Pathology - Theses
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ItemThe use of SELDI-TOF MS in the search for biological markers of Alzheimer’s disease( 2013)Alzheimer’s disease (AD) is an insidious, neurodegenerative disease characterised by progressive decline in memory and cognitive performance. However, despite more than a century passing since the discovery of AD, two major challenges in the field remain unmet: the ability to identify individuals with preclinical AD using large-scale population screens and the ability to delay or halt its progression. The advent of more sensitive technologies, such as surface enhanced laser desorption / ionisation time-of-flight mass spectrometry (SELDI-TOF MS), has enabled a more thorough investigation of Aβ profiles in AD-affected tissue. These investigations have revealed that the AD brain is besieged by an array of N-terminally truncated monomeric Aβn-42 peptides. However, despite the ability to detect synthetic Aβ dimers using this method, spectral evidence for the naturally occurring low-order oligomeric Aβ observable using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was not found. The formation of low-order oligomers of Aβ is believed to be a central component of AD pathogenesis; however, further examination using SELDI-TOF MS and SDS-PAGE revealed that the dimeric Aβ observable using SDS-PAGE was an SDS induced artefact. Attempts to regulate Aβ oligomerisation and aggregation within the AD brain and CSF have formed the basis of AD therapeutics and led to the development of a number of monoclonal anti-Aβ antibodies, such as bapineuzumab, solanezumab and crenezumab. All three antibodies demonstrated selective affinity for synthetic Aβ peptides, corresponding to their respective epitopes, when analysed using surface plasmon resonance (SPR). However, SELDI-TOF MS analysis of AD brain using these antibodies indicated that the selectivity of all three compounds was lost in complex biological samples, with each antibody binding a comparable array of Aβ species. These findings highlight the difficulty of targeting Aβ in AD, as complex interactions between different Aβ isoforms makes selective targeting of individual peptides inherently difficult. The central role of Aβ in AD has led to expansive research into its viability as a peripheral marker of disease. In this thesis, an investigation of Aβ levels in blood samples obtained from the Australian Imaging, Biomarkers and Lifestyle (AIBL) Flagship Study of Aging was conducted using the WO2 antibody in conjunction with SELDI-TOF MS. This investigation revealed that observable levels of Aβ in the blood cellular fraction deteriorated over time, with variations in basic sample collection and handling procedures significantly affecting the stability of the peptide. An examination of the prevailing literature revealed a lack of consensus regarding the optimal pre-analytical methodology for blood-based investigations and an overwhelming failure amongst researchers in the field to adequately report basic methodologies, thus preventing accurate comparisons across investigations. Despite the instability of Aβ in the AIBL samples, further analysis was conducted using immobilised metal affinity capture and found that peripheral levels of α-defensin were elevated in AD. In the absence of Aβ levels, the α-defensin levels did not have the necessary power to produce a predictive model; however, the finding did act to support the growing body of literature reporting increases in peripheral inflammation as an early indicator of AD.