Date on Master's Thesis/Doctoral Dissertation

5-2021

Document Type

Master's Thesis

Degree Name

M. Eng.

Department

Bioengineering

Degree Program

JB Speed School of Engineering

Committee Chair

Fuqua, Joshua

Committee Member

O'Toole, Martin

Committee Member

Steinbach-Rankins, Jill

Committee Member

Hamorsky, Krystal

Author's Keywords

Protein; ELISA; Serology; Trimerization; Spike; Coronavirus

Abstract

Serological tests are conducted to assess humoral response against viral protein antigens, to assess viral exposure and protection from pathogens. The rapid development and modularity of serological assays have proven critical to managing the current SARS-CoV-2 pandemic. The receptor binding domain of SARS-CoV-2 is within the trimeric Spike protein and serves as a highly immunogenic target for potentially neutralizing antibodies. Current receptor binding domain serological assays use recombinant monomers or dimers of the receptor binding domain. The receptor binding domain is presented to the immune system natively in the context of the Spike protein trimer. Therefore, a recombinant trimeric receptor binding domain may be predictive of protection and improve antibody binding. For this thesis, using the trimerization domain from Bacteriophage T4 Fibritin, called Foldon, fused to the receptor binding domain, a novel antigen was produced. The antigen was expressed in and extracted from Nicotiana benthamiana plants, purified through immobilized metal affinity FPLC, and used to develop a serological ELISA. The antigen was tested with hospitalized (n=46), non-hospitalized (n=36), and negative (n=46) patient sera sample lots and batch reproducibility was examined. From these studies, it was concluded that this trimeric antigen can be consistently expressed, extracted, and purified and can be used to reliably detect responses to SARS-CoV-2 in sera. Additionally, fusion of the Foldon trimerization domain to trimeric viral proteins serves as a platform for the development of plant produced viral antigens to better detect host response.

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