S-RBD IgG and surrogate virus neutralization test (sVNT)
The SARS-CoV-2 S receptor-binding domain (S-RBD) IgG enzyme-linked immunosorbent assay (ELISA) were carried out as previously described and validated.15,25 sVNT was conducted according to the manufacturer’s instructions (GenScript Inc, Piscataway, USA) and as described in our previous publications.25,39 All sera were heat-inactivated at 56° C for 30 minutes before testing.
In brief, S-RBD IgG ELISA plates were coated overnight with 100 ng/well of purified recombinant S-RBD in PBS buffer, followed by addition of 100 µL Chonblock Blocking/Sample Dilution (CBSD) ELISA buffer (Chondrex Inc, Redmond, USA). This was incubated at room temperature (RT) for 2 hours. Serum was tested at a dilution of 1:100 in CBSD ELISA buffer, then added to the wells for 2 hours at 37°C. After washing with PBS containing 0.1% Tween 20, horseradish peroxidase (HRP)-conjugated goat anti-human IgG (1:5,000) (GE Healthcare, Chicago, USA) was added for 1 hour at 37°C, followed by washing five times with PBS containing 0.1% Tween 20. HRP substrate (Ncm TMB One, New Cell & Molecular Biotech Co. Ltd, China) of 100 µL was added for 15 minutes, and the reaction was stopped by 50 µL of 2 M H2SO4. The OD was analysed in a Sunrise absorbance microplate reader (Tecan, Männedorf, Switzerland) at 450 nm wavelength. The background OD in PBS-coated control wells with the participant’s serum was subtracted from each OD reading. Values at or above an OD450 of 0.5 were considered positive and values below were imputed as 0.25.
The sVNT was performed using 10 µL of each serum, positive and negative controls, which were diluted at 1:10 and mixed with an equal volume HRP conjugated to the WT or BA.1 SARS-CoV-2 S-RBD (6 ng). The mixture was incubated for 30 minutes at 37°C, then 100 μL of each sample was added to microtitre plate wells coated with angiotensin-converting enzyme-2 (ACE-2) receptor. This plate was sealed for 15 minutes at 37°C and then washed with wash-solution, tapped dry, and 100 μL of 3,3’,5,5’-tetramethylbenzidine (TMB) was added and incubated in the dark at RT for 15 minutes. This reaction was stopped with 50 µL of Stop Solution and the absorbance at 450 nm was detected by a microplate reader. The % inhibition of each serum was calculated as (1 - sample OD value/negative control OD value) x100%. Inhibition (%) of at least 30%, the limit of quantification (LOQ), was regarded as positive, and values below 30% were imputed as 15%.