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.