Document Type

Dissertation

Degree

Doctor of Philosophy

Major

Chemistry, Biochemistry

Date of Defense

2-7-2023

Graduate Advisor

Michael R. Nichols

Committee

James K. Bashkin

Keith J. Stine

Chung F. Wong

Abstract

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder. The AD brain is characterized by significant neuronal loss and accumulation of insoluble fibrillar amyloid-β protein (Aβ) plaques and tau protein neurofibrillary tangles in the brain. However, over the last decade, many studies have shown that the neurodegenerative effect of Aβ may in fact be caused by various soluble oligomeric forms as opposed to the insoluble fibrils. Furthermore, the data suggest that a pre-fibrillar aggregated form, termed protofibrils, mediates direct neurotoxicity, and triggers a robust neuroinflammatory response.

Antibodies targeting the various conformation of Aβ are important therapeutic agents to prevent the progression of AD. We have generated conformationally-selective monoclonal antibody St. Louis (mAbSL) that selectively targets Aβ42 protofibrils compared to Aβ42 monomers and fibrils. The development aspects of these antibodies include the cloning of HC and LC variable fragments into the plasmid vector, transfection of the plasmids into 293 F cells, collection of the supernatant and purification using Protein A or protein G affinity chromatography. Sequencing of the heavy and light chain variable regions for multiple antibodies identified sequence characteristics that may impart conformational selectivity to the antibodies. Thus, I have successfully developed, expressed, and characterized these conformationally selective antibodies using various ELISA formats.

Exploration of Aβ42 aggregation in the presence a selective (mAbSL 113) and a non-selective antibody (mAb Ab 513) using spectroscopic and microscopic techniques is quintessential to looking at the effect of these antibodies on Aβ42 monomer aggregation and protofibril dynamics. It yielded a unique inhibitory mechanism on Aβ42 monomer aggregation offered by mAbSL antibodies. Aβ42 protofibril dynamics were prominently altered in the presence of mAbSL 113 with an insoluble complex formation by the antibody at low sub-stoichiometric molar ratios.

We focused on accurately determining the conformational epitope of our developed antibodies on Aβ42 protofibrils. The conformational epitope on Aβ42 protofibril was detected using a monoclonal antibody in various experimental formats like antibody competition ELISA, HDX-MS, and FPOP analysis. Our findings demonstrated new insights into monoclonal antibodies that target AD progression.

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