Ebola Zaire ELASA
Base Pair has been collaborating with Dr. Misaki Wayengera of Makerere University in Uganda to explore aptamers for the development of simpler, more sensitive assays for the diagnosis of Ebolavirus*.
Ebolavirus is a filovirus that causes severe hemorrhagic fever with a high rate of mortality. Believed to originate with the fruit bat, the virus can be transferred from infected animals and insects and also through the transfer of blood, mucus, or interaction with infected materials. On August 1, 2018, the Ministry of Health of the Democratic Republic of the Congo in Africa declared a new outbreak of Ebola Virus Disease (EVD) in the North Kivu Province. As of June 9, 2019, there have been 1,968 confirmed cases and 1,296 confirmed deaths1. An experimental vaccine is being administered and a clinical trial for a new therapeutic is underway2.
GP1 glycoprotein appears as spikes on the surface of the Ebolavirus virion3. The glycoprotein is a heterodimer consisting of a GP1 and GP2 subunit. The glycoprotein forms a trimer consisting of three heterodimers on the virion surface. The GP1 subunit is involved in attachment to host cells and includes a receptor-binding domain. The smaller GP2 subunit includes a fusion peptide, a transmembrane domain and a short cytoplasmic tail. Glycoprotein residues involved in host entry are highly conserved between Zaire, Sudan, Cote d’Ivoire and Reston species of Ebolavirus4.
*The current work and Dr. Wayengera’s filovirus laboratory at Makerere University are funded by the European & Developing Countries Clinical Trials Partnership ( EDCTP). Grand Challenges Canada provided the initial seed funding for the pan filovirus RDT innovation in 2013. **Heinz Feldmann, Peter Jahrling, Elizabeth Fischer, and Anita Mora, National Institute of Allergy and Infectious Diseases, National Institutes of Health
- WHO, Ebola situation reports: Democratic Republic of the Congo, accessed June 11, 2019, https://www.who.int/ebola/situation-reports/drc-2018/en/
- WHO, Ebola virus disease fact sheet, accessed June 11, 2019, https://www.who.int/news-room/fact-sheets/detail/ebola-virus-disease
- Nehls, J., et al. Release of immunomodulatory ebola virus glycoprotein-containing microvesicles is suppressed by tetherin in a species-specific manner. 2019. Cell Reports. 26: 1841-1853.
- Lee, J.E. and Saphire, E.L. Ebolavirus glycoprotein structure and mechanism of entry. Future Virology. 2009. 4(6):621-635.
Base Pair has developed a prototype sandwich assay for the detection of Zaire Ebolavirus GP1 glycoprotein based on the use of selective DNA aptamers. A chart showing signal (in relative fluorescence units) versus concentration of Zaire Ebolavirus GP1 glycoprotein is shown at right. This preliminary test included 1:4 dilutions of GP1 glycoprotein, with concentrations of 250 nM, 62.5 nM, and 15.6 nM falling within the preliminary detectable range. Aptamer concentrations and buffer formulations have not yet been optimized for this assay.
Ebola Assay Protocol
A sequential sandwich assay was performed, with wash steps separating binding of the capture aptamer, GP1 protein, detecting aptamer, and secondary detecting antibody.
- Fold Capture and Detecting Aptamers.
- Add 100 µL of diluted Capture Aptamer to each well. Incubate for 1 hour with shaking. Decant.
- Add 200 µL of Blocking Buffer to each well. Incubate for 1 hour with shaking.
- Wash 3x with Assay Buffer.
- Add 50 µL of diluted Standards and Samples to each well. Incubate for 1 hour with shaking.
- Wash 3x with Wash Buffer.
- Add 50 µL of diluted Detecting Aptamer to each well. Incubate for 1 hour with shaking.
- Wash 3x with Wash Buffer.
- Add 50 µL of diluted Detecting Antibody to each well. Incubate for 30 minutes with shaking.
- Wash 3x with Wash Buffer
- Add 100 µL of Substrate to each well. Incubate for 10 minutes in the dark.
- Read fluorescence at excitation 560 and emission 590.
A series of 1:4 dilutions of culture supernatant containing inactivated Zaire Ebolavirus virion (supplied by John Klena, Ph.D. of the CDC) were tested in the assay. 1:54 and 1:256 dilutions fell within the detectable range of the current assay. Because the concentration of inactivated Zaire Ebolavirus virion present in the neat supernatant is unknown, it is not possible to determine a minimum detectable concentration of Zaire Ebolavirus virion for the prototype assay.
Optimization of this assay, comparison to existing diagnostic tests, and evaluation of other species of Ebolavirus virion in the assay are potential next steps in the development of an aptamer-based sandwich assay. The aptamers have been shown to detect the GP1 glycoprotein on the surface of the Zaire Ebolavirus virion. Further testing is required to determine the effect of these aptamers on GP1-mediated binding of virion to host cells and evaluate therapeutic potential.
Contact Base Pair for more information about the Zaire Ebolavirus aptamers and detection assay.