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 shown 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 and some may be cross-reactive. There is a need for easy-to-use SARS-CoV-2 neutralizing assays that allow 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 the cytopathic effect, then lysed and RNA RT-PCR of SARS-CoV-2. The 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, while 4 samples gave neutralization at lower dilution, while one sample did not give any neutralization. The correlation between RBD OD and neutralization potential was found to be statistically correlated. Conclusion We describe a rapid RT-PCR based SARS-CoV-2 microneutralization assay for detection of neutralizing antibodies. This can effectively be used to test anti-viral activity of serum antibodies for investigation of both disease-driven and vaccine-induced responses.

BBIBP-CorV (Sinopharm) vaccination- induced immunity is affected by age, gender and prior COVID-19 and activates responses to spike and other antigens

“…IgG to RBD is associated with neutralizing activity (45) (46). We found IgG to RBD measured in both COVID-19 convalescent and healthy individuals to be associated with neutralizing activity to SARS-CoV-2 (47). Aijaz et al reported from Pakistan that antibodies to RBD after BBIBP-CorV vaccination were associated with neutralizing antibodies (29).…”

Development of a Novel Medium Throughput Flow-Cytometry Based Micro-Neutralisation Test for SARS-CoV-2 with Applications in Clinical Vaccine Trials and Antibody Screening

“…Alternatively, recombinant SARS-CoV-2 virus expressing Green Fluorescent Protein can be detected directly without the need for further processing (25). Another approach is to detect viral genetic material in cell lysates through RT-qPCR following RNA extraction (26, 27) or to quantify replicating virus (viral load) by enzyme-linked immunosorbent assay (ELISA) (28). Other assays score the presence or absence of CPE under a microscope (29, 30) using a cell imager (31), or using colorimetric cellular dyes (29).…”