Mers cov

Aptamers were generated by in vitro selection, from a diverse starting library of 10¹⁴ different sequences. Aptamer selection was performed by Aptamer Group (York, UK) according to proprietary automated selection methods. These aptamers are commercially known as Optimer binders. Briefly, DNA aptamers were selected against the S1 domain of the SARS‐Cov‐2 Spike protein (Sino Biological, Eschborn, Germany) through 8 successive rounds of selection and preferential amplification. Following identification of the best performing individual aptamer sequences, the minimal functional fragment of the aptamers (the Optimers), were identified and assessed for binding to the SARS‐CoV‐2 S1 domain and the SARS‐Cov‐2 Spike protein trimer (Peak Protein, Macclesfield, UK) by Bio‐Layer Interferometry (BLI) using an Octet Red 384 system (Sartorius, Goettingen, Germany). Cross‐reactivity to the homologous SARS‐CoV, and MERS‐CoV (Sino Biological), was also assessed.

MaxiSorp 96‐well plates (Thermo Fisher) were coated with 0.1 μg recombinant spike [S1, S2, or full‐length S (S1 + S2), as indicated] or nucleocapsid proteins of OC43, 229E, SARS‐CoV‐2, or MERS‐CoV (Sino Biological) per well overnight at 4°C. The plates were washed with PBST (PBS containing 0.05% Tween 20) and blocked with blocking buffer (5% nonfat milk in PBST) for 2 hours. Human sera were heat treated at 56°C for 30 min and were serially threefold diluted from 1:100 to 1:2700 with blocking buffer. Diluted sera (100 μL per well) were added in duplicate to the plate and incubated for 1 hour, followed by detection using 1:10000 diluted HRP‐conjugated goat anti‐human IgG secondary antibody (100 μL per well). TMB substrate (100 μL per well) (Thermo Scientific) was added to the plate for colorimetric signal formation for 10 min and stopped by adding 50 μL per well of 2 M sulphuric acid. Plates were read at wavelength of 450 nm for absorbance (OD 450 nm). In each ELISA plate, the mean OD 450 nm from wells without human sera (n = 8 per plate) was calculated as the background. The area under the curve (AUC) was calculated for each serially diluted sera after subtracting the background.

The recombinant S, S1, RBD and S2 proteins derived from the wild-type SARS-CoV-2, and S1 proteins or S proteins of SARS-CoV-1 and MERS-CoV (Sino Biological), were diluted to final concentrations of 0.5 or 2 μg ml⁻¹, coated onto 96-well plates and incubated overnight at 4 °C. The plates were washed with PBS-T (PBS containing 0.05% Tween 20) and blocked with blocking buffer (PBS containing 5% skim milk and 2% BSA) at room temperature for 1 h. Serially diluted plasma samples or mAbs were added to the plates and incubated at 37 °C for 1 h. After extensive washing, the plates were then incubated with secondary anti-human IgG labeled with HRP (1:5,000 dilution) (ZSGB-BIO) at 37 °C for 30 min or 1 h before incubation with TMB substrate (Kinghawk) at room temperature for 5 min or 20 min. Optical density was measured by a spectrophotometer at 450 nm.