Abstract
BackgroundIndividuals 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.MethodsThe 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.ResultsOut 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.ConclusionWe 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.
Highlights
The first cases of SARS-CoV-2 infection were reported in the city of Wuhan, China on December 1, 2019, as pneumonia with an unknown etiology [1, 2]
A rapid RT-PCR-based live virus microneutralization assay for antibody detection against SARS-CoV-2
Studies have shown that patients who recovered from COVID-19 or, those who are vaccinated, maintain a high antibody titer against the Spike/receptor-binding domain (RBD) protein [3,4,5,6,7]
Summary
The first cases of SARS-CoV-2 infection were reported in the city of Wuhan, China on December 1, 2019, as pneumonia with an unknown etiology [1, 2]. Studies have shown that patients who recovered from COVID-19 or, those who are vaccinated, maintain a high antibody titer against the Spike/RBD protein [3,4,5,6,7]. There are several live virus neutralization assays in use, where the most common one is based on a plaque reduction neutralization test [4]. These assays require an agarose overlay, which makes the assay laborious to perform. Other live-virus neutralization assays are ELISA-based which are more effective than the plaque assays but still involve antibody labeling and processing steps [4]. We describe a PCRbased micro-neutralization assay that can be used to evaluate the viral neutralization titers of serum from SARS-CoV-2 infected individuals.
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