Develop an ELISA to Screen COVID-19 Inhibitors

Abstract

Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2) outbreak has resulted in the death of several million people across the globe. However, there is currently no vaccine, no drug for the treatment of the disease resulting from the infection of the virus. The search for effective treatment agents for the disease as well as increased understanding of the binding properties of the virus lead to the suggestion that agents, such as heparin (which has been reported to have the ability to bind to the same receptor as the virus), can be developed as a therapeutic agent for the infection. This study aims to develop an ELISA technique for the assessment of the binding of heparin to the receptor binding spike protein of the virus. Recombinant DNA technology was initially used to produce a recombinant version of the spike protein. Following its purification by affinity chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), the secondary structure of the expressed protein was analysed by circular dichroism spectroscopy. The IC50 of the spike protein, heparin and enoxaparin were determined prior to the optimisation of the competitive ELISA between SARS-CoV-2 S1 receptor binding protein (RBD), heparin and the angiotensin-converting enzyme 2 receptor. Results obtained indicated changes in the secondary structure of the recombinant spike protein. BestSel analysis revealed that the percentage of the alpha-helix in the 24 h sample was significantly reduced (P<0.001). The IC50 values obtained for ACE2, heparin and enoxaparin were 26.35 μM, 1.317 μM and 3.163 μg/ml, respectively. The competitive ELISA conducted in this study revealed that the RBD protein binding affinity with ACE2 protein is more potent than the binding with heparin. Moreover, a better ELISA profile was obtained by using RBD as the capture agent and ACE2 protein as the detection material. These results indicate that ELISA can be used to assess the interaction between RDB, heparin and ACE2. It also encourages further research towards the development of therapeutic usage of heparin in COVID-19 patients.