Chicken scFvs and bivalent scFv-CH fusions directed against HSP65 of Mycobacterium bovis

Abstract

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.

Avian IgY antibodies and their recombinant equivalents in research, diagnostics and therapy

“…Moreover, the enormous diversity of the synthetic antibody libraries available today means that immune animals are not necessarily needed to derive suitable binders. Indeed, frameworks of synthetic human [64] and avian [65] variable regions have been successfully converted to their IgY derivatives and produced in eukaryotic hosts.…”

Mapping of antigenic determinants on a SAT2 foot-and-mouth disease virus using chicken single-chain antibody fragments

“…Of the three binders identified, only (Fehrsen et al, 2005;Rakabe, 2008;Sixholo, 2008;Wemmer et al, 2010). This is the first time the selection of SAT2-specific scFvs from this highly diverse and naïve, semi-synthetic IgY library is being described.…”

The antigenicity and cholesteroid nature of mycolic acids determined by recombinant chicken antibodies

“…Single chain variable fragments antibody inserts were prepared for cloning following methods previously described [ 45 ]. Briefly, scFv inserts were amplified from plasmid templates by PCR using primers to introduce a BsiW ( ) and an AscI ( ) cleavage site (Inqaba Biotech, South Africa).…”