Development of an antigen detection assay for early point-of-care diagnosis of Zaire ebolavirus

Abstract

The 2013-2016 Ebola virus (EBOV) outbreak in West Africa and the ongoing cases in the Democratic Republic of the Congo have spurred development of a number of medical countermeasures, including rapid Ebola diagnostic tests. The likelihood of transmission increases as the disease progresses due to increasing viral load and potential for contact with others. Early diagnosis of EBOV is essential for halting spread of the disease. Polymerase chain reaction assays are the gold standard for diagnosing Ebola virus disease (EVD), however, they rely on infrastructure and trained personnel that are not available in most resource-limited settings. Rapid diagnostic tests that are capable of detecting virus with reliable sensitivity need to be made available for use in austere environments where laboratory testing is not feasible. The goal of this study was to produce candidate lateral flow immunoassay (LFI) prototypes specific to the EBOV glycoprotein and viral matrix protein, both targets known to be present during EVD. The LFI platform utilizes antibody-based technology to capture and detect targets and is well suited to the needs of EVD diagnosis as it can be performed at the point-of-care, requires no cold chain, provides results in less than twenty minutes and is low cost. Monoclonal antibodies were isolated, characterized and evaluated in the LFI platform. Top performing LFI prototypes were selected, further optimized and confirmed for sensitivity with cultured live EBOV and clinical samples from infected non-human primates. Comparison with a commercially available EBOV rapid diagnostic test that received emergency use approval demonstrates that the glycoprotein-specific LFI developed as a part of this study has improved sensitivity. The outcome of this work presents a diagnostic prototype with the potential to enable earlier diagnosis of EVD in clinical settings and provide healthcare workers with a vital tool for reducing the spread of disease during an outbreak.

Fig 1. Western immunoblot analysis to evaluating monoclonal antibody (mAb) reactivity.

mAb reactivity was assessed using 1 μg recombinant GP (Panel A) or 0.5 μg recombinant VP40 (Panel B).

Fig 2. Sensitivity of EBOV lateral flow immunoassay (LFI) with live cultured EBOV.

The GP-specific LFI prototype, VP40-specific LFI prototype and ReEBOV Antigen Rapid Test (Zalgen Labs) were evaluated for sensitivity with two-fold dilutions of EBOV (variant Makona) culture supernatant; positive (+) or negative (-) results are reported below each LFI. Viral titer of the supernatant was quantified using a TCID50 assay.

Fig 3. EBOV lateral flow immunoassay (LFI) prototype testing of infected non-human primate (NHP) serum.

Panel A–The GP-specific LFI prototype, VP40-specific LFI prototype and ReEBOV Antigen Rapid Test (Zalgen Labs) were tested with serum samples taken 4, 5, 6, and 7.5 days post-infection (DPI) from two EBOV (variant Makona) infected NHPs; positive (+) or negative (-) results are reported below each LFI. Panel B–Quantitative viremia determined via qRT-PCR for 0, 3, 4, 5, 6, 7, and 7.5 days dpi from the same two NHPs; results are reported as genome equivalents (GEQ) per mL of sample.