JP5235212B2 - 溶血した全血試料中の分析物の検出 - Google Patents
溶血した全血試料中の分析物の検出 Download PDFInfo
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- JP5235212B2 JP5235212B2 JP2010510690A JP2010510690A JP5235212B2 JP 5235212 B2 JP5235212 B2 JP 5235212B2 JP 2010510690 A JP2010510690 A JP 2010510690A JP 2010510690 A JP2010510690 A JP 2010510690A JP 5235212 B2 JP5235212 B2 JP 5235212B2
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
- G01N33/538—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody by sorbent column, particles or resin strip, i.e. sorbent materials
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- G—PHYSICS
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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
- G01N33/5002—Partitioning blood components
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- G—PHYSICS
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- 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
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Description
試料中に多くの成分が存在すればするほど、含まれる標的分析物の分析はより困難になる。赤血球は、全血のような生物学的液体からの目的の分析物の検出を潜在的に妨げる多量のタンパク質および低分子量の成分を含む。これが、臨床の日常業務で、好ましくは血液血漿もしくは単に血漿と呼ばれる(即ち、抗凝固全血試料; 細胞および赤血球を除く)または血液血清もしくは単に血清と呼ばれる(即ち、凝固全血; 細胞、赤血球および凝固系の大半のタンパク質、特にフィブリン/フィブリノーゲンを除く)それぞれが使用されることの主な理由の1つである。また、全血試料は、例えば血清または血漿と比較して取り扱うことが困難である傾向がある。全血は安定でない傾向があり、赤血球の緩やかな破壊は目的の多くの分析物の信頼性の高い測定を損なう。
第一態様において、本発明は、溶血した全血試料中の分析物の検出方法であって、目的の分析物を含むことが公知である溶血した全血試料または目的の分析物を含むことが疑われる溶血した全血試料を、制限アクセスクロマトグラフィー物質(RAM)を含むカラムに適用して分析物を結合させる工程、RAMから分析物を溶出する工程、および分析物を検出する工程を含む方法に関し、少なくとも該試料を該RAMに適用する工程において8.0より高いpHを有するバッファが使用される。
本発明の方法は、ヒトまたは動物の体でなく、インビトロで行われる。
1. 正常相クロマトグラフィーは、非極性(分散性)移動相と共に極性固定相の使用を要する。
2. 逆相クロマトグラフィー、反対の可能性は、非極性固定相および極性移動相(1つ以上の極性溶媒、例えば水、メタノール、アセトニトリルおよびテトラヒドロフランからなる)の使用を要する。
3. イオン交換クロマトグラフィーはイオン相互作用を伴う。この場合、移動相は、イオン性溶質の溶解を確実にするためにイオン化を支持する必要がある。固定相も、ある程度の保留を促進するために部分的にイオン性である必要がある。
4. サイズ排除クロマトグラフィーは分子の大きさのみに基づいた分離を伴い、理想的には溶質と固定相との吸着相互作用が存在しないことを必要とする。
5. アフィニティークロマトグラフィーは特異的な相互作用、例えば抗原と対応する抗体、またはレセプターと対応するリガンドのような特異的な結合ペアの間の相互作用に基づく。例えば、結合ペアの第1のパートナーは適切な固定相に結合し、結合ペアの第2のパートナーを捕捉するために使用される。第2のパートナーは適切な手段により放出および単離することができる。
(式中mは0または1であり、nは4または6である)から選択される。
(式中、mは0または1であり、nは4または6である)から選択され、好ましくはアニオンが塩化物、テトラフルオロボレート、オクチル硫酸塩、ヨウ化物およびチオシアネートから選択される塩である。
異なるpH値での種々のRAMの評価
RAMカラムを分析HPLCシステムに直接取り付けた。10μLのヘモグロビン溶液を注入した。6.6およびpH10.7それぞれの異なるpHの試料適用バッファ(溶出バッファA)を用いて第1の試験を行った。それぞれのpH値について、カラムを流れるおよびカラムに保持されるヘモグロビンの量を計算した。ヘモグロビンの注入に連続してブランク注入を実施して持ち越しも評価した。
MerckのLiChrospher(登録商標) RP-18 ADSカラムを使用した。10μLの試料(5mM酢酸アンモニウムにpH6.6で溶解した150mg/mLのヘモグロビン)をカラムに適用した。ヘモグロビンの溶出のために2種類のバッファ(A)H2O中5mM酢酸アンモニウム(pH6.6)および(B)アセトニトリル(ACN)中5mM酢酸アンモニウム(pH6.6)をそれぞれ使用した移動相を使用した。100%の(A)で15.0分間定組成溶出を行い、次いで100%(A)〜0%(B)から0%(A)〜100%(B)の直線的勾配を30分間使用して、その後100%(B)で2.0分間の洗浄工程によりRAMに可逆的に結合した任意のヘモグロビンを検出した。流速は1.5mL/分であり、全ての工程は室温でおこなった。
MerckのLiChrospher(登録商標) RP-18 ADSカラムを使用した。10μLの試料(pH10.7で10mMのエタノールアミン中に溶解した150mg/mLのヘモグロビン)をカラムに適用した。RAM結合ヘモグロビンの溶出のために、2種類のバッファ(A)H2O中10mMエタノールアミン(pH10.7)および(B)ACN中10mMエタノールアミン(pH10.7)それぞれを用いる移動相を使用した。100%(A)で15.0分間定組成溶出を行い、次いで100%(A)〜0%(B)から0%(A)〜100%(B)の直線勾配を30分使用して、その後100%Bで2.0分間洗浄工程を行った。流速は1.5mL/分であり、全ての工程は室温で行った。pH10.7では、フロースルー中にヘモグロビンは優先的に存在し(100%に設定)、20%(B)周辺で部分的に溶出し(20%)、洗浄ピークでは有意なレベルでは全く存在しない(図2参照)。これらのバッファ条件下ではヘモグロビンはメモリー効果を何ら生じない、つまり同じカラムを使用する次の測定においてヘモグロビンは存在しないことを示すために、100%(B)の適用後に観察された際の洗浄ピーク中のヘモグロビンの非存在は非常に重要である。実際にpH10.7では持ち越しは観察されなかった。このpH値はRAMへの異なる注入の間にヘモグロビンの持ち越しを回避すること、およびそれによりメモリー効果を回避することに適切であると思われる。
ヒト全血溶血物中の塩酸テトラサイクリンの定量
この一連の実験では、Biotrap 500 MS(登録商標)カラムを使用した。全血を特異的に溶血して、潜在的目的物の分析物に加えるための試料またはマトリックスとして使用した。実施例1で記載された肯定的な実験のために、10.7のpHを有する適用バッファおよび溶出バッファを使用した。具体的な分析物の検出について、直線四重極質量分析器を使用した。
抗凝固全血を分析まで+4℃に保存した。0.7g C13H28ClN(塩化1-メチル-オクチル-ピロリジニウム)および0.3g KSCNを25mL H2O中で混合して特異的溶血のための試薬を調製した。
分析設定を示すスキームを図4に示す。Biotrap 500 MS、pH10.7で試料を注入して、試料に含まれたヘモグロビンがカラムを流れた後、目的の分析物をHPLCに移して、6方向バルブを切替えて質量分析検出に移した。負荷ポンプはDionex, GermanyのP680 HPLCポンプであり、注入システムは2.5mL外部ループが装着されたRheodyne 6つ口バルブ(7725)からなった。ヘモグロビンモニタリングはBiotrap 500 MS トラップカラムの後に取り付けたダイオードアレイ検出器UVD340U(Dionex)で実施した。切替えユニットはRheodyne6つ口バルブ(7000)からなった。溶出はBischoff Analysentechnik und Gerate Leonberg, GermanyのProntosil 300-5-C18-H 5μm(125x2.0mm)分析カラムで行った。溶出物の送達はFlux Instruments, SwitzerlandのRheos 2000ポンプで行った。分析物のMS検出はThermo Finnigan, CA, USAの直線四重極Surveyor MSQで行った。
2種類の試料を使用して、対応するクロマトグラムを互いに比較した。第1の試料はテトラサイクリンが添加されていない純粋な溶血物であった。第2の試料はテトラサイクリンが加えられた溶血物であった。第2の試料について、30ngの塩酸テトラサイクリンを3mLの全血溶血物(150μL血液+1,350μLの150mM NaCl+1,500μLの溶解試薬)に加えた。得られた塩酸テトラサイクリンの濃度は全血中200pg/μLに相当するはずである。
Biotrap 500 MS上のヘモグロビンの持ち越しを調べるために、2.5mLの全血溶血物を注入した。Biotrap 500 MSにおける負荷工程は10mMエタノールアミンで、3.2mL/分で18分間実施した。次いで6つ口バルブを切替えてACN+0.05%TFAの勾配(10〜30%(B)で7.5分、30〜100%(B)で2.5分、100%(B)で2.0分、および0%(B)で7.9分)で逆流溶出を実施した。
血液溶血物に加えられた塩酸テトラサイクリンからなる5種類の試料を調製した。全血中の塩酸テトラサイクリンの濃度は200pg/μL、600pg/μL、1,000pg/μL、1,500pg/μLおよび2,000pg/μLであった。注入される溶血溶液中の対応する濃度は、9.5pg/μL、28.5pg/μL、47.5pg/μL、70.9pg/μLおよび94.3pg/μLであった。それぞれの試料について、2.5mLを注入して、上述の(c)に記載されるように分析した。試料は2または3回分析した。
全血溶血物からの免疫抑制薬の抽出
実施例2において、Biotrap 500 MS RAMが全血溶血物から抗生物質を抽出する能力が示された。これらの肯定的な結果によって助長されたために、該方法が目的の他の分析物にも適用可能であり得るかどうかについてさらなる調査を開始した。免疫抑制薬は非常に重要な臨床的に関連のある分析物であるので、この種類の分析物も、実施例2に記載され使用された方法に基づく良好なオンライン検出法に適用可能であるかどうか調べた。
Claims (10)
- a) 目的の分析物を含むことが分かっているかまたは疑われる溶血された全血の試料を、制限アクセスクロマトグラフィー物質(RAM)を含むカラムに適用して、分析物を結合させる工程、
b) RAMから分析物を溶出する工程、および
c) 分析物を検出する工程
を含む、溶血された全血試料中の分析物を検出する方法であって、少なくとも工程(a)において8.5以上のpHを有するバッファが使用される、方法。 - RAMが、多孔質シリカまたは多孔質ポリマー系粒子から選択される、請求項1記載の方法。
- RAMの内部表面が、疎水性相互作用、イオン相互作用または極性相互作用により目的の分析物と結合する、請求項1または2記載の方法。
- 多孔質シリカまたは多孔質ポリマー系粒子が、孔の疎水性内部表面および孔の外部の親水性コーティングを有する、請求項3記載の方法。
- RAMの内部表面が、疎水性相互作用(内部表面=C4、C8、C18)により目的の分析物と結合する、請求項1〜4いずれか記載の方法。
- RAMの孔径が60〜120Åである、請求項1〜5いずれか記載の方法。
- タンパク質画分が15kDa以上のポリペプチドを含む、請求項1〜4いずれか記載の方法。
- RAMカラムから溶出した後の分析物が、逆相HPLCによりさらに分離される、請求項1〜7いずれか記載の方法。
- 分析物が10kDa以下の分子量を有する、請求項1〜8いずれか記載の方法。
- 分析物が、免疫抑制薬、乱用薬物、葉酸および葉酸ビタマーからなる群より選択される、請求項1〜9いずれか記載の方法。
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