Validation and Establishment of the SARS-CoV-2 Lentivirus Surrogate Neutralization Assay as a Prescreening Tool for the Plaque Reduction Neutralization Test

Abstract

Neutralization studies play an important role in providing guidance and justification for vaccine administration and helping prevent the spread of diseases. The neutralization data generated in our laboratory have been included in the decision-making process of the National Advisory Committee on Immunization (NACI) in Canada.

Plaque Reduction Neutralization Test (PRNT) Accuracy in Evaluating Humoral Immune Response to SARS-CoV-2

“…Our optimized assay was able to cover a wide range of NAb titers in reciprocal of the dilution from 10 (1.43 log 5 ) to 31,250 (6.43 log 5 ), which permitted the construction of a serological panel (negative, low, medium, and high levels of NAbs to SARS-CoV-2), which is essential to run the PRNT validation. This range proved to be wider than the ranges previously demonstrated, with titers from 1:10 to 1:600 [60], 1:20 to 1:1280 [50], 1:10 to 1:4000 [61], and 1:20 to 1:10,000 [62]. Moreover, this panel demonstrated an excellent repeatability of our PRNT-SARS-CoV-2, showing a reliable test with a positive correlation (r = 0.9826, p < 0.0001) between NAb titers in two independent assays.…”

High-Content Imaging-Based Assay for SARS-CoV-2-Neutralizing Antibodies

“…In this work, we used the imaging analysis to evaluate pseudovirus neutralization; however, a pseudovirus based on pHAGE-CMV-Luc2-IRES-ZsGreen-W vector also integrates luciferase reporter gene in the host cell, making it possible to validate neutralization by using other methods, such as a bioluminescence plate reader [ 26 , 27 ]. Therefore, neutralizing protocols like ours should be widespread in different countries to monitor and characterize the immune response by neutralizing antibodies in a specific population.…”

“…Repeatability (intra-assay) was determined by one researcher through the performance of three independent experiments (three viral batches) in triplicate. Reproducibility (inter-assay) was determined by two researchers who performed three independent experiments with five replicates [ 27 ]. The coefficient of variation (CV) was calculated by the ratio between the standard deviation and the pNT 50 mean values.…”

tANCHOR-cell-based assay for monitoring of SARS-CoV-2 neutralizing antibodies rapidly adaptive to various receptor-binding domains

“… 39 , 40 Pseudotyped-virus-based assays require BSL-2 conditions, but this method is not suitable for high-throughput screening (HTS) and cannot be rapidly adapted to upcoming variants. 41 , 42 Therefore, neutralization assays that are suitable for HTS without the use of infectious virus preparation are still needed. These are the reasons why ELISA-based assays mimicking the ACE-RBD interaction were developed.…”