Magnabind streptavidin

The nanobodies binding to SARS-CoV-2 RBD were selected by phage display. Briefly, 60 µg biotinylated RBD was immobilized onto 20 µL MagnaBind Streptavidin (Thermo Fisher Scientific, Waltham, MA, USA) in PBS. After washing the beads with PBST (PBS containing 0.1% Tween20), the phage library (10¹¹ phages/selection round) and the beads (bioRBD-coated or naked) were pre-blocked in PBS/milk 2% (w/v) and allowed to associate for 60 min at room temperature with mixing. Unbound phages were removed by 6 washes with PBST and 2 washes with PBS. The elution of phage particles bound to the RBD-coated magnetic beads was performed with a 10 mM DTT solution. Aliquots of the eluted phage suspensions from bioRBD-coated or naked beads were used to infect E. coli TG1 cells for evaluating the enrichment, which was at least 100-fold (bioRBD vs. naked beads). Eluted phages were then used to infect exponentially growing E. coli TG1 cells in order to amplify and prepare phage particles for the next selection round. For library 1, one panning round with RBD Wu as the target was performed, whereas library 2 was panned once against BQ.1.1, followed by the XBB.1.5 RBD.

Mice at age between 7 to 10 weeks were sacrificed and the spleens were isolated. After red blood cell lysis, cells were incubated with biotin anti-mouse CD8α (100,704, BioLegend), biotin anti-mouse I-A/I-E (107,604, BioLegend), biotin anti-mouse NK1.1 (108,704, BioLegend), biotin anti-mouse/human B220 (103,204, BioLegend), biotin anti-mouse CD49b (103,522, BioLegend), biotin anti-mouse CD19 (115,504, BioLegend), biotin anti-mouse/human CD11b (101,204, BioLegend), and biotin anti-mouse CD11c (117,304, BioLegend). Antibody-bound cells were then negatively selected by using MagnaBind™ Streptavidin (21,344, Thermo Fisher Scientific). Subsequently, Biotin anti-mouse CD25 (102,004, BioLegend) and MojoSort™ streptavidin nanobeads (480,016, BioLegend) were used for positive selection. The cells were purified by magnetic separation using LS Columns (130–042-401, Miltenyi Biotec) according to the manufacturer’s instructions.
The isolated cells were activated with plate-bound anti-CD3ε (145-2C11; 5 μg/ml) and soluble anti-CD28 (37.51; 2 μg/ml). For tTreg culture, mouse recombinant IL-2 (50 ng/ml), human recombinant TGF-β1 (5 ng/ml), XMG1.2 (10 μg/ml), and 11B11 (10 μg/ml) were added to the cell culture medium. All cytokines for differentiation were purchased from eBioscience.