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Using magnetic beads and signal amplifiers to produce short and simple immunoassays: Application to MMP-9 detection in plasma samples
Analytica Chimica Acta ( IF 5.7 ) Pub Date : 2018-01-01 , DOI: 10.1016/j.aca.2017.11.013
Manel Ben Ismail , Erica de la Serna , Gisela Ruiz-Vega , Teresa García-Berrocoso , Joan Montaner , Mohammed Zourob , Ali Othmane , Eva Baldrich

Magnetic beads (MB) and signal amplifiers, such as horseradish peroxidase polymers (poly-HRP), have been used before for the production of highly sensitive immunoassays. However, most of the examples reported previously entailed long and tedious multi-step procedures, which were not necessarily shorter or simpler than classical paths such as Enzyme-Linked Immunosorbent Assay (ELISA). Here, instead of exploiting the combination of MB and poly-HRP to ameliorate sensitivity, we show that they conform a powerful tool that can be used to shorten the incubation times, which allows optimizing extremely simple, fast and efficient immunoassays with minimal technical requirements. In order to do so, here we used the highly sensitive and specific pair of antibodies of a commercial ELISA kit to optimize a magneto-ELISA for the detection of matrix metallopeptidase 9 (MMP-9). Three signal amplifiers were then tested and the best performing one was implemented in the magneto-assay to shorten the incubation times and improve assay performance. As we show, the shortened magneto-assay could be carried out in about 35 min, which included two 5-min incubations, washing, and incubation with enzyme substrate for 20 min before colorimetric detection. Moreover, the quantification of MMP-9 provided by the shortened assay in 12 plasma samples collected from patients was comparable to that generated by the 5-h ELISA, which was 8.5 times longer.

中文翻译:

使用磁珠和信号放大器进行简短而简单的免疫分析:在血浆样品中 MMP-9 检测中的应用

磁珠 (MB) 和信号放大器,例如辣根过氧化物酶聚合物 (poly-HRP),以前曾用于生产高度灵敏的免疫测定。然而,之前报道的大多数示例都需要冗长乏味的多步骤程序,这些程序不一定比酶联免疫吸附测定 (ELISA) 等经典路径更短或更简单。在这里,我们没有利用 MB 和 poly-HRP 的组合来提高灵敏度,而是表明它们符合一种强大的工具,可用于缩短孵育时间,从而以最低的技术要求优化极其简单、快速和高效的免疫测定。为了做到这一点,在这里,我们使用商业 ELISA 试剂盒的高灵敏度和特异性抗体对来优化用于检测基质金属肽酶 9 (MMP-9) 的磁 ELISA。然后测试了三个信号放大器,并在磁检测中实施了性能最好的一个,以缩短孵育时间并提高检测性能。正如我们所展示的,缩短的磁检测可以在大约 35 分钟内进行,其中包括两次 5 分钟的孵育、洗涤和在比色检测之前与酶底物孵育 20 分钟。此外,从患者收集的 12 份血浆样本中,由缩短的测定提供的 MMP-9 定量与 5 小时 ELISA 产生的结果相当,后者是 8.5 倍。然后测试了三个信号放大器,并在磁检测中实施了性能最好的一个,以缩短孵育时间并提高检测性能。正如我们所展示的,缩短的磁检测可以在大约 35 分钟内进行,其中包括两次 5 分钟的孵育、洗涤和在比色检测之前与酶底物孵育 20 分钟。此外,从患者收集的 12 份血浆样本中,由缩短的测定提供的 MMP-9 定量与 5 小时 ELISA 产生的结果相当,后者是 8.5 倍。然后测试了三个信号放大器,并在磁检测中实施了性能最好的一个,以缩短孵育时间并提高检测性能。正如我们所展示的,缩短的磁检测可以在大约 35 分钟内进行,其中包括两次 5 分钟的孵育、洗涤和在比色检测之前与酶底物孵育 20 分钟。此外,从患者收集的 12 份血浆样本中,由缩短的测定提供的 MMP-9 定量与 5 小时 ELISA 产生的结果相当,后者是 8.5 倍。
更新日期:2018-01-01
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