S1 s2 ecd

The amino acid sequence of the SARS-CoV-2 strain (MN908947) was analyzed and the 20-mer peptides with an overlap of 10 amino acid (aa) residues, partially covering four structural proteins of SARS-CoV-2 (i.e., Spike, Envelope, Membrane, and N proteins), were chemically synthesized by GenScript (Jiangsu, China). The microarray yielded 131 peptides (Table S1). In order to assess the protein-peptide interactions, a peptide and protein hybrid microarray (PPHM) was designed using RBD (GenScript, Jiangsu, China), (S1+S2) ECD (Sino Biological, Beijing, China) and the Nucleotide protein (N protein, VACURE Biotechnology, Sichuan, China) and 131 peptides of SARS-CoV-2, which finally yielded 136 peptides and proteins in total. Each well of the chip had a 4x4 rectangular microarray, with three human immunoglobulin G (IgG)-positive controls and one negative control in the four corners. The PPHM was screened to obtain the indicated peptides for detecting COVID-19 patients with both high sensitivity and high specificity, detailed descriptions was showed in supplementary material (Appendix 1). The probes included in the center of the microarray were M1, N16, N24, S15, S39, S44, S64, S82, S95, S104, and S115 (see Table 1).

Serum IgG antibodies against SARS-CoV-2 Spike protein were measured by an ELISA developed and standardized in our laboratory. Briefly, 96-well microplates (Immulon 4HBX, Thermo Scientific) were coated with recombinant NCP-CoV (2019-nCoV) Spike protein (S1+S2 ECD) (Sino Biological #40589-V08B1) at 2 μg/mL for 1 hr at room temperature. Plates were then washed with Tween-20 0.05% in PBS (PBST) and blocked with assay buffer (1% BSA in PBS) for 1 hr at 37°C. After blocking, all subsequent steps were performed by a DYNEX DS2® Automated ELISA system (DYNEX Technologies, Chantilly, VA, USA). Serum samples diluted 1:50,000 in assay buffer were added in duplicate, and plates were incubated for 2 hrs. Plates were washed with PBST and 100 μL per well of a peroxidase-conjugated goat anti-human IgG (Jackson ImmunoResearch #109-036-098), diluted 1:10,000 in assay buffer, were added. After 1 hr incubation, plates were washed, and a stabilized 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (Sigma) was added to the wells. The enzymatic reaction was stopped after 20 min with a Stop solution (1 M sulfuric acid), and absorbance at 450 nm was read by the DYNEX DS2 instrument.