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.
Keywords: Gaussia Luciferase; SARS CoV-2; protein-fragment complementation assay; recombinant antibodies; spike (S) protein.
【저자키워드】 SARS CoV-2, recombinant antibodies, spike (S) protein, Gaussia Luciferase, protein-fragment complementation assay, 【초록키워드】 COVID-19, SARS CoV-2, Treatment, spike, antibody, threat, diagnostic, SARS-CoV-2 virus, Protein, sensitivity, specificity, Epitopes, Viral, SARS-CoV-2 antigen, virus-like particles, reporter, epitope, Incubation, binding, S-protein, recombinant, PCA, complementary, Virus-like particle, other diseases, enzyme, SARS CoV, high affinity, promise, pathogenic, viral spike, Express, luciferase, catalytic activity, Complementation, reporters, Trimers, develop, assays, detectable, applied, C-terminal, steric, the SARS-CoV-2 virus, 【제목키워드】 SARS-CoV-2, Specific,