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Abidi SH, Imtiaz K, Kanji A, et al. A rapid real-time polymerase chain reaction-based live virus microneutralization assay for detection of neutralizing antibodies against SARS-CoV-2 in blood/serum. PLoS One. 2021;16(12):e0259551doi: 10.1371/journal.pone.0259551.
Abidi, S. H., Imtiaz, K., Kanji, A., Qaiser, S., Khan, E., Iqbal, K., Veldhoen, M., Ghias, K., Simas, J. P., & Hasan, Z. (2021). A rapid real-time polymerase chain reaction-based live virus microneutralization assay for detection of neutralizing antibodies against SARS-CoV-2 in blood/serum. PloS one, 16(12), e0259551. https://doi.org/10.1371/journal.pone.0259551
Abidi, Syed Hani, et al. "A rapid real-time polymerase chain reaction-based live virus microneutralization assay for detection of neutralizing antibodies against SARS-CoV-2 in blood/serum." PloS one vol. 16,12 (2021): e0259551. doi: https://doi.org/10.1371/journal.pone.0259551
Abidi SH, Imtiaz K, Kanji A, Qaiser S, Khan E, Iqbal K, Veldhoen M, Ghias K, Simas JP, Hasan Z. A rapid real-time polymerase chain reaction-based live virus microneutralization assay for detection of neutralizing antibodies against SARS-CoV-2 in blood/serum. PLoS One. 2021 Dec 10;16(12):e0259551. doi: 10.1371/journal.pone.0259551. eCollection 2021. PMID: 34890401; PMCID: PMC8664206.
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PLoS One. 2021 Dec 10;16(12):e0259551. doi: 10.1371/journal.pone.0259551. eCollection 2021.

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

PloS one

Syed Hani Abidi, Kehkashan Imtiaz, Akbar Kanji, Shama Qaiser, Erum Khan, Kiran Iqbal, Marc Veldhoen, Kulsoom Ghias, J Pedro Simas, Zahra Hasan

Affiliations

  1. Department of Biological and Biomedical Sciences, Aga Khan University, Karachi, Pakistan.
  2. Department of Pathology and Laboratory Medicine, Aga Khan University, Karachi, Pakistan.
  3. Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal.
  4. Católica Biomedical Research; Católica Medical School, Universidade Católica Portuguesa, Lisboa, Portugal.

PMID: 34890401 PMCID: PMC8664206 DOI: 10.1371/journal.pone.0259551

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.

Conflict of interest statement

The authors have declared that no competing interests exist.

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