Development and Validation of Novel Cell‐free Direct Neutralization Assay for SARS-CoV-2

Neutralizing antibody titer elicited through infection or vaccination is widely accepted as a reliable surrogate for protection from SARS-CoV-2 infection. The gold standard for measuring these titers viz. live-virus neutralization assay suffers from the following drawbacks: the need for a BSL-3 laboratory, limited reproducibility, and difficulty adapting to emerging variants. Using the Ancestral and BA.5 SARS-CoV-2 strains as model systems, we developed the Q-NAb IgG Test for the quantitative determination of neutralizing antibodies against SARS-CoV-2 variants, traceable to WHO International Standards. The test utilizes a novel Fusion Protein that mimics the Spike receptor binding domain docked to the human ACE2 protein and effectively blocks non-neutralizing antibodies in the sample. After pre-blocking samples with Fusion Protein, direct binding of the residual neutralizing antibodies to variant RBDs coated in the wells of the microtiter plate is measured with a fluorescent secondary antibody. Results of the Q-NAb IgG Test agree with a live-virus microneutralization assay for both the Ancestral strain (WA1–2020) and the Omicron BA.5 (COR-22-063113/2022) variant (Spearman’s correlation, ρ = 0.87 and 0.92, respectively). The analytical performance (sensitivity, linearity, precision, and interference) of the Q-NAb IgG Test was established along with specificity using a panel of monoclonal neutralizing and non-neutralizing anti-SARS-CoV-2 antibodies. Clinical sensitivity and specificity using pre-pandemic, convalescent, and vaccinated serum and plasma samples is also reported. The advantages of the Q-NAb IgG Test are its strong correlation to live virus neutralization tests, traceability to WHO International Standards, convenient microtiter plate format, low sample volume requirements, and suitability for a BSL-2 laboratory.