Expression and engineering of recombinant antibodies against a heat-shock protein of Mycobacterium bovis

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dc.contributor.advisor Du Plessis, Dion H. en
dc.contributor.advisor Fehrsen, Jeanni en
dc.contributor.postgraduate Wemmer, Susan en
dc.date.accessioned 2013-09-07T14:25:29Z
dc.date.available 2009-04-16 en
dc.date.available 2013-09-07T14:25:29Z
dc.date.created 2008-10-28 en
dc.date.issued 2009-04-16 en
dc.date.submitted 2008-10-21 en
dc.description Dissertation (MSc)--University of Pretoria, 2009. en
dc.description.abstract In the medical and veterinary diagnostic fields there is an ongoing need for stable and specific antibodies. There is also a requirement for simple, robust and cost-effective diagnostic assays to be used in the developing world. Recombinant antibodies from phage displayed libraries are economical to produce and can often be engineered to improve affinity, avidity and stability. While recombinant antibody fragments are useful in immunoassays, they are not strictly comparable to normal immunoglobulins and may under-perform in certain assays. Converting monovalent single-chain antibody fragments (scFvs) to bivalent immunoglobulin-like formats could conceivably provide a more suitable molecular scaffold for use in immunoassays. Two scFvs that recognised the 65 kDa heat-shock protein (HSP65) of Mycobacterium bovis were used in this study. They were originally derived from the Nkuku® repertoire, a phage displayed antibody library based on the immune repertoire of the chicken, Gallus gallus. The genes coding for these scFvs were subcloned in expression vectors containing chicken IgY constant-heavy domains, to create bivalent constructs which were designated ‘gallibodies’. Expression of these constructs was attempted in three heterologous systems. While they were successfully produced in adherent mammalian cell cultures, the growth requirements of these cultures complicated subsequent purification. Bacteria and yeasts were investigated as alternative expression systems, but antibodies were not produced in either system. The gallibodies were compared to their monovalent scFv counterparts for stability as well as their applicability in ELISAs and gold-conjugated immunochromatographic lateral-flow assays. As gallibodies, both retained their functionality after exposure to different conditions and they were capable of immunocapture in ELISA. This was in contrast to their performance as scFvs. Furthermore, these antibody-like molecules could be stably conjugated to colloidal gold and used in lateral flow tests where positive and specific signals were obtained. This confirmed that recombinant single-chain monomeric antibody fragments could be reconstituted as bivalent immunoglobulin-like molecules and that they are a potentially useful platform for developing practical, robust immunodiagnostic reagents. It appeared from these experiments that the antibodies could act as a pair in which one captures, and the other detects HSP65. To find out whether they recognised discrete regions on the protein, their epitopes were mapped using a phage displayed peptide library in combination with computer-based algorithms. The presumptive epitope of one was mapped to residues 350 to 370 on HSP65 of M. bovis. The sequences selected from the peptide library by the other corresponded to three separate regions on the target protein. These recombinant antibody recognition sites are analogous to some of those that have been mapped by others using traditional monoclonal antibodies. en
dc.description.availability unrestricted en
dc.description.department Veterinary Tropical Diseases en
dc.identifier.citation 2008 en
dc.identifier.other E1230/gm en
dc.identifier.upetdurl http://upetd.up.ac.za/thesis/available/etd-10212008-153754/ en
dc.identifier.uri http://hdl.handle.net/2263/28886
dc.language.iso en
dc.publisher University of Pretoria en_ZA
dc.rights ©University of Pretoria 2008 E1230/ en
dc.subject Antibody engineering en
dc.subject Chicken immunoglobulins en
dc.subject Single-chain antibodies en
dc.subject Immunochromatographic tests en
dc.subject Colloidal gold en
dc.subject UCTD en_US
dc.title Expression and engineering of recombinant antibodies against a heat-shock protein of Mycobacterium bovis en
dc.type Dissertation en


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