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Development of diagnostic immunoassays for tularemia and leptospirosis
AuthorHannah, Emily Elizabeth
AdvisorAuCoin , David P
Cell and Molecular Biology
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The zoonotic bacteria Francisella tularensis and Leptospira species are the causative agents for tularemia and leptospirosis respectively. Both of these diseases have significant deficits in the diagnostic tools available for efficient early diagnosis, which can result in delayed treatment of these potentially life-threatening infections. Both bacteria are extremely challenging to isolate from patients for definitive diagnosis and instead serological techniques for detection are relied on. Antibody titers can take up to two weeks to reach diagnostic significance, and the assays used to measure this often require extensive laboratory equipment and trained staff to perform. To address the lack of diagnostic options available for these bacteria, libraries of monoclonal antibodies to potential diagnostic targets were produced for inclusion in diagnostic immunoassays. Mice were immunized with purified F. tularensis lipopolysaccharide and ten monoclonal antibodies were isolated and confirmed to be reactive to pathogenic strains of the bacteria. The antibodies were screened, and reactive pairs identified to develop a sensitive, quantitative enzyme-linked immunosorbent assay (ELISA) and prototype lateral flow immunoassay (LFI) for rapid point of care detection. The ELISA was optimized in human serum and urine and used to quantify LPS in filtered tularemia patient serum samples. Eight samples contained quantifiable levels of F. tularensis LPS, showing that LPS is a viable diagnostic antigen for tularemia. An LFI prototype was developed and determined to be specific for pathogenic F. tularensis. A limit of detection of 5 ng/mL purified LPS in normal human serum and urine was determined. Leptospiral lipoprotein LipL32 was selected as the diagnostic target for production of antibodies specific for pathogenic Leptospira. This antigen is conserved in pathogenic Leptospira and is not present in saprophytic strains. Mice were immunized with purified recombinant LipL32 and fifteen monoclonal antibodies were isolated. Reactivity of these antibodies was confirmed with heat inactivated pathogenic Leptospira species. These antibodies were used to develop a Western blot for direct detection of LipL32 in patient urine samples. A limit of detection of between 2.72x103 and 1.36x103 colony forming units/mL heat inactivated L. interrogans spiked into pooled normal human urine. Previously described clinical ranges indicate that this sensitivity would likely be sufficient for detection of leptospires in symptomatic leptospirosis patients.