基于单分子阵列技术的血清中Fn14蛋白的超灵敏检测
首发时间:2019-05-15
摘要:成纤维细胞生长因子诱导型-14(Fn14)是一种受体蛋白,在癌症和其他一些疾病的进展中起重要作用。在此,使用基于磁珠的数字式夹心酶联免疫吸附测定(dELISA),开发了用于检测Fn14的超灵敏测定法。将含有单个免疫复合物的珠子加载到微孔中(~46fL)并通过酶催化反应产生荧光,并通过测量排列在圆盘上的阵列的荧光微孔的数量,确定Fn14的浓度。为了获得更好的Fn14检测性能,优化了包括试剂浓度和测量参数在内的测定条件,并筛选了44种不同的抗体对。Fn14的检测范围为1.26 pg/mL至3,683 pg/mL,检测下限可低至0.32 pg/mL,远低于常规ELISA的检测限。本文中我们所发展的测定的操作是自动化的,仅需要大约一个小时来完成。此外,该测定法成功应用于血清样品中加标Fn14的测定,并具有令人满意的性能。
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Ultrasensitive detection of Fn14 in human serum using a single molecule assay
Abstract:Fibroblast growth factor inducible-14 (Fn14) is a receptor protein that plays an important role in the progression of cancer and some other diseases. Here, an ultrasensitive assay was developed for the detection of Fn14 based on a digital sandwich enzyme-linked immunosorbent bead-based assay (dELISA). Beads containing a single immunocomplex are loaded into microwells (~46 fL) and produce fluorescence through enzyme-catalyzed reactions in extremely small volumes. By measuring the number of fluorescent microwells in arrays arranged on a circular Disc, the concentration of Fn14 was determined. To obtain better performance for Fn14 detection, assay conditions including reagent concentrations and measurement parameters were optimized and 44 different antibody pairs were screened. The detection range of Fn14 is 1.26 pg/mL to 3,683 pg/mL with a lower detection limit of 0.32 pg/mL, which is much lower than that of conventional ELISAs. In addition, the total operation of this assay is automated and only takes approximately an hour to accomplish. Furthermore, this assay was successfully applied to the determination of spiked Fn14 in serum samples with satisfactory performance.
Keywords: Single Molecule Array Fn14 Ultrasensitive detection
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基于单分子阵列技术的血清中Fn14蛋白的超灵敏检测
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