Matching Items (3)
Description
The research objective is to maintain the A4 nanobody stability during dialysis. Various dialysis buffers were tested and compared, including PBS with varying amounts of the detergent, Tween: low, high, none. Furthermore, PBS, Tris, and HEPES, were tested and compared. PBS without Tween was the worst for preserving A4 stability. PBS was determined to be a better dialysis buffer than Tris or HEPES. To find the optimum buffer, other buffers will be tested and compared with PBS; methods such as gravity filtration and lyophilization will be considered as alternatives to dialysis.
ContributorsTao, Kevin Huang (Author) / Sierks, Michael (Thesis director) / Williams, Stephanie (Committee member) / Barrett, The Honors College (Contributor) / Chemical Engineering Program (Contributor)
Created2015-05
Description
Alzheimer's disease (AD), which currently affects approximately 5.4 million Americans, is a type of dementia, which causes memory, cognitive, and behavioral problems. AD is among the top 10 leading causes of death in the United States, typically affecting people ages 65 and older. Beta-Amyloid (Aβ) is an Alzheimer's target protein, which starts as a single protein, but can misfold and bind to itself, forming larger chains and eventually fibrils and plaques of Aβ in the brain. Antibodies that bind to different regions and sizes of Aβ may prevent progression into a more toxic stage. The antibody worked with in this thesis, A4 scFv, binds to oligomeric Aβ. The objective of this antibody research is to optimize the production of functional antibodies, specifically A4, through modifications in the scFv growth process, in order to enhance the discovery of possible diagnostics and therapeutics for Alzheimer's disease. In order to produce functional A4 antibody, four complex sugars were tested in the E. Coli bacterial culture growth media that expresses the desired antibody. The sugars: sucrose, glucose, mannitol, and sorbitol were used in the growth process to improve the yield of functional antibody. Through the steps of growth, purification, and dialysis, the sugar sorbitol was found to provide the optimal results of ending functional antibody concentration. Once an ample amount of functional A4 scFv is produced, it can be used in assays as a biomarker for Alzheimer's disease.
ContributorsDolberg, Taylor Brianne (Author) / Sierks, Michael (Thesis director) / Nielsen, David (Committee member) / Barrett, The Honors College (Contributor) / Chemical Engineering Program (Contributor) / School of Sustainability (Contributor)
Created2014-05
Description
The following paper discusses the potential for Designed Ankyrin Repeat Proteins (DARPin) use as a diagnostic tool for neurodegenerative diseases in particular Alzheimer's disease (AD) and Parkinson's disease (PD). The two structures investigated for AD and PD were ADC7 and PDC1. Plasmid transformation was performed in order to grow the DARPin in E. coli for simple expression. Following growth and purification the proteins were validated using SDS-PAGE, Western Blot, BCA and indirect sandwich ELISA using transgenic mouse brain tissue. Targeted functionality of the DARPin structure was utilized during characterization methods to ensure the efficacy of the protein as a diagnostic for the respective disease targets. Both the ADC7 and PDC1 demonstrated improved binding with transgenic mice compared to wild type with a maximum 1.8 and 1.7 relative ratio, respectively. Additionally, both of the proteins demonstrated exclusive binding to their disease target and did not provide false positive results.
ContributorsTindell, John (Co-author) / Card, Emma (Co-author) / Sierks, Michael (Thesis director) / Nannenga, Brent (Committee member) / Chemical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12