Development of Monoclonal Antibodies Against SARS-CoV-2 Nucleocapsid Protein for COVID-19 Antigen Detection

Background The coronavirus disease 2019 (COVID-19) pandemic underscored the global need for reliable diagnostic tools with quick turnaround time for effective patient management and mitigation of virus spread. This study aimed to express severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein and produce monoclonal antibodies (mAbs) against the expressed protein. Methods Following successful expression and purification of His-tagged SARS-CoV-2 N protein using a wheat germ cell-free protein expression system (WGCFS), BALB/c mice were immunized, and generated hybridomas screened for mAb production. Indirect and sandwich ELISA were used to screen the reactivity of the monoclonal antibody against both our recombinant antigen and commercial antigen. The mAbs were also assessed for their performance using RT-PCR confirmed positive samples with varying cycle threshold (CT) values and their specificity screened using intracellular fluid (ICF) of other respiratory viruses. Results Our mAb demonstrated high reactivity against our recombinant antigen, commercial antigen, SARS-CoV-2 Beta and Omicron variants. There was no significant difference in the binding affinity of our mAb and commercial mAb against the study recombinant (p = 0.12) and commercial (p = 0.072) antigens. Our mAb detected SARS-CoV-2 from clinical samples with varying CT values and exhibited no cross-reactivity against other respiratory viruses. Conclusion We successfully expressed SARS-CoV-2 N protein leveraging WGCFS in a resource-limited setting. Our mAb had a high binding affinity to the recombinant antigen, making it a suitable candidate for antigen detection kit development. Beyond diagnostics, the mAb holds potential for therapeutic applications as well as use in clinical and environmental surveillance platforms.