tetano
Editor, Senior Moderator
J Virol Methods
. 2022 Jan 30;114486.
doi: 10.1016/j.jviromet.2022.114486. Online ahead of print.
Development of magnetic particle-based chemiluminescence immunoassay for measurement of SARS-CoV-2 nucleocapsid protein
Ye Xu[SUP] 1 [/SUP], Chuan Xia[SUP] 2 [/SUP], Xuan Zeng[SUP] 1 [/SUP], Yilan Qiu[SUP] 3 [/SUP], Minjing Liao[SUP] 1 [/SUP], Qing Jiang[SUP] 1 [/SUP], Meifang Quan[SUP] 3 [/SUP], Rushi Liu[SUP] 4 [/SUP]
Affiliations
Abstract
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.
Keywords: Antigen detection; COVID-19; Chemiluminescence enzyme immunoassay; Monoclonal antibody; Nucleocapsid protein; SARS-CoV-2.
. 2022 Jan 30;114486.
doi: 10.1016/j.jviromet.2022.114486. Online ahead of print.
Development of magnetic particle-based chemiluminescence immunoassay for measurement of SARS-CoV-2 nucleocapsid protein
Ye Xu[SUP] 1 [/SUP], Chuan Xia[SUP] 2 [/SUP], Xuan Zeng[SUP] 1 [/SUP], Yilan Qiu[SUP] 3 [/SUP], Minjing Liao[SUP] 1 [/SUP], Qing Jiang[SUP] 1 [/SUP], Meifang Quan[SUP] 3 [/SUP], Rushi Liu[SUP] 4 [/SUP]
Affiliations
- PMID: 35108595
- PMCID: PMC8801064
- DOI: 10.1016/j.jviromet.2022.114486
Abstract
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.
Keywords: Antigen detection; COVID-19; Chemiluminescence enzyme immunoassay; Monoclonal antibody; Nucleocapsid protein; SARS-CoV-2.