Date of Award
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a virus that infects cells in the lungs, nasal passages, and intestines via the ACE2 receptors of the host, and leads to the coronavirus disease (COVID-19). Since the occurrence of the pandemic in December 2019, there have been 114 million cases worldwide in which 2.5 million lives have unfortunately been taken away.
Being diagnosed with a past infection stems questions among those that have tested positive through antibody testing. The problem here is that patients were notified that they have antibodies resulting from a SARS-CoV-2 infection, but in fact they may not possess these antibodies which lead to a false positive result. This is detrimental, as patients may believe they are immune to the virus due to a perception of acquired immunity. Patients will then go out in public at free will, which can result in the contraction and further transmission of the virus.
This thesis follows the goal to develop a protein purification protocol and an immunoblot technique that can successfully identify the presence of human antibodies in response to the nucleocapsid (N), spike (S), and receptor binding domain (RBD) proteins of the SARS-CoV-2 virus. The immunoblots, in theory, will help towards the goal of eliminating false positives that arise from other antibody tests.
To achieve the goal, the N, S, and RBD proteins were subjected to purification. E. coli cells were transformed with DNA genetic material that coded the proteins. The proteins were expressed, a cell lysate was formed, and the proteins were then purified through his-tag affinity column chromatography. The purified proteins were subjected to SDS page gel electrophoresis and membrane transfer. The transferred proteins were used in a series of immunoblots with specific rabbit sera to confirm protein purification. Sera from humans were used to detect COVID-19 through antibody interactions with the purified proteins.
Results showed that human antibodies from COVID-19 patients were able to bind to the N and S protein, but not the RBD. Therefore, with more human sera immunoblot data, an immunoblot protocol can then be put into effect to diagnose patients for past infections.
Desai, Shivum, "Development of an Immunoblot to Detect Human Antibodies Against SARS-COV-2 Virus Proteins" (2021). Honors Theses. 1919.
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