27th April 2020

Currently, viral infection is a serious threat for human beings. The world is facing a global pandemic associated with the COVID-19 virus, for which virulence and infectious dose data are still emerging. With almost 3,025,732 confirmed cases of COVID-19 worldwide and 209,001 deaths as of 27th April 2020 as per WHO, accurate and early detection of such viruses is highly crucial for clinical diagnosis and therapeutics.

Currently there are few specific antiviral strategies, but several potent candidates of antiviral agents are under urgent investigation. Most biomedical studies have focused primarily on measuring ultra-low or low abundance serum markers based on the enzyme-linked immunosorbent assay (ELISA) platform because it is a highly sensitive detection method and the use of commercial antibodies is attractive.  However, it comes with limitations due to the highly non-specific interaction and signal interference from the most abundant serum proteins, which generate a non-measurable signal.

A recent method that has evolved from the ELISA is the Enzyme linked oligonucleotide assay (ELONA), which has similar characteristics in quantifying and detecting an analyte. Aptamer-based ELONA assays for virus detection have shown to improve these drawbacks and have proven to be of great potential as a feasible tool in virus detection. Such target-binding aptamers selected using SELEX technology offers various advantages over antibodies in terms of:

Taking advantage of highly specific aptamers, Lee and Zeng, 2017 successfully isolated ssDNA aptamers against Zika NS1 protein and developed an aptamer mediated ELISA assay for highly specific and sensitive detection of Zika NS1 antigen in buffer and human serum.

With the extraordinary high binding affinity coupled with high specificity, the aptamer – antibody pairing were successfully used to construct an ELISA based assay with detection limit of 0.1 ng/ml in buffer and as low as 1 and 10 ng/ml in 10 % and 100 % human serum respectively (Figure 1). The results overall suggested that aptamers would be useful for medical diagnosis of Zika virus infection in various aptamer-based diagnostic devices including ELISA assay.

Figure 1. (A) Schematic representation of Aptamer based ELISA assay. (B) Biotin tagged aptamer specific for Zika NS1 showed high specificity for the target with negligible binding to Dengue NS1 serotypes along with IFN- γ and BSA using BLItz. (C) Sandwich complex-based ELISA assay for Zika NS1 detection.

Aptamers have shown several advantages over antibodies in sandwich format assays. In this view, Kang et al., 2019 demonstrated a new SELEX method to select aptamers specific for influenza A viral nucleoprotein (InfA NP) that bound to the antibody – antigen complex in a sandwich form. The aptamers selected by this method allowed highly sensitive and accurate detection of the target virus without interference signals from viruses of other types, as confirmed by ELONA assay.

The aptamers showed the strongest binding affinity to InfA NP with the detection limit of 1.02 ng/mL, and the detection range was 1–300 ng/mL (Figure 2A). The ELONA assay also showed high signal intensity from the InfA NP sample as compared to the closely related target, InfB NP (Figure 2B). The visual detection limit of this method was five times greater than that of the colorimetric assay in a sandwich format using an antibody as a detecting agent.

Figure 2. (A) Detection limit of InfA NP as determined by naked eye (a) titration curve (b) using ELONA assay. (B) Specificity of selected InfA NP aptamer towards the target InfA.

Overall, aptamers could provide a strong tool for the expansion of new diagnostic factors including ELISA and various biosensors in virus infections with higher signal to noise ratio as compared to traditional antibodies. Since the outbreak of the novel coronavirus disease COVID-19, caused by the SARS-CoV-2 virus, this disease has spread rapidly around the globe. The poor clinical outcome proves the high severity of the disease. Aptamers can offer great resources for improving global healthcare by early detection and thereby preventing the spread of highly contagious diseases.

At Aptamer Group Ltd, our scientists are continuously working to develop an aptamer binder selective for the spike protein in SARS CoV-2 which we expect to complete in 2-3 weeks time. Our aim is to work this into a point-of-use test for the detection of virus itself, and have a proof-of-concept in two months. Please contact us using the form below if you would like more information and if this is something that you’d like to get involved in.

References:

  • Kang J. et al. Development of a DNA aptamer selection method based on the heterogeneous sandwich form and its application in a colorimetric assay for influenza A virus detection. New Journal of Chemistry, 43(18). DOI: 10.1039/C8NJ06458J.
  • Lee, K. H., & Zeng, H. (2017). Aptamer-Based ELISA Assay for Highly Specific and Sensitive Detection of Zika NS1 Protein. Analytical Chemistry, 89(23), 12743–12748.doi:10.1021/acs.analchem.7b02862.

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