Editing Alpaca Antibody (section)

== Screening and selection of phage antibodies ==

<br/>Selection was carried out by “panning” of VHH-displayed phage libraries for phage that bind to immunotubes (Nunc) coated overnight at 4° C with 5 μg/ml soluble ovTNF. The tubes were then washed three times with PBS, and blocked with 4% non-fat dried milk in PBS (MPBS) at 37° C for 2 h. A 4 ml suspension of phage in MPBS was prepared containing 5.0×1011 CFU was incubated in an immunotube at room temperature for 30 min with continuous rotation, and then for a further 90 min without rotation. The tubes were washed 20 times with PBS containing 0.1% Tween 20 (PBST) followed by 20 times with PBS. Bound phage were eluted by continuous rotation with 1 ml of 100 mM triethanolamine (Sigma) for 10 min, then, recovered and neutralised with 0.2 ml of 1 M Tris–HCl, pH 4.5. A 0.75 ml aliquot of the eluted phage was used to infect 10ml culture of log-phase E. coli TG1 cells. A small aliquot of the infected bacteria was used in serial dilutions to titrate the number of phage eluted while the remainder was processed as described above to amplify the phagemid for further selection or analysis. The binding of selected VHHs encoded by phagemid clones to ovTNFα was tested by phage ELISA using anti-M13 antibody (GE Healthcare) for detection. Positive clones were “fingerprinted” by analysis of their BstN1 digestion patterns (Tomlinson et al., 1992).