Development of an Affordable ELISA Targeting the SARS-CoV-2 Nucleocapsid and Its Application to Samples from the Ongoing COVID-19 Epidemic in Ghana

Abstract

Introduction The true nature of the population spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in populations is often not fully known as most cases, particularly in Africa, are asymptomatic. Finding the true magnitude of SARS-CoV-2 spread is crucial to provide actionable data about the epidemiological progress of the disease for researchers and policymakers. This study developed and optimized an antibody enzyme-linked immunosorbent assay (ELISA) using recombinant nucleocapsid antigen expressed in-house using a simple bacterial expression system. Methods Nucleocapsid protein from SARS-CoV-2 was expressed and purified from Escherichia coli. Plasma samples used for the assay development were obtained from Ghanaian SARS-CoV-2 seropositive individuals during the pandemic, while seronegative controls were plasma samples collected from blood donors before the coronavirus disease 2019 (COVID-19) pandemic. Another set of seronegative controls was collected during the COVID-19 pandemic. Antibody detection and levels within the samples were validated using commercial kits and Luminex. Analyses were performed using GraphPad Prism, and the sensitivity, specificity and background cut-off were calculated. Results and Discussion This low-cost ELISA (£0.96/test) assay has a high prediction of 98.9%, and sensitivity and specificity of 97% and 99%, respectively. The assay was subsequently used to screen plasma from SARS-CoV-2 RT-PCR-positive Ghanaians. The assay showed no significant difference in nucleocapsid antibody levels between symptomatic and asymptomatic, with an increase of the levels over time. This is in line with our previous publication. Conclusion This study developed a low-cost and transferable assay that enables highly sensitive and specific detection of human anti-SARS-CoV-2 IgG antibodies. This assay can be modified to include additional antigens and used for continuous monitoring of sero-exposure to SARS-CoV-2 in West Africa.

Transient Expression in HEK-293 Cells in Suspension Culture as a Rapid and Powerful Tool: SARS-CoV-2 N and Chimeric SARS-CoV-2N-CD154 Proteins as a Case Study

“…An analysis of the results published demonstrated that in several cases the N protein showed lower molecular bands probably caused by degradation, similar to those observed in our work. This phenomenon was seen in the four expression systems: E. coli [34,38,39,[42][43][44][45][46][47][48][49], mammalian cells [19,20,51,58,59], plants [39,60,61] and insects [63]; it was more obvious in eukaryotics. However, most of them did not mention and none of them explained the degradation pattern of the expressed SARS-CoV-2 N protein.…”

“…The SARS-CoV-2 N protein has mainly been produced in E. coli. There are at least 42 scientific publications related to this topic [31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49]. The other expression system most used for this purpose is mammalian cells, specifically HEK-293 [19,20,[50][51][52][53][54][55][56][57] and CHO cells [58,59], with intracellular and/or extracellular protein expression.…”

Formation of metal-biomaterials nanocomposites and their application for the development of immunosensors

“…In 1969, Guilbault and Montalvo developed the first potentiometric biosensor for urea detection (Guilbault & Montalvo, 1969). In 1971, Engvall and Perlman independently discovered the enzyme-linked immunosorbent assay method (ELISA) for detecting hormones and viruses, a technique widely utilized to this day (Alfie et al, 2023;Engvall & Perlmann, 1971;Tapela et al, 2023;Van Weemen & Schuurs, 1971). Subsequently, the first commercial biosensor entered the market in 1975, introduced by Yellow Spring Instruments (Yoo & Lee, 2010).…”