Abstract
Francisella tularensis is the causative agent of tularemia, a zoonotic bacterial infection that is often fatal if not diagnosed and treated promptly. Natural infection in humans is relatively rare, yet persistence in animal reservoirs, arthropod vectors, and water sources combined with a low level of clinical recognition make tularemia a serious potential threat to public health in endemic areas. F. tularensis has also garnered attention as a potential bioterror threat, as widespread dissemination could have devastating consequences on a population. A low infectious dose combined with a wide range of symptoms and a short incubation period makes timely diagnosis of tularemia difficult. Current diagnostic techniques include bacterial culture of patient samples, PCR and serological assays; however, these techniques are time consuming and require technical expertise that may not be available at the point of care. In the event of an outbreak or exposure a more efficient diagnostic platform is needed. The lipopolysaccharide (LPS) component of the bacterial outer leaflet has been identified previously by our group as a potential diagnostic target. For this study, a library of ten monoclonal antibodies specific to F. tularensis LPS were produced and confirmed to be reactive with LPS from type A and type B strains. Antibody pairs were tested in an antigen-capture enzyme-linked immunosorbent assay (ELISA) and lateral flow immunoassay format to select the most sensitive pairings. The antigen-capture ELISA was then used to detect and quantify LPS in serum samples from tularemia patients for the first time to determine the viability of this molecule as a diagnostic target. In parallel, prototype lateral flow immunoassays were developed, and reactivity was assessed, demonstrating the potential utility of this assay as a rapid point-of-care test for diagnosis of tularemia.
Highlights
Ten monoclonal antibodies (mAbs)-producing hybridoma cell lines were created from female CD1 mice immunized with LPS purified from the CDC Live Vaccine Strain of F. tularensis subsp. holarctica (BEI Resources) conjugated to BSA and administered with or without Alhydrogel® adjuvant 2%. mAbs were purified and subclass was determined by indirect enzyme-linked immunosorbent assay (ELISA)
The importance of developing a panel of mAbs to test different pairs can be seen in the variation between pairs shown in positive test for a given antibody pair, or limit of detection (LOD), for LPS in PBS was calculated at 3× background OD 450 nm value and an average of two rows taken to rank the pairs to proceed with optimization (Table S1)
This need is compounded by the status of F. tularensis as a potential biothreat
Summary
Respiratory tularemia resulting from inhalation of aerosolized F. tularensis is the most severe of the organ-specific infections, involving Type A strains. Confirmation of tularemia diagnosis can be made by measuring the fold change in serological response to infection via detection of antibodies to F. tularensis lipopolysaccharide (LPS) in patient serum. This approach is limited in that often antibodies do not reach diagnostically significant levels until approximately two weeks post-infection [25], and can persist for decades in endemic areas, potentially complicating later diagnosis and meaning that changes in titers over time need to be monitored [26]. Prototype LFIs were constructed and reactivity with clinically relevant strains was determined
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