Editing Antibody Design by AI (section)

== Phage Display panning against target molecule ==

Panning of the library against target etanercept, trastuzumab, bevacizumab, and ranibizumab (in-house expression and purification) was done in solid phase mode 23. 96-well maxisorb plates (Nunc) were coated with the target using 500 nM in first and second round and 200 nM in the third round and then blocked with PBS/5% (w/v) dried milk protein. '''1E12 '''phages (approximately '''50x''' the library size) were blocked in 0.5x Chemiblocker (Millipore)/2.5% (w/v) dried milk protein containing 0.05% (v/v) Tween-20 and applied on the coated wells. The washing regimen was intensified during the selection rounds with 5 cycles (3x quick and 2x 5 minutes) using PBS with 0.05% Tween 20 followed by 5 cycles (3x quick and 2x 5 minutes) using PBS in the first round, 5 cycles (1x quick and 4x 5 minutes) using PBS with 0.05% Tween 20 followed by 5 cycles (1x quick and 4x 5 minutes) using PBS in the second round and 15 cycles (10x quick and 5x 5 minutes) using PBS with 0.05% Tween 20 followed by 15 cycles (10x quick and 5x 5 minutes) using PBS in the third round. For the stringent washing condition the same number of washing cycles were used but with a buffer containing 0.75M NaCl, 2% Octyl β-D-glucopyranoside and 0.1M Tris, pH 7.4 while the last two steps of each regimen were done with PBS. Phages were eluted with 10mM Glycine/HCl pH 2.0. E.coli TG1F+ were infected with eluted phages which were neutralized beforehand using 1M Tris/HCl, pH8.0. Propagation of phages between rounds was performed using '''VCSM13''' helper phage (Agilent).