Colorimetric stack pad immunoassay for bacterial identification
Colorimetric stack pad immunoassay for bacterial identification
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DOI：
10.1016/j.bios.2016.08.044
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发表时间：
2017-01-15
影响因子：
12.6
通讯作者：
Marks, Robert S.
中科院分区：
文献类型：
--
作者：
Eltzov, Evgeni;Marks, Robert S.
一种新的比色免疫测定概念，利用传统的横向流动膜（例如，偶联膜、样品膜、吸收膜和硝化纤维膜），以堆叠方式放置在不同的配置中，其中可能含有分析物的液体样品从底部扩散到最上层。这项专利技术的关键要素是捕获层，其中硝化纤维素膜被感兴趣的目标分析物修饰，即在本研究中目标大肠杆菌。在免疫分析操作过程中，被目标细菌污染的样品将与等待的相应hrp抗体结合，免疫目标测量复合物通过毛细作用向最上层流动，通过酶促反应产生比色信号（阳性答案）。在无目标样品中，先前固定在捕获层上的目标细菌将阻止hrp标记的抗目标抗体迁移到最上层，酶底物在那里等待。优化后，发现该方法的灵敏度比elisa(10（2)个细胞mL(-1)）高1000倍。叠垫法的优点包括：小型化、操作简单、反应时间快（小于5分钟）、灵敏度高。(C) 2016 Elsevier B.V.版权所有
A new colorimetric immunoassay concept, utilizing conventional lateral flow membranes (e.g., conjugation, sample, absorption and nitrocellulose), were placed in a different configuration in a stacking manner, where the liquid sample that may contain the analyte diffuses from the bottom to the uppermost layer. The key element of this proprietary technology is a capture layer, where a nitrocellulose membrane is modified with the target analyte of interest, namely in this study target Escherichia coli. During the immunoassay operation, samples contaminated with the target bacteria will conjugate to their corresponding HRP-antibodies laying in wait and the immune-target measurand complex flows by capillarity towards the upper-most layer to generate a colorimetric signal (positive answer) through an enzymatic reaction. In target-free samples, previously immobilized target bacteria on the capture layer will prevent the HRP-labeled anti-target antibodies from migrating to the upper-most layer, where the enzymatic substrate lays in wait. After optimization, the sensitivity of this approach was found to be 1,000 folds higher than ELISAs (10(2) cells mL(-1)). The advantages of the stacked pad assay include: miniaturization, operational simplicity, fast response time (less than 5 min), useful sensitivity. (C) 2016 Elsevier B.V. All rights reserved.