Evaluating Humoral Immunity against SARS-CoV-2: Validation of a Plaque-Reduction Neutralization Test and a Multilaboratory Comparison of Conventional and Surrogate Neutralization Assays

Microbiol Spectr. 2021 Dec 22;9(3):e0088621. doi: 10.1128/Spectrum.00886-21. Epub 2021 Nov 17.


The evaluation of humoral protective immunity against SARS-CoV-2 remains crucial in understanding both natural immunity and protective immunity conferred by the several vaccines implemented in the fight against COVID-19. The reference standard for the quantification of antibodies capable of neutralizing SARS-CoV-2 is the plaque-reduction neutralization test (PRNT). However, given that it is a laboratory-developed assay, validation is crucial in order to ensure sufficient specificity and intra- and interassay precision. In addition, a multitude of other serological assays have been developed, including enzyme-linked immunosorbent assay (ELISA), flow cytometry-based assays, luciferase-based lentiviral pseudotype assays, and commercially available human ACE2 receptor-blocking antibody tests, which offer practical advantages in the evaluation of the protective humoral response against SARS-CoV-2. In this study, we validated a SARS-CoV-2 PRNT to assess both 50% and 90% neutralization of SARS-CoV-2 according to guidelines outlined by the World Health Organization. Upon validation, the reference-standard PRNT demonstrated excellent specificity and both intra- and interassay precision. Using the validated assay as a reference standard, we characterized the neutralizing antibody response in specimens from patients with laboratory-confirmed COVID-19. Finally, we conducted a small-scale multilaboratory comparison of alternate SARS-CoV-2 PRNTs and surrogate neutralization tests. These assays demonstrated substantial to perfect interrater agreement with the reference-standard PRNT and offer useful alternatives to assess humoral immunity against SARS-CoV-2. IMPORTANCE SARS-CoV-2, the causal agent of COVID-19, has infected over 246 million people and led to over 5 million deaths as of October 2021. With the approval of several efficacious COVID-19 vaccines, methods to evaluate protective immune responses will be crucial for the understanding of long-term immunity in the rapidly growing vaccinated population. The PRNT, which quantifies SARS-CoV-2-neutralizing antibodies, is used widely as a reference standard to validate new platforms but has not undergone substantial validation to ensure excellent inter- and intraassay precision and specificity. Our work is significant, as it describes the thorough validation of a PRNT, which we then used as a reference standard for the comparison of several alternative serological methods to measure SARS-CoV-2-neutralizing antibodies. These assays demonstrated excellent agreement with the reference-standard PRNT and include high-throughput platforms, which can greatly enhance capacity to assess both natural and vaccine-induced protective immunity against SARS-CoV-2.

Keywords: COVID-19; SARS-CoV-2; immunity; immunoserology; neutralizing antibodies.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Angiotensin-Converting Enzyme 2
  • Animals
  • Antibodies, Neutralizing
  • Antibodies, Viral / immunology
  • COVID-19 / diagnosis*
  • COVID-19 / immunology*
  • COVID-19 Serological Testing / methods*
  • COVID-19 Vaccines / immunology
  • Chlorocebus aethiops
  • Diagnostic Tests, Routine
  • Enzyme-Linked Immunosorbent Assay / methods
  • HEK293 Cells
  • Humans
  • Immunity
  • Immunity, Humoral / immunology*
  • Neutralization Tests / methods*
  • SARS-CoV-2 / immunology*
  • SARS-CoV-2 / isolation & purification
  • Sensitivity and Specificity
  • Vero Cells


  • Antibodies, Neutralizing
  • Antibodies, Viral
  • COVID-19 Vaccines
  • ACE2 protein, human
  • Angiotensin-Converting Enzyme 2