The use of Endopep-MS for the detection of botulinum toxins A B, E, and F in serum and stool samples
The use of Endopep-MS for the detection of botulinum toxins A B, E, and F in serum and stool samples
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DOI：
10.1016/j.ab.2006.01.027
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发表时间：
2006-04-01
影响因子：
2.9
通讯作者：
Barr, JR
中科院分区：
文献类型：
--
作者：
Kalb, SR;Moura, H;Barr, JR
肉毒杆菌神经毒素（BoNT）导致肉毒杆菌中毒，如果不治疗可能致命。我们实验室以前的工作集中在开发Endopep-MS，这是一种基于质谱的内肽酶方法，用于检测和区分BoNT血清型。我们已经扩大了这一努力，包括抗体捕获方法，以部分纯化和浓缩BoNT从血清和粪便提取物样品的Endopep-MS测定。因为复杂的基质如血清和粪便含有丰富的内源性蛋白酶，需要这种技术从样品中除去大多数蛋白酶，同时从100 μ l至500 μ l至20 μ l的样品中浓缩BoNT。当该抗体捕获方法与Endopep-MS反应结合时，在500 μ l加标人血清中的检测限为10小鼠LD 50（20小鼠LD 50/ml），BoNT A，0.5小鼠LD 50/ml BoNT B（1只小鼠LD 50/ml），0.1只小鼠LD 50（0.2小鼠LD 50/ml），和0.5小鼠LD 50（1小鼠LD 50/ml）的BoNT F的加标粪便提取物中的检测限略高，这是由于高-粪便提取物的蛋白酶环境也需要使用蛋白酶抑制剂。整个方法可以在短至4小时的时间内进行。(c)2006年爱思唯尔公司All rights reserved.
Botulinum neurotoxin (BoNT) causes the disease botulism, which can be lethal if untreated. Previous work in our laboratory focused on developing Endopep-MS, a mass spectrometric-based endopeptidase method for the detection and differentiation of BoNT serotypes. We have expanded this effort to include an antibody capture method to partially purify and concentrate BoNT from serum and stool extract samples for the Endopep-MS assay. Because complex matrices such as serum and stool contain abundant endogenous proteases, this technique was needed to remove most proteases from the sample while concentrating BoNT from a sample size of 100 to 500 mu l to 20 mu l. When this antibody capture method is combined with the Endopep-MS reaction, limits of detection in 500 mu l of spiked human serum are 10 mouse LD50 (20 mouse LD50/ml) for BoNT A, 0.5 mouse LD50 (1 mouse LD50/ml) for BoNT B, 0.1 mouse LD50 (0.2 mouse LD50/ml) for BoNT E, and 0.5 mouse LD50 (1 mouse LD50/ml) for BoNT F The limits of detection in spiked stool extracts are somewhat higher due to the high-protease environment of stool extract that also requires use of protease inhibitors. The entire method can be performed in as short a time as 4 h. (c) 2006 Elsevier Inc. All rights reserved.