The potential bioterrorism agents, Francisella tularensis and Burkholderia pseudomallei, are the etiologic agents of two life-threatening diseases, termed tularemia and melioidosis, respectively. An early diagnosis and a timely treatment regimen are crucial for successful therapeutic outcomes. However, bacterial isolation, which is known to be time-consuming and have low sensitivity, remains the 'gold standard' for diagnosis of these infections. Therefore, in the first study in this dissertation, we developed an antigen-capture enzyme-linked immunosorbent assay (ELISA) targeting bacterial surface-expressed lipopolysaccharide (LPS) for rapid F. tularensis detection. A murine monoclonal antibody (mAb) specific to F. tularensis LPS, 1A4 IgG3, and its subclass family (1A4 IgG1 and 1A4 IgG2b, bearing the same antigen-binding site as mAb 1A4 IgG3) were isolated and used for assay development. Surface plasmon resonance (SPR) and competition ELISA were used to assess the binding affinities of the mAbs. We found that the assay developed using 1A4 IgG1 or IgG2b had better assay sensitivity compared to when the IgG3 was used. Interestingly, while the assay sensitivity was improved, we also found a decrease in functional affinity as a result of subclass switching. Direct ELISA and SPR suggested that the higher affinity of 1A4 IgG3 might be related to self-association, which correlated to high assay background and low assay sensitivity. Altogether, we demonstrated that IgG subclass switch could improve assay sensitivity by reduction of the assay background (through elimination of IgG3 self-association).
As for melioidosis, a rapid diagnostic targeting B. pseudomallei CPS (Active Melioidosis Detect (AMD™) rapid test) has already been developed and is currently being assessed. However, a rapid immunoassay for differentiation between typical and atypical LPS strains has never been developed. This is important to advance our understanding of the epidemiology and pathology of melioidosis. Thus, in the second project, we developed antigen-capture immunoassays for typical and atypical LPS strain typing using CPS-specific mAb 4C4 for bacterial capture, and mAbs 4C7 and 3A2 for detection of typical and atypical LPS strains, respectively. In this study, two atypical LPS-specific mAbs (3A2 and 5B4) were successfully isolated; SPR results suggested that 3A2 is preferable for the assay. B. pseudomallei (174 strains) was used to evaluate the assay, and the results showed the assays have 98.8% accuracy, suggesting that they are effective and applicable for B. pseudomallei LPS typing.
Additionally, in the present work, we also investigate the in vivo fate of B. pseudomallei CPS using a murine model. The goal of this study was to improve our understanding of the appropriate clinical use of the AMD™ test. We found that CPS has a short serum half-life and is eliminated predominantly through the urine, suggesting that i) the presence of CPS in serum and/or urine may be indicative of active melioidosis, ii) CPS may be used as a marker to monitor and assess melioidosis treatment outcome, and iii) in addition to serum, urine (a noninvasive sample) has the potential to be used as a clinical specimen for the diagnosis of melioidosis.
|Advisor:||AuCoin, David P.|
|Commitee:||Hunter, Kenneth, Kozel, Thomas R., Shintani, David, Verma, Subhash C.|
|School:||University of Nevada, Reno|
|Department:||Cell and Molecular Biology|
|School Location:||United States -- Nevada|
|Source:||DAI-B 77/12(E), Dissertation Abstracts International|
|Subjects:||Microbiology, Health sciences, Immunology|
|Keywords:||Burkholderia pseudomallei, Capsular polysaccharide, Francisella tularensis, Immunoassay, Lipopolysaccharide|
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