Evaluation of Norovirus Detection in Dilute Solutions

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.