Development of an Immunochromatography Assay (QuickNavi-Ebola) to Detect Multiple Species of Ebolaviruses

Abstract

The latest outbreak of Ebola virus disease (EVD) in West Africa has highlighted the urgent need for the development of rapid and reliable diagnostic assays. We used monoclonal antibodies specific to the ebolavirus nucleoprotein to develop an immunochromatography (IC) assay (QuickNavi-Ebola) for rapid diagnosis of EVD. The IC assay was first evaluated with tissue culture supernatants of infected Vero E6 cells and found to be capable of detecting 10³-10⁴ focus-forming units/mL of ebolaviruses. Using serum samples from experimentally infected nonhuman primates, we confirmed that the assay could detect the viral antigen shortly after disease onset. It was also noted that multiple species of ebolaviruses could be detected by the IC assay. Owing to the simplicity of the assay procedure and absence of requirements for special equipment and training, QuickNavi-Ebola is expected to be a useful tool for rapid diagnosis of EVD.

Keywords: EVD; Ebola virus; diagnosis; ebolavirus; filovirus; immunochromatography assay; monoclonal antibody; nucleoprotein.

Figure 1.

Design of the immunochromatography (IC) assay. QuickNavi-Ebola is a lateral flow–based IC kit (85 mm × 21 mm × 6.9 mm) for the direct detection of ebolavirus nucleoprotein (NP) antigens. For each assay, 30 μL of serum/plasma or 10–20 μL of whole-blood samples can be used. When a sample is added to the sample window of the kit, it migrates through the reagent pads via capillary action. One of the reagent pads is a conjugate pad on which latex conjugated with an anti–ebolavirus NP monoclonal antibody (mAb) has been dried. The ebolavirus NP antigens present in the sample bind to the latex-conjugated mAbs on the pad. Another ebolavirus NP-specific mAb is also immobilized on a nitrocellulose membrane at the test line position to capture the complexes of NP antigens and mAbs conjugated with latex. As the assay develops, those complexes deposit a visible blue line.

Figure 2.

Hematological changes and 50% tissue culture infective doses (TCID₅₀) of nonhuman primates (NHPs) infected with Ebola virus (EBOV). Virus titers in the blood samples were determined as TCID₅₀ in Vero E6 cells (please also see NHPs 1 and 2 in Table 3). Platelet counts were determined in whole-blood samples. Alanine aminotransferase levels (ALT) were determined in serum specimens. The arrows indicate the time at which the NHPs tested had positive results of the immunochromatography assay.