Fig. 1

Generation of Nbs. A Schematic diagram of the generation of the Nbs. First, a nanobody library was prepared to obtain Nbs (a), and then, human CD19 or CD20 was added to bind the Nb (b). Finally, streptomycin-labeled magnetic beads were mixed with those complexes (c), followed by several washes (d). A total of 4 rounds of panning was performed. B Amino acid sequence of anti-CD19 and anti-CD20 Nbs. The framework (FR) and complementarity-determining region (CDR) sequences were defined according to the IMGT. CDR1, CDR2 and CDR3 are highlighted in green, blue and red, respectively. C Simulation of the three-dimensional crystal structure of anti-CD19 Nb1-3 and anti-CD20 Nb1-2 using Swiss-model. D Nb’s molecular weight (Mw) and grand average of hydropathicity (GRAVY) were analysed by the online soft ExPAsy-ProtParam Tool. E SDS–PAGE analysis of the molecular weights of the purified anti-CD19 Nb1 and anti-CD20 Nb1 antibodies. F, G Cell binding capacity of nanobodies detected by QD immunofluorescence. Fluorescence was captured by fluorescence microscope, and TBS was used as a control. H, I The affinity between CD19/CD20 and Nbs was determined by SPR binding assays