Ultrasensitive point-of-care immunoassay for secreted glycoprotein detects Ebola infection earlier than PCR

Abstract

Ebola virus (EBOV) hemorrhagic fever outbreaks have been challenging to deter due to the lack of health care infrastructure in disease-endemic countries and a corresponding inability to diagnose and contain the disease at an early stage. EBOV vaccines and therapies have improved disease outcomes, but the advent of an affordable, easily accessed, mass-produced rapid diagnostic test (RDT) that matches the performance of more resource-intensive polymerase chain reaction (PCR) assays would be invaluable in containing future outbreaks. Here, we developed and demonstrated the performance of a new ultrasensitive point-of-care immunoassay, the EBOV D4 assay, which targets the secreted glycoprotein of EBOV. The EBOV D4 assay is 1000-fold more sensitive than the U.S. Food and Drug Administration–approved RDTs and detected EBOV infection earlier than PCR in a standard nonhuman primate model. The EBOV D4 assay is suitable for low-resource settings and may facilitate earlier detection, containment, and treatment during outbreaks of the disease.

Synthetic Nanobody-Functionalized Nanoparticles for Accelerated Development of Rapid, Accessible Detection of Viral Antigens

“…Its sensing performance drops ~10 times to 100 pg/mL when using a customized fluorescent system, which costs ~$1,000 and occupies ~3,000 cm 3 . The performance further decreases to 6,000 pg/mL when using LFA with colorimetric readout 18 . Clearly standing apart from the rest, Nano2RED utilizes miniaturized and low-cost semiconductor devices for signal readout rather than a fluorescent system.…”

“…There are several notable observations. First, using sGP sensing as an example and comparing to previously reported results 18,39 (Supplementary Table S2),…”

“…Nano2RED with spectrometric and electronic readout consistently produced ~130 fM to 1.3 pM LOD (or ~10 to ~100 pg/mL) in PBS, FBS, and HPS. It is noted that a very recently reported cobinder-based D4 assay format reported ~30 pg/mL LOD in human serum, and was able to detect the Ebola virus earlier than PCR in a monkey model 18 . In LOD comparison, the sensitivity of Nano2RED (~10 pg/mL with electronic readout) is even better, indicating its competitiveness in high-precision diagnostics.…”

“…sGP is believe to act as decoys to disrupt the host immune system by absorbing anti-GP antibodies 14,22 . Given its abundance in blood stream upon infection and its quantitative correlation with disease progression and humoral response, sGP is widely used as a circulating biomarker in EBOV diagnostics 22,23 .…”

“…sGP is believed to act as a decoy to disrupt the host immune system by absorbing anti-GP antibodies 16,17 . Given its abundance in the blood stream upon infection and its quantitative correlation with disease progression and humoral response, sGP is widely used as a circulating biomarker in EBOV diagnostics 17,18 . The chosen SARS-CoV-2 antigen, RBD, engages the viral receptor, human angiotensin-converting enzyme 2 (ACE2), causing conformational changes that trigger a cleavage needed for viral infection 19,20 .…”