Recombinant antibodies can be engineered to improve their binding or other characteristics. A chicken
single chain variable fragment (scFv) phage display library was panned against the mycobacterial 16 kDa
antigen. Three fusion phages which bound specifically to the antigenwere selected, each of which produced
low signals in ELISA when secreted as a soluble scFv. One scFv was therefore chosen to be modified in an
attempt to improve its binding. Firstly, a mutant sublibrary was created by random mutagenesis. High
stringency panning of this sublibrary yielded binders which produced ELISA signals up to eleven times
higher than the parent scFv. An increase in the intrinsic affinity was confirmed by surface plasmon resonance.
Secondly, the flexible linker between the heavy and light chains of the parent scFv was either
shortened to one glycine residue or deleted entirely. No ELISA signal was obtained when the linker was
absent, but the glycine-linked scFv showed enhanced binding. Size exclusion chromatography revealed
that the enhanced binder had aggregated to form tetramers. This study confirms that the strategies used to
improve the binding of human and mouse scFvs can also enhance chicken scFvs.