Abstract
Two chicken single-chain variable region antibody fragments (scFvs) that recognised the 65 kDa heat-shock 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.