JP2018506985A - 細胞内へのボツリヌス神経毒素の特異的取り込みを強化するための方法 - Google Patents
細胞内へのボツリヌス神経毒素の特異的取り込みを強化するための方法 Download PDFInfo
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Abstract
Description
神経毒素中毒を受けやすい細胞を、神経毒素ポリペプチドとともに、神経毒素ポリペプチドがその生物学的活性を発揮することを可能にする時間及び条件下でインキュベートすることを含み、インキュベーションが、(i)細胞のK+媒介型脱分極工程、(ii)短縮された神経毒素ポリペプチド曝露時間の工程及び/又は(iii)神経毒素ポリペプチド曝露中の細胞の撹拌工程のうちの少なくとも1つを含み、それによって細胞内への神経毒素ポリペプチドの特異的取り込みを強化する、方法に関する。好ましくは、本方法は、インビトロ法である。
a)神経毒素中毒を受けやすい細胞を、神経毒素ポリペプチドとともに、神経毒素ポリペプチドがその生物学的活性を発揮することを可能にする時間及び条件下でインキュベートすることであって、インキュベーションが、(i)細胞のK+媒介型脱分極工程、(ii)短縮された神経毒素ポリペプチド曝露時間の工程及び/又は(iii)神経毒素ポリペプチド曝露中の細胞の撹拌工程のうちの少なくとも1つを含むことと、
b)細胞を固定し、任意選択で、界面活性剤を用いて細胞を透過性にすることと、
c)細胞を、未切断基質及び神経毒素により切断された基質に特異的に結合する少なくとも1つの第1の捕捉抗体並びに神経毒素により切断された基質の切断部位に特異的に結合する少なくとも1つの第2の捕捉抗体と、捕捉抗体と基質との結合を可能にする条件下で接触させることと、
d)細胞を、第1の捕捉抗体に特異的に結合する少なくとも1つの第1の検出抗体と、第1の検出抗体と第1の捕捉抗体との結合を可能にする条件下で接触させ、それによって第1の検出複合体を形成すること、及び第2の捕捉抗体に特異的に結合する少なくとも1つの第2の検出抗体と、第2の検出抗体と第2の捕捉抗体との結合を可能にする条件下で接触させ、それによって第2の検出複合体を形成することと、
e)工程d)の第1及び第2の検出複合体の量を決定することと、
f)細胞中の神経毒素ポリペプチドによって切断された基質の量を、第2の検出複合体を用いて計算し、それによって、細胞における神経毒素ポリペプチドの生物学的活性を決定することと、を含む、方法を提供する。
Amplex UltraRed:励起540nm、放出600nm
DiFMUP:励起360nm、放出450nm
1.培地/毒素溶液を除去する。100μL/ウェルの氷冷メタノール(−20℃)を添加し、−20℃で20分間インキュベートする。
備考:後続の工程はすべて、室温で行う。
1.メタノール溶液を除去し、100μL/ウェルのPBS緩衝液を添加する。より長く保管する場合(>1日)、300μL//ウェルのPBS緩衝液を添加し、プレートをパラフィルムで封止すべきである。プレートは、冷蔵庫で保管すべきである。
励起540nm、放出600nm。
励起360nm、放出450nm。
正規化のために、切断されたSNAP−25のRFU値(600nmでの蛍光)を、各ウェルにおいて全SNAP−25(450nm)のRFUに対して正規化する。図でRFUをより良好に示すために、すべての値に、以下の式を用いて係数1000を乗じる:
RFU(600nm)/RFU(450nm)・1000
続いて、得られたRFU値を、各標準物又は試料について平均する。
PBS緩衝液(10mM):
リン酸緩衝食塩水(Sigma、#P5368)(pH7.4)
クエンチ緩衝液:
10mM PBS緩衝液(pH7.4)中の0.6%H2O2
ブロッキング緩衝液:
10mM PBS緩衝液(pH7.4)+0.05%Triton X−100中の2%BSA
HEPES緩衝液:
50mM HEPES(pH7.4)
HRP基質:
50mM HEPES(pH7.4)
0.007%のH2O2
150pM Amplex UltraRed
AP基質:
25mM ジエタノールアミン(pH9.8)
2mM MgCl2
100μL M DiFMUP
a)iCell(登録商標)ニューロンを解凍し、Cellular Dynamics International(CDI)ユーザマニュアルに従って、4つの異なる細胞バッチから96ウェルプレートに播種した。播種の24時間後に、ユーザマニュアルに記載のように、培地を新しい維持培地と置き換えた。
まとめると、細胞のK+媒介型脱分極、短縮された神経毒素ポリペプチド曝露時間又は神経毒素ポリペプチド曝露中の細胞の撹拌により、マウス50%致死量バイオアッセイと比較したときに、実施例1の細胞に基づくアッセイの同程度の安定性を示す動態が促進される。
Claims (15)
- 細胞内への神経毒素ポリペプチドの特異的取り込みを強化するための方法であって、
神経毒素中毒を受けやすい細胞を、神経毒素ポリペプチドとともに、前記神経毒素ポリペプチドがその生物学的活性を発揮することを可能にする時間及び条件下でインキュベートすることを含み、前記インキュベーションが、(i)前記細胞のK+媒介型脱分極工程、(ii)短縮された神経毒素ポリペプチド曝露時間の工程及び/又は(iii)神経毒素ポリペプチド曝露中の前記細胞の撹拌工程のうちの少なくとも2つを含み、それによって前記細胞内への前記神経毒素ポリペプチドの前記特異的取り込みを強化する、方法。 - 続いて、前記細胞における前記神経毒素ポリペプチドの前記生物学的活性を決定することによる、請求項1に記載の方法。
- 細胞における神経毒素ポリペプチドの生物学的活性を直接決定するための方法であって、
a)神経毒素中毒を受けやすい細胞を、神経毒素ポリペプチドとともに、前記神経毒素ポリペプチドがその生物学的活性を発揮することを可能にする時間及び条件下でインキュベートすることであって、前記インキュベーションが、(i)前記細胞のK+媒介型脱分極工程、(ii)短縮された神経毒素ポリペプチド曝露時間の工程及び/又は(iii)神経毒素ポリペプチド曝露中の前記細胞の撹拌工程のうちの少なくとも2つを含むことと、
b)前記細胞を固定し、任意選択で、界面活性剤を用いて前記細胞を透過性にすることと、
c)前記細胞を、未切断基質及び神経毒素により切断された基質に特異的に結合する少なくとも1つの第1の捕捉抗体、並びに前記神経毒素により切断された基質の切断部位に特異的に結合する少なくとも1つの第2の捕捉抗体と、前記捕捉抗体と前記基質との結合を可能にする条件下で接触させることと、
d)前記細胞を、前記第1の捕捉抗体に特異的に結合する少なくとも1つの第1の検出抗体と、前記第1の検出抗体と前記第1の捕捉抗体との結合を可能にする条件下で接触させ、それによって第1の検出複合体を形成すること、及び前記第2の捕捉抗体に特異的に結合する少なくとも1つの第2の検出抗体と、前記第2の検出抗体と前記第2の捕捉抗体との結合を可能にする条件下で接触させ、それによって第2の検出複合体を形成することと、
e)工程d)の前記第1及び第2の検出複合体の量を決定することと、
f)前記細胞中の前記神経毒素ポリペプチドによって切断された基質の量を、前記第2の検出複合体を用いて計算し、それによって、前記細胞における前記神経毒素ポリペプチドの前記生物学的活性を決定することと、
を含む、方法。 - 前記細胞の前記K+媒介型脱分極が、約20mM〜約55mMの追加のK+濃度で、少なくとも2時間行われる、請求項1〜3のいずれか一項に記載の方法。
- 前記細胞の前記K+媒介型脱分極及び/又は前記神経毒素ポリペプチド曝露が、GT1bの存在下で行われる、請求項1〜4のいずれか一項に記載の方法。
- GT1bが、15〜50μM、好ましくは20μMの濃度で使用される、請求項1〜5のいずれか一項に記載の方法。
- 前記短縮された神経毒素ポリペプチド曝露時間が、少なくとも24時間かつ96時間未満、好ましくは、少なくとも48時間かつ最大72時間の、前記神経毒素ポリペプチドへの前記細胞の曝露である、請求項1〜6のいずれか一項に記載の方法。
- 神経毒素ポリペプチド曝露中の前記細胞の撹拌が、磁気撹拌子、回転式スピナーフラスコを用いて、又は振盪器によって前記細胞を好ましくは約25cm/分〜約300cm/分の平均培地流動速度で振盪させることによって、行われる、請求項1〜7のいずれか一項に記載の方法。
- 前記細胞の前記K+媒介型脱分極が、少なくとも約20mM〜約55mMの追加のK+濃度で、少なくとも2時間、20μMのGT1b及び神経毒素ポリペプチドの存在下において行われ、続いて、神経毒素ポリペプチド曝露が、撹拌しながら更に70時間行われる、請求項1〜8のいずれか一項に記載の方法。
- 神経毒素ポリペプチドなしのインキュベーション時間が、前記神経毒素ポリペプチド曝露に先行する、請求項1〜8のいずれか一項に記載の方法。
- 前記神経毒素ポリペプチドなしのインキュベーション時間が、16〜48時間である、請求項10に記載の方法。
- 蛍光法である、請求項1〜11のいずれか一項に記載の方法。
- 前記神経毒素ポリペプチドが、BoNT/A、BoNT/B、BoNT/C1、BoNT/D、BoNT/E、BoNT/F、BoNT/G、BoNT/H、TeNT又はこれらのサブタイプである、請求項1〜12のいずれか一項に記載の方法。
- 前記基質が、VAMP/シナプトブレビン、SNAP−25又はシンタキシンである、請求項1〜13のいずれか一項に記載の方法。
- 前記細胞が、初代神経細胞、神経芽腫細胞、P19細胞など神経細胞に分化することができる腫瘍細胞又は人工多能性幹細胞(IPS)由来のニューロンからなる群から選択される、神経細胞又は神経分化細胞である、請求項1〜14のいずれか一項に記載の方法。
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