Development of magnetic particle-based chemiluminescence immunoassay for measurement of SARS-CoV-2 nucleocapsid protein.

Autor: Xu Y; Department of Medical Laboratory, School of Medicine, Hunan Normal University, Changsha, 410013, China; Immunodiagnostic Reagents Engineering Research Center of Hunan Province, Hunan Normal University, Changsha, 410013, China., Xia C; Department of Medical Laboratory, School of Medicine, Hunan Normal University, Changsha, 410013, China; Department of Clinical Laboratory, the First People's Hospital of Chenzhou, Chenzhou, 423000, China., Zeng X; Department of Medical Laboratory, School of Medicine, Hunan Normal University, Changsha, 410013, China; Immunodiagnostic Reagents Engineering Research Center of Hunan Province, Hunan Normal University, Changsha, 410013, China., Qiu Y; Immunodiagnostic Reagents Engineering Research Center of Hunan Province, Hunan Normal University, Changsha, 410013, China., Liao M; Department of Medical Laboratory, School of Medicine, Hunan Normal University, Changsha, 410013, China; Immunodiagnostic Reagents Engineering Research Center of Hunan Province, Hunan Normal University, Changsha, 410013, China., Jiang Q; Department of Medical Laboratory, School of Medicine, Hunan Normal University, Changsha, 410013, China; Immunodiagnostic Reagents Engineering Research Center of Hunan Province, Hunan Normal University, Changsha, 410013, China., Quan M; Immunodiagnostic Reagents Engineering Research Center of Hunan Province, Hunan Normal University, Changsha, 410013, China., Liu R; Department of Medical Laboratory, School of Medicine, Hunan Normal University, Changsha, 410013, China; Immunodiagnostic Reagents Engineering Research Center of Hunan Province, Hunan Normal University, Changsha, 410013, China. Electronic address: liurushi@hunnu.edu.cn.
Jazyk: angličtina
Zdroj: Journal of virological methods [J Virol Methods] 2022 Apr; Vol. 302, pp. 114486. Date of Electronic Publication: 2022 Jan 30.
DOI: 10.1016/j.jviromet.2022.114486
Abstrakt: Background: Recently, the Coronavirus Disease 2019 (COVID-19) caused by SARS-CoV-2 infection has spread rapidly around the world, becoming a new global pandemic disease. Nucleic acid detection is the primary method for clinical diagnosis of SARS-CoV-2 infection, with the addition of antibody and antigen detection. Nucleocapsid protein (NP) is a kind of conservative structural protein with abundant expression during SARS-CoV-2 infection, which makes it an ideal target for immunoassay.
Methods: The coding sequence for SARS-CoV-2-NP was obtained by chemical synthesis, and then inserted into pET28a(+). The soluble recombinant NP (rNP) with an estimated molecular weight of 49.4 kDa was expressed in E. coli cells after IPTG induction. Six-week-old BALB/c mice were immunized with rNP, and then their spleen cells were fused with SP2/0 cells, to develop hybridoma cell lines that stably secreted monoclonal antibodies (mAbs) against NP. The mAbs were preliminarily evaluated by enzyme-linked immunosorbent assay (ELISA), and then used to develop a magnetic particle-based chemiluminescence enzyme immunoassay (CLEIA) for measurement of SARS-CoV-2-NP.
Results: mAb 15B1 and mAb 18G10 were selected as capture and detection antibody respectively to develop CLEIA, due to the highest sensitivity for rNP detection. The proposed CLEIA presented a good linearity for rNP detection at a working range from 0.1 to 160 μg/L, with a precision coefficient of variance below 10 %.
Conclusion: The newly developed mAbs and CLEIA can serve as potential diagnostic tools for clinical measurement of SARS-CoV-2-NP.
(Copyright © 2022. Published by Elsevier B.V.)
Databáze: MEDLINE
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