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- All Subjects: Detection
- Creators: Diehnelt, Chris
- Member of: Theses and Dissertations
Description
In this project, biochemical characteristics of peptide binding agents, synthetic antibodies or synbodies, were examined with respect to the capture efficiency and specific binding ability to norovirus. Norovirus, although generally not a deadly pathogen, is the most common cause of acute gastroenteritis and outbreaks present a large social and financial burden to the healthcare and food service industries. With Dr. Diehnelt's laboratory group, a platform has been developed that enables us to rapidly construct peptide-based affinity ligands that can be characterized for binding to norovirus. The design needed to display clear results, be simple to operate, and be inexpensive to produce and use. Four synbodies, originally engineered with a specificity to the GII.4 Minerva genotype were tested with different virus strains varying in similarity to the GII.4 Minerva between 43% and 95.4%. Initial assays utilized norovirus-like particles to qualitatively compare the capture efficiency of the different synbodies without utilizing limited resources. To quantify the amount of actual virus captured by the synbodies, western blots with RT-PCR and RT-qPCR were utilized. The results indicated the synbodies were able to enrich the dilute solutions of the different noroviruses utilizing a magnetic bead pull-down assay. The capture efficiencies of the synbodies were comparable to currently utilized binding agents such as aptamers and porcine gastric mucine magnetic beads. This thesis presents data collected over nearly two years of research at the Center for Innovations in Medicine at the Biodesign Institute located at Arizona State University.
ContributorsSlosky, Rachael Marie (Author) / Diehnelt, Chris (Thesis director) / Stafford, Phillip (Committee member) / Chemical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
The influenza virus, also known as "the flu", is an infectious disease that has constantly affected the health of humanity. There is currently no known cure for Influenza. The Center for Innovations in Medicine at the Biodesign Institute located on campus at Arizona State University has been developing synbodies as a possible Influenza therapeutic. Specifically, at CIM, we have attempted to design these initial synbodies to target the entire Influenza virus and preliminary data leads us to believe that these synbodies target Nucleoprotein (NP). Given that the synbody targets NP, the penetration of cells via synbody should also occur. Then by Western Blot analysis we evaluated for the diminution of NP level in treated cells versus untreated cells. The focus of my honors thesis is to explore how synthetic antibodies can potentially inhibit replication of the Influenza (H1N1) A/Puerto Rico/8/34 strain so that a therapeutic can be developed. A high affinity synbody for Influenza can be utilized to test for inhibition of Influenza as shown by preliminary data. The 5-5-3819 synthetic antibody's internalization in live cells was visualized with Madin-Darby Kidney Cells under a Confocal Microscope. Then by Western Blot analysis we evaluated for the diminution of NP level in treated cells versus untreated cells. Expression of NP over 8 hours time was analyzed via Western Blot Analysis, which showed NP accumulation was retarded in synbody treated cells. The data obtained from my honors thesis and preliminary data provided suggest that the synthetic antibody penetrates live cells and targets NP. The results of my thesis presents valuable information that can be utilized by other researchers so that future experiments can be performed, eventually leading to the creation of a more effective therapeutic for influenza.
ContributorsHayden, Joel James (Author) / Diehnelt, Chris (Thesis director) / Johnston, Stephen (Committee member) / Legutki, Bart (Committee member) / Barrett, The Honors College (Contributor) / Department of Psychology (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2014-05