Introduction: The gold standard for diagnosing SARS-CoV-2 infection remains RT-PCR; however serological testing has an important role in epidemiology and assessment of vaccine responses. We expressed the receptor binding domain (RBD) of the spike (S) protein as a glycosylphosphatidylinositol (GPI)-anchored protein on CHO cells transfected with a generated construct and determined binding of RBD specific antibodies by immunofluorescence (IF) and flow cytometry (FACS) assays.
Methods and results: IF and FACS demonstrated cell surface RBD expression using anti-S RBD mAb and macaque immune sera. GPI anchored expression was confirmed through loss of positive cells on FACS post GPI anchor cleavage with phosphoinositide phospholipase C (PI-PLC). Binding of antibodies from COVID-19 convalescent sera to RBD- and mock-transfectants was performed by FACS. Positive results on serial dilutions of patient sera down to 1:3200 were observed and compared to in-house RBD ELISA which lost positivity at a 1:800 dilution. Soluble recombinant ACE2 bound to RBD-CHOs and binding was inhibited by COVID-19 convalescent sera indicating the RBD was expressed in a functional conformation and that neutralising antibodies were detected.
Conclusion: Mammalian cells expressing GPI-anchored RBD can successfully be used to detect previous SARS-CoV-2 infection and likely indicate the presence of neutralising antibodies in COVID-19 survivors.
|Number of pages||2|
|Issue number||Suppl. 1|
|Publication status||Published - Jul 2021|
|Event||Australasian Society for Infectious Diseases Annual Meeting - |
Duration: 24 Mar 2021 → 26 Mar 2021
- COVID-19 testing
- GPI-anchored RBD