Structure-based Design of a Specific, Homogeneous Luminescence Enzyme Reporter Assay for SARS-CoV-2. Issue 13 (25th June 2021)

• Record Type: Journal Article
• Authors: Fellouse, Frederic A.; Miersch, Shane; Chen, Chao; Michnick, Stephen W.
• Is Part Of: Journal of molecular biology. Volume 433:Issue 13(2021)
• Publication Date: 2021-06-25
• Subjects: recombinant antibodies -- SARS CoV-2 -- spike (S) protein -- protein-fragment complementation assay -- Gaussia Luciferase; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2021.166983
• Languages: English
• ISSNs: 0022-2836

Abstract

Graphical abstract: Highlights: Structure-based design of a fast, simple and highly specific SARS-CoV-2 reporter. High specificity assured by two epitope recombinant antibody recognition of Spike antigen. Reporter created from antibodies coupled to a luciferase fragment complementation assay. Resulting reporter can detect SARS-CoV-2 pseudo-virus within minutes. 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.