Antibody fragments derived from naive heavy chain antibody libraries to detect listeriolysin O and Listeria monocytogenes.

摘要

Listeria monocytogenes is an emerging foodborne pathogen that is responsible for 28% of the food-related deaths in the United States. It causes meningitis, septicaemia and in pregnant women, abortions and stillbirths. It secretes the toxin listeriolysin O (LLO) that allows the bacteria to enter the cytoplasm of host cells, where they can replicate and cause further infection. The rapid and sensitive detection of L. monocytogenes and/or LLO in food samples is key to monitoring and prevention of listeriosis. This thesis describes the development of a platform for detecting L. monocytogenes in enrichment culture and food using llama heavy chain antibody variable fragments (VHHs). VHHs were raised against both the L. monocytogenes cells and the LLO toxin. In the course of this work, a method was devised for producing a large amount of functional LLO by expressing the hlyA gene (encoding LLO) in Escherichia coli and purifying the recombinant LLO using a one-step purification method. LLO constructs containing His tags at both the N- and C-termini of LLO were created, with levels of expression ranging between 2.5 to 8 mg/l of bacterial culture. Naive VHH phage and ribosome display libraries were panned to select fragments with affinity for either LLO, and L. monocytogenes cells. By phage-display, five LLO- and three L. monocytogenes-specific VHHs were identified. Preliminary surface plasmon resonance (SPR) studies confirmed the interaction of listeriolysin with four of the anti-LLO VHH clones. The naive phage display library provided a means of raising antibodies as detection agents, but the antibody fragments obtained displayed equilibrium dissociation constants in the muM range and were thus not of high affinity. Panning of the ribosome display library yielded four anti-LLO VHH clones. Each of these VHH constructs bound to His-LLO, but not to the related toxin streptolysin O or to an unrelated His-tagged protein. Two of the VHHs were further characterized using dot blotting and colony blotting methods to detect the presence L. monocytogenes via the presence of LLO, thus providing the initial work required for creating an immunoassay-based detection platform.