Development of a double‐antibody sandwich ELISA for detection of SARS‐CoV‐2 variants based on nucleocapsid protein‐specific antibodies

Abstract The COVID‐19 pandemic, driven by the SARS‐CoV‐2 virus, has posed a severe threat to global public health. Rapid, reliable, and easy‐to‐use detection methods for SARS‐CoV‐2 variants are critical for effective epidemic prevention and control. The N protein of SARS‐CoV‐2 serves as an ideal target for antigen detection. In this study, we achieved soluble expression of the recombinant SARS‐CoV‐2 N protein using an Escherichia coli expression system and generated specific monoclonal antibodies by immunizing BALB/c mice. We successfully developed 10 monoclonal antibodies against the N protein, designated 5B7, 5F2‐C11, 5E2‐E8, 6C3‐D8, 7C8, 9F2‐E9, 12H5‐D11, 13G2‐C10, 14E9‐F6, and 15H3‐E10. Using these antibodies, we established a sandwich ELISA with 6C3‐D8 as the capture antibody and 5F2‐C11 as the detection antibody. The assay demonstrated a sensitivity of 0.78 ng/mL and showed no cross‐reactivity with MERS‐CoV, HCoV‐OC43, HCoV‐NL63, and HCoV‐229E. Furthermore, this method successfully detected both wild‐type SARS‐CoV‐2 and its variants, including Alpha, Beta, Delta, and Omicron. These findings indicate that our sandwich ELISA exhibits excellent sensitivity, specificity, and broad‐spectrum applicability, providing a robust tool for detecting SARS‐CoV‐2 variants.