Development and characterization of monoclonal antibodies recognizing nucleocapsid protein of multiple SARS-CoV-2 variants

Rapid antigen test (RAT) is widely used for SARS-CoV-2 infection diagnostics. However, test sensitivity has decreased recently due to the emergence of the Omicron variant and its sublineages. Here we developed a panel of SARS-CoV-2 nucleocapsid protein (NP) specific mouse monoclonal antibodies (mAbs) and assessed their sensitivity and specificity to important SARS-CoV-2 variants. We identified seven mAbs that exhibited strong reactivity to SARS-CoV-2 variants and recombinant NP (rNP) by Western immunoblot or ELISA. Their specificity to SARS-CoV-2 was confirmed by negative or low reactivity to rNPs from SARS-CoV-1, MERS, and common human coronaviruses (HCoV-HKU1, HCoV-CO43, HCoV-NL63, and HCoV-229E). These seven mAbs were further tested by immunoplaque assay against selected variants of concern (VOCs), including two Omicron sublineages, and five mAbs (F461G13, F461G7, F459G7, F457G3, and F461G6), showed strong reactions, warranting further suitability testing for the development of diagnostic assay.