Senior Research Scientist Promega Corporation Madison, WI, United States
Abstract: The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was initially detected in December 2019, and became a worldwide pandemic of viral respiratory illness. SARS-CoV-2 virus enters target cells through the interaction between its surface Spike protein (S) and the Angiotensin Converting Enzyme 2 (ACE2) receptor on human cells. The importance of the Spike protein for viral internalization is highlighted by the efforts to identify molecules that would interfere with its binding to ACE2, thus preventing its cellular internalization resulting in an effective therapeutic treatment against COVID-19. Towards that goal, an assay that monitors the interaction of SARS-CoV-2 Spike RBD and human ACE2 would be a useful tool both for high throughput screening for inhibitory molecules as well as for the screening and characterization of clinical specimens that may contain virus neutralizing antibodies (Nabs). Commonly used methods such as neutralizing tests using live virus or virus like particles to detect Nabs in patient derived samples and ELISA for monitoring RBD: ACE2 interactions are labor intensive, time consuming and low throughput. Therefore, we developed Lumit SARS-CoV 2 Spike RBD: hACE2 Immunoassay, a bioluminescent no-wash, add-and-read assay that relies on the detection of recombinant RBD and ACE2 protein-protein interaction (PPI) that is based on NanoBiT assisted complementation technology. In this immunoassay, detection of the interaction between RBD and ACE2, using rabbit Fc-domain tagged RBD and a mouse Fc-domain tagged ACE2 can be monitored by the addition of SmBiT- and LgBiT-conjugated secondary antibodies (Lumit Anti-Mouse Ab-LgBiT and Lumit Anti-Rabbit Ab-SmBiT). Interaction of RBD and ACE2 along with binding of the Lumit secondary antibodies to their corresponding epitopes on the Fc-domains brings NanoBiT subunits into proximity to form an active NanoLuc luciferase that generates light in proportion to the PPI level. The Lumit SARS-CoV 2 Spike RBD: hACE2 Immunoassay is solution-based, and does not require washing, liquid transfer, or immobilization steps. The assay takes less than 2 hours to complete in a homogeneous “add-and-read” format. This assay is simple, making it suitable for high-throughput screening and for detection of not only small molecules interfering with the RBD: ACE2 PPI but also was successfully used as a surrogate assay for detecting neutralizing antibodies in buffer, patient serum, and plasma samples. This method could have broad utility, from enabling the screening of novel viral entry inhibitors, to monitoring the efficacy of newly developed antibodies against the virus.