Zaire ebolavirus (EBOV) and Burkholderia pseudomallei are the causative agents of Ebola virus disease (EVD) and melioidosis, respectively. Early diagnosis is critical for halting the spread of the disease or providing treatment of the disease. Current diagnosis for EBOV includes polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) for the detection of EBOV antigen or EBOV reactive antibodies present in patient serum. For B. pseudomallei, culturing the bacterium and bacterial isolation is the gold standard for diagnosis. These techniques can take days to complete, need specialized training to perform, expensive equipment or have low sensitivity. Hence, in the first study, we developed two lateral flow immunoassay (LFI) prototypes targeting two known EBOV biomarkers including the glycoprotein (GP) and matrix protein (VP40) for rapid diagnosis of EVD. First, a library of high affinity monoclonal antibodies (mAbs) was established by immunizing mice with EBOV antigens, including EBOV recombinant VP40 (rVP40), and EBOV virus-like particles (eVLP), which express both VP40 and GP. Thirty-two new mAbs, specific to either GP or VP40, were isolated. Western blot was used to assess the reactivity of the mAbs. Surface plasmon resonance (SPR) and antigen-capture ELISA techniques were used to assess binding affinity of the mAbs. MAb pairs successful at capturing eVLP were determined by antigen-capture ELISA and initial LFI testing. Top prototype LFIs for both GP and VP40 were selected based on reactivity with eVLP. Furthermore, top prototype LFIs were tested with live EBOV and non-human primate (NHP) EBOV infected serum. The GP-specific LFI prototype showed increased sensitivity compared to the VP40-specific LFI prototype and current FDA emergency approved ReEBOV Antigen Rapid Test, when tested with live EBOV. As for melioidosis, a mouse/human chimeric antibody specific to B. pseudomallei capsular polysaccharide (CPS) was developed. This is important because it could potentially be used as a prophylaxis treatment for melioidosis. A murine monoclonal antibody (m4C4) that binds to B. pseudomallei CPS was previously developed by our laboratory. The m4C4 variable region was used to design the mouse/human chimeric version of 4C4 (c4C4). This included the m4C4 variable region and a human IgG1 constant region. c4C4 was characterized for reactivity and binding affinity by antigen-capture ELISA, Western blot, and SPR. Both antibodies show similar reactivity and affinity in all methods tested. m4C4 and c4C4 also showed promise as therapeutics for melioidosis. This novel chimeric treatment could increase the survival rate of melioidosis. A rapid diagnostic targeting B. pseudomallei CPS, Active Melioidosis Detect (AMDTM) rapid test has already been developed by our laboratory in collaboration with InBios International Inc. (Seattle, WA). In the final study we investigated the concentration of CPS present in urine of suspected melioidosis patients. This study would lead to a better understanding of the use of CPS as a biomarker of melioidosis. Urine samples were collected from 42 suspected melioidosis patients and the presence of CPS was analyzed by AMDTM lateral flow immunoassay and quantitative antigen-capture ELISA. We determined that the concentration of CPS within patient urine samples is variable, but most appear higher than concentrations previously described in blood. Many samples contained CPS that is well within the detection range of both the AMDTM LFI and the quantitative antigen-capture ELISA. However, some patient samples appeared to have low concentrations of CPS that are currently below the limit of detection for one or both of these assays.
|Advisor:||AuCoin, David P.|
|Commitee:||Burkin, Dean J., Kozel, Thomas R., Rossetto, Cyprian C., Verma, Subhash C.|
|School:||University of Nevada, Reno|
|Department:||Cell and Molecular Biology|
|School Location:||United States -- Nevada|
|Source:||DAI-B 79/12(E), Dissertation Abstracts International|
|Subjects:||Microbiology, Health sciences, Immunology|
|Keywords:||Antibodies, Burkholderia pseudomallei, Lateral flow immunoassay, Zaire ebolavirus|
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