SARS-CoV-2 Nucleocapsid Protein ELISA

The native recombinant nucleocapsid proteins (NPs) of SARS-CoV, SARS-CoV-2 or variants, MERS-CoV, HKU1, OC43, NL63, and 229E (Sino Biological) (2 μg/mL) or denatured by treatment with the denaturing buffer containing 0.5% SDS and 40 mM DTT [18 (link)] (New England Biolabs) at 95 °C for 10 min were coated into 96-well half-area plates overnight at 4 °C. The plates were then blocked with blocking buffer (PBS containing 4% skim milk) at 37 °C for 1 h. Five-fold or three-fold serial-diluted NP-specific mAbs or Rabbit pAb against NP of SARS-CoV-2 (Sino Biological) were added to the plates in duplicate and incubated for 1 h at 37 °C. And then HRP-conjugated Goat anti-Human IgG (ZSGB-BIO) or Goat anti-Rabbit IgG (TransGen Biotech) secondary antibody was added to the plates and incubated at 37 °C for 1 h. For competitive ELISA, after blocking, serially diluted P301-F7, P301-H5, and human IgG1 were mixed with HRP-conjugated P301-F7, and then added to the plates and incubated at 37 °C for 1 h. The enzymatic reaction was developed with TMB substrate (BD Biosciences) and stopped by 2 M H₂SO₄. The optical density was measured at 450 nm (OD. 450 nm) with a Varioskan™ LUX Multimode Microplate Reader (Thermo Scientific).