JP2015531489A - 低レベルのlal反応性物質を検出するための高感度かつ迅速な方法 - Google Patents
低レベルのlal反応性物質を検出するための高感度かつ迅速な方法 Download PDFInfo
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Abstract
Description
(Pはカスケード生成物(発色指示薬又は凝固したコアグロゲン)であり、P0は初期レベルであり、tは反応時間であり、かつa及びbは定数である)。
以下の実施例は、LAL測定試験の相関法を記載する。LAL測定試験手順及びこれらの試験手順の結果も記載する。カイネティック発色法のように試料を調製した。この方法については、エンドトキシン水溶液を種々の濃度(0.005エンドトキシン単位(「EU」/ml、0.05EU/ml、0.5EU/ml及び5EU/ml)で調製した。いかなるエンドトキシンも含まない溶液、すなわちブランクも調製し、対照溶液として使用した。次いで、溶液を37℃で10分間インキュベートした。インキュベーション後、各エンドトキシン濃度及びブランクを、NaOH及びデオキシコレートで洗浄した試料ホルダーに分配(3連で)した。
上に論じられるように、エンドトキシンとグルカンの両方の速度論サインを監視することにより、使用者が、試料中に存在し得る任意のエンドトキシン又はグルカンの濃度を予想することに加えて、試料分析において異常が存在するかどうかを検証又は評価することが可能になる。実際のカスケード反応も、酵素の各々を阻害する1つ又は複数の因子を有する。エンドトキシンカスケードは4ステップカスケードであり、グルカンカスケードは2ステップカスケードである。しかしながら、エンドトキシン反応とグルカン反応の両方が、その速度論に影響を及ぼす阻害因子を有する。カブトガニでは、閾値に達するまでこれらの因子が凝固を防止し、このことが、閾値が感度を制限するためにこれらの因子を分析システムにとって望ましくないものにしている。試薬の処理の一部は、製造業者及びロットによって変化するこれらの因子を除去することである。速度論への効果は、各反応の順序へのわずかな調整として最も単純にモデル化される。実際、反応速度論がモデルである検出力関数の挙動を利用する種々の数値技法を使用することができるので、正確な程度は問題でない。
発色又は比濁カイネティック法のいずれについても、光学濃度を経時的に測定する。エンドトキシンレベルの測定基準として使用する開始時間は、LAL反応の光学濃度又は光吸収が指定された量変化した時間である。通常、この時間は、このレベルを超過した測定ポイント間の補間によって計算される。反応がまだエンドポイントに達していないが、その初期状態から、背景ノイズと区別可能なほど十分な変化を示した場合、その結果を検出力関数にあてはめて、開始時間を含む将来の性能を予測することができる。
エンドポイント予測のために検出力関数を用いることの単純であるがより正確なバリエーションは、エンドトキシンレベルのC因子活性への移行によるカスケード開始の見かけの時間遅延、及びカスケードステップ遅延を含めることである。全てのロットが異なるので、一定の時間遅延の使用は、変化し得るものより望ましくない。
関数がデータストリームを十分に記載する場合、新たなデータポイントは適合関数を解答に収束させる。解答が収束しない場合には、関数はデータを十分に記載していない。このことを、データストリームの妥当性についてチェックする単純な方法の1つとして使用することができる。
Claims (20)
- 水性試料中のLAL反応性物質の濃度を測定する方法であって、
(a)水性試料を検出試薬と接触させて、調製試料を形成するステップと、
(b)調製試料の物理特性を測定して、調製試料に特徴的な1以上の試料測定値を得るステップと、
(c)曲線あてはめを使用して、試料測定値及び既知の濃度のLAL反応性物質を中に含む溶液の物理特性の1以上の基準測定値の間で発達した相関に基づいて、水性試料が将来の指定された時間に有するLAL反応性物質の濃度を予測し、履歴データ及び/又は基準測定値を用いて、試料測定値の品質を検証するステップと
を含む方法。 - 物理特性が透過率、吸光度、濁度、光散乱、光学濃度、色、色値、色相、スペクトル、化学発光、蛍光、経時的な吸光度の磁気共鳴、吸光度の一定の変化までの時間、経時的な吸光度の最大勾配、及び一定時間後の吸光度からなる群から選択される光学特性である、請求項1記載の方法。
- 物理特性が試料測定値の反応速度論及び品質からなる群から選択される非線形特性であり、非線形特性を用いて検証される、請求項1記載の方法。
- 1以上の反応がエンドトキシン反応、グルカン反応及びこれらの組合せからなる群から選択される、請求項3記載の方法。
- 反応のカスケード開始の1以上の見かけの時間遅延を変数として曲線あてはめステップに含めるステップをさらに含む、請求項4記載の方法。
- 水性試料中のLAL反応性物質の濃度を測定する方法であって、
(a)水性試料を検出試薬と接触させて、調製試料を形成するステップと、
(b)調製試料の物理特性を測定して、調製試料に特徴的な2以上の試料測定値を得るステップと、
(c)試料測定値及び既知の濃度のLAL反応性物質を中に含む溶液の物理特性の3以上の基準測定値の非線形挙動間で発達した相関に基づいて、水性試料が将来の指定された時間に有するLAL反応性物質の濃度を予測するステップと
を含む方法。 - 物理特性が透過率、吸光度、濁度、光散乱、光学濃度、色、色値、色相、スペクトル、化学発光、蛍光、経時的な吸光度の磁気共鳴、吸光度の一定の変化までの時間、経時的な吸光度の最大勾配、及び一定時間後の吸光度からなる群から選択される光学特性である、請求項6記載の方法。
- 物理特性が非線形特性であり、非線形特性が検出試薬とLAL反応性物質との間の反応の既知の反応速度論に基づく、請求項6記載の方法。
- 1以上の反応がエンドトキシン反応、グルカン反応及びこれらの組合せからなる群から選択される、請求項8記載の方法。
- 水性試料中のLAL反応性物質の濃度を測定するシステムであって、
(a)メモリと、
(b)メモリに作動的に接続されたプロセッサであって、
(I)試料測定値を含む1以上の入力を受信し、試料測定値は、水性試料に特徴的な測定された物理特性であり、
(II)曲線あてはめを使用して1以上の出力を生成し、出力は、試料測定値及び既知の濃度のLAL反応性物質を中に含む溶液の物理特性の1以上の基準測定値間で発達した相関に基づいて、水性試料が将来の指定された時間に有するLAL反応性物質の予測された濃度、及び検証された試料測定値を含み、試料測定値の品質は履歴データ及び/又は基準測定値を用いて検証され、かつ
(III)メモリ中に出力を報告及び/又は保存する
ようプログラムされたプロセッサとを含むシステム。 - プロセッサに作動的に接続された光学読取装置をさらに含む、請求項10記載のシステム。
- 物理特性が透過率、吸光度、濁度、光散乱、光学濃度、色、色値、色相、スペクトル、化学発光、蛍光、経時的な吸光度の磁気共鳴、吸光度の一定の変化までの時間、経時的な吸光度の最大勾配、及び一定時間後の吸光度からなる群から選択される光学特性である、請求項10記載のシステム。
- 物理特性が非線形特性であり、非線形特性が反応速度論からなる群から選択される、請求項12記載のシステム。
- 1以上の反応がエンドトキシン反応、グルカン反応及びこれらの組合せからなる群から選択される、請求項13記載のシステム。
- プロセッサが、反応のカスケード開始の1以上の見かけの時間遅延を変数として曲線あてはめステップに含めるようさらにプログラムされた、請求項14記載のシステム。
- 水性試料中のLAL反応性物質の濃度を測定するシステムであって、
(a)メモリと、
(b)メモリに作動的に接続されたプロセッサであって、
(I)2以上の試料測定値を含む1以上の入力を受信し、試料測定値は、水性試料に特徴的な測定された物理特性であり、かつ
(II)既知の濃度のLAL反応性物質を中に含む溶液の物理特性の3以上の基準測定値の非線形挙動間で発達した相関を使用して1以上の出力を作成し、出力は、試料測定値に基づいて水性試料が将来の指定された時点で有するLAL反応性物質の予測された濃度を含み、かつ
(III)メモリ中に出力を報告及び/又は保存する
ようプログラムされたプロセッサとを含むシステム。 - プロセッサに作動的に接続された光学読取装置をさらに含む、請求項16記載のシステム。
- 物理特性が透過率、吸光度、濁度、光散乱、光学濃度、色、色値、色相、スペクトル、化学発光、蛍光、経時的な吸光度の磁気共鳴、吸光度の一定の変化までの時間、経時的な吸光度の最大勾配、及び一定時間後の吸光度からなる群から選択される光学特性である、請求項16記載のシステム。
- 物理特性が非線形特性であり、非線形特性が検出試薬とLAL反応性物質との間の反応の既知の反応速度論に基づく、請求項18記載のシステム。
- 1以上の反応がエンドトキシン反応、グルカン反応及びこれらの組合せからなる群から選択される、請求項19記載のシステム。
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