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.
【저자키워드】 COVID-19, monoclonal antibody, SARS-CoV-2., nucleocapsid protein, antigen detection, Chemiluminescence enzyme immunoassay, 【초록키워드】 coronavirus disease, SARS-CoV-2, Coronavirus disease 2019, antibody, SARS-COV-2 infection, monoclonal antibody, diagnostic, monoclonal antibodies, ELISA, nucleocapsid protein, global pandemic, Spread, Protein, immunoassay, nucleic acid, sensitivity, enzyme-linked immunosorbent assay, Nucleic acid detection, cells, enzyme immunoassay, structural protein, antigen detection, disease, Clinical diagnosis, expression, chemiluminescence, spleen, mAbs, mAb, capture, Precision, Coefficient of variance, enzyme, variance, pandemic disease, cell line, BALB/c mice, coding sequence, E. coli cells, detection antibody, molecular weight, Cell, immunized, enzyme-linked immunosorbent, IPTG, selected, highest, develop, caused, addition, evaluated, expressed, kDa, was obtained, fused, E. coli cell, secreted, 【제목키워드】 Protein, chemiluminescence immunoassay, development, SARS-CoV-2 nucleocapsid,