試薬を含む要素、
を備える、複数の多孔性要素;ならびに
(ii)(a)該HDL試験パッドが該サンプル分配マトリクスと流体連絡しない、サンプル分配位置と、
(b)該試験パッドおよび分配マトリクスが互いに流体連絡している、試験位置との間に
該デバイスを合わせるために有効な取り付け手段、
を備える、アッセイデバイス。 - 請求項1に記載のデバイスであって、前記固定化試薬は、前記サンプル分配マトリクス内の第1のサンプル収集領域に固定化され、該領域は、該デバイスが前記試験位置にある場合に、前記HDL試験パッドと流体連絡して配置される、デバイス。
- 請求項1に記載のデバイスであって、前記固定化試薬は、前記分配マトリクスの下流かつ前記HDL試験パッドの上流に位置した多孔性試薬パッドに固定化され、該試薬パッドは、該デバイスが前記試験位置にある場合に、該HDL試験パッドと流体連絡して配置される、デバイス。
- 請求項3に記載のデバイスであって、前記試薬パッドは、前記HDL試験パッドに取り付けられる、デバイス。
- 請求項1に記載のデバイスであって、反応バーをさらに備え、該反応バーに前記HDL試験パッドが取り付けられている、デバイス。
- 請求項5に記載のデバイスであって、前記取り付け手段は、前記サンプル分配位置と前記試験位置との間に、前記反応バーおよびカセット本体の相対位置を合わせるに有効である、デバイス。
- 請求項1に記載のデバイスであって、前記取り付け手段は、(c)前記試験位置から、前記HDL試験パッドが前記サンプル分配マトリクスと流体連絡していない位置へと該デバイスを移すためにさらに有効である、デバイス。
- 請求項1に記載のデバイスであって、前記固定化試薬は、ポリアニオン試薬を含む、デバイス。
- 請求項8に記載のデバイスであって、前記固定化ポリアニオン試薬は、スルホン化多糖を含む、デバイス。
- 請求項1に記載のデバイスであって、前記HDLアッセイパッドは、バイオセンサを備える、デバイス。
- 請求項10に記載のデバイスであって、前記バイオセンサは、酸素もしくは過酸化水素の生成を電気化学的に測定するに有効であり、該生成は、前記アッセイパッド内のHDL関連コレステロール濃度に依存する、デバイス。
- 請求項1に記載のデバイスであって、前記試薬パッドは、多孔性ポリマー膜を備える、デバイス。
- 請求項12に記載のデバイスであって、前記ポリマー膜は、カチオン性表面基を含む、デバイス。
- 請求項1に記載のデバイスであって、前記試薬パッドは、多層積み重ね膜を備え、該多層積み重ね膜のうちの少なくとも1層は、非HDLリポタンパク質を結合するに有効な固定化試薬を含む、デバイス。
- 請求項1に記載のデバイスであって、前記HDL試験パッドおよび前記試薬パッドの各々は、多孔性ポリマー膜である、デバイス。
- 請求項4に記載のデバイスであって、前記HDL試験パッドおよび試薬パッドは、ともに積層されている、デバイス。
- 低密度リポタンパク質(LDL)または超低密度リポタンパク質(VLDL)もまた含む血液流体サンプル中の高密度リポタンパク質(HDL)と関連する血清コレステロールを測定するためのアッセイデバイスであって、該デバイスは、以下:
該血液流体サンプルを分配するためのサンプル分配マトリクス;
該流体サンプルから非HDLリポタンパク質を選択的に除去するに有効な固定化試薬を含む試薬パッド;および
該試薬パッドと流体連絡しているHDL試験パッドであって、該HDL試験パッドにおいて、HDL濃度がアッセイされ得るHDL試験パッド;
を備え、
ここで該試薬パッドは、該サンプルマトリクスと流体連絡した状態にされ得る、デバイス。 - 請求項17に記載のデバイスであって、前記固定化試薬は、スルホン化多糖を含む、デバイス。
- 請求項17に記載のデバイスであって、前記試薬パッドは、カチオン性表面基を有する多孔性ポリマー膜を備える、デバイス。
- 低密度リポタンパク質(LDL)または超低密度リポタンパク質(VLDL)もまた含む血液流体サンプル中の高密度リポタンパク質(HDL)と関連する血清コレステロールを測定する方法であって、該方法は、以下の工程:
(a)該サンプルと、吸収性サンプル分配マトリクスとを接触させる工程であって、ここで該サンプル分配マトリクスは、アッセイデバイス内に含まれる複数の多孔性要素のうちの1つであり、該要素を通って、該血液流体サンプルは、毛細管作用および/または重力によって順番に流れ得、該複数の要素は、
該サンプル分配マトリクスの下流にあるHDL試験パッドであって、このHDL試験パッドにおいて、HDL濃度がアッセイされ得る、HDL試験パッド;および
該HDL試験パッドの上流に、流体サンプルから非HDLリポタンパク質を選択的に結合して除去するに有効な、該流体サンプルに不溶性である固定化試薬を含む要素、
をさらに備える、工程;
(b)該サンプルと、該固定化試薬を含む該要素とを接触させる工程;
(c)該マトリクスを、該HDL試験パッドと流体連絡させ、それにより、該サンプルを、該要素から該HDL試験パッドへと移す工程;ならびに
(d)該血液流体サンプル中のHDLリポタンパク質の含有量を決定する工程;
を包含し、ここで工程(c)の前に、該マトリクスは、該HDL試験パッドと流体連絡していない、方法。 - 請求項20に記載の方法であって、前記要素は、前記分配マトリクスの下流かつ前記HDL試験パッドの上流に位置する、多孔性試薬パッドであり、該多孔性試薬パッドは、工程(c)において該HDL試験パッドと流体連絡状態にされる、方法。
- 請求項21に記載の方法であって、工程(b)の前に、前記マトリクスは、前記試薬パッドと流体連絡していない、方法。
- 請求項21に記載の方法であって、工程(b)において、前記マトリクスと、前記試薬パッドとを接触させる際に、該試薬パッドは、前記HDL試験パッドと同時に流体連絡している、方法。
- 請求項23に記載の方法であって、前記試薬パッドは、前記HDL試験パッドに取り付けられている、方法。
- 請求項20に記載の方法であって、前記マトリクスは、前記固定化試薬を含み、その結果、工程(a)および工程(b)の接触は、同時に起こる、方法。
- 請求項20に記載の方法であって、前記固定化試薬は、前記マトリクスの上流の篩分けマトリクス内に含まれ、かつ該マトリクスと接触し、その結果、工程(a)および工程(b)の接触は、逆の順番で起こる、方法。
- 請求項20に記載の方法であって、該方法は、所望の量のサンプルが移された場合に、前記マトリクスと前記試験パッドとの間の前記流体連絡を中断する工程をさらに包含する、方法。
- 請求項20に記載の方法であって、前記固定化試薬は、スルホン化多糖を含む、方法。
- 請求項21に記載の方法であって、前記試薬パッドは、カチオン性表面基を有する多孔性ポリマー膜を備える、方法。
- 請求項21に記載の方法であって、前記試薬パッドは、多層積み重ね膜を備え、該多層積み重ね膜のうちの少なくとも1層は、非HDLリポタンパク質を結合するに有効な固定化試薬を含む、方法。
- 請求項20に記載の方法であって、前記HDL試験パッドは、バイオセンサを備える、方法。
- 請求項31に記載の方法であって、前記バイオセンサは、酸素もしくは過酸化水素の生成を電気化学的に測定するに有効であり、該生成は、前記試験パッド内のHDL関連コレステロール濃度に依存する、方法。
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JP2006521823A (ja) * | 2003-04-02 | 2006-09-28 | コレステック コーポレイション | 高密度リポタンパク質に関連する血清コレステロールを測定するためのアッセイデバイス中の多孔性および生物学的活性を保持する接着膜 |
JP4641303B2 (ja) * | 2003-04-02 | 2011-03-02 | コレステック コーポレイション | 高密度リポタンパク質に関連する血清コレステロールを測定するためのアッセイデバイス中の多孔性および生物学的活性を保持する接着膜 |
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Publication number | Publication date |
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JP2005522681A (ja) | 2005-07-28 |
AU2003220669A1 (en) | 2003-10-27 |
EP1357383B1 (en) | 2005-11-09 |
ATE309544T1 (de) | 2005-11-15 |
DE60207196T2 (de) | 2006-07-20 |
US20030224471A1 (en) | 2003-12-04 |
EP1357383A1 (en) | 2003-10-29 |
HK1058397A1 (en) | 2004-05-14 |
AU2003220669B2 (en) | 2008-09-18 |
CA2480491A1 (en) | 2003-10-23 |
TWI306510B (en) | 2009-02-21 |
WO2003087401A1 (en) | 2003-10-23 |
ES2252358T3 (es) | 2006-05-16 |
TW200305020A (en) | 2003-10-16 |
US7795038B2 (en) | 2010-09-14 |
DE60207196D1 (de) | 2005-12-15 |
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