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A Toolbox of Humanized Recombinant Antibody Fragments for Prion Diseases
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- Author / Creator
- Rathod, Vineet S.
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Prion diseases are rare, inexorably progressive, and fatal neurodegenerative disorders, with no therapy other than palliation. The key event in the pathogenesis of prion diseases is the conformational conversion of the cell-surface glycoprotein (PrPC), from a predominantly α-helical structure into an infectious, aggregated isoform (PrPSc), with β-sheet oligomers and amyloid fibrils. Multiple abnormal PrP structures have been observed both in vivo and in vitro, and determining which are relevant to disease, that is, infectious and/or toxic, is a major challenge. Recently high-resolution structures of brain-derived prion strains revealed a parallel in-register intermolecular β-sheet (PIRIBS) structure, which is in conflict with brain-derived X-ray fiber diffraction patterns proposing a four-rung β-solenoid (4RβS) architecture. Antibodies are powerful tools for protein purification and molecular detection. Although conventional antibodies are ideal for most applications, the performance of certain assays is enhanced by using antibody fragments. Fab fragments (Fabs) are small antibody derivatives that maintain antigen-binding capacity. One of the advantages of Fabs over intact antibodies is they are small in size, which allows them to penetrate deeper into protein aggregates, such as amyloid fibrils. However, having antibodies that are conformational dependent on proteins such as the pathologic conformation of PrPSc recognizing the toxic oligomers and fibrils has enormous value for biomedical research and therapeutics of prion diseases. I established a pipeline to produce recombinant humanized antibody fragments for prions derived from phage display technology and hybridoma clones. The fully functional and soluble antibody fragments were characterized extensively using linear PrP peptides and via structural epitope mapping of PrPSc-specific antibody fragments. This allowed me to build a toolbox of various recombinant antibody fragments, including engineered conformational PrPSc-specific antibody fragments and small conjugated antibody derivatives. I used these to perform ultrastructural analyses of animal and human prions using immunogold labelling, direct detection of prions, and in-vitro structural analysis of genetic prions using time-dependent folding immunoassay.Taken together, these recombinant humanized antibody fragments provide an alternate approach to understand the structural relationships and pathophysiological roles of various prion ultrastructures and offer potential therapeutic candidates for prion diseases.
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- Graduation date
- Spring 2023
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- Type of Item
- Thesis
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- Degree
- Doctor of Philosophy
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- License
- This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.