Date of Award
The emergence of the coronavirus disease (COVID-19) has instigated one of the most influential pandemics, causing unique and detrimental effects throughout various parts of the world. By developing a stronger understanding of the viral genome and structural components, researchers are driven towards medical advancements and the implementation of improved testing methods. This research aims to provide a reliable COVID-19 antibody test by immunoblot detection, which can deliver more accurate results than the currently used enzyme linked immunosorbent assay (ELISA). Within this protocol, differing SARS-CoV-2 viral proteins were detected through western blotting analysis and were evaluated based upon differing strengths in antibody detection. The SARS-CoV-2 spike (S) glycoprotein and nucleocapsid (N) protein were expressed within an E. Coli histidine tag plasmid and subjected to column purification. The purified proteins were fractionated by SDS-PAGE gel electrophoresis and transferred to a PVDF membrane by western blotting. The separated membranes were exposed to primary antibodies, gathered from human and rabbit sera, and secondary antibodies, gathered from goat anti-human and goat anti-rabbit IgG Horse Radish Peroxidase Conjugate. Detection by chemiluminescent western blotting confirmed the presence of antibodies to the S and N proteins within positive SARS-CoV-2 patient sera, and the absence of antibodies to the S and N proteins within SARS-CoV-2 seronegative sera. The produced imaging displays the strength of antibody detection against both proteins, promoting the use of the viral nucleocapsid protein as a valuable target for antibody detection. This study conclusively demonstrates the reliability of antibody detection by immunoblot testing and promotes the implementation of western blotting analysis in future COVID- 19 research efforts.
Gammon, Virginia Kate, "The Detection of Human Antibodies Against Differing SARS-CoV-2 Virus Proteins by Immunoblot Testing" (2022). Honors Theses. 2488.