Generation of Dual Functional Nanobody-Nanoluciferase Fusion and Its Potential in Bioluminescence Enzyme Immunoassay for Trace Glypican-3 in Serum

Overview

Journal Sens Actuators B Chem

Date 2022 Mar 7

PMID 35250176

Authors

Sheng Yu

Zhenfeng Li

Jingzhang Li

Shimei Zhao

Shanguang Wu

Hongjing Liu

Xiongjie Bi

Dongyang Li

Jiexian Dong

Siliang Duan

Bruce D Hammock

Affiliations

Soon will be listed here.

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.

Citing Articles

Strategies for the Immobilization and Signal Amplification of a Double Nanobody Sandwich ELISA for Human Microsomal Epoxide Hydrolase.

He Q, Pan B, McCoy M, Pan J, Xu Z, Morisseau C Anal Chem. 2024; 96(49):19605-19614.

PMID: 39576950 PMC: 11635756. DOI: 10.1021/acs.analchem.4c04505.

NANOBODIES®: A Review of Diagnostic and Therapeutic Applications.

Jin B, Odongo S, Radwanska M, Magez S Int J Mol Sci. 2023; 24(6).

PMID: 36983063 PMC: 10057852. DOI: 10.3390/ijms24065994.

Mix-and-Read Nanobody-Based Sandwich Homogeneous Split-Luciferase Assay for the Rapid Detection of Human Soluble Epoxide Hydrolase.

He Q, McCoy M, Yang H, Lin M, Cui X, Zhao S Anal Chem. 2023; 95(14):6038-6045.

PMID: 36972550 PMC: 10335774. DOI: 10.1021/acs.analchem.3c00079.

H-rGO-Pd NPs Nanozyme Enhanced Silver Deposition Strategy for Electrochemical Detection of Glypican-3.

Li G, Wang B, Li L, Li X, Yan R, Liang J Molecules. 2023; 28(5).

PMID: 36903516 PMC: 10004744. DOI: 10.3390/molecules28052271.

Production of Ribosomal Protein S12/Renilla Luciferase Fusion and Development of a Bioluminescent Method for Detection of Aminoglycosides in Pork and Studying Its Recognition Mechanism.

Xia W, Zhang L, Wang J Foods. 2023; 12(2).

PMID: 36673376 PMC: 9858597. DOI: 10.3390/foods12020284.

References

Lu Q, Li X, Zhao J, Zhu J, Luo Y, Duan H . Nanobody‑horseradish peroxidase and -EGFP fusions as reagents to detect porcine parvovirus in the immunoassays. J Nanobiotechnology. 2020; 18(1):7. PMC: 6945459. DOI: 10.1186/s12951-019-0568-x. View

Xu D, Su C, Sun L, Gao Y, Li Y . Performance of Serum Glypican 3 in Diagnosis of Hepatocellular Carcinoma: A meta-analysis. Ann Hepatol. 2019; 18(1):58-67. DOI: 10.5604/01.3001.0012.7863. View

Ishiguro T, Sugimoto M, Kinoshita Y, Miyazaki Y, Nakano K, Tsunoda H . Anti-glypican 3 antibody as a potential antitumor agent for human liver cancer. Cancer Res. 2008; 68(23):9832-8. DOI: 10.1158/0008-5472.CAN-08-1973. View

Traister A, Shi W, Filmus J . Mammalian Notum induces the release of glypicans and other GPI-anchored proteins from the cell surface. Biochem J. 2007; 410(3):503-11. DOI: 10.1042/BJ20070511. View

Feng M, Gao W, Wang R, Chen W, Man Y, Figg W . Therapeutically targeting glypican-3 via a conformation-specific single-domain antibody in hepatocellular carcinoma. Proc Natl Acad Sci U S A. 2013; 110(12):E1083-91. PMC: 3607002. DOI: 10.1073/pnas.1217868110. View

Haruyama Y, Kataoka H . Glypican-3 is a prognostic factor and an immunotherapeutic target in hepatocellular carcinoma. World J Gastroenterol. 2016; 22(1):275-83. PMC: 4698492. DOI: 10.3748/wjg.v22.i1.275. View

Ma R, Wang S, Qin S, Wang X, Li G, Li M . Preparation and characterization of monoclonal antibody against glypican-3. Hybridoma (Larchmt). 2012; 31(6):455-61. DOI: 10.1089/hyb.2012.0030. View

Yasuda E, Kumada T, Toyoda H, Kaneoka Y, Maeda A, Okuda S . Evaluation for clinical utility of GPC3, measured by a commercially available ELISA kit with Glypican-3 (GPC3) antibody, as a serological and histological marker for hepatocellular carcinoma. Hepatol Res. 2010; 40(5):477-85. DOI: 10.1111/j.1872-034X.2010.00624.x. View

Yu J, Xu X, Ma R, Qin S, Wang C, Wang X . Development of a clinical chemiluminescent immunoassay for serum GPC3 and simultaneous measurements alone with AFP and CK19 in diagnosis of hepatocellular carcinoma. J Clin Lab Anal. 2014; 29(2):85-93. PMC: 6806998. DOI: 10.1002/jcla.21733. View

10.

Xie C, Tiede C, Zhang X, Wang C, Li Z, Xu X . Development of an Affimer-antibody combined immunological diagnosis kit for glypican-3. Sci Rep. 2017; 7(1):9608. PMC: 5575301. DOI: 10.1038/s41598-017-10083-w. View

11.

Wu Y, Liu H, Ding H . GPC-3 in hepatocellular carcinoma: current perspectives. J Hepatocell Carcinoma. 2016; 3:63-67. PMC: 5108608. DOI: 10.2147/JHC.S116513. View

12.

Bray F, Ferlay J, Soerjomataram I, Siegel R, Torre L, Jemal A . Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018; 68(6):394-424. DOI: 10.3322/caac.21492. View

13.

Mu W, Jiang D, Mu S, Liang S, Liu Y, Zhang N . Promoting Early Diagnosis and Precise Therapy of Hepatocellular Carcinoma by Glypican-3-Targeted Synergistic Chemo-Photothermal Theranostics. ACS Appl Mater Interfaces. 2019; 11(26):23591-23604. DOI: 10.1021/acsami.9b05526. View

14.

Hippo Y, Watanabe K, Watanabe A, Midorikawa Y, Yamamoto S, Ihara S . Identification of soluble NH2-terminal fragment of glypican-3 as a serological marker for early-stage hepatocellular carcinoma. Cancer Res. 2004; 64(7):2418-23. DOI: 10.1158/0008-5472.can-03-2191. View

15.

Huo J, Li Z, Wan D, Li D, Qi M, Barnych B . Development of a Highly Sensitive Direct Competitive Fluorescence Enzyme Immunoassay Based on a Nanobody-Alkaline Phosphatase Fusion Protein for Detection of 3-Phenoxybenzoic Acid in Urine. J Agric Food Chem. 2018; 66(43):11284-11290. PMC: 6442738. DOI: 10.1021/acs.jafc.8b04521. View

16.

Bever C, Dong J, Vasylieva N, Barnych B, Cui Y, Xu Z . VHH antibodies: emerging reagents for the analysis of environmental chemicals. Anal Bioanal Chem. 2016; 408(22):5985-6002. PMC: 4983233. DOI: 10.1007/s00216-016-9585-x. View

17.

Kim H, McCoy M, Majkova Z, Dechant J, Gee S, Tabares-da Rosa S . Isolation of alpaca anti-hapten heavy chain single domain antibodies for development of sensitive immunoassay. Anal Chem. 2011; 84(2):1165-71. PMC: 3264785. DOI: 10.1021/ac2030255. View

18.

Li D, Cui Y, Morisseau C, Gee S, Bever C, Liu X . Nanobody Based Immunoassay for Human Soluble Epoxide Hydrolase Detection Using Polymeric Horseradish Peroxidase (PolyHRP) for Signal Enhancement: The Rediscovery of PolyHRP?. Anal Chem. 2017; 89(11):6248-6256. PMC: 5611449. DOI: 10.1021/acs.analchem.7b01247. View

19.

Capurro M, Wanless I, Sherman M, Deboer G, Shi W, Miyoshi E . Glypican-3: a novel serum and histochemical marker for hepatocellular carcinoma. Gastroenterology. 2003; 125(1):89-97. DOI: 10.1016/s0016-5085(03)00689-9. View

20.

Chen J, Xie C, Wang C, Wan Y, Dong Z, Li M . Development of a Time-Resolved Fluorescence Immunoassay for the Diagnosis of Hepatocellular Carcinoma Based on the Detection of Glypican-3. J Fluoresc. 2017; 27(4):1479-1485. DOI: 10.1007/s10895-017-2087-1. View