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Development of a Rapid Point of Care Diagnostic Immunoassay for the Detection of P. aeruginosa Infections in Cystic Fibrosis Patients

Martin, Andrew (2019) Development of a Rapid Point of Care Diagnostic Immunoassay for the Detection of P. aeruginosa Infections in Cystic Fibrosis Patients. Doctor of Philosophy (PhD) thesis, University of Kent. (Access to this publication is currently restricted. You may be able to access a copy if URLs are provided) (KAR id:79904)

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Language: English

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Abstract

Pseudomonas aeruginosa infections are the leading cause of morbidity and mortality in cystic fibrosis (CF) patients and it is the predominant pathogen found in patients by adulthood. During the initial stages of infection, isolates are mostly planktonic, express high levels of virulence factors, and can often be eradicated via aggressive antibiotic treatment. However, once chronic infection is established, P. aeruginosa may develop a mucoid phenotype, leading to development of a bacterial biofilm, the emergence of antibiotic resistance, a worsening of symptoms and an increased frequency of pulmonary exacerbations. There is therefore an urgent need for a reliable biomarker of initial P. aeruginosa infection that will allow early intervention/application of antibiotics in CF patients and monitoring of treatment. In the work described here, a potential biomarker of early infection has been identified as elastase, a secreted protease encoded by the lasB structural gene under regulation of the las quorum sensing pathway, which plays a key role in virulence and infection via digestion of a wide range of substrates including collagen and elastin. Development of a rapid point of care diagnostic assay targeting elastase in CF patient samples, such as sputum, may allow for early identification of acute P. aeruginosa infection and thus earlier treatment. A recombinant form of elastase was produced in the E. coli strain BL21*(DE3)pLysS and subsequently purified and characterised to assess its conformational stability and specific activity. This recombinant material was used to immunise rabbits and sheep, and elastase-specific polyclonal antibodies were purified from the resulting antisera. Attempts were made to generate high affinity monoclonal antibody fragments from peripheral blood lymphocytes of the immunised animals via antibody phage display, but were unsuccessful. Prototype sandwich ELISA and lateral flow format immunoassays (LFIAs) were developed incorporating purified polyclonal antibodies, with lower limits of detection of 27 pg/mL (rabbit polyclonal ELISA) and 1 ng/mL (rabbit polyclonal LFIA). Specificity of the assay was assessed via application of culture supernatants from clinical isolates of P. aeruginosa and other bacterial strains commonly associated with CF. When the assay was used to analyse CF patient isolates, 29/31 first isolates and 12/17 chronic isolates returned a positive test, whereas 0/20 non Pseudomonas strains returned a positive test, demonstrating the highly specific nature of the assay and its utility as an indicator of early infection. In conclusion, the work presented here demonstrates the proof of concept development of a highly specific and sensitive point of care diagnostic assay for the detection of acute P. aeruginosa infection in CF patients, with scope for further development via generation of monoclonal antibodies and signal enhancement techniques.

Item Type: Thesis (Doctor of Philosophy (PhD))
Thesis advisor: Smales, Mark
Thesis advisor: Davis, Paul
Uncontrolled keywords: Immunoassay Cystic Fibrosis Lateral Flow Assay Diagnostics Point-of-Care Phage Display Antibody Discovery Recombinant Protein Pseudomonas aeruginosa Elastase
Subjects: Q Science > QP Physiology (Living systems) > QP517 Biochemistry
Divisions: Faculties > Sciences > School of Biosciences
SWORD Depositor: System Moodle
Depositing User: System Moodle
Date Deposited: 31 Jan 2020 13:10 UTC
Last Modified: 04 Feb 2020 10:02 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/79904 (The current URI for this page, for reference purposes)
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