2.

Characterization of anti-CD45 nanobodies. (A) Purification of anti-CD45 nanobodies. Nanobodies were expressed in E. coli and purified by SpySwitch affinity chromatography, followed by SDS-PAGE ± dithiothreitol (DTT) and Coomassie staining to assess disulfide bond formation. The experiment was conducted once. (B) Schematic of the organization of CD45, including extracellular domains d1–d4. (C) Binding of nanobodies to purified CD45. Nanobodies were coated on a plate and incubated with the indicated concentration of biotinylated human CD45 domains 1 and 2 (CD45d1d2), followed by colorimetric ELISA detection (absorbance at 450 nm). Anti-HER2 nanobody was used as a negative control. Each triplicate data point is shown with a line connecting the mean. Representative ELISA data were obtained from two independent experiments. (D) Binding of anti-CD45 nanobodies at the cell surface by flow cytometry. Anti-CD45 nanobodies were incubated with Expi293F, NK92, or YTS cells. Nanobody binding was detected using anti-VHH-Alexa Fluor 647. Anti-CD45 antibody was used as a positive control, with anti-HER2 nanobodies or unstained (no binder) cells as negative controls. Representative flow cytometry data were obtained from two independent experiments for Expi293F and YTS and one experiment with all three cell lines.