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. 2021 Apr 27;22(5):e52325. doi: 10.15252/embr.202052325

Figure 2. Biochemical characterization of RBD‐specific Nbs.

Figure 2

  1. Amino acid sequences of the complementarity determining region (CDR) 3 from unique Nbs selected after two rounds of biopanning are listed (upper panel). Phylogenetic tree based on a ClustalW alignment of the Nb sequences is shown (lower panel).
  2. Recombinant expression and purification of Nbs using immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC). Coomassie staining of 2 µg of purified Nbs is shown.
  3. For biolayer interferometry (BLI)‐based affinity measurements, biotinylated RBD was immobilized on streptavidin biosensors. Kinetic measurements were performed by using four concentrations of purified Nbs ranging from 15.6 nM to 2 µM. As an example, the sensogram of NM1230 at indicated concentrations is shown (upper panel). The table summarizes affinities (K D), association (k on), and dissociation constants (k off) determined for individual Nbs (lower panel).