Detection of Francisella tularensis in biological specimens using a capture enzyme-linked immunosorbent assay, an immunochromatographic handheld assay, and a PCR

Abstract

The early detection of Francisella tularensis, the causative agent of tularemia, is important for adequate treatment by antibiotics and the outcome of the disease. Here we describe a new capture enzyme-linked immunosorbent assay (cELISA) based on monoclonal antibodies specific for lipopolysaccharide (LPS) of Francisella tularensis subsp. holarctica and Francisella tularensis subsp. tularensis. No cross-reactivity with Francisella tularensis subsp. novicida, Francisella philomiragia, and a panel of other possibly related bacteria, including Brucella spp., Yersinia spp., Escherichia coli, and Burkholderia spp., was observed. The detection limit of the assay was 10(3) to 10(4) bacteria/ml. This sensitivity was achieved by solubilization of the LPS prior to the cELISA. In addition, a novel immunochromatographic membrane-based handheld assay (HHA) and a PCR, targeting sequences of the 17-kDa protein (TUL4) gene of F. tularensis, were used in this study. Compared to the cELISA, the sensitivity of the HHA was about 100 times lower and that of the PCR was about 10 times higher. All three techniques were successfully applied to detect F. tularensis in tissue samples of European brown hares (Lepus europaeus). Whereas all infected samples were recognized by the cELISA, those with relatively low bacterial load were partially or not detected by PCR and HHA, probably due to inhibitors or lack of sensitivity. In conclusion, the HHA can be used as a very fast and simple approach to perform field diagnosis to obtain a first hint of an infection with F. tularensis, especially in emergent situations. In any suspect case, the diagnosis should be confirmed by more sensitive techniques, such as the cELISA and PCR.

FIG. 1

Western blot of LPS-specific MAbs to F. tularensis. Whole bacterial antigen of F. tularensis was separated by SDS-polyacrylamide gel electrophoresis with 4 to 20% Tris-glycine gel and blotted onto a nitrocellulose membrane. The MAbs Ft-27 and Ft-11 were used at 5 μg/ml in PBS (A). For specificity control, the MAbs were preincubated with extracted LPS from 10⁴ (B), 10⁶ (C), and 10⁸ (D) bacteria/ml (lane E, molecular mass markers).

FIG. 2

Comparison of detection of whole bacteria and extracted LPS of F. tularensis by cELISA. Serial dilutions of whole bacteria and extracted LPS antigen of F. tularensis LVS from spiked PBS and pooled human sera were tested in the LPS-specific cELISA. The results are given as OD₄₅₀.

FIG. 3

Sensitivity of cELISA, HHA, and PCR for the detection of F. tularensis. Serial dilutions of the solubilized LPS antigen of F. tularensis in PBS and in pooled human normal sera (NS-P) were tested in the specific cELISA (upper diagram). The results are given as OD₄₅₀. The same dilutions of bacteria were tested in the immunochromatographic HHA and in the PCR (lower table). The results are expressed as relative intensity of obtained visible bands. +, light; ++, medium; +++, dark; neg, negative; n.t., not tested.