Lipopolysaccharide (LPS) is a major consituent of the outer membrane of all Gram-negative bacteria and is known to be responsible for the range of pathophysiological features of endotoxic shock. This thesis considers the expression of <i>Escherichia coli</i> LPS under different environmental conditions and its detection in the serum of septic patients by means of monoclonal antibody (MAb) probes. From an existing panel of MAbs, reactive with either Lipid A, core oligosaccharide and O-polysaccharide components of LPS, eleven were selected and characterised in a number of assay systems. Immunoblotting established that core reactive MAbs were reactive against either core not substituted with O-antigen or both substituted and unsubstituted core material. Core-glycolipid reactive MAbs demonstrated either full cross reactivity against all <i>E. coli</i> core types, or preferential binding to selective <i>E. coli</i> core types. Flow cytometric and ELISA analysis on whole bacteria showed that the absence of O-antigens on rough mutants increased accessibility of core-glycolipid LPS to antibodies. Analysis of the whole cell ELISA technique established that the expression of whole cells on ELISA plates differed from those in suspension. Sandwich ELISA methods employing suitable combinations of solid phase and biotinylated secondary MAbs were developed for the detection of <i>E. coli</i> types R1-R4, specific core types R1 and R3 and <i>E. coli</i> O18, O-antigen. The sensitivity of the assay using the two most cross-reactive MAbs was between 0.01 and 10 ng ml<SUP>-1</SUP> <i>E. coli</i>, depending on the core type. The sensitivity of assays for the detection of specific core and O-types was between 0.01 and 0.1 ng ml<SUP>-1</SUP> <i>E. coli</i> LPS. Assay sensitivity was significantly reduced for the detection of LPS in spiked serum. Methods to improve the sensitivity were investigated.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:659922 |
| Date | January 1991 |
| Creators | Nelson, David |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/20061 |
Page generated in 0.0019 seconds