Structure-based Design of a Specific, Homogeneous Luminescence Enzyme Reporter Assay for SARS-CoV-2.
J Mol Biol
; 433(13): 166983, 2021 06 25.
Article
in English
| MEDLINE | ID: covidwho-1174385
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
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
SARS-CoV-2
/
COVID-19
/
Antibodies, Viral
/
Antigens, Viral
Type of study:
Diagnostic study
Limits:
Humans
Language:
English
Journal:
J Mol Biol
Year:
2021
Document Type:
Article
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