Background: Antibody fragments selected from large combinatorial libraries have numerous applications in diagnosis and therapy. Most existing antibody repertoires are derived from human immunoglobulin genes. Genes from other species can, however, also be used. Because of the way in which gene conversion introduces diversity, the naïve antibody repertoire of the chicken can easily be accessed using only two sets of primers.

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a b s t r a c tTwo chicken single-chain variable region antibody fragments (scFvs) that recognised the 65 kDa heatshock protein (HSP65) of Mycobacterium bovis were selected from a large semi-synthetic phage displayed library. Both recognised HSP65 in indirect enzyme-linked immunosorbent assay (ELISA) and immunoblots and retained their activity during storage. Neither, however, could function as the capture reagent in a sandwich ELISA when immobilised on polystyrene. To establish whether they could be engineered for general use in immunotests, the genes coding for these scFvs were subcloned in expression vectors that contained sequences encoding chicken IgY heavy-chain constant region domains. This resulted in larger bivalent constructs which more closely resembled IgY molecules. The engineered fragments were evaluated in ELISAs and gold-conjugated immunochromatographic tests (ICTs). In contrast to their previous behaviour as scFvs, the modified fragments (designated ''gallibodies'') could be used for immunocapture in ELISA and could be readily conjugated to colloidal gold nanoparticles. A sandwich ICT that could detect recombinant HSP65 was also devised. Although converting the recombinant single-chain monomeric antibody fragments to bivalent immunoglobulin-like molecules did not entirely 'standardise' the behaviour of the scFvs, this approach remains potentially useful for developing practical, robust, immunodiagnostic reagents.

a b s t r a c tRecombinant 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.

There is an ongoing need for standardized, easily renewable immunoreagents for detecting African horsesickness virus (AHSV). Two phage displayed single-chain variable fragment (scFv) antibodies, selected from a semi-synthetic chicken antibody library, were used to develop double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) to detect AHSV. In the DAS-ELISAs, the scFv previously selected with directly immobilized AHSV-3 functioned as a serotype-specific reagent that recognized only AHSV-3. In contrast, the one selected with AHSV-8 captured by IgG against AHSV-3 recognized all nine AHSV serotypes but not the Bryanston strain of equine encephalosis virus. Serving as evidence for its serogroup-specificity. These two scFvs can help to rapidly confirm the presence of AHSV while additional serotype-specific scFvs may simplify AHSV serotyping.