Rapid and Highly Sensitive Non-Competitive Immunoassay for Specific Detection of Nodularin.
Rapid and Highly Sensitive Non-Competitive Immunoassay for Specific Detection of Nodularin.
复制标题
DOI：
10.3390/microorganisms5030058
复制
发表时间：
2017-09-12
期刊：
影响因子：
4.5
通讯作者：
Lamminmäki U
中科院分区：
文献类型：
--
作者：
Akter S;Vehniäinen M;Kankaanpää HT;Lamminmäki U
节球藻毒素（NOD）是一种主要由泡沫节球藻（Nodularia spumigena）产生的环状五肽肝毒素，在世界各地的咸水水体中均有报道。它可以在食物链中积累，出于安全原因，不仅水体中的NOD水平，而且食物基质中的NOD水平也值得关注。在这里，我们报告了一种非竞争性免疫测定的特异性检测NOD。利用噬菌体展示技术来询问合成抗体噬菌体文库中识别与抗ADDA（3-氨基-9-甲氧基-2，6，8-三甲基-10-苯基癸-4（E），6（E）-二烯酸）单克隆抗体（Mab）结合的NOD的结合剂。所获得的免疫复合物结合剂之一，命名为SA 32 C11，相对于测试的10种不同微囊藻毒素（微囊藻毒素-LR、-dmLR、-RR、-dmRR、-YR、-LY、-LF、-LW、-LA、-WR），显示出对节球藻毒素-R（NOD-R）非常高的特异性。它在大肠杆菌中表达为单链抗体片段（scFv）融合蛋白，并与抗ADDA Mab联合用于建立基于时间分辨荧光法的测定。总测定时间为1小时10分钟，免疫测定的检测限（空白+ 3SD）为0.03 µg/L NOD-R。这是迄今为止报道的特异性检测NOD的最灵敏的免疫测定方法。使用加标（0.1至3 µg/L NOD-R）水样品（包括微咸水和沿海水）测试了该方法检测NOD的性能，回收率范围为79至127%。此外，一组环境样品，包括来自不同来源的水，鱼和其他海洋组织标本，分析NOD使用的测定。该方法具有潜在的快速筛选工具，用于分析大量的水样中NOD的存在。它还可用于分析海洋生物和食物链中NOD的生物累积性。
Nodularin (NOD) is a cyclic penta-peptide hepatotoxin mainly produced by Nodularia spumigena, reported from the brackish water bodies of various parts of the world. It can accumulate in the food chain and, for safety reasons, levels of NOD not only in water bodies but also in food matrices are of interest. Here, we report on a non-competitive immunoassay for the specific detection of NOD. A phage display technique was utilized to interrogate a synthetic antibody phage library for binders recognizing NOD bound to an anti-ADDA (3-Amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4(E),6(E)-dienoic acid) monoclonal antibody (Mab). One of the obtained immunocomplex binders, designated SA32C11, showed very high specificity towards nodularin-R (NOD-R) over to the tested 10 different microcystins (microcystin-LR, -dmLR, -RR, -dmRR, -YR, -LY, -LF, -LW, -LA, -WR). It was expressed in Escherichia coli as a single chain antibody fragment (scFv) fusion protein and used to establish a time-resolved fluorometry-based assay in combination with the anti-ADDA Mab. The detection limit (blank + 3SD) of the immunoassay, with a total assay time of 1 h 10 min, is 0.03 µg/L of NOD-R. This represents the most sensitive immunoassay method for the specific detection of NOD reported so far. The assay was tested for its performance to detect NOD using spiked (0.1 to 3 µg/L of NOD-R) water samples including brackish sea and coastal water and the recovery ranged from 79 to 127%. Furthermore, a panel of environmental samples, including water from different sources, fish and other marine tissue specimens, were analyzed for NOD using the assay. The assay has potential as a rapid screening tool for the analysis of a large number of water samples for the presence of NOD. It can also find applications in the analysis of the bioaccumulation of NOD in marine organisms and in the food chain.
登录
查看更多内容
影响因子：
4.5
作者：
Karjalainen, M;Reinikainen, M;Viitasalo, M
通讯作者：
Viitasalo, M
影响因子：
6.6
作者：
Akcaalan, Reyhan;Mazur-Marzec, Hanna;Albay, Meric
通讯作者：
Albay, Meric
影响因子：
4.4
作者：
CARMICHAEL, WW;ESCHEDOR, JT;MOORE, RE
通讯作者：
MOORE, RE
影响因子：
6.1
作者：
Lankoff, A;Banasik, A;Nowak, M
通讯作者：
Nowak, M
DOI：
10.1016/j.mrgentox.2004.06.010
发表时间：
2004-11-14
影响因子：
1.9
作者：
Maatouk, I;Bouaïcha, N;Périn, F
通讯作者：
Périn, F