Nanobody medicated immunoassay for ultrasensitive detection of cancer biomarker alpha-fetoprotein
Nanobody medicated immunoassay for ultrasensitive detection of cancer biomarker alpha-fetoprotein
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用于超灵敏检测癌症生物标志物甲胎蛋白的纳米抗体药物免疫分析
DOI：
10.1016/j.talanta.2015.10.027
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发表时间：
2016-01-15
期刊：
影响因子：
6.1
通讯作者：
Liu, Yuan-yuan
中科院分区：
文献类型：
--
作者：
Chen, Jing;He, Qing-hua;Liu, Yuan-yuan
肿瘤生物标志物的免疫检测在癌症预防和早期诊断中起着重要作用。对于免疫分析的发展而言，所用抗体的质量和稳定性是获得免疫分析可靠性和高灵敏度的关键之一。本研究的主要目的是建立一种基于抗甲胎蛋白纳米体超灵敏检测肿瘤生物标志物甲胎蛋白(AFP)的免疫分析方法。通过生物扫描策略从噬菌体展示纳米体库中筛选出两个与APP结合的纳米体。所制备的纳米体具有可克隆、热稳定性好的特点，可用于夹心酶联免疫分析和免疫-聚合酶链式反应检测甲胎蛋白。优化后的纳米体双抗体夹心ELISA法的最低检测限为0.48ngmL(-1)，饱和浓度(SC50)为6.68+/-0.56ngmL(-1)。建立了甲胎蛋白的免疫-聚合酶链式反应检测方法，检测下限为0.005-10,000 ng-1，线性范围为0.01-10,000 ng-1。这些建立在纳米抗体基础上的免疫分析对APP具有高度的特异性，与其他被测试的癌症生物标记物的交叉反应可以忽略不计。此外，纳米体介导免疫分析的这一新概念可能在各种癌症生物标志物的超灵敏检测的通用方法中提供潜在的应用。(C)2015爱思唯尔B.V.保留所有权利。
Immunoassay for cancer biomarkers plays an important role in cancer prevention and early diagnosis. To the development of immunoassay, the quality and stability of applied antibody is one of the key points to obtain reliability and high sensitivity for immunoassay. The main purpose of this study was to develop a novel immunoassay for ultrasensitive detection of cancer biomarker alpha-fetoprotein (AFP) based on nanobody against AFP. Two nanobodies which bind to APP were selected from a phage display nanobody library by biopanning strategy. The prepared nanobodies are clonable, thermally stable and applied in both sandwich enzyme linked immunoassay (ELISA) and immuno-PCR assay for ultrasensitive detection of AFP. The limit detection of sandwich ELISA setup with optimized nanobodies was 0.48 ng mL(-1), and the half of saturation concentration (SC50) value was 6.68 +/- 0.56 ng mL(-1). These nanobodies were also used to develop an immuno-PCR assay for ultrasensitive detection of AFP, its limit detection values was 0.005 ng mL(-1), and the linear range was 0.01-10,000 ng mL(-1). These established immunoassays based on nanobodies were highly specific to APP and with negligible cross reactivity with other tested caner biomarkers. Furthermore, this novel concept of nanobodies mediated immunoassay may provide potential applications in a general method for the ultrasensitive detection of various cancer biomarkers. (C) 2015 Elsevier B.V. All rights reserved.