The Development and Application of Ultra-sensitive Protein Aggregate Detection Techniques

Abstract

There are more than 55 million patients suffering from dementia worldwide, yet there is no definitive diagnostic tool or effective treatment for the disease. The thesis focuses on the development and application of ultra-sensitive techniques for detecting protein aggregates. It begins by studying the role of apolipoprotein E (apoE) in amyloid- β (Aβ) pathology in Alzheimer’s disease (AD). The isoform-specific and lipidation-specific behaviours of apoE and the co-aggregates formed with A β are explored. The discussion proposes potential therapeutic strategies involving the promotion of apoE lipidation. Then, the focus shifts to the development of ultra-sensitive techniques for quantifying protein aggregates in human biofluid. The resulting single molecule array (SiMoA)-based assays exhibit high sensitivity in the lower picomolar range with satisfactory precision. The aggregate quantification assay developed was then used in biomarker discovery for AD diagnosis. The multifaceted approach, combining individual assay measurements and ratios, exhibited a more reliable blood-based prediction for the disease than individual measurements. The SiMoA assays were also employed to evaluate the effectiveness of MEDI1341 in a clinical trial, providing insights into antibody interference and the drug’s impact on physiological protein aggregate concentration. In addition, the thesis introduces a fluorescence image analysis suite, ACT, which proves to be a valuable tool in protein aggregate studies. ACT was rigorously tested and found to be particularly user-friendly. It demonstrated great performance in identifying and characterising aggregate in fluorescence microscopy image.