A rapid real-time polymerase chain reaction-based live virus microneutralization assay for detection of neutralizing antibodies against SARS-CoV-2 in blood/serum

Abstract

Background Individuals recovering from COVID-19 are known to have antibodies against the Spike and other structural proteins. Antibodies against Spike have been shown to display viral neutralization. However, not all antibodies against Spike have neutralizing ability although they may be cross-reactive. There is a need for easy-to-use SARS-CoV-2 neutralizing assays for the determination of virus-neutralizing activity in sera of individuals. Here we describe a PCR-based micro‐neutralization assay that can be used to evaluate the viral neutralization titers of serum from SARS-CoV-2 infected individuals. Methods The SARS-CoV-2 strain used was isolated from a nasopharyngeal specimen of a COVID-19 case. The limiting dilution method was used to obtain a 50% tissue culture infective dose (TCID50) of Vero cells. For the micro‐neutralization assay, 19 serum samples, with positive IgG titers against Spike Receptor-Binding Domain (RBD) were tested. After 24 hours, infected cells were inspected for the presence of a cytopathic effect, lysed and RNA RT-PCR conducted for SARS-CoV-2. PCR target Ct values were used to calculate percent neutralization/inhibition of SARS-CoV-2. Results Out of 19 samples, 13 samples gave 100% neutralization at all dilutions, 1 sample showed neutralization at the first dilution, 4 samples showed neutralization at lower dilutions, while one sample did not demonstrate any neutralization. The RBD ODs and neutralization potential percentages were found to be positively correlated. Conclusion We describe a rapid RT-PCR-based SARS-CoV-2 microneutralization assay for the detection of neutralizing antibodies. This can effectively be used to test the antiviral activity of serum antibodies for the investigation of both disease-driven and vaccine-induced responses.

Investigating the impact of prior COVID‐19 on IgG antibody and interferon γ responses after BBIBP‐CorV vaccination in a disease endemic population: A prospective observational study

“…A study from Sri Lanka by Jeewandara et al showed that BBIBP‐CorV vaccination resulted in 95% seroconversion at 6 weeks and also found antibodies associated with neutralizing activity to SARS‐CoV‐2 27 . Earlier, we reported IgG to RBD measured in both COVID‐19 convalescent and healthy individuals to be associated with neutralizing activity to SARS‐CoV‐2 28 . Here, we found IgG seropositivity at 8 weeks postvaccination in participants without a history of COVID‐19 to be lower at, 89.1% to Spike and 64.9% to RBD.…”

“…27 Earlier, we reported IgG to RBD measured in both COVID-19 convalescent and healthy individuals to be associated with neutralizing activity to SARS-CoV-2. 28 Here, we found IgG seropositivity at 8 weeks postvaccination in participants without a history of COVID-19 to be lower at, 89.1% to Spike and 64.9% to RBD. Our data correlates with previous reports from Pakistan that showed 78% of individuals to display antibody responses to RBD after vaccination with BBIBP-CorV.…”

IgG antibodies to SARS-CoV-2 in asymptomatic blood donors at two time points in Karachi

“…In this data set, we were not able to directly test all samples here for virus neutralizing activity. However, we have shown IgG to RBD as detected by the assay for neutralizing activity against SARS-CoV-2 and found this to correlation with IgG titers [ 33 ]. Therefore, we speculate that RBD antibodies found in our healthy blood donors reflected previous SARS-CoV-2 infection whilst a higher frequency of spike antibodies could be associated with cross-reactive antibodies.…”

Development of a Rapid Live SARS-CoV-2 Neutralization Assay Based on a qPCR Readout

“…This general test principle was previously demonstrated to be suitable also for other RNA viruses ( 41 ). Moreover, Abidi et al recently reported that RT-PCR can detect antiviral activity in selected sera with high anti-RBD antibody titers in a SARS-CoV-2 neutralization assay ( 42 ). However, the authors did not evaluate if their method can be used to determine neutralization titers, since only three different serum dilutions were used, and 13 out of the 19 sera exhibited 100% virus neutralization for all tested concentrations.…”