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The Development of Diagnostic Immunoassays for Melioidosis and Ebola Virus Disease
AuthorGreen, Heather Rayne
AdvisorAuCoin, David P
Cell and Molecular Biology
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Burkholderia pseudomallei and five species of Ebola virus (EBOV) are the causative agents of melioidosis and Ebola virus disease (EVD), respectively. Melioidosis and EVD are deadly infectious diseases with high mortality rates. Furthermore, both pathogens are regulated under the United States of America Federal Select Agent Program due to their potential to be used in bioterrorism. Early diagnosis is imperative for treating the specific disease as well as minimizing the spread of the pathogen. Diagnostic measures for both diseases rely on identification of the causative agent or identifying a humoral response against the causative agent. For melioidosis, the current gold standard for diagnosing an infection is through isolating the bacteria by culturing. For EVD, the current diagnostic measures include polymerase chain reaction (PCR) to identify viral nucleic acid and enzyme-linked immunosorbent assay (ELISA) for the detection of viral antigens or antibodies reactive to EBOV antigens. All three diagnostic assays can take several hours to days to get a result, require specialized training, and access to expensive equipment. The areas in which both pathogens cause the highest global burden are typically resource poor areas including Southeast Asia and northern Australia for B. pseudomallei and West Africa for Ebola virus. More rapid diagnostic methods would greatly improve the morbidity and mortality associated with these diseases. Lateral flow immunoassays (LFIs) are rapid, point-of-care (POC) diagnostic tools that utilize antibodies and colorimetry to detect a biomarker for a specific disease or other condition. This dissertation outlines alternative approaches to enhance the detection of B. pseudomallei capsular polysaccharide (CPS) on an LFI as well as outlines the development of a prototype LFI for the rapid diagnosis of early EVD through detection of Ebola virus soluble glycoprotein (sGP). The Active Melioidosis DetectTM Lateral Flow Immunoassay (AMD LFI) was developed by our laboratory in collaboration with InBios International, Inc. (Seattle, WA) to diagnose melioidosis through the detection of CPS, a biomarker of the disease. The AMD LFI has high specificity and analytical sensitivity, but the clinical sensitivity can be low depending on the sample type. testing of some patient samples on this assay indicated that it was not sensitive enough to detect all clinically relevant concentrations of CPS. Here we developed a magnetic immunoprecipitation method for the pre-concentration of CPS from melioidosis patient urine samples to enrich these samples for CPS. CPS reactive monoclonal antibody (mAb) 4C4 was conjugated to magnetic particles and a protocol was developed utilizing acid-base elution neutralization chemistry. We have shown that this protocol resulted in increased AMD LFI positivity when testing melioidosis patient urine samples.Passive concentration of Burkholderia pseudomallei CPS was also explored as an alternative method to enrich the sample for CPS prior to evaluating on the AMD LFI for a less labor-intensive approach. Following a melioidosis diagnosis, patients were actively enrolled into a study in which they provided urine and/or serum samples to be tested on the AMD LFI. These samples underwent extensive testing, including culturing to identify if B. pseudomallei was present at the time of sample collection as well LFI testing. Urine samples were tested on the AMD LFI, as well as serum samples from some patients for a direct comparison. Urine was found to be the optimal matrix for CPS detection in confirmed melioidosis patient samples as in general it appeared urine contained higher concentrations of CPS when compared to blood samples from the same patient taken at nearly the same time. Furthermore, we found that passive concentration of urine resulted in stronger test line intensity on the AMD LFI as well as provided more positive results than when testing serum or unconcentrated urine.Lastly, antibody-based immunoassays were developed for detection of EBOV sGP, a well-established biomarker of an Ebola virus infection. Current diagnostic assays for EVD focus on detection of glycoprotein (GP) and viral matrix protein (VP40), but sGP may have the potential to be a superior biomarker. sGP and GP are encoded by the same gene, GP, and sGP is the main product of GP. Furthermore, large amounts of sGP can be detected in the bloodstream early during an Ebola virus disease infection. Splenocytes were isolated from mice immunized with either sGP or Ebola virus-like particles (VLPs), and hybridoma technology was utilized to establish immortalized cell lines that produced sGP reactive antibodies. This work resulted in the isolation of a library of seventeen high affinity mAbs that are reactive to EBOV sGP. These mAbs were characterized via Western immunoblotting, direct and antigen-capture ELISA, surface plasmon resonance, and lateral flow immunoassay. Prototype antigen-capture ELISA and LFIs were developed and optimized using pairs of mAbs isolated in this study.