A high-throughput neutralizing antibody assay for COVID-19 diagnosis and vaccine evaluation

Abstract

Virus neutralization remains the gold standard for determining antibody efficacy. Therefore, a high-throughput assay to measure SARS-CoV-2 neutralizing antibodies is urgently needed for COVID-19 serodiagnosis, convalescent plasma therapy, and vaccine development. Here, we report on a fluorescence-based SARS-CoV-2 neutralization assay that detects SARS-CoV-2 neutralizing antibodies in COVID-19 patient specimens and yields comparable results to plaque reduction neutralizing assay, the gold standard of serological testing. The fluorescence-based neutralization assay is specific to measure COVID-19 neutralizing antibodies without cross reacting with patient specimens with other viral, bacterial, or parasitic infections. Collectively, our approach offers a rapid platform that can be scaled to screen people for antibody protection from COVID-19, a key parameter necessary to safely reopen local communities.

Fig. 1. A high-throughput neutralizing antibody assay for COVID-19 diagnosis.

a Diagram of the cDNA constructs of wild-type (WT) SARS-CoV-2 (top panel) and mNG SARS-CoV-2 (bottom panel). The nucleotide positions of viral genome where mNG is engineered are indicated. b Assay flowchart. mNG SARS-CoV-2 was neutralized with COVID-19 patient sera. Vero CCL-81 cells were infected with the reporter virus/serum mixture with an MOI of 0.5. The fluorescence of infected cells was quantified to estimate the NT₅₀ value for each serum. c Representative images of reporter virus-infected Vero CCL-81 cells. Images for a positive neutralizing serum (top panel) and no serum control (bottom panel) are presented. Scale bar, 100 μm. d Neutralization curves. Representative neutralization curves are presented for three positive sera and one negative sera. The means and standard deviations from two independent experiments are presented. e Correlation analysis of NT₅₀ values between the reporter virus and PRNT assays. The Pearson correlation efficiency R² and p-value (two-tailed) are indicated.