Pseudomonas aeruginosa and Burkholderia cepacia are opportunistic human pathogens. Hazards pose to immunocompromited patient groups, most of whom are patients suffering from cystic fibrosis, for which is the infection caused by Pseudomonas aeruginosa or Burkholderia cepacia, often lethal. Bacteria exhibit resistance to most antibiotics and the immunization of patients, paradoxically, worsens the patients' condition. Hen antibodies, unlike mammalian do not activate the complement cascade and do not cause the inflammatory response, therefore, seem to be a suitable tool to protect high risk populations as a means of passive immunization. In this work were prepared specific chicken antibodies against significant virulent factor - bacterial lectins PAIIL and BClA responsible for adhesion. Antibody specificity was demonstrated by ELISA and Western blot. Antibodies were affinity purified and cleaved to fragments. Pneumocytes type II from the lungs of patients with Cystic Fibrosis were isolated and cultured. Tests of the adhesion of bacteria cells were performed on them using ELISA. As an alternative model rat pneumocytes and tumor cell line A549 has been used. Prepared antibodies specifically detect bacterial lectins on the surface of bacteria. Antibody has been shown to reduce adhesion of bacteria to...

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. / Dissertation (MSc)--University of Pretoria, 2008. / Veterinary Tropical Diseases / unrestricted