Chronic respiratory colonisation by the adaptable opportunistic pathogen <i>Pseudomonas aeruginosa</i> is a major debilitating feature of the inherited disease cystic fibrosis (CF). This thesis considers various aspects of the pathogenesis of <i>P.aeruginosa</i> in CF, including the serological response to bacterial colonisation, and possible factors involved in early colonisation. Anti-<i>P.aeruginosa</i> lipopolysaccharide (LPS) antibodies in sera, saliva and sputa from patients with CF were measured by enzyme-linked immunosorbent assay (ELISA) incorporating either a polyvalent pseudomonas smooth LPS extract vaccine, or <i>P.aeruginosa</i> core, rough LPS. Elevated levels of anti-LPS IgG antibodies in serum, and IgA antibodies in saliva and sputum were demonstrated in patients chronically colonised by <i>P.aeruginosa</i>. Low levels of serum anti-LPS IgG antibodies were detected in some patients intermittently colonised by <i>P.aeruginosa</i>, but not in non-<i>P.aeruginosa</i> colonised patients. Anti-LPS IgA antibodies were detected in some of both intermittently and non-colonised patients. Immunoblot analysis of serum IgG and sputum IgA antibodies to <i>P.aeruginosa</i> LPS revealed a response directed towards O-antigenic LPS in the initial stages of pulmonary colonisation with non-mucoid <i>P.aeruginosa</i> and a response towards common core LPS during subsequent chronic infection with mucoid <i>P.aeruginosa</i>. Flagellar preparations from <i>P.aeruginosa</i> strains were characterised and used in ELISA and immunoblot studies to detect anti-<i>P.aeruginosa</i> flagellar antibodies in sera, saliva and sputum. Serum anti-flagellar IgG antibodies were detected, particularly in those CF patients intermittently or chronically colonised by <i>P.aeruginosa</i>. Antibodies to both type -a and -b flagella were detected; in some patients a pronounced antibody response to only one of the flagellar types was evident. Anti-<i>P.aeruginosa</i> LPS monoclonal antibodies (MAbs) were produced for use in a sandwich ELISA for the detection of <i>P.aeruginosa</i> in respiratory secretions of patients with CF. LPS defective mutants expressing only common core LPS were used to immunise mice for preparation of MAbs. Antibodies were screened in ELISA and the antigenic component(s) of LPS recognised by the most cross-reactive MAbs was checked by immunoblotting. Five IgG MAbs were characterised and found to recognise the core component of <i>P.aeruginosa</i> LPS. Two of the MAbs were particularly reactive against core LPS from all O-antigenic serotypes of <i>P.aeruginosa</i> and were included in the sandwich ELISA for detection of <i>P.aeruginosa</i> LPS. A biotin-streptavidin amplification system was used to increase assay sensitivity. The sensitivity of the assay was 0.1 ng/ml <i>P.aeruginosa</i> LPS; the assay was able to detect <i>P.aeruginosa</i> LPS in the respiratory secretions from patients with CF.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:659926 |
| Date | January 1991 |
| Creators | Nelson, J. W. |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/19187 |
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