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首页> 外文期刊>Journal of Pharmacological and Toxicological Methods >Detection of biologically active botulinum neurotoxin-A in serum using high-throughput FRET-assay
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Detection of biologically active botulinum neurotoxin-A in serum using high-throughput FRET-assay

机译：使用高通量FRET测定检测血清中的生物活性肉毒杆菌神经毒素-A

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Introduction: 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. Methods: 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. Results: 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 0.1. nM 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 256. min. Discussion: 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 0.1. nM 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.
机译：简介：该项目的目标是使用具有市售的FRET基板（SNAPTIDE）的定制FRET基板（衬底 - 基板-A，SSA）进行比较荧光共振能量转移（FRET）测定;优化SSA的测定条件，以获得最低水平的检测;并涂抹SSA以检测血清样品中的肉毒杆菌神经毒素-A（Bonta）。方法：在使用前使用小鼠膈神经 - 血管瘤生物测定和蛋白质印迹，术前验证了Bonta和轻链-A（LCA）的生物活性。优化反应条件以确定可以检测到的最小量的毒素。研究了一系列血清样品，用于干扰SSA的基于SSA的FRET测定。随着时间的推移，进行来自大鼠血清样品的Bonta的检测。结果：我们发现Bonta和LCA能够切割基板，而突变的LCA和不同的血管型，Bontb，不能。与FRET底物SNAPTIDE相比，SSA具有明显的任意荧光单元，并且SSA测定可以检测0.1。在20μl反应中舒适（p = <0.05）的NM舒适（p = <0.05）。当血清存在于反应缓冲液中时，没有观察到显着的干扰。由于近噪声噪音可忽略不计，即使在256℃后，SSA FRET测定也可能检测来自尖刺大鼠血清的植物。讨论：FRET测定的最大优势是其极其速度，其成本效益和与ELISA不同，其检测生物活性毒素的能力。 SSA是用于检测植物毒素的更好的褶皱基质（检测到0.1.nm浓度）。因为在测定反应中存在的血清没有引起任何可观的干扰，所以可以使用测定来检测血清样品中的Bonta。因此，SSA FRET测定可用于药代动力学和药效学研究，筛选抑制剂和检测血清样品中的植物。

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