SARS-CoV-2 N Protein and Epitope ELISA

Ninety-six-well, flat-bottomed microplates were coated with 100 ng/well of N protein (40588-V08B, Sino Biological Inc., Eschborn, Germany) or Ep9 epitope (ANNAAIVLQLPQGTTLPKGFY) [43 (link)] in 50 μL of carbonate–bicarbonate buffer (C-3041, Sigma, St. Louis, MO, USA) and incubated at 4 °C overnight. After washing with PBS-T, the wells were blocked with 200 μL of 25% Block Ace solution for 1 h at RT. Microplates were washed after incubation, 50 µL of patient serum diluted 100× with PBS-T was then added to each well, and the plates were incubated for 1 h at RT. After washing with PBS-T, 50 μL of the secondary antibody solution consisting of peroxidase-conjugated AffiniPure alpaca anti-human IgG (H+L) (609-035-213, Jackson ImmunoResearch, Pennsylvania, PA, USA) or peroxidase-labeled goat anti-mouse IgG (H+L) (5220–0341, CeraCare, Milford, MA, USA) was added to each well and incubated for 1 h at RT. A TMB substrate kit (34021, Thermo Fisher Scientific, Waltham, MA, USA) was used for colorimetric detection, and the optical density at 450 nm was measured using a multigrading microplate reader (SH-9500Lab, Corona, Hitachinaka, Ibaraki, Japan).