Tetramethylbenzidine substrate

SARS-CoV-2 NP and S-RBD specific IgG antibodies in plasma were measured by in-house ELISA separately. Purified NP and S-RBD antigens were coated onto MaxiSorp ELISA plates (Corning Costar, Acton, MA, USA) in 0.1 M carbonate buffer (pH 9·6) at concentration of 0.2 μg/mL overnight at 4 °C, respectively. Plates were washed 4 times with phosphate-buffered saline (PBS) containing 0.1% vol/vol Tween-20 (PBST) and blocked with 1% bovine serum albumin for 2 h at 37 °C. The plates were then washed with PBST. The serum samples were diluted to 80-fold into PBS as initial concentration, and then serial 2-fold diluted until 81920-fold. The serial dilutions of serum samples were added to the plate wells and incubated, followed by wash and incubation with anti-human IgG HRP-conjugated coat secondary antibody (Abcam, Cambridge, UK). After 4 washes, plates were developed by tetramethylbenzidine substrate (TianGen Biotech Co., Beijing, China) at room temperature in the dark. The absorbance was measured at 450 nm using a microplate reader (Molecular Devices Co., Sunnyvale, CA, USA) after adding the stop solution (2 M sulphuric acid). All samples were run in duplicate. The titers of NP and S-RBD specific IgG antibodies were defined as the highest dilution giving an absorbance value of more than 2.1 times that of the negative control.