Humoral-response assessment
Enzyme-linked immunosorbent assay (ELISA) COVID-19 receptor-binding
domain (RBD) IgG (TestLine Clinical Diagnostics, Brno, Czech Republic)
kits were used to measure anti- SARS-CoV-2 IgG titers (positive cutoff
value > 18 U/mL), and anti-nucleocapsid (NCP), anti-Spike
2, anti-protein E, anti-ACE2, and RBD antibodies (positive cutoff value
> 180 U/mL) were measured using the immunoblot assay (IB,
Microblot-Array COVID-19 IgG, TestLine Clinical Diagnostics, Brno, Czech
Republic).
The virus neutralization test (VNT) was performed according to a
previously published protocol using the SARS-CoV-2 strain extracted from
a clinical sample (hCoV-19/Czech
Republic/NRL_9640/2020|EPI_ISL_626593) and CV-1 cells
(African green monkey kidney fibroblasts). Serum samples were diluted to
a final serum concentration of 1/10-/2560. Thereafter, uninfected cells
were stained with neutral red dye. VNT results were expressed in the
form of a virus-neutralization titer, representing an inverted value of
the highest sample dilution neutralizing the virus’s cytopathic effect
by > 50%. Positivity was determined using a titer ≥ 20.
Responders were defined as individuals in whom anti-RBD SARS-CoV-2
specific antibodies exceeding the positive cutoff level were detected by
IB and ELISA at month 1. The humoral response was also determined by
measuring serum B-cell-activating factor (BAFF), a
proliferation-inducing ligand (APRIL), and interferon-α (IFN-ɑ) levels
using ELISA (BAFF, R&D Systems, Minneapolis, MN, USA; APRIL, Abcam,
Cambridge, UK; and IFNɑ, Thermo Fisher Scientific).
Anti-RBD SARS-CoV-2 specific antibody presence was also tested in
immunoglobulin replacement therapy (IRT) solutions used for
immunoglobulin substitution in the enrolled patients with CVID (Kiovig
and HyQvia, Takeda Manufacturing, Vienna, Austria; Hizentra, CSL
Behring, Marburg, Germany). All solutions were diluted with a 5% bovine
serum albumin (BSA, lyophilized IgG-free powder, Merck KGaA, Darmstadt,
Germany) solution to a 1% concentration, corresponding to the IgG
concentration in human plasma.