Rapid and sensitive detection of biological warfare agents using time-resolved fluorescence assays
Rapid and sensitive detection of biological warfare agents using time-resolved fluorescence assays
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DOI：
10.1016/s0022-1759(02)00030-3
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发表时间：
2002-05-01
影响因子：
2.2
通讯作者：
Peruski, LF
中科院分区：
文献类型：
--
作者：
Peruski, AH;Johnson, LH;Peruski, LF
我们已经实现了敏感，快速和可重复的检测三种生物威胁剂在各种生物和环境矩阵使用的DELFIA时间分辨荧光（TRF）测定系统（珀金埃尔默生命科学，阿克伦，OH）。将现有的用于检测土拉热弗朗西丝氏菌、肉毒梭菌A/B神经毒素（BotNT A/B）和金黄色葡萄球菌肠毒素B（SE B）的ELISA测定转化为TRY测定。每个测试孔使用100穆尔阳性对照或未知物，运行时间仅需2小时，荧光信号读取时间为每孔几分之一秒。该测定形式包括利用生物素化捕获抗体的捕获ELISA，该捕获抗体预先结合至链霉亲和素包被的96孔板和镧系元素（铕，Eu 3+）标记的检测抗体。结合的Eu标记的检测抗体在加入增强溶液后产生荧光信号。该信号是由于铕从抗体上解离而产生的，产生了胶束，从而将信号放大了近一百万倍。通过这些测定实现的灵敏度在缓冲液中为4至20 pg/ml。此外，我们还在不同的基质中测试了该系统，如血清，尿液，污垢和污水。从标准溶液生成的浓度曲线产生了宽的线性范围，使得未知样品的系列稀释没有必要。DELFIA TRF检测在灵敏度、线性范围和运行时间方面明显优于标准捕获ELISA，应有助于临床和环境样品中潜在生物战剂的早期检测。(C)2002 Elsevier Science B.V保留所有权利。
We have achieved sensitive, rapid and reproducible detection of three biological threat agents in a variety of biological and environmental matrices using the DELFIA time-resolved fluorometry (TRF) assay system (Perkin-Elmer Life Sciences, Akron, OH). Existing ELISA assays for the detection of Francisella tularensis, Clostridium botulinum A/B neurotoxin (BotNT A/B), and Staphylococcus aureus enterotoxin B (SEB) were converted to TRY assays. They use 100 mul of positive control or unknown per test well and require just over 2 h to run, Fluorescent signal read time is a fraction of a second per well. The assay format consists of a capture ELISA utilizing a biotinylated capture antibody, prebound to a streptavidin-coated 96-well plate and a lanthanide (Europium, Eu3+)-labeled detector antibody. The bound Eu-labeled detector antibody produces a fluorescent signal upon the addition of an enhancement solution. The signal results from the dissociation of the Europium from the antibody, creating a micelle, thus amplifying the signal nearly one million-fold. Sensitivities achieved by these assays were between 4 and 20 pg/ml in buffer. Additionally, we have tested this system in different matrices such as serum, urine, dirt, and sewage. Concentration curves generated from standard solutions produced a wide linear range making serial dilutions of unknown samples unnecessary. DELFIA TRF assays are significantly better in terms of sensitivity, linear range, and run time than standard capture ELISAs and should facilitate early detection of potential biological warfare agents in clinical and environmental samples. (C) 2002 Elsevier Science B.V All rights reserved.