Serological assays for SARS-CoV-2 are being utilized at an exponential rate for surveillance programs. This enterprise was designed to develop and validate a qualitative immunochromatographic test, via the Lateral Flow Assay (LFA), for detection of immunoglobulins M and G (IgM and IgG) against both nucleocapsid (N) and the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2. Both targeted proteins were cloned and expressed in baculovirus expression system utilizing insect cells Sf9. The recombinant RBD and N proteins were purified and conjugated with gold nanoparticles (AuNPs) to set up the coating antigens pad. Both anti-human IgG and IgM were dispensed on nitrocellulose membrane to capture human antibodies in serum samples. A home-made dispensing system was developed to draw identical test and control lines. The validity of the developed LFA was verified by testing serum samples from 103 convalescent COVID-19 patients who were PCR positive for SARS-CoV-2 along with 28 control serum samples. The developed strips showed distinctive bands for IgM and IgG of both proteins (RBD and N) in positive samples. The sensitivity of RBD-based LFA was 70.9% and 39.8% for IgG and IgM, respectively, with a specificity of 100% for both. The N-based LFA exhibited a sensitivity of 73.8% and 35.9% for IgG and IgM, respectively, while its specificity was 75% and 100% for IgG and IgM, respectively. Our developed LFA could afford a tool for surveillance programs in low-resource countries. Moreover, it might be functional for rapid and inexpensive monitoring of the anti-SARS-CoV-2 antibodies in the sera of vaccinated individuals.

Background: SARS-CoV-2 genome encodes different kinds of structural and nonstructural proteins. The spike structural protein is the prime target for vaccine manufacturing, drug development, and diagnostic assays. Methods: In this cross-sectional study, we assessed the developed Receptor Binding Domain (RBD) protein in 520 serum samples. The samples were stratified according to vaccine status into two groups: Group 1 consisted of 274 pre-vaccinated samples collected from April to December 2020 (109 were asymptomatic contacted with COVID-19-patients; 100 patients had positive RT-PCR test for SARS-CoV-2 infection, and 65 were convalescent-COVID-19 cases), and group 2 consisted of 246 post-vaccinated participants with or without past-infection recruited from students, coworkers, and staff members from November 2021 to March 2022. Indirect Enzyme-Linked Immunosorbent Assay (ELISA) was performed to evaluate the seroprevalence of the SARS-CoV-2 immunoglobulin G (IgG) across both groups. Results: The sensitivity and specificity of the RBD-developed protein were 88.4% (95% CI: 84% - 92%) and 100% (95% CI: 88%- 100%), respectively in the pre-vaccinated group. 213 out of 274 of the pre-vaccinated samples were positive. Meanwhile, the RBD-developed protein provided 100% RBD IgG seropositivity in the recovered COVID-19 cases. Moreover, the results were seropositive in all post-vaccinated candidates (n=246) and the results were reliable in 70 negative pre-pandemic sera specimens. Conclusion: The developed S-RBD protein has the potential to be a successful screening method in COVID-19-vaccinated or infected individuals.