| This dissertation describes the development of new tailored methods for the discriminative detection of amphiphilic lipopolysaccharide (LPS) antigens, so as to improve screening methodologies for food-safety applications, and detection of amphiphiles in general. LPS is associated with the outer membrane of Gram-negative bacteria, and is a primary virulence biomarker of several pathogens. Direct detection of amphiphilic LPS in the aqueous matrices of the host/sample requires an appreciation of the complex biochemistry of the molecule, and forms the basis for this research. The unique structure of this molecule can be used for identification of both the serogroup and strain of pathogen. However, current detection methods lack sensitivity, and are also not serogroup specific. To achieve discriminative detection, we have first created a unique repertoire of associated reagents by isolating amphiphilic LPS from seven strains of Shiga toxin-producing Escherichia coli, and developing highly specific monoclonal antibodies against the O antigen regions of the same. We demonstrate the use of a targeted detection technique, called membrane insertion, which facilitates the physiological presentation of LPS by inserting the hydrophobic lipid A portion of the molecule into a lipid bilayer, leaving the O antigen exposed. This method is advantageous because it minimizes exposure of the highly conserved lipid A epitopes, and maximizes exposure of the serogroup specific O antigens. In addition, we present the first comprehensive biophysical analysis of the interaction of LPS with supported lipid bilayer architectures, and identify several novel and interesting effects of the same. Further characterization of these effects reveals the role or impact of membrane proteins and complexity on the interactions between host and pathogen biomarkers and significantly questions the design and execution of cell studies and in vitro platforms for amphiphilic targets like LPS. Cell studies clearly reveal that presentation of LPS either in buffer or in serum dramatically alters associated cytokine profiles. Our conclusions indicate that the biochemistry of amphiphilic molecules, like LPS, and their presentation, should always be considered when interfacing with physiological systems. |
