Attomolar detection of botulinum toxin type A in complex biological matrices.
Attomolar detection of botulinum toxin type A in complex biological matrices.
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DOI：
10.1371/journal.pone.0002041
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发表时间：
2008-04-30
期刊：
影响因子：
3.7
通讯作者：
Kalkum M
中科院分区：
文献类型：
--
作者：
Bagramyan K;Barash JR;Arnon SS;Kalkum M
为了1)对抗潜在的生物恐怖威胁2)加强食品安全3)在利用BoNT的医学应用中进行未来的药代动力学研究，需要一种高灵敏度、快速和经济高效的方法来检测复杂生物样品（如食品或血清）中的活性肉毒杆菌神经毒素（BoNT）。在这里，我们描述了一种具有大免疫吸附表面积（BoNT/ a ALISSA）的肉毒杆菌神经毒素血清a型测定方法，该方法捕获少量毒素分子并使用荧光底物测量其内在金属蛋白酶活性。与“金标准”小鼠生物测定法直接比较，ALISSA的灵敏度要高4到5个数量级，而且速度要快得多。我们的方法在血清、牛奶、胡萝卜汁和小鼠实验中使用的稀释液中达到了原子摩尔灵敏度。在与其他BoNT血清型和胰蛋白酶等替代蛋白酶进行测试时，ALISSA对目标A型毒素具有高特异性，并且可以检测全毒素以及BoNT/A的多蛋白复合物形式。该实验对温度、底物浓度、免疫吸附基质的大小和体积比、富集和反应时间进行了优化。最后，提出了一个与观测到的灵敏度提高相一致的动力学模型。BoNT ALISSA的灵敏度、特异性、速度和简单性使该方法在诊断、生物防御和药理学应用方面具有吸引力。
A highly sensitive, rapid and cost efficient method that can detect active botulinum neurotoxin (BoNT) in complex biological samples such as foods or serum is desired in order to 1) counter the potential bioterrorist threat 2) enhance food safety 3) enable future pharmacokinetic studies in medical applications that utilize BoNTs. Here we describe a botulinum neurotoxin serotype A assay with a large immuno-sorbent surface area (BoNT/A ALISSA) that captures a low number of toxin molecules and measures their intrinsic metalloprotease activity with a fluorogenic substrate. In direct comparison with the “gold standard” mouse bioassay, the ALISSA is four to five orders of magnitudes more sensitive and considerably faster. Our method reaches attomolar sensitivities in serum, milk, carrot juice, and in the diluent fluid used in the mouse assay. ALISSA has high specificity for the targeted type A toxin when tested against alternative proteases including other BoNT serotypes and trypsin, and it detects the holotoxin as well as the multi-protein complex form of BoNT/A. The assay was optimized for temperature, substrate concentration, size and volume proportions of the immuno-sorbent matrix, enrichment and reaction times. Finally, a kinetic model is presented that is consistent with the observed improvement in sensitivity. The sensitivity, specificity, speed and simplicity of the BoNT ALISSA should make this method attractive for diagnostic, biodefense and pharmacological applications.
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