BLI-based competitive assay
The ability of the sera of the COVID convalescent patient to compete
with ACE2 for binding to RBDWT and
RBDTRIP was tested in a sandwich format assay on the
Octet RED96e (Fortebio). Anti-penta-His (HIS1K) biosensors were loaded
for 10 min with RBDWT and RBDTRIP at a
concentration of 7.5 µg/ml in BLI assay buffer followed by addition of
samples (diluted 1:20 in BLI assay buffer) from convalescent human sera.
To assess whether the sera can inhibit the binding of ACE2 to
RBDWT and RBDTRIP, ACE2-mFc (50 nM) was
added to biosensor. For control two additional sensors with BLI buffer
were used, one for baseline and one without serum sample to determine
binding of ACE2-mFc alone. The results are expressed of single
individual. The response data were normalized using ForteBio data
analysis software version1.2.0.1.55.