To Catch a Virus: the Development of Immunoassays to Detect Viral Pathogens
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Authors
Hill, Haydon James
Issue Date
2025
Type
Dissertation
Language
en_US
Keywords
Hepatitis B virus , Immunoassay , Monoclonal antibodies , Rapid antigen test , Rift Valley fever virus , SARS-CoV-2
Alternative Title
Abstract
Viral pathogens make up a large percentage of global infectious disease. Diagnostic immunoassays play a critical role in curbing the threat of emerging and endemic viruses and contributing to improved disease detection and surveillance. There is a need for global access to accurate, affordable diagnostic assays to facilitate outbreak response and clinical management. This dissertation will explore the development of immunoassays for three such viral diseases: coronavirus disease 2019 (COVID-19), Rift Valley fever (RVF), and hepatitis B. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for COVID-19 and the COVID-19 pandemic from 2020-2023. The widespread availability and usage of immunoassays, particularly in the form of lateral flow immunoassays (LFIs), were instrumental in stymying the spread of the virus and leading to better disease surveillance. The natural mutation rate of the virus led to new SARS-Cov-2 variants appearing which detrimentally affected commercial diagnostic performance. In this work, 18 monoclonal antibodies (mAbs) were developed following immunizations with SARS-CoV-2 nucleoprotein. Two of these mAbs (1CV7 and 1CV14) were utilized in LFI development. Experimentation indicated that this prototype LFI is both sensitive and specific for SARS-CoV-2. Additionally, in a direct comparison analysis, the LFI prototype outperformed the commercially available BinaxNOW COVID-19 Antigen Card. Rift Valley fever virus (RVFV) is the etiological agent of RVF. RVF is a severe disease affecting both humans and animals. Due to its aerosol spread and significant public health risk, the United States Centers for Disease Control and Prevention (CDC) and the United States Department of Agriculture (USDA) deemed RVFV a Select Agent with the capability of being used as a bioweapon. The World Health Organization (WHO) also listed RVFV as a Blueprint Priority Disease, indicating that more research needs to be done to prevent, treat, and detect RVFV around the globe. RVFV is endemic to sub-Saharan Africa and the Arabian Peninsula. Existing diagnostics for RVFV are primarily reliant on nucleic acid amplification tests (NAATs) such as polymerase chain reaction (PCR). NAATs require dedicated laboratory infrastructure and trained personnel, making the largescale implementation of these tests impractical in the resource-limited settings RVFV is endemic to. The described work explores the utilization of rapid immunoassays for the diagnosis and detection of RVFV in austere environments. An LFI was developed that demonstrated low limits of detection for the RVFV nucleocapsid (NP) biomarker. Additionally, collaborators at the University of Arizona Zenhausern laboratory were able to further implement the LFI pair into a rapid vertical flow immunoassay capable of sub-nanogram/milliliter levels of NP detection. Chronic hepatitis B (CHB) is a lifelong infection caused by the hepatitis B virus (HBV). CHB was responsible for 1.1 million deaths in 2022 and is additionally responsible for a majority of hepatocellular carcinomas (HCC). CHB has a worldwide prevalence; however, it is most abundant in sub-Saharan Africa and southeast Asia. CHB is readily treated with antiviral therapies. These antivirals significantly lower the risk of CHB complications such as cirrhosis and HCC. The key to successful CHB treatment is effective CHB monitoring and management. NAATs are frequently implemented to monitor disease state by assaying serum HBV DNA levels; however, these are expensive and inaccessible for many suffering from CHB. Hepatitis B core-related antigen (HBcrAg) is a recently defined biomarker with high correlations to serum HBV DNA levels. Here we investigated the implementation of HBcrAg specific mAbs into immunoassays for the sensitive detection of the antigen. A colorimetric LFI, fluorescent LFI, and antigen-capture ELISA were developed to facilitate the point-of-care (POC) detection and management of CHB. Furthermore, an acid/base pretreatment protocol to expose HBcrAg epitopes was developed to allow the assays to be potentially performed in low-resource settings. All the assays were shown to have clinically relevant limits of detection, indicating they have promise for future diagnostic applications.