Large-Scale Antibody Production and Characterization

To produce large-scale antibodies, selected hybridomas were media-adapted in serum-free media (Gibco, USA) and introduced into a 1 L CELLine bioreactor (Corning, USA). The harvested culture supernatants from the bioreactors were concentrated using a Jumbosep™ concentrator (PALL Corporation, USA) and purified on a Protein-G column (Thermo Scientific, USA). The purified antibodies were dialyzed/concentrated on a Vivaspin column 20 concentrator (Cytiva, USA). The purities of the mAbs were analyzed using capillary gel electrophoresis (Agilent system, USA). The antibody reactivity toward the recombinant (S) was assessed using conventional ELISA and Western blot performance. A 96-well ELISA plate was coated overnight at 4°C with 50 µL of (S) diluted to 1 µg/mL in 0.05 M carbonate buffer (pH 9.6). The next day, the wells were washed with PBS and blocked with 150 µL of 5% non-fat milk protein for 1 hour at room temperature (20 - 25°C). After blocking, milk protein was removed from the plate, washed briefly with PBS, and duplicates of each mAb sample were added to test and control wells. The plates were subsequently washed with PBS-T, and bound antibodies were detected by HRP-conjugated goat anti-mouse IgG (H + L) secondary antibody (1:1000, Bethyle Lab, USA), and colorized with KPL SureBlue TMB Microwell Peroxidase Substrate (Seracare, USA). The enzyme-substrate reaction was stopped by adding 1 N sulfuric acid. Finally, the optical densities (OD) were measured at 450 nm, and the mean OD of the control wells was deducted from the mean OD of the test wells to obtain the final OD value for each sample. Western blot analysis was performed using SARS-CoV-2 full-length recombinant S, HCOV-HKU1, Middle East respiratory syndrome (MERS) coronavirus, SARS-Related Coronavirus-2, SARS-CoV-2 S receptor binding domain (RBD) recombinant protein, and Vero-Cell E6 Lysate as a negative control. Samples were separated on NuPAGE Novex 4–12% Bis-Tris Gels (Invitrogen, USA) under reducing conditions with the addition of NuPAGE Reducing Agent (Invitrogen, USA) and heating of the samples for 5 min at 95 C. After electrophoresis, protein bands were transferred onto nitrocellulose membranes. The membrane was blocked with 5% non-fat milk protein and probed with selected anti-S mAbs followed by an IRDye conjugated goat anti-mouse IgG (H + L) secondary antibody (LICOR, USA). The specific bands were visualized using the LICOR Odyssey system.