title: Improving the characteristics of a mycobacterial 16 kDa-specific chicken scFv.
abstract: 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 antigen were 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.
authors.affiliation: Immunology Section, Onderstepoort Veterinary Institute, Private Bag x5, Onderstepoort 0110, South Africa.
authors.forename: Joy
authors.initials: J
authors.lastname: Sixholo
authors.forename: Wouter
authors.initials: W
authors.lastname: Van Wyngaardt
authors.forename: Cordelia
authors.initials: C
authors.lastname: Mashau
authors.forename: Janine
authors.initials: J
authors.lastname: Frischmuth
authors.forename: Dion H
authors.initials: DH
authors.lastname: Du Plessis
authors.forename: Jeanni
authors.initials: J
authors.lastname: Fehrsen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000917:Antibody Formation / Q000235:genetics / Q000502:physiology; D000918:Antibody Specificity / Q000235:genetics; D000942:Antigens, Bacterial / Q000276:immunology*; D002645:Chickens / Q000276:immunology*; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000276:immunology; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification
chemical_list: D000942:Antigens, Bacterial; D050505:Mutant Proteins; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; C072681:Mycobacterium tuberculosis antigens