Detection of Francisella tularensis-Specific Antibodies in Patients with Tularemia by a Novel Competitive Enzyme-Linked Immunosorbent Assay

Abstract

A novel competitive enzyme-linked immunosorbent assay (cELISA) was developed and evaluated for detection of antibodies against Francisella tularensis in humans. The assay is based on the ability of serum antibodies to inhibit the binding of monoclonal antibodies (MAbs) directed against F. tularensis lipopolysaccharide antigens. The assay was evaluated using serum samples of tularemia patients, inactivated F. tularensis-immunized rabbits, and F. tularensis-infected mice. Antibodies against F. tularensis were successfully detected in serum samples of tularemia patients as well as the immunized and infected animals. The cELISA method was compared to indirect ELISA (iELISA) and the commonly used microagglutination test (MA) using serum samples of 19 tularemia patients and 50 healthy individuals. The sensitivity and specificity of cELISA were 93.9 and 96.1%, respectively, in comparison to the iELISA. MA was less sensitive than cELISA with a sensitivity and specificity of only 81.8 and 98.0%, respectively. A high degree of correlation (R 2 ‫؍‬ 0.8226) was observed between cELISA and iELISA results. The novel cELISA developed in this study appears to be highly sensitive and specific for serodiagnosis of human tularemia. The potential of the MAb-based cELISA to be used in both human and animal samples emphasizes its usefulness for serological survey of tularemia among multiple animal species.

Serosurveillance forFrancisella tularensisAmong Wild Animals in Japan Using a Newly Developed Competitive Enzyme-Linked Immunosorbent Assay

“…and Yersinia spp., was observed (Sharma et al 2013). In the present study, the cutoff value in the cELISA for animal specimens was set as 40%, thus cross-reaction of antibodies to F. novicida was negligible.…”

“…cELISA cELISA was performed to detect antibodies to F. tularensis in the blood samples of the wild animals, using previously described protocols with some modifications (Sharma et al 2013). In brief, 96-well microtiter plates (Greiner BioOne, Frickenhausen, Germany) were coated with purified lipopolysaccharide (LPS) from F. tularensis holarctica (strain NVF1, a Japanese isolate from a wild hare in 2009) in carbonate-bicarbonate buffer (pH 9.6) (2.5 lg/50 lL per a Only 45 of the 142 Japanese hare samples were tested using the MA test.…”

“…The indirect enzyme-linked immunosorbent assay (iELISA) is frequently used for serological surveys of tularemia and has high sensitivity (Al Dahouk et al 2005); however, its usefulness in seroepidemiological studies of various wild animals is limited because of the unavailability of species-specific secondary antibodies. We recently developed a highly sensitive and specific monoclonal antibody (mAb)-based competitive ELISA (cELISA) for use in tularemia patients (Sharma et al 2013). In the present study, we used this novel cELISA to examine the seroprevalence of tularemia among wild animals in Japan.…”

In Vitro Antibiotic Susceptibility of <i>Francisella tularensis</i> Isolates from Japan

“…These isolates were originated from humans (27 isolates), hares (3 isolates), ticks (3 isolates), and Japanese shrew mole (1 isolate) (6). Two recent isolates, KU-1, a generous gift from Prof. H. Sato (Kitasato University, Aomori, Japan) (5), and NVF1, were isolated from wild hare carcasses in 2008 and 2009, respectively (12). All the F. tularensis isolates were grown on Eugon agar plates containing 8z (w/w) chocolatized sheep blood (13) and passaged twice in Mueller-Hinton broth (BD, Sparks, Md., USA) supplemented with 0.1z (w/v) glucose, 2z (v/v) IsoVitalX (BD), and 0.025z (w/v) ferric pyrophosphate (14).…”

Antibodies to Both Terminal and Internal B-Cell Epitopes of Francisella tularensisO-Polysaccharide Produced by Patients with Tularemia

“…The results also build on a recent report that used a mouse MAb specific for F. tularensis LPS as a reporter in competition ELISA with human blood serum samples from tularemia patients to demonstrate the potential use of the assay for the surveillance of tularemia (39). Another recent report used two mouse MAbs specific for invasion-inhibitory or noninhibitory epitopes of the Plasmodium falciparum protein apical membrane antigen 1 as reporters in competition ELISA to detect antibodies that inhibit the antigen binding of each MAb in the serum samples of malaria patients (40).…”