Phage display

Fab libraries displayed on phage coat protein IX were panned against biotinylated human TF ECD. Phage was produced by helper phage infection. Binders were retrieved by addition of streptavidin beads to form a bead/antigen/phage complex. After the final wash, phage was rescued by infection of exponentially growing Escherichia coli TG-1 cells. Phage was again produced and subjected to additional rounds of panning.

The pIX gene was excised by NheI/SpeI digestion from the selected clones to allow for secreted soluble Fab production. After religation, the DNA was transformed into TG-1 cells and grown on LB/Agar plates overnight. The cultures were used for (1) colony PCR and sequencing of the V-regions, and (2) soluble Fab production. The soluble Fabs were captured onto plates by a polyclonal anti-human Fd antibody. After appropriate washing and blocking, biotinylated TF ECD was added at 0.2 nM and was detected by HRP-conjugated streptavidin and chemiluminescence. The variants were ranked with respect to the binding of the parent M59 Fab, which was present as a control in all plates.