A novel design of low cost, ready to use cell based surrogate SARS-CoV-2 Neutralizing antibody assay (CENA) kit: for surveying mass scale anti-COVID-19 protective antibody
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Regular surveillance of herd immunity and individual protection by measuring neutralizing antibody against SARS-Cov-2 would be instrumental to control future COVID-19 pandemic. The SARS-COV-2 live virus neutralization tests or other surrogate virus neutralization tests could not be used for mass scale clinical application. A robust cell based surrogate SARS-Cov-2 neutralization assay (CENA) have been designed, based on blocking of GFP-tagged RBD (RBD-GFP) to its interaction with cell surface ACE2 receptor and detected by flow cytometry. A stable and high binding of RBD-GFP on cell surface was achieved using ammonium containing buffer inhibiting receptor-ligand endocytosis. Using DMSO containing frozen cells instead of conventional fresh cultured cells in the assay, enable further reduction of endocytosis as well as simple PI stanning of the nucleus for identifying cells in ordinary flow cytometry without additional staining protocol with antibody or any washing steps.. CENA were compared with (i) commercially available surrogate neutralization kit (cPass) and (ii) live virus neutralization assay showing sensitivity 97% and 100% respectively. Specificity of CENA using pre-COVID-19 pandemic samples were 100%. CENA were highly corelated with both live virus neutralization assay (r=0.9.p=0.0016) as well as with cPass kit(r=0.9,p=0.00001 respectively). Very poor correlation with conventional binding anti-Spike protein IgG level detected by ELISA (r=0.14), suggests that the conventional ELISA tests could not be a substitute for detecting neutralizing antibody. This is first ready to use cell based neutralization assay design, to be carried out without requirement of cell culture facility at end user’s site and could be completed in two hours. Besides surveying anti-SARS-COV-2 protective antibody, the assay could also be used for low budget high throughput screening of blocking molecules.
