Yu Sheng, Li Zhenfeng, Li Jingzhang, Zhao Shimei, Wu Shanguang, Liu Hongjing, Bi Xiongjie, Li Dongyang, Dong Jiexian, Duan Siliang, Hammock Bruce D
Medical College, Guangxi University of Science and Technology, Liuzhou, Guangxi, 545005, China.
The Second Clinical Medical College of Guangxi University of Science and Technology, Liuzhou, Guangxi, 545005, China.
Sens Actuators B Chem. 2021 Jun 1;336. doi: 10.1016/j.snb.2021.129717. Epub 2021 Mar 2.
Glypican-3 (GPC3) is a serological biomarker for the diagnosis of Hepatocellular carcinoma (HCC), but it is a challenging task to develop a bioassay for determination of the trace GPC3 in serum. In this study, Bioluminescense immunoassay based on bifunctional nanobody-nanoluciferase fusion was developed with the ultra-sensitive feature to achieve this goal. First, nanobodies special against GPC-3 binder as biological recognition element were generated by immunization and phage display technology. Second, The best clone GPN2 was fused with nanoluciferase as a dual-functional immunoreagent to establish an ultra-sensitive bioluminescence enzyme immunoassay (BLEIA), which is 30 and 5 times more sensitive than the traditional colorimetric assay and fluorescent assay, respectively. The cross-reactivity analysis of BLEIA showed that there was no cross-reactivity with HCC related tumor markers AFP, CEA, CA19-9 and GPC1/GPC2. The limit of detection (LOD) of developed BLEIA was 1.5 ng/mL, which assured its application in the diagnosis of GPC3 in 94 serum samples. This study indicates that BLEIA based on nanobody-nanoluciferase fusion could be used as a useful tool for the diagnosis of HCC patients.
磷脂酰肌醇蛋白聚糖-3（GPC3）是诊断肝细胞癌（HCC）的一种血清生物标志物，但开发一种用于测定血清中痕量GPC3的生物测定方法是一项具有挑战性的任务。在本研究中，基于双功能纳米抗体-纳米荧光素酶融合构建了生物发光免疫测定法，以实现超灵敏检测痕量GPC3的目标。首先，通过免疫和噬菌体展示技术制备了针对GPC-3结合物的纳米抗体作为生物识别元件。其次，将最佳克隆GPN2与纳米荧光素酶融合，构建一种双功能免疫试剂，建立了一种超灵敏生物发光酶免疫测定法（BLEIA），其灵敏度分别比传统比色法和荧光法高30倍和5倍。BLEIA的交叉反应分析表明，其与HCC相关肿瘤标志物甲胎蛋白（AFP）、癌胚抗原（CEA）、糖类抗原19-9（CA19-9）和GPC1/GPC2无交叉反应。所构建的BLEIA的检测限（LOD）为1.5 ng/mL，这确保了其在94份血清样本中GPC3诊断中的应用。本研究表明，基于纳米抗体-纳米荧光素酶融合的BLEIA可作为诊断HCC患者的一种有用工具。
