Background: Diagnosis is an important factor in controlling disease. Single-chain fragment variables (scFvs) can be used for diagnosis; however, due to their immobilization issues, their application has been limited. Herein, we isolated a SARS-CoV-2 nucleocapsid phosphoprotein (NP)-specific scFv and propose it as a diagnostic tool in the scFv-displaying phage format to overcome the immobilization issue.

Method: Spleen from NP-immunized BALB/c mice was isolated, total RNA was extracted, and cDNA was synthesized. An scFv library was constructed, using the splicing by overlap extension (SOE) PCR technique, which was cloned into the pCANTAB5E phagemid. The phage library was panned against the NP antigen, and the output phages with the highest binding capability were screened for the most qualified scFv, which was later assessed in terms of sensitivity and specificity.

Results: The scFv-displaying phage library was panned against the recombinant NP in three rounds and 40 randomly selected colonies from the third round's outputs were screened. Alongside several clones, clone #31 was chosen as the most qualified scFv, which later exhibited favorable sensitivity and specificity against NP in further ELISA-based experiments.

Conclusions: Clone #31 could be utilized to develop diagnostic tools and therapeutics against SARS-CoV-2.

Keywords: COVID-19; SARS-CoV-2; nucleocapsid; phage display; recombinant antibodies.