Virus Reduction Neutralization Test: A Single-Cell Imaging High-Throughput Virus Neutralization Assay for Dengue

Abstract

Abstract.Vaccine immunogenicity and clinical efficacy are often assessed by the measure of serum-neutralizing antibodies. The present gold standard for detecting neutralizing antibodies against many viruses, including dengue, is the plaque/focus reduction neutralization test (P/FRNT). The FRNT is a cell-based assay that inherits high variability, resulting in poor precision and has lengthy turnaround times. The virus reduction neutralization test (VRNT) is a high-throughput alternative to the standard low-throughput and laborious FRNT. The VRNT is similar to FRNT using unaltered wild-type virus and immunostaining, yet uses imaging cytometry to count virus-infected cells 1 day post-infection, reducing assay time and increasing overall throughput 15-fold. In addition, the VRNT has lowered variability relative to FRNT, which may be explained in part by the observation that foci overlap alters foci count and titer over time, in the FRNT. The ability to count one infected cell, rather than waiting for overlapping foci to form, ensures accuracy and contributes to the precision (7–25% coefficient of variation) and sensitivity of the VRNT. Results from 81 clinical samples tested in the VRNT and FRNT show a clear positive relationship. During sample testing, a 96-well plate edge effect was noted and the elimination of this edge effect was achieved by a simple plate seeding technique. The VRNT is an improvement to the current neutralization assays for its shortened assay time, increased precision and throughput, and an alternative to the P/FRNT.

Parameters of immunoglobulin extraction from dried blood spot cards and immunoassays for detection of antibody response to pathogens including the novel SARS-CoV-2

“…VN is considered as the gold standard test for measurement of protective immunity against viruses following vaccination or natural infection ( Haralambieva et al, 2008 ; Whiteman et al, 2018 ). Potential caveats in VN performed on DBS specimens could be extraction conditions and smaller extraction volumes in order to run the assay at lower dilutions.…”

A Novel In-Cell ELISA Assay Allows Rapid and Automated Quantification of SARS-CoV-2 to Analyze Neutralizing Antibodies and Antiviral Compounds

“…The actual data acquisition is conducted within seconds, using standard ELISA plate readers present in most routine diagnostics departments. We believe that this is advantageous compared to other NT assays that rely on more sophisticated and more expensive devises, e.g., for imaging cytometry ( 39 ). The icELISA and icNT provide increased data quality and precision by generating continuous data sets.…”

Current coronavirus (SARS-CoV-2) epidemiological, diagnostic and therapeutic approaches: An updated review until June 2020

“…Current COVID-19 diagnostic tools and techniques are shown in Table 1 (Tab. 1) (References in Table 1: Cai et al, 2020[ 7 ]; Carter et al, 2020[ 11 ]; Chan et al, 2020[ 16 ]; Chen et al, 2010[ 19 ]; Park et al, 2009[ 52 ]; Wang et al, 2005[ 65 ]; Whiteman et al, 2018[ 70 ]) and a diagnostic model for COVID-19 in Figure 5 (Fig. 5) .…”