*4.1. Nb Production*

The cAbVCAM1-5 Nb was generated by immunisation of a dromedary in the context of a previous study, was produced in E. coli as a *C*-terminal His-tagged (six histidine residues) protein and was purified by immobilised metal affinity chromatography and size exclusion chromatography according to standard procedures as described elsewhere [14].

### *4.2. Random-Conjugation of the RESCA Chelator*

The cAbVCAM1-5 Nb was modified by incubation with a 12-fold molar excess of TFP-RESCA (135 mM stock in DMSO) in 0.05 M sodium carbonate buffer (120 μM of Nb, pH 8.5–8.7) for 2 h at RT. The modified Nb was subsequently purified by SEC on a Superdex Peptide 10/300 GL column (GE Healthcare, Belgium) and eluted in 0.1 M NH4OAc pH 7 on a medium-pressure chromatography system (Bio-Rad NGC, Belgium). The average number of chelators per Nb was determined by ESI-Q-ToF-MS (GIGA Proteomics, Liège, Belgium).
