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%.