Abstract:
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 virus isolates 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.
Conclusions 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.
