Development of a high-sensitivity vertical flow immunoassay for the detection of Rift Valley fever virus

Abstract

Rift Valley fever (RVF) is a vector-borne, zoonotic infectious disease with a proven history of morbidity and mortality in both humans and animals. Rift Valley fever virus (RVFV) is categorized as a high-priority biothreat agent by the Centers for Disease Control and Prevention and poses a serious national threat due to its ease of dissemination and potential for social disruption. RVF often presents as a febrile disease without specific symptoms, making early-stage detection particularly challenging. As such, it is critical that rapid, sensitive, and specific diagnostics are available for the detection of RVFV. While lateral flow immunoassays (LFIs) have been developed and validated for point-of-care (POC) diagnostics, vertical flow immunoassays (VFIs) provide enhanced analytical sensitivity and are equally suitable for POC use. In this study, we developed a VFI system for the detection of RVFV, achieving a limit of detection of 0.78 ng/mL, which is a 2.5-fold increase in analytical sensitivity compared to an LFI prototype. Furthermore, minimal cross-reactivity was demonstrated when performing the assay with target analytes of other high-priority biothreats and one other common viral nucleoprotein. This high-sensitivity VFI has the potential to prove useful for the detection of RVFV and other high-priority biothreat agents at the POC.IMPORTANCEIn this study, we have developed a rapid, sensitive vertical flow immunoassay (VFI) for the detection of Rift Valley fever virus (RVFV) in spiked human serum. The prototype diagnostic described in this research was shown to be more sensitive than traditional methods, such as lateral flow dipstick tests. Moreover, the VFI is readily deployable at the point of care in resource-limited settings. The ability of the described diagnostic to accurately and rapidly detect RVFV in samples could expedite the delivery of life-saving care and thus improve patient outcomes.

Keywords: Rift Valley fever virus; biothreat agents; colorimetric detection; rapid antigen test; vertical flow device.

Fig 1

Western blot analysis of RVFV mAb library. Anti-RVFV mAbs 1RV1, 1RV2, 1RV3, 1RV4, and 1RV5 (250 ng/mL) were used in lanes 1–5, respectively. Reactivity to probe non-reduced RVFV rNP (A) and reduced RVFV rNP (B) is demonstrated. HRP-conjugated goat anti-mouse IgG was used as the detection marker.

Fig 2

Sensitivity and specificity testing of the RVFV LFI prototype. RVFV rNP suspended in 1× DPBS (A) or NHS (B) was applied to the sample pad of the LFI prototype at varying concentrations. Presence or absence of a test line, as interpreted by the eye, is depicted as (+) or (−) to indicate the visual result. Gamma-irradiated RVFV lysate suspended in NHS (1:200) was assayed on the RVFV LFI prototype (C). Antigen biomarkers from other pathogens were assayed for cross-reactivity (100 ng/mL) in NHS (D).

Fig 3

Spot size, density of cAb, and signal intensity reported as functions of the volume of 1RV4 immobilized; VFI membranes were run with 2.5 ng/mL of RVFV rNP in 5 mL of buffer (A and B). Image of 1RV4 cAb optimization membrane after assay (C). Optimization of dAb concentration involved measuring signal intensity with varying levels of AuNP-1RV5 (D).

Fig 4

Calibration curve for detection of RVFV rNP in spiked buffer samples (A). LOD study for the detection of RVFV rNP in spiked 10% NHS samples (B). Background (mean negative + 3σ) signal is shown by red dotted line for control samples, and the LOD is indicated as “*” in each graph.

Fig 5

Evaluation of VFI specificity for Rift Valley fever virus nucleoprotein. All antigens were spiked at 10 ng/mL into 10% NHS.