A highly sensitive immuno-polymerase chain reaction assay for Clostridium botulinum neurotoxin type A
A highly sensitive immuno-polymerase chain reaction assay for Clostridium botulinum neurotoxin type A
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DOI：
10.1016/j.toxicon.2003.10.013
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发表时间：
2004-01-01
期刊：
影响因子：
2.8
通讯作者：
Liu, HW
中科院分区：
文献类型：
--
作者：
Chao, HY;Wang, YC;Liu, HW
我们的目标是建立一种检测A型肉毒梭菌神经毒素(BONT/A)的灵敏方法。我们能够使用间接免疫-聚合酶链式反应和间接夹心免疫-聚合酶链式反应检测到Femtogram(10(-15)g)范围内的BoNT/A。间接免疫-聚合酶链式反应(EL ISA)板上包被抗BONT/A单抗识别的BONT/A。在间接夹心免疫-聚合酶链式反应中，将单抗固定在酶标板上，以检测其多克隆抗体识别的BoNT/A。用生物素标记的5‘-引物进行聚合酶链式反应，制备报告DNA，通过链霉亲和素将其与生物素标记的抗体偶联。为了提高灵敏度和降低背景噪声，对报告DNA(50fg~50 ng)和链霉亲和素(0.125~8 ng)的用量进行了优化。利用优化的报告DNA和链霉亲和素浓度，间接和间接夹心免疫-聚合酶链式反应均可检测到低至50fg的BoNT/A。这些结果比传统的间接ELISA和间接夹心ELISA法提高了10(5)倍。我们开发的检测方法是目前检测BONT/A最灵敏的方法。(C)2003爱思唯尔有限公司。版权所有。
Our goal was to develop a sensitive method for detecting Clostridium botulinum neurotoxin type A (BoNT/A). We were able to detect BoNT/A in the femtogram (10(-15) g) range using an indirect immuno-polymerase chain reaction (immuno-PCR) assay and an indirect sandwich immuno-PCR assay. For the indirect immuno-PCR assay, enzyme-linked immunosorbent assay (ELISA) plates were coated with BoNT/A that was recognized by anti-BoNT/A monoclonal antibody. For the indirect sandwich immuno-PCR assay, the monoclonal antibody was immobilized on ELISA plates for detecting BoNT/A that was recognized by its polyclonal antibodies. Reporter DNA was prepared by PCR amplification using biotinylated 5'-primers, and it was coupled with biotinylated antibodies through streptavidin. In order to increase sensitivity and reduce background noise, the amounts of reporter DNA (ranging from 50 fg to 50 ng) and streptavidin (ranging from 0.125 ng to 8 ng) were optimized. Using the optimized concentration of reporter DNA and streptavidin, both indirect and indirect sandwich immuno-PCR assays detected BoNT/A as low as 50 fg. These results are a 10(5)-fold improvement over conventional indirect ELISA and indirect sandwich ELISA methods. The assays we developed are currently the most sensitive methods for detecting BoNT/A. (C) 2003 Elsevier Ltd. All rights reserved.