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

Neutralizing antibody titer elicited through infection or vaccination is accepted as a reliable surrogate for protection from SARS-CoV-2 infection, hospitalization, and mortality. The gold standard for measuring neutralizing antibody levels relies on culturing live virus in the presence of a target cell and quantitating the level where 50% of the target cells are infected. These assays have numerous technical challenges, not the least is the requirement for a BSL-3 laboratory to perform the live virus testing. 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 sequesters the non-neutralizing antibodies from the samples, 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 (Spearmans correlation, ρ = 0.87 and 0.92, respectively). The analytical performance (LoB, LoD, LoQ, linearity, precision, and interference) of the Q-NAb IgG Test was established along with sensitivity and 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.