The work presented in this thesis describes a rational design approach to the rapid
detection of Bacillus anthracis spores. A range of assay formats are available for detection
of B. anthracis, however none fulfil the criteria of speed, sensitivity or specificity. Optical
biosensors have shown promise for the near real time detection of low molecular weight
analytes, although exploitation of these systems for bacterial detection has been restricted
due to a comparative lack of sensitivity. Electroporation was unsuitable as a disruption
method for immunoassay based techniques, whereas preliminary experiments suggested
sonication could be utilised. A novel tubular sonicator designed to operate in a continuous
flow format, and to minimise antigen denaturation was shown to remove the exosporium
and some of the underlying spore coat of B. anthracis spores, resulting in a significant
increase in assay sensitivity (up to 500 fold). A range of methods were employed to
characterise the structural and antigenic components of the spore, focusing on those
released by sonication. Many of these were not suitable for detection purposes due to the
high levels of identity with homologous proteins within closely related species. However,
BxpA and EAI and two novel B. anthracis spore proteins, a homologues of B. cereus
ExsA and MntA, were identified as potential targets. Computer modelling techniques
mapped the unique regions of these proteins to potential binding pockets, indicating that
they could be utilised for the production of specific recognition elements. Due to the
inherent problems with the use of traditional antibodies, single chain antibodies (scFv)
were developed. EAI was used as a target as it was consistently identified using a range of
methods. Results indicated that it was predominantly associated with the spore and was not
completely removed by Urografin purification and subsequent wash steps, in variance to
previous work. Elimination of previously observed cross reactivity of scFv produced using
a standard biopanning procedure was possible through the development of a one step
competitive panning strategy. Finally, through the use of the scFv produced, combined
with flow through sonication prior to the assay, near real time specific detection of B.
anthracis spores was demonstrated through the use of a known protein target.
Date of Award | 2006 |
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Original language | English |
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Awarding Institution | |
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IDENTIFICATION OF STRUCTURAL AND ANTIGENIC COMPONENTS OF BACILLUS ANTHRACIS SPORES AND THEIR APPLICATION TO RAPID DETECTION USING BIOSENSORS
LOVE, T. E. (Author). 2006
Student thesis: PhD