One-step non-competitive fluorescence polarization immunoassay based on a Fab fragment for C-reactive protein quantification
One-step non-competitive fluorescence polarization immunoassay based on a Fab fragment for C-reactive protein quantification
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DOI：
10.1016/j.snb.2020.128982
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发表时间：
2021-01-01
影响因子：
8.4
通讯作者：
Tokeshi, Manabu
中科院分区：
文献类型：
--
作者：
Nishiyama, Keine;Fukuyama, Mao;Tokeshi, Manabu
开发了使用Fab片段的非竞争性荧光偏振免疫测定（FPIA）用于大分子定量。大多数常规FPIA是均相和竞争性免疫测定。对于竞争性FPIA，当靶向大分子如蛋白质时，难以获得足够的检测灵敏度。为了克服这个根本的缺点，我们报告了一个非竞争性FPIA使用荧光标记的Fab片段。C-反应蛋白（CRP）被用作基于Fab的非竞争性FPIA验证的模型物质。成功实现了磷酸盐缓冲液（PBS）中CRP的定量分析，PBS中CRP的检测限（LOD）为207 ng/mL。此外，使用远红外发射荧光染料（HiLyte Fluor（TM）647）作为Fab片段的荧光标记物质，我们在CRP的截止值（10 μ g/mL）附近10分钟内测量了人血清中的CRP，而没有对样品进行预处理。人血清中CRP的LOD为1.58 μ g/mL。在我们提出的方法中，反应在仅使用一种类型的荧光标记Fab片段试剂的简单的一步混合中完成，并且不需要洗涤操作。因此，快速蛋白质定量实现了一个大大简化的程序。
A non-competitive fluorescence polarization immunoassay (FPIA) using a Fab fragment was developed for large molecule quantification. Most conventional FPIAs are homogeneous and competitive immunoassays. With competitive FPIAs, it has been difficult to obtain sufficient detection sensitivity when targeting large molecules like proteins. To overcome this fundamental drawback, we report a non-competitive FPIA using a fluorescence-labeled Fab fragment. C-reactive protein (CRP) was used as model substance for validation of the Fab-based non-competitive FPIA. Quantitative analysis of CRP in phosphate buffered saline (PBS) was successfully achieved and the limit of detection (LOD) of CRP in PBS was 207 ng/mL. Moreover, using far-red emitting fluorescent dye (HiLyte Fluor (TM) 647) as a fluorescent labeling substance of Fab fragment, we measured CRP in human serum without pretreatment of a sample in 10 min around the cut-off value of CRP (10 mu g/mL). The LOD of CRP in human serum was 1.58 mu g/mL. In our proposed method, the reaction was completed in a simple one-step mixing with only one type of fluorescence-labeled Fab fragment reagent, and no washing operation was required. Therefore, rapid protein quantification was achieved with a greatly simplified procedure.