Rational design and high-yield expression of SARS-CoV-2 nucleocapsid antigen for a sensitive and specific in-house ELISA

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a global pathogen with a high prevalence rate. Among diagnostic methods, serological assays have been effective for rapid screening and identifying a history of infection or vaccination. The nucleocapsid protein is the most conserved and immunogenic antigen of SARS-CoV-2, making it a key target for such tests. Although commercial kits are available for this purpose, a custom design with superior specificity, sensitivity, and accuracy can be both clinically and economically advantageous. In the present study, we designed a recombinant nucleocapsid protein (rNP) using a multi-parameter suite of bioinformatic approaches, including codon optimization, ribosomal binding site engineering, mRNA secondary structure analysis, and optimized expression of the final construct in an E. coli system. The purified rNP was then applied in an in-house ELISA to compare its overall diagnostic efficacy with that of a commercially available kit on a pool of 37 predetermined positive and 15 negative sera for SARS-CoV-2. Results showed that both assays were 100% specific, but the in-house design showed higher sensitivity (91.9% vs. 83.8%) and a greater area under the curve (0.94 vs. 0.84) compared to the commercial ELISA. This demonstrates that our optimized rNP production platform enables the development of a high-performance serological assay with superior diagnostic accuracy compared to a commercial alternative, providing a powerful and economical tool for surveillance and research in the post-pandemic era.