Sars cov 2 rbd

The affinity of antibody binding SARS-Cov-2-S-RBD was measured via the Biacore X100 platform. The CM5 chip (GE Healthcare) was coupled with an anti-human IgG-Fc antibody to capture 9000 RU antibodies. Gradient concentrations of SARS-Cov-2 RBD (Sino Biological Inc.) were diluted (2-fold dilution, from 50 nM to 0.78 nM) with HBS-EP⁺ Buffer (0.01 M HEPES, 0.15 M NaCl, 0.003 M EDTA and 0.05% (v/v) Surfactant P20, pH 7.4), then injected into the human IgG capturing chip. The sensor surface was regenerated with 3 M magnesium chloride at the end of each cycle. The affinity was calculated using a 1:1 binding fit model in Biacore X100 Evaluation software (Version:2.0.2).

Spike subunit ELISA was based on the method described by Rodda et al (2021 (link)). 96‐well plates (Corning) were coated with 2 µg/ml of recombinant SARS‐CoV‐2 S1+S2 (Sino Biological 40589‐V08H4), SARS‐CoV‐2 S1 (Sino Biological 40591‐V08H), SARS‐CoV‐2 S2 (Sino Biological 40590‐V08H1), SARS‐CoV‐2 RBD (Sino Biological 40592‐V08H), or SARS‐CoV‐2 NTD (Sino Biological 40591‐V49H) diluted in PBS and incubated at 4°C overnight. Plates were washed with PBS‐T (PBS containing 0.05% Tween‐20) and blocked in 2% BSA for 1h at 37°C. Monoclonal antibodies diluted at 10 µg/ml in 0.1% BSA were added and incubated at 37°C for 1 h. After washing three times in PBS‐T, secondary antibodies diluted in 0.1%BSA were added to the wells. Secondary antibodies used were: anti‐human IgG‐HRP (Southern Biotec 2014‐05), anti‐IgM‐HRP (Southern Biotec 2023‐05), anti‐IgA‐HRP (Southern Biotec 2053‐05). Plates were incubated with secondary antibodies for 1 h at 37°C. Plates were then washed three times with PBS‐T and developed with TMB ELISA substrate (SureBlue Reserve TMB Microwell Peroxidase Substrate, REF 53‐00‐00) until a blue color was visible; the reaction was stopped with sulfuric acid and plates were read at 450 nm immediately after stopping.

Analysis of RBD-IgG reaction kinetics was performed on a MASS-16 biosensor instrument (Bruker, Hamburg, Germany). Anti-Human IgG (Fc) (Cytiva, Uppsala, Sweden) was diluted to 25 μg/ml in 10 mM sodium acetate buffer pH 5 and immobilized on a High Capacity Amine Sensor chip (Bruker) (time of interaction: 7 min; flow rate: 10 µl/min). S-protein-specific IgG was diluted in running buffer (Dulbecco’s PBS (HyClone, South Logan, UT, USA) containing 0.01% Tween 20) and allowed to bind during a 90 s long injection (flow rate: 10 µl/min). Its capture level was set to be below 140 RU. The antigen (SARS-CoV-2 RBD (SinoBiological, Beijing, China; product number 40592-V08H) at 0.7-180 nM or Spike protein at 0.4-90 nM in running buffer) was subsequently injected (time of interaction: 2 min; flow rate: 30 µl/min). Dissociation was subsequently allowed to proceed for 5-15 min. The sensor chip was regenerated by treatment with 3 M magnesium chloride solution (Cytiva). All interactions were performed at 25°C. Apparent reaction rate kinetics was determined using a Langmuir 1:1 model using the Sierra Analyser software version 3.4.3 (Bruker).

Serum samples from SARS-CoV-2 infected animals were inactivated by γ-irradiation and used in BSL2 according to IBC-approved SOPs. NUNC Maxisorp Immuno plates were coated with 50 μl of 1 μg/mL of recombinant SARS-CoV-2 S (S1+S2), SARS-CoV-2 RBD (Sino Biological) or EBOV GP at 4°C overnight and then washed three times with PBS containing 0.05% Tween 20 (PBST). The plates were blocked with 3% skim milk in PBS for 3 hours at room temperature, followed by three additional washes with PBST. The plates were incubated with 50 μl of serial dilutions of the samples in PBS containing 1% skim milk for 1 hour at room temperature. After three washes with PBST, the bound antibodies were labeled using 50 μl of 1:2,500 peroxidase anti-hamster IgG (H+L) (SeraCare Life Sciences) diluted in 1% skim milk in PBST. After incubation for 1 h at room temperature and three washes with PBST, 50 μl of KPL ABTS peroxidase substrate solution mix (SeraCare Life Sciences) was added to each well, and the mixture was incubated for 30 min at room temperature. The optical density (OD) at 405 nm was measured using a GloMax® explorer (Promega) plate reader. The OD values were normalized to the baseline samples obtained with naïve hamster serum and the cutoff value was set as the mean OD plus standard deviation of the blank.