Construction and next-generation sequencing analysis of a large phage-displayed V(NAR) single-domain antibody library from six naïve nurse sharks

Feng, Mingqian; Bian, Hejiao; Wu, Xiaolin; Fu, Tianyun; Fu, Ying; Hong, Jessica; Fleming, Bryan D; Flajnik, Martin F; Ho, Mitchell

The phage panning protocol has been described previously [44, 45] . Briefly, a 96 well Maxisorp ELISA plate (Nunc/Thermo Fisher Scientific, Rochester, NY) was used to capture various antigens (100 μg/ml) in PBS buffer at 4 • C overnight. After the coating buffer was decanted, the plate was treated with blocking buffer (2% bovine serum albumin in PBS) at room temperature for 1 h. Then 30 μl pre-blocked phage supernatant (typically contained 10 10 -10 11 cfu) in 30 μl blocking buffer was added per well for 1 h at room temperature to allow binding. After four washes with PBS containing 0.05% Tween-20, bound phages were eluted with 100 mM triethylamine. After four rounds of panning, single colonies were picked and identified by using phage ELISA.