Two 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.