4.

Optimization of NanoBondy clamp site and linker length. (A) AlphaFold prediction of 2H5 NanoBondy (purple) bound to CD45d1d2 (green). The reactive anhydride is shown in cyan, alternative clamp sites are shown in magenta, and linkers are shown in orange. (B) Clamp-site variant reactivity. 2H5 NanoBondy variants were incubated with CD45d at 10.5 μM each for 1 h at 37 °C in HBS ± Ca²⁺, followed by SDS-PAGE with Coomassie staining. Anti-IgG NanoBondy was used as a negative control. The leftmost lane represents CD45d without PNGase F treatment. The experiment was conducted once. (C) Linker variant reactivity for 2H5 R72C anti-CD45 NanoBondy, analyzed as in (B). Representative gels were obtained from two independent experiments.