Fig. 1: Process flow for B-cell purification, V-region amplification, and cloning into pDAN5 phage display vector.

a Schematics of B-cell purification of a single donor repertoire from LeukoPak. b FACS characterization of the CD19⁺ paramagnetically isolated cells for CD37⁺ (left) and CD20⁺ (right) staining. c Rescue of V domain diversity from donor and creation of the scFv phage antibody library: specific constant domain primers are used for the RT-reaction, followed by V domain amplification with IGKV/IGLV and IGHV-specific forward and reverse primers (PCR1). A second PCR introduces vector and linker overlaps (PCR2) that are exploited for the assembly of the scFv genes (PCR3). The genes are cloned into the pDAN5 phagemid vector by restriction enzyme digestion and transformed into bacteria. Upon superinfection with M13K07 helper phage, the phage is produced (primary library) and used to infect the recombinase positive bacteria (Cre+) at a multiplicity of infection of 1:200. The presence of incompatible lox sites between IGKV/IGLV and IGHV domains and downstream g3p (in black and white) allows recombination to occur between different VH and VL genes carried by different phagemid vectors. The process yields a phage population (secondary library) that has greater recombinatorial diversity than the primary library. A final phage production step, at a multiplicity of infection ≤1 restores the essential phenotype/genotype coupling (tertiary library/final library).