Development of Immunoassays for Detection of Francisella tularensis Lipopolysaccharide in Tularemia Patient Samples

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.

Keywords: Francisella tularensis; LPS; antibodies; diagnostic; enzyme-linked immunosorbent assay; lateral flow immunoassay; lipopolysaccharide; monoclonal antibodies; patient samples; tularemia.

Figure 1

mAb reactivity with purified F. tularensis LPS and F. tularensis strains (type A and type B). Purified, HRP-conjugated mAbs were used to probe 1 μg/lane purified LPS from F. tularensis subsp. holarctica LVS. Previously isolated F. tularensis antibody 1A4 IgG1 was included as a positive control (A), 1 × 10⁸ CFU F. tularensis subsp. tularensis Schu S4 (type A strain) (B) and F. tularensis subsp. holarctica (FRAN-012) (type B strain) loaded across 11 wells (9.09 × 10⁶ cfu/lane) (C) by direct Western blot.

Figure 2

Sensitivity of optimized F. tularensis LPS antigen-capture ELISA. Limit of detection (LOD) of capture antibody 1Ft5 and detection antibody 1Ft7-HRP was assessed. The standard curve of the optimized antigen capture ELISA with F. tularensis LPS spiked into normal human serum and urine is shown. LOD of the assay in each matrix was calculated using a cutoff value of 2× background. LOD in serum was 0.18 ± 0.067 ng/mL and 0.13 ± 0.028 ng/mL (n = 3).

Figure 3

Reactivity of LFIs with clinically relevant F. tularensis strains and near neighbors, and purified LPS from different bacteria. Prototype LFIs were run with a panel of killed whole cells to determine potential usefulness as a diagnostic for tularemia. Brain Heart Infusion broth supplemented with cysteine (BHI-c) is included as a negative control. 100 ng/mL F. tularensis subsp. holarctica LVS LPS was included as a positive control. Whole cells from F. tularensis subsp. holarctica LVS (LVS), F. tularensis subsp. tularensis NIH-B38 (NIH-B38), F. tularensis subsp. holarctica isolate FRAN-012 (FRAN-012), F. tularensis subsp. tularensis Schu S4 (SchuS4), F. novicida, F. philomiragia were also tested for reactivity (A). Prototype LFIs were also tested with 100 ng/mL purified LPS from other species of bacteria to determine potential for cross-reactivity (B).

Figure 4

LFI prototype sensitivity in pooled normal human serum (A) and urine (B) spiked with purified F. tularensis LPS. Visual LOD is indicated (*) as assessed by three blinded readers. Test line intensity is shown as given by the ESE-Quant lateral flow reader.