Figure 1.

Identification and characterization of nanobodies (Nbs) against human CD4 (hCD4). (A) Amino acid sequences of the complementarity determining region (CDR) 3 from unique CD4-Nbs selected after two rounds of biopanning are listed. (B) Recombinant expression and purification of CD4-Nbs using immobilized metal ion affinity chromatography (IMAC) and size exclusion chromatography (SEC). Coomassie-stained SDS-PAGE of 2 µg of purified Nbs is shown. (C) Representative images of live CHO-hCD4 cells stained with CD4-Nbs for 30 min at 4°C (top row) or 60 min at 37°C (bottom row); scale bar: 50 µm. (D) For biolayer interferometry (BLI)-based affinity measurements, biotinylated hCD4 was immobilized on streptavidin biosensors. Kinetic measurements were performed using four concentrations of purified Nbs ranging from 15.6 to 1,000 nM. As an example, the sensogram of CD4-Nb1 at indicated concentrations is shown. (E) EC₅₀ determination by flow cytometry. Exemplarily shown for CD4-Nb1, the percentage of positively stained HEK293-hCD4 (frequency of parent) was plotted against indicated concentrations of CD4-Nbs.