• Authors: Fellouse FA. et al.
  • Year: 2021
  • Journal: J Mol Biol 433 166983
  • Applications: in vitro / DNA / FectoPRO
  • Cell type: Expi293F
    Description: Human embryonic kidney Fibroblast
    Known as: Expi 293-F, Expi, HEK-293 Expi


- IgG and Glu-IgG fusions were produced in Expi293F cells by transient transfection, mixing equivalent amounts of expression construct DNA encoding the heavy and light chains in OptiMem serum-free media before complexation with FectoPro for 10 min. - Complexed DNA complex was then added to Expi293F cells and expressed for 5 days. - Expressed Abs were purified using rProtein A Sepharose, then buffer exchanged into PBS and concentrated using Amicon Ultra-15 Centrifugal Filter devices before storage.


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.