JP6458087B2 - 院内感染に対する易感染性を判定するための方法 - Google Patents
院内感染に対する易感染性を判定するための方法 Download PDFInfo
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Description
・上記患者から採取した生体サンプル又は被験サンプル中のsCD127の発現を測定する工程と、
・sCD127の発現を参照値と比較した後に、院内感染に対する易感染性が高いと結論づける工程と
を含む方法である。
・上記生体サンプル中のsCD127の発現を測定することを可能にする特異的ツール又は試薬と、
・院内感染していることが分かっている患者から採取したサンプルのプールで測定された平均量に相当する量のsCD127を含むように調整されたサンプルである陽性対照サンプル、及び/又は、院内感染していないことが分かっている患者から採取したサンプルのプールで測定された平均量に相当する量のsCD127を含むように調整されたサンプルである陰性対照サンプルと
を含むキットである。
<IL−7の血漿中濃度の測定>
IL−7の血漿中濃度は、Bio−Rad社から市販されているLUMINEX(TM)法を利用したキット(Bio−Plex Pro サイトカイン/ケモカイン/成長因子アッセイ:BioPlex Pro試薬キット(Bio−Rad、#171−304070)及びシングルプレックスIL−7)を用いて、サプライヤーの使用説明書に従って測定した。
手短に言うと、全血50μlを、フィコエリトリン−テキサスレッド(ECD)と結合した抗CD4抗体(Beckman Coulter社、#6604727)5μl、又は、ECDを結合した抗CD8抗体(Beckman Coulter社、#737659)5μlと、フィコエリトリン(PE)と結合した抗CD127抗体(Beckman Coulter社、#IM1980U)10μlとの存在下、室温、暗所で15分間インキュベートした。次に赤血球を低張溶解液により溶血させ、自動TQ−Prepシステム(Beckman Coulter社)を用いて自動溶血により細胞を固定した。CD4+又はCD8+Tリンパ球の表面上でのCD127の膜発現は、フローサイトメトリーによって測定した。
コーティング
500mlの水中に0.8gのNa2CO3、1.4gのNaHCO3、0.1gのNaN3を含むように、コーティング緩衝液(pH9.6)を調製した。
各ウェルごとに150μLのブロッキング緩衝液(10%ウシ胎児血清(FBS)/0.05%PBS−Tween20)を用いて非特異的な固定をブロッキングした後、プレートを37℃で1時間インキュベートした。
C.Janot−Sardet et al.Journal of Immunological Methods,2010,28,115−123に従って、下記表1に示すように、5%FCS含有PBS希釈用緩衝液で希釈した組換えヒトIL−7Rα/CD127 Fcキメラ(R&D Systems社、カタログ番号:306−IR)を用いて参照範囲を得た。
PBS/5%FBSで希釈した検出抗体(1%TBS−BSA1mLで再構成したビオチン化ポリクローナル抗CD127ヤギ抗体(R&D Systems(R)社))100μLを各ウェルに加え([Ab]=200ng/mL)、次にプレートを37℃で1時間インキュベートした。
100μLのストレプトアビジン−HRPを各ウェルに添加した([ストレプトアビジン−HRP]=8μL/mL)。プレートにカバーをかけて室温で30分間インキュベートした。
測定は、フローサイトメトリー法を用いてEDTA加全血の直接染色によって行った。
・5μLのフルオレセイン結合抗CD14抗体(Beckman Coulter Immunotech社、Ref.:IM0645)。これにより、白血球の中から単球を同定することができる。
・10μLのフィコエリトリン結合抗HLA−DR抗体(Becton Dickinson社、Ref.:347401)。これにより、細胞表面上でのHLA−DR発現を定量できる。
敗血症性ショック発症後1〜2日目及び3〜4日目の35人の敗血症性ショック患者から血漿サンプルを採取した後、保管した(後ろ向きコホート)。IL−7及びsCD127の各血漿中濃度、CD4+T細胞でのCD127の発現、単球でのHLA−DRの発現などの異なるパラメータやマーカーを測定した。敗血症性ショックで集中治療室に入院してから28日後時点で、35人の患者のうち13人(37%)の患者は死亡し(“NS”)、22人の患者は生存していた(“S”)。6人(17%)の患者が院内感染し(“NI”)、29人の患者は院内感染の発症を免れていた(“No NI”)。
Claims (17)
- 院内感染に対する患者の易感染性を判定するための方法であって、
前記患者から採取した生体サンプル又は被験サンプル中のsCD127の発現を測定する工程と、
被験サンプルで測定されたsCD127の発現が、被験サンプルの採取よりも前に同一患者から採取した生体サンプルで測定された参照値と比較して有意に減少していない場合に、院内感染に対する易感染性が高いと結論づける工程と
を含む方法。 - 前記被験サンプルで測定されたsCD127の発現の減少率が前記参照値と比較して25%以下の場合に、院内感染に対する易感染性が高いと結論づけることを特徴とする、請求項1に記載の方法。
- 前記被験サンプルで測定されたsCD127の発現の減少率が前記参照値と比較して20%以下の場合に、院内感染に対する易感染性が高いと結論づけることを特徴とする、請求項1に記載の方法。
- 前記生体サンプルは、集中治療室入院患者、及び/又は、手術、やけど、外傷などの、全身性炎症反応症候群(SIRS)を生じさせる傷害を受けた患者から採取したものであることを特徴とする、請求項1〜3のいずれかに記載の方法。
- 前記患者は、敗血症患者であることを特徴とする、請求項4に記載の方法。
- 前記患者は、重症敗血症患者であることを特徴とする、請求項4に記載の方法。
- 前記生体サンプルは、敗血症性ショック患者から採取したものであることを特徴とする、請求項1〜4のいずれかに記載の方法。
- 前記被験サンプル中のsCD127の発現の測定は、敗血症性ショック発症後10日目又は約10日目(D10)に行うことを特徴とする、請求項7に記載の方法。
- 前記被験サンプル中のsCD127の発現の測定は、敗血症性ショック発症後7日目又は約7日目(D7)に行うことを特徴とする、請求項7に記載の方法。
- 前記被験サンプル中のsCD127の発現の測定は、敗血症性ショック発症後4日目又は約4日目(D4)に行うことを特徴とする、請求項7に記載の方法。
- 前記事前のサンプル採取は、敗血症性ショック発症後48時間以内又は48時間経過時であって、前記被験サンプルの採取の24時間以上前に行うことを特徴とする、請求項7〜10のいずれかに記載の方法。
- 前記事前のサンプル採取は、敗血症性ショック発症後48時間以内又は48時間経過時に行われ、前記被験サンプルの採取は、前記事前のサンプル採取後48時間以内又は48時間経過時に行われることを特徴とする、請求項11に記載の方法。
- 前記患者から採取した前記被験サンプルは血漿サンプル又は血清サンプルであることを特徴とする、請求項1〜12のいずれかに記載の方法。
- sCD127の発現をタンパク質レベルで測定することを特徴とする、請求項1〜13のいずれかに記載の方法。
- sCD127の発現をタンパク質レベルでELISA法を用いて測定することを特徴とする、請求項14に記載の方法。
- sCD127の発現を抗sCD127抗体を用いて測定することを特徴とする、請求項1〜15のいずれかに記載の方法。
- sCD127の発現をモノクローナル抗sCD127抗体を用いて測定することを特徴とする、請求項1〜16のいずれかに記載の方法。
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