JP6553020B2 - アレルギー及び自己免疫疾患に関する診断分析を行うための自動化された免疫分析計システム - Google Patents
アレルギー及び自己免疫疾患に関する診断分析を行うための自動化された免疫分析計システム Download PDFInfo
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Description
培養ステップの間にビードを懸濁状態に保持するために、反応媒質の粘度を増加するために部分的に機能することが理解されるべきである。加えて、高いHSAはまた、この培養の間に非特異的な結合を低減し、患者サンプルの希釈に対して相対光ユニット(RLU)の線形性を改善する。
抗ヒトIgE又はアレルゲン抽出物のビオチン化
DMSO中の250 mMの濃度のNHS-PEG12-Biotin (Pierce) 2μLが、リン酸緩衝食塩水(PBS)中の5.0 mg/mLの濃度の精製された親和性抗ヒトIgE(免疫試薬)1mLに加えられる。又は、DMSO中の250 mMの濃度のNHS-PEG12-Biotin (Pierce) 1.6μLが、リン酸緩衝食塩水(PBS)中の1.0 mg/mLの濃度のアレルゲン抽出物1mLに加えられる。
流体ビードの準備
ddH2O中の1 mMの濃度のBiotin-Fluo (Alexa Fluor 594 Biocytin、Sodium Salt、Life Technologies) 5μLが、PBSTHP Buffer (10 mM リン酸ナトリウム、pH 7.4、0.9% (w/v) NaCl、0.05% (v/v) Tween-20、10 mg/mL HSA、1% (v/v) ProClin 950) 45 mLに加えられる。よく混合する。
アレルゲン特異的IgEレベルのための分析
ビード濃度1mg/mLにおける10μL流体ビード(蛍光標識化された常磁性体微粒子)は、反応キュベットに分配される。40μLのビオチン−アレルゲン(例えば、卵白、ミルク、ピーナッツ等)又はビオチン−抗IgE抗体は、流体ビードに分配され、混合される、37℃で1〜10分、培養される。洗浄後、アレルゲン又は抗IgEで被覆されたビードは40μLの反応バッファーで再懸濁される。アトピー及びアトピーでない個人から得られた血清サンプルがアレルゲンに対して分析される。10μLのサンプルが反応キュベットにおいて40μLの懸濁されたアレルゲンで被覆されたビードに加えられた。6点標準曲線(six point standard curve)に関して、10μLの血清標準(WHOIgE標準75/502に対して較正された第2標準)がそれぞれ反応キュベットにおける40μLの抗IgEで被覆されたビードに加えられる。様々な異なる標識化された抗IgE共役体が本教示に従って利用され得るが、ある教示に従って、以下の抗IgE共役体が利用される。アレルギーの分析のために−抗IgE-HRP、自己免疫分析のために−抗IgA-HRP、抗IgG-HRP及び 抗IgM-HRP、ECPのために−抗ECP-HRP、及びトリプターゼのために− 抗Tryptase-HRP。その上、ここで用いられるように、それぞれの共役体は化学的性質において使用のための最適化HRP取り込み比率を有する。本教示のある特徴に従って、リストアップされた共役体のために使用されるHRPの取り込み比率の範囲は、約1.2〜約5.4の間である。加えて、本教示はまた、アルカリ性ホスファターゼ共役体及びb−ガラクトシダーゼ共役体を含み、これらに限られない共役伝達システムの他のタイプの組み込みを予期する。
ビード:流体ビード(SA-Speed Bead、Atto 590 labeled)、1 mg/mL、
捕捉試薬希釈剤:IgE:10 mMリン酸ナトリウム、pH 7.4、0.9% (w/v) NaCl、0.05% Tween-20、1% (w/v) ヒト血清アルブミン、1%(v/v) ProClin 950、最大で5% (v/v) グリセロール、
ANA:10 mM リン酸ナトリウム、pH 7.4、0.9% (w/v) NaCl、0.05% Tween-20、1% (w/v) ウシ血清アルブミン、1% プロテアーゼ阻害剤混合物、0.1 mM DTT、1%(v/v) ProClin 950、25% (最大で30%) (v/v) グリセロール、
洗浄バッファー濃縮物(5×):50 mM リン酸ナトリウム、pH 7.4、4.5% (w/v) NaCl、0.05% Tween-20、0.05%(v/v) ProClin 950、0.02% (v/v) Antifoam-C v/v、
試薬希釈剤(反応希釈剤及びサンプル希釈剤)IgE −10 mM リン酸ナトリウム、pH 7.4、500 mM NaCl、0.02% Tween-20、1% (w/v) ヒト血清アルブミン、1% (v/v)ヒトIgG、1%(v/v) ProClin 950、0.005% Antifoam-B v/v、2% (w/v) PEG 6,000、
ANA−10 mM リン酸ナトリウム、pH 7.4、500 mM NaCl、0.02% Tween-20、25% (w/v) ヒト血清アルブミン、1%(v/v) ProClin 950、
較正器及び制御器:較正器:サンプル希釈剤に希釈された患者サンプル、制御器:患者サンプルプール、
共役体:共役希釈剤:50 mMリン酸ナトリウム、pH 6.7、150 mM NaCl、0.05% Tween-20、1% BSA、5% (w/v) PEG 6,000、1%(v/v) ProClin 950、
IgE:100 ng/mL 抗IgE-HRP、100μg/mL apo-HRP、希釈剤中、0.015% Antifoam-B v/v、及び
基質:PS-atto A & B、0.01% Antifoam-B v/v。
[付記]
[付記1]
固相複合体を形成するためにストレプトアビジンで被覆された媒質と共に捕捉試薬を培養すること、
余分な捕捉試薬を除去するために前記固相複合体を洗浄すること、
免疫複合体を形成するために血清サンプルと共に前記固相複合体を培養すること、
いかなる結合していないサンプルを除去するために前記免疫複合体を洗浄すること、
免疫共役複合体を生成するために共役体と共に前記免疫複合体を培養すること、
いかなる結合していない共役体を除去するために前記免疫共役複合体を洗浄すること、
定量化可能な反応を生成することができる基質を導入すること、及び、
前記基質の導入により生成された前記反応を較正すること、
を含む、自動化された診断分析を行うための定量的方法。
[付記2]
前記ストレプトアビジンで被覆された媒質と共に前記捕捉試薬を培養するステップは、ビオチン化捕捉試薬と共に前記ストレプトアビジンで被覆された媒質を培養することを含む、付記1に記載の方法。
[付記3]
前記血清サンプルと共に前記固相複合体を培養するステップは、前記血清サンプルに存在するアレルゲン特異的ヒト免疫グロブリンE(IgE)をビオチン化捕捉試薬に結合することを含む、付記1に記載の方法。
[付記4]
前記血清サンプルと共に前記固相複合体を培養するステップは、前記血清サンプルに存在する自己免疫特異的ヒト免疫グロブリンG(IgG)、自己免疫特異的ヒト免疫グロブリンM(IgM)、又は自己免疫特異的ヒト免疫グロブリンA(IgA)をビオチン化捕捉試薬に結合することを含む、付記1に記載の方法。
[付記5]
前記捕捉試薬は、精製されたアレルゲン、プロテイン、酵素、又は抗体のビオチン化に由来する、付記1に記載の方法。
[付記6]
前記捕捉試薬は、多数のアレルゲンを含むアレルゲン抽出物のビオチン化に由来する、付記1に記載の方法。
[付記7]
前記捕捉試薬は、精製されたアレルゲン、プロテイン、酵素、抗体、及びアレルゲン抽出物から選択される多数のビオチン化捕捉試薬の混合物として存在する、付記1に記載の方法。
[付記8]
前記ストレプトアビジンで被覆された媒質と共に前記捕捉試薬を培養するステップは、万能な(universal)蛍光標識磁気微粒子を含む付記1に記載の方法。
[付記9]
1又は複数の洗浄するステップは、反応キュベットの制限された領域内で洗浄されている前記複合体を磁気的に隔離することにより前記複合体を洗浄することを含む、付記1に記載の方法。
[付記10]
前記ストレプトアビジンで被覆された媒質と共に前記捕捉試薬を培養するステップは、高濃度のヒト血清アルブミン(HSA)を含む反応希釈剤により懸濁状態に保たれた前記捕捉試薬を培養することを含む、付記1に記載の方法。
[付記11]
前記共役体と共に前記免疫複合体を培養するステップは、わずかな濃度のポリエチレングリコールを含む共役希釈剤により懸濁状態に保たれた免疫複合体を培養することを含む、付記1に記載の方法。
[付記12]
結合していないサンプルを除去するために前記免疫複合体を洗浄するときに、間接標識としてホースラディッシュペルオキシダーゼ(HRP)を用いるステップをさらに含む、付記11に記載の方法。
[付記13]
蛍光信号及び化学発光信号の両方が定量化される光学箱に前記基質と前記免疫共役複合体を移すこと、及び、
伝達値を計算するために、最後の蛍光に対する最初の蛍光の比率を採用して、前記定量化された化学発光信号を調整すること、により、
ビード保持のための前記定量化可能な反応を調整するステップをさらに含む、付記1に記載の方法。
[付記14]
前記光学箱に前記基質と前記免疫共役複合体を移すステップは、前記サンプルを吸引するための再利用可能なピペットチップを有する自動化されたピペットアームを用いることを含む、付記13に記載の方法。
[付記15]
ビード保持を決定するために前記光学箱内で蛍光を測定すること、及び、
生成された相対光ユニット信号を検出するために前記光学箱内で発光を測定すること、
をさらに含む、付記13に記載の方法。
[付記16]
ビード保持が調整された相対光ユニット信号を生成するためのアルゴリズムに前記蛍光及び発光測定結果を入力すること、をさらに含む付記15に記載の方法。
[付記17]
前記生成されたビード保持が調整された相対光ユニット信号を較正曲線の相対光ユニット信号と比較すること、をさらに含む付記16に記載の方法。
Claims (14)
- ストレプトアビジンで共役された万能な(universal)蛍光標識磁気微粒子の量と関連している第一の蛍光信号を測定すること、
万能な蛍光標識磁気微粒子を含むストレプトアビジンで被覆された媒質を、アレルゲン抽出物、単一アレルギー誘発タンパク質、及び自己免疫疾患に関与しているヒトタンパク質の少なくとも1つに由来するビオチン化捕捉試薬と共に培養することにより固相複合体を形成すること、
余分な捕捉試薬を除去するために前記固相複合体を洗浄すること、
免疫複合体を形成するために血清サンプルと共に前記固相複合体を培養すること、
いかなる結合していないサンプルを除去するために前記免疫複合体を洗浄すること、
免疫共役複合体を生成するために共役体と共に前記免疫複合体を培養すること、
いかなる結合していない共役体を除去するために前記免疫共役複合体を洗浄すること、
定量化可能な反応を生成することができる基質を導入すること、
前記免疫共役複合体と関連している第二の蛍光信号を測定し、ビード保持比を取得するために、第二の蛍光信号に対する第一の蛍光信号の比を算出すること、
前記基質と関連している化学発光信号を測定し、反応を定量化すること、及び、
前記ビード保持比によって前記反応を調整することによって伝達値を算出すること、
を含む、自動化された診断分析を行うための定量的方法。 - 前記血清サンプルと共に前記固相複合体を培養するステップは、前記血清サンプルに存在するアレルゲン特異的ヒト免疫グロブリンE(IgE)を前記ビオチン化捕捉試薬に結合することを含む、請求項1に記載の方法。
- 前記血清サンプルと共に前記固相複合体を培養するステップは、前記血清サンプルに存在する自己免疫特異的ヒト免疫グロブリンG(IgG)、自己免疫特異的ヒト免疫グロブリンM(IgM)、又は自己免疫特異的ヒト免疫グロブリンA(IgA)を前記ビオチン化捕捉試薬に結合することを含む、請求項1に記載の方法。
- 前記ビオチン化捕捉試薬は、精製されたアレルゲン、プロテイン、酵素、又は抗体のビオチン化に由来する、請求項1に記載の方法。
- 前記アレルゲン抽出物は、多数のアレルゲンを含む、請求項1に記載の方法。
- 前記ビオチン化捕捉試薬は、精製されたアレルゲン、プロテイン、酵素、抗体、及びアレルゲン抽出物から選択される多数のビオチン化捕捉試薬の混合物として存在する、請求項1に記載の方法。
- 1又は複数の洗浄するステップは、反応キュベットの制限された領域内で洗浄されている前記複合体を磁気的に隔離することにより前記複合体を洗浄することを含む、請求項1に記載の方法。
- 前記ストレプトアビジンで被覆された媒質を培養することにより前記固相複合体を形成するステップは、高濃度のヒト血清アルブミン(HSA)を含む反応希釈剤により懸濁状態に保たれた前記ビオチン化捕捉試薬を培養することを含む、請求項1に記載の方法。
- 前記共役体と共に前記免疫複合体を培養するステップは、ポリエチレングリコールを含む共役希釈剤により懸濁状態に保たれた免疫複合体を培養することを含む、請求項1に記載の方法。
- 結合していないサンプルを除去するために前記免疫複合体を洗浄するときに、間接標識としてホースラディッシュペルオキシダーゼ(HRP)を用いるステップをさらに含む、請求項9に記載の方法。
- 光学箱に前記基質と前記免疫共役複合体を移すことにより、
ビード保持のための前記定量化可能な反応を調整するステップをさらに含む、請求項1に記載の方法。 - 前記光学箱に前記基質と前記免疫共役複合体を移すステップは、前記サンプルを吸引するための再利用可能なピペットチップを有する自動化されたピペットアームを用いることを含む、請求項11に記載の方法。
- ビード保持が調整された相対光ユニット信号を生成するためのアルゴリズムに蛍光及び発光測定結果を入力すること、をさらに含む請求項11に記載の方法。
- 前記生成されたビード保持が調整された相対光ユニット信号を較正曲線の相対光ユニット信号と比較すること、をさらに含む請求項13に記載の方法。
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