Maeno Hinata, Wong Pooi-Fong, AbuBakar Sazaly, Yang Ming, Sam Sing-Sin, Jamil-Abd Juraina, Shunmugarajoo Anusha, Mustafa Mahiran, Said Rosaida Md, Mageswaren Eashwary, Azmel Azureen, Mat Jelani Anilawati
Department of System Design, Tokyo Metropolitan University, Tokyo 191-0065, Japan.
Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia.
Micromachines (Basel). 2021 Nov 30;12(12):1503. doi: 10.3390/mi12121503.
Serum is commonly used as a specimen in immunoassays but the presence of heterophilic antibodies can potentially interfere with the test results. Previously, we have developed a microfluidic device called: 3D Stack for enzyme-linked immunosorbent assay (ELISA). However, its evaluation was limited to detection from a single protein solution. Here, we investigated the sensitivity of the 3D Stack in detecting a severe dengue biomarker-soluble CD163 (sCD163)-within the serum matrix. To determine potential interactions with serum matrix, a spike-and-recovery assay was performed, using 3D Stacks with and without surface modification by an EDC-NHS (N-ethyl-N'-(3-(dimethylamino)propyl)carbodiimide/N-hydroxysuccinimide) coupling. Without surface modification, a reduced analyte recovery in proportion to serum concentration was observed because of the Vroman effect, which resulted in competitive displacement of coated capture antibodies by serum proteins with stronger binding affinities. However, EDC-NHS coupling prevented antibody desorption and improved the sensitivity. Subsequent comparison of sCD163 detection using a 3D Stack with EDC-NHS coupling and conventional ELISA in dengue patients' sera revealed a high correlation (R = 0.9298, < 0.0001) between the two detection platforms. Bland-Altman analysis further revealed insignificant systematic error between the mean differences of the two methods. These data suggest the potentials of the 3D Stack for further development as a detection platform.
血清常用于免疫测定，但嗜异性抗体的存在可能会干扰检测结果。此前，我们开发了一种名为“用于酶联免疫吸附测定（ELISA）的3D堆栈”的微流控装置。然而，其评估仅限于从单一蛋白质溶液中进行检测。在此，我们研究了3D堆栈在血清基质中检测严重登革热生物标志物可溶性CD163（sCD163）的灵敏度。为了确定与血清基质的潜在相互作用，进行了加标回收试验，使用了经过和未经过1-乙基-3-(3-二甲氨基丙基)碳二亚胺/ N-羟基琥珀酰亚胺（EDC-NHS）偶联进行表面修饰的3D堆栈。未经表面修饰时，由于弗罗曼效应，观察到分析物回收率随血清浓度降低，这导致具有更强结合亲和力的血清蛋白竞争性取代包被的捕获抗体。然而，EDC-NHS偶联可防止抗体解吸并提高灵敏度。随后，在登革热患者血清中使用经过EDC-NHS偶联的3D堆栈和传统ELISA对sCD163进行检测比较，结果显示两个检测平台之间具有高度相关性（R = 0.9298，< 0.0001）。布兰德-奥特曼分析进一步表明两种方法的平均差异之间不存在显著的系统误差。这些数据表明3D堆栈作为检测平台具有进一步开发的潜力。
