Development and Evaluation of Quantitative Immunoglobulin G Enzyme-Linked Immunosorbent Assay for the Diagnosis of Coronavirus Disease 2019 Using Truncated Recombinant Nucleocapsid Protein as Assay Antigen

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Real-time RT-PCR is the most commonly used method for COVID-19 diagnosis. However, serological assays are urgently needed as complementary tools to RT-PCR. Hachim et al. 2020 and Burbelo et al. 2020 demonstrated that anti-nucleocapsid(N) SARS-CoV-2 antibodies are higher and appear earlier than the spike antibodies. Additionally, cross-reactive antibodies against N protein are more prevalent than those against spike protein. We developed a less cross-reactive immunoglobulin G (IgG) indirect ELISA by using a truncated recombinant SARS-CoV-2 N protein as assay antigen. A highly conserved region of coronaviruses N protein was deleted and the protein was prepared using an E. coli protein expression system. A total of 177 samples collected from COVID-19 suspected cases and 155 negative control sera collected during the pre-COVID-19 period were applied to evaluate the assay’s performance, with the plaque reduction neutralization test and the commercial SARS-CoV-2 spike protein IgG ELISA as gold standards. The SARS-CoV-2 N truncated protein-based ELISA showed similar sensitivity (91.1% vs. 91.9%) and specificity (93.8% vs. 93.8%) between the PRNT and spike IgG ELISA, as well as also higher specificity compared to the full-length N protein (93.8% vs. 89.9%). Our ELISA can be used for the diagnosis and surveillance of COVID-19.

A systematic review and meta-analysis comparing the diagnostic accuracy tests of COVID-19

“…Sensitivity and specificity are reported as a mean (95% confidence limits). The forest plot represents the estimated sensitivity and specificity (black squares) and their 95% confidence limits (horizontal black line) [73][74][75][76][77][78][79][80][81][82][83][84][85][86][87][88]. Sensitivity and specificity are reported as a mean (95% confidence limits).…”

“…Sixteen studies were selected using IgG-detecting ELISA as a diagnostic technique [73][74][75][76][77][78][79][80][81][82][83][84][85][86][87][88]. A total of 9,221 subjects were studied.…”

Chemiluminescent Nanogels as Intensive and Stable Signal Probes for Fast Immunoassay of SARS-CoV-2 Nucleocapsid Protein

“…Coronavirus disease 2019 (COVID-19) is a viral disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) . To date, the spread of the virus has not been controlled, although huge efforts are being made to counter the virus.…”

“…Coronavirus disease 2019 (COVID-19) is a viral disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). 18 To date, the spread of the virus has not been controlled, although huge efforts are being made to counter the virus. Developing sensitive, accurate and fast detection methods for rapid identification of infected individuals is benefited to stop the spread of the outbreak.…”

“…However, RT-PCR requires skilled technicians, time-consuming amplification steps, and expensive reagents . Therefore, convenient, fast and economical testing methods are urgently needed as complementary tools to RT-PCR, such as antibody or antigen testing . However, antibodies in serum samples increased after 7 days of infection with the virus, which makes the antibody test unsuitable for early diagnosis of COVID-19. , The nucleocapsid protein of SARS-CoV-2 (N protein) plays a significant role in the replication of the virus.…”