Scalable, Micro-Neutralization Assay for Assessment of SARS-CoV-2 (COVID-19) Virus-Neutralizing Antibodies in Human Clinical Samples

Abstract

As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic expanded, it was clear that effective testing for the presence of neutralizing antibodies in the blood of convalescent patients would be critical for development of plasma-based therapeutic approaches. To address the need for a high-quality neutralization assay against SARS-CoV-2, a previously established fluorescence reduction neutralization assay (FRNA) against Middle East respiratory syndrome coronavirus (MERS-CoV) was modified and optimized. The SARS-CoV-2 FRNA provides a quantitative assessment of a large number of infected cells through use of a high-content imaging system. Because of this approach, and the fact that it does not involve subjective interpretation, this assay is more efficient and more accurate than other neutralization assays. In addition, the ability to set robust acceptance criteria for individual plates and specific test wells provided further rigor to this assay. Such agile adaptability avails use with multiple virus variants. By February 2021, the SARS-CoV-2 FRNA had been used to screen over 5000 samples, including acute and convalescent plasma or serum samples and therapeutic antibody treatments, for SARS-CoV-2 neutralizing titers.

Keywords: COVID; COVID-19; SARS-CoV; SARS-CoV-2; antibodies; coronavirus; diagnosis; neutralization.

Figure 1

Sample dilution plate map for standard assay. (A). Up to seven samples were diluted twice for six dilution points on each plate and run on two duplicate plates. The yellow and blue shading indicate different replicates of each sample tested. (B). If more dilution points were required, samples were diluted across the entire plate for 12 dilution points and subsequently run on four plates.

Figure 2

Immunofluorescence staining of SARS-CoV-2-infected cells. (A). Non-infected cells stained with Hoechst nuclear stain (blue). (B). Cells infected with SARS-CoV-2 and probed with a SARS-CoV N-protein-specific antibody and Alexa594 secondary antibody (red). Cells were counterstained with Hoechst nuclear stain (blue).

Figure 3

Box plot results of virus control observations.

Figure 4

Box plot results of cell control observations showing variability between controls in individual experimental runs.

Figure 5

A histogram of the variability of virus control observations with fitted normal and beta distributions.

Figure 6

A histogram of the virus control observations (Figure 5) after outliers were removed.

Figure 7

A histogram of the variability of cell control observations with fitted normal and beta distributions.

Figure 8

A histogram of the cell control observations (Figure 7) after outliers were removed.