Title: Generation of Dual functional Nanobody-Nanoluciferase Fusion and its potential in Bioluminescence Enzyme Immunoassay for trace Glypican-3 in Serum.
Authors: Yu, Sheng; Li, Zhenfeng; Li, Jingzhang; Zhao, Shimei; Wu, Shanguang; Liu, Hongjing; Bi, Xiongjie; Li, Dongyang; Dong, Jiexian; Duan, Siliang; Hammock, Bruce D
Published In Sens Actuators B Chem, (2021 Jun 01)
Abstract: 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.
PubMed ID: 35250176
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