Journal: Pharmaceutics

Article Title: Sustained Delivery of a Monoclonal Antibody against SARS-CoV-2 by Microencapsulated Cells: A Proof-of-Concept Study

doi: 10.3390/pharmaceutics14102042

Figure Lengend Snippet: SARS-CoV-2 monoclonal antibody production in cells transfected with the CR3022 heavy and light chains. For western blot analysis ( A ) different amounts of Spike (50–400 ng) and nucleocapsid (Nc, 200–400 ng) proteins were separated on a 10% SDS–PAGE gel, transferred onto a PVDF membrane and then blotted with G-8 cell culture supernatant; probing was done using an HRP-conjugated rabbit IgG specific to both the heavy and light chains of human IgG. Lanes M: protein ladder; Spike lanes: S protein at 50, 100, 200, and 400 ng. Lanes N: N protein at 200 and 400 ng. kDA = kilodaltons. ( B ) CR3022 expression in cultured G-8 cells quantified using receptor binding domain (RBD) ELISA and normalized to the untransfected supernatant readout. Cell media were changed every 24 h. Lines and brackets represent the geometric means and 95% confidence intervals, respectively. ( C ) Microphotographs of G-8 cell-containing alginate microcapsules obtained using light (top left panel at ×4 and top right panel at ×10 magnification) and electron microscopy (bottom left panel at ×800 magnification). ( D ) Supernatants collected from free and encapsulated G-8 cells stably expressing CR3022 at 48 h after last media change. Supernatants (at 1/100 dilution) were incubated on RBD-coated ELISA plates. Lines and brackets represent the geometric means and 95% confidence intervals, respectively. In ( B , D ): mAb concentrations were calculated using a standard curve generated using a CR3022 mAb (NR-52481) from BEI on experimental readouts normalized to the corresponding baseline control (untransfected supernatant) OD readouts (see Methods).

Article Snippet: Spike (NR-722, BEI Resources, NIAID, NIH, Bethesda, MD, USA) and nucleocapsid (NR-48761, BEI Resources) proteins were separated on a 10% SDS–PAGE gel and transferred onto a PVDF (Polyvinylidene fluoride) membrane (#116-2807, Bio-Rad, Munich, Germany) for 1 h at 350 mA.

Techniques: Transfection, Western Blot, SDS Page, Membrane, Cell Culture, Expressing, Binding Assay, Enzyme-linked Immunosorbent Assay, Electron Microscopy, Stable Transfection, Incubation, Generated, Control