Structure-based Design of a Specific,Homogeneous Luminescence Enzyme Reporter Assay for SARS-CoV-2 |
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Affiliation: | 1. The Donnelly Centre, University of Toronto, Toronto, Canada;2. Department of Molecular Genetics, University of Toronto, Toronto, Canada;3. Département de Biochimie, Université de Montréal, Montréal, Canada |
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Abstract: | Recombinant antibodies (Abs) against the SARS-CoV-2 virus hold promise for treatment of COVID-19 and high sensitivity and specific diagnostic assays. Here, we report engineering principles and realization of a Protein-fragment Complementation Assay (PCA) detector of SARS-CoV-2 antigen by coupling two Abs to complementary N- and C-terminal fragments of the reporter enzyme Gaussia luciferase (Gluc). Both Abs display comparably high affinities for distinct epitopes of viral Spike (S)-protein trimers. Gluc activity is reconstituted when the Abs are simultaneously bound to S-protein bringing the Ab-fused N- and C-terminal fragments close enough together (8 nm) to fold. We thus achieve high specificity both by requirement of simultaneous binding of the two Abs to the S-protein and also, in a steric configuration in which the two Gluc complementary fragments can fold and thus reconstitute catalytic activity. Gluc activity can also be reconstituted with virus-like particles that express surface S-protein with detectable signal over background within 5 min of incubation. Design principles presented here can be readily applied to develop reporters to virtually any protein with sufficient available structural details. Thus, our results present a general framework to develop reporter assays for COVID-19, and the strategy can be readily deployed in response to existing and future pathogenic threats and other diseases. |
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Keywords: | recombinant antibodies SARS CoV-2 spike (S) protein protein-fragment complementation assay Gaussia Luciferase |
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