Generation of Dual functional Nanobody-Nanoluciferase Fusion and its potential in Bioluminescence Enzyme Immunoassay for trace Glypican-3 in Serum.

Journal: Sensors and actuators. B, Chemical
Published Date: Mar 2, 2021

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.

Authors

  • Sheng Yu
    Medical College, Guangxi University of Science and Technology, Liuzhou, Guangxi, 545005, China.
  • Zhenfeng Li
    Department of Entomology and Nematology and UCD Comprehensive Cancer Center, University of California, Davis, California 95616.
  • Jingzhang Li
    Liuzhou People's Hospital, Liuzhou, Guangxi, 545005, China.
  • Shimei Zhao
    Medical College, Guangxi University of Science and Technology, Liuzhou, Guangxi, 545005, China.
  • Shanguang Wu
    Medical College, Guangxi University of Science and Technology, Liuzhou, Guangxi, 545005, China.
  • Hongjing Liu
    Medical College, Guangxi University of Science and Technology, Liuzhou, Guangxi, 545005, China.
  • Xiongjie Bi
    The First Affiliated Hospital of Guangxi University of Science and Technology, Liuzhou, Guangxi, 545005, China.
  • Dongyang Li
    CAS Key Laboratory of Molecular Imaging, Institute of Automation, Chinese Academy of Sciences, Beijing 100190, China.
  • Jiexian Dong
    Department of Entomology and Nematology and UCD Comprehensive Cancer Center, University of California, Davis, California 95616.
  • Siliang Duan
    Medical College, Guangxi University of Science and Technology, Liuzhou, Guangxi, 545005, China.
  • Bruce D Hammock
    Department of Entomology and Nematology, UCD Comprehensive Cancer Center, University of California, Davis, CA 95616, USA.

Keywords

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