Joshi Suresh G
Center for Research in Botulism and Biodefense, Jefferson Medical College, Philadelphia, USA.
J Pharmacol Toxicol Methods. 2012 Jan;65(1):8-12. doi: 10.1016/j.vascn.2011.11.002. Epub 2011 Dec 3.
The goals of this project were to compare fluorescent resonance energy transfer (FRET) assays using a customized FRET substrate (substrate-substrate-A, SSA) with a commercially available FRET substrate (SNAPtide); optimize the assay conditions for SSA for lowest level of detection; and apply SSA to detect botulinum neurotoxin-A (BoNTA) in serum samples.
Biological activity of BoNTA and light-chain-A (LCA) was verified by murine phrenic nerve-hemidiaphragm bioassay and western blot before use in both FRET assays. The reaction conditions were optimized to determine the smallest amount of toxin that could be detected. A range of serum samples was investigated for interference in the SSA-based FRET assay. Detection of BoNTA from rat serum samples was performed over time.
We found that BoNTA and LCA were able to cleave the substrates whereas mutated LCA and a different serotype of BoNT, BoNTB, could not. SSA had significantly more arbitrary fluorescing units compared to the FRET substrate SNAPTide, and the SSA assay could detect 0.1nM of BoNTA or LCA comfortably (p=<0.05) in a 20-μl reaction. No significant interference was observed when serum was present in the reaction buffer. Due to negligible background noise, the SSA FRET assay could detect BoNTA from spiked rat serum even after 256min.
The greatest advantage of the FRET assay is its extreme rapidity, its cost effectiveness, and unlike ELISA, its ability to detect biologically active toxin. SSA is a better FRET substrate for detecting BoNTA toxin (detected 0.1nM concentration). Because serum present in the assay reaction did not cause any appreciable interference, the assay can be used to detect BoNTA in serum samples. Therefore, the SSA FRET assay can be used for pharmacokinetic and pharmacodynamic studies, screening inhibitors, and detecting BoNTA in serum samples.
本项目的目标是将使用定制的荧光共振能量转移（FRET）底物（底物-底物-A，SSA）的FRET检测方法与市售FRET底物（SNAPtide）进行比较；优化SSA的检测条件以实现最低检测水平；并应用SSA检测血清样本中的肉毒杆菌神经毒素A（BoNTA）。
在两种FRET检测中使用之前，通过小鼠膈神经-半膈肌生物测定法和蛋白质印迹法验证BoNTA和轻链A（LCA）的生物活性。优化反应条件以确定可检测到的最小毒素量。研究了一系列血清样本对基于SSA的FRET检测的干扰。对大鼠血清样本中的BoNTA进行了随时间的检测。
我们发现BoNTA和LCA能够切割底物，而突变的LCA和不同血清型的肉毒杆菌神经毒素BoNTB则不能。与FRET底物SNAPtide相比，SSA具有明显更多的任意荧光单位，并且在20μl反应中，SSA检测能够轻松检测到0.1nM的BoNTA或LCA（p<0.05）。当反应缓冲液中存在血清时，未观察到明显干扰。由于背景噪声可忽略不计，即使在256分钟后，SSA FRET检测仍能从添加了BoNTA的大鼠血清中检测到BoNTA。
FRET检测的最大优点是其速度极快、成本效益高，并且与酶联免疫吸附测定（ELISA）不同，它能够检测生物活性毒素。SSA是检测BoNTA毒素（检测浓度为0.1nM）的更好的FRET底物。由于检测反应中存在的血清不会引起任何明显干扰，该检测可用于检测血清样本中的BoNTA。因此，SSA FRET检测可用于药代动力学和药效学研究、筛选抑制剂以及检测血清样本中的BoNTA。
