Reconciliation of titer differences between SARS-CoV-2 neutralizing antibody assays

ABSTRACT SARS-CoV-2 antibody neutralization is a correlate of protection for COVID-19 and has been used to guide public health decisions. However, there is no standard neutralization assay, and absolute titers differ between assays. To investigate mechanisms causing these absolute titer differences, we compared 28 samples with three assays: a live virus cytopathic effect (CPE)-based microneutralization assay, a live virus focus reduction (immunospot, IS) assay, and a pseudotyped virus luciferase expression reduction (pseudovirus, PV) assay. We found significant correlations between all assays (ρ > 0.95 and P < 0.0001 for all pairwise comparisons), but also significant differences in absolute titers by assay (paired t -tests P < 0.001). IS and PV assays that assess neutralization as the titer required to reduce foci/luminescence by 50% had the smallest difference, with 1.2-fold higher IS assay titers (95% confidence interval, CI: 1.2–1.2). CPE assay titers were 5.5-fold lower (95% CI: 5.4–5.5) than in the IS assay. We show that these differences can be explained (i) by accounting for the size of inoculum being neutralized (half the infectious virions in the IS and PV assays, “per virion,” and the entire inoculum half the time in the CPE assay, “per well”) and (ii) by removing serum during the 5-day culture in the CPE assay (so serum can only neutralize the inoculum, rather than acting on the inoculum and slowing viral growth during culture). These findings highlight areas for more precise and harmonized terminology and particular assay elements that must be considered in efforts to harmonize neutralization assays. IMPORTANCE Protection against COVID-19 is related to how well serum antibodies neutralize SARS-CoV-2. This neutralization can be assessed using different assays, which often provide significantly different estimates. To better understand why assay results vary, we compared three commonly used types of neutralization assay. We found both strong correlations and significant differences in measured neutralization between the different assays. Differences in the type of virus (live SARS-CoV-2 virus compared to a pseudotyped virus with SARS-CoV-2 spike protein), how long cells, virus, and antibody were allowed to interact, and whether neutralization of a batch of virions (“per well” neutralization) or individual infectious virions (“per virion”) was measured, all impacted virus neutralization. Understanding which factors have the largest impact on the differences between neutralization assays is an important first step toward harmonization of neutralization assays across the field.