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.

Development of a dual antigen lateral flow immunoassay for detecting Yersinia pestis

“…Evaluation of LFI and ELISA mAb pairs for Ebola glycoprotein and F . tularensis in our laboratory have also shown differences between the two formats [ 64 , 65 ]. Though both LFIs and ELISAs are antigen-capture immunoassays, there are notable differences in the assay formats.…”

Digital image analysis for biothreat detection via rapid centrifugal microfluidic orthogonal flow immunocapture

“…Prototype LFI strips were prepared according the protocol outlined in DeMers et al 28 As described above, 2.5 μl of eVLP-specific antibodies (1HK7 at 5.1 mg ml −1 ) was manually spotted onto a 4 mm NC membrane cutout (BioRad). Fully assembled discs were dried and cured as described above.…”

“…On-disc, vertical ow with sandwich-type immunocapture EBOV and Y. pestis reagents were prepared per the protocol described in DeMers et al and Hau et al, respectively. 28,29 Monoclonal antibodies specic to EBOV glycoprotein (GP) or to Y. pestis F1 (YpF1) capsular antigen were used as capture antibodies herein; each 4 mm NC membrane punch was manually spotted with 2.5 ml of 1HK7 (EBOV GP-specic mAb at 5.1 mg ml −1 ) or Yp11C7 0416 IgG1 (YpF1 specic-mAb at 5.95 mg ml −1 ). Fully assembled discs were dried and cured as described above.…”

“…Recombinantly produced EBOV virus-like particles (eVLP) expressing both EBOV GP and VP40 proteins were used for EBOV assay evaluation; eVLPs are non-infectious and mimic the morphology of the live virus. 28 The YpF1 protein used as antigen for the Y. pestis assay was also recombinantly expressed as described previously. 29 Antigen dilutions (10.0 and 1.0 mg ml −1 ) were prepared by mixing stock eVLP and YpF1 solutions (eVLP = 10 mg ml −1 and YpF1 = 430 mg ml −1 ) with assay buffer.…”

COVID-19 diagnosis in a Senegalese company: A model for COVID-19 vaccination?

“…Our first local response to the outbreak was to established a free, rapid medical screening consultation in our company located in the town of Saint-Louis (northern Senegal) because the nearest existing laboratory for PCR detection was over 100 km away and the result time was at least a week. Simple, rapid antigen detection assays have already proved their worth in epidemic and endemic situations [1,2] thus we have been using the Abbott Panbio TM COVID-19 Ag Rapid Test to detect SARS-CoV-2 nucleocapsid protein in naso-pharyngeal samples. This assay is rapid, giving results in 20 min, and has been approved by the WHO.…”