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 shark single-domain soluble protein PE38-B6-His was buffer exchanged in PBS buffer after purification. The binding kinetics of PE38-B6-His was measured with ForteBio Octet RED96 located at the Biophysics Core in National Heart, Lung and Blood Institute. PE38-B6-His diluted in assay buffer PBS supplemented with 0.1% Tween-20 and 1% (w/v) BSA. The Octet RED96 program was as follows: 10 min presoak, 120 s baseline establishment, 300 s antigen loading, 60 s baseline re-establishment after antigen loading, 600 s PE38-B6-His association, 30 min dissociation. A total of 1 μg/ml PE38-B6-his was used to load the Ni-NTA biosensor, and serial diluted antigen protein PE38 was used for binding assay. The binding kinetics was calculated with forteBio Octet RED96 software.