IMMUNO-COV v2.0: Development and Validation of a High-Throughput Clinical Assay for Measuring SARS-CoV-2-Neutralizing Antibody Titers

Rianna Vandergaast, Timothy Carey, Samantha Reiter, Chase Lathrum, Patrycja Lech, Clement Gnanadurai, Michelle Haselton, Jason Buehler, Riya Narjari, Luke Schnebeck, Anne Roesler, Kara Sevola, Lukkana Suksanpaisan, Alice Bexon, Shruthi Naik, Bethany Brunton, Scott C. Weaver, Grace Rafael, Sheryl Tran, Alina BaumChristos A. Kyratsous, Kah Whye Peng, Stephen J. Russella

Research output: Contribution to journalArticlepeer-review


Neutralizing antibodies are key determinants of protection from future infection, yet well-validated high-throughput assays for measuring titers of SARS-CoV-2-neutralizing antibodies are not generally available. Here, we describe the development and validation of IMMUNO-COV v2.0, a scalable surrogate virus assay, which titrates antibodies that block infection of Vero-ACE2 cells by a luciferase-encoding vesicular stomatitis virus displaying SARS-CoV-2 spike glycoproteins (VSVSARS2-Fluc). Antibody titers, calculated using a standard curve consisting of stepped concentrations of SARS-CoV-2 spike monoclonal antibody, correlated closely (P, 0.0001) with titers obtained from a gold standard 50% plaque-reduction neutralization test (PRNT50%) performed using a clinical isolate of SARS-CoV-2. IMMUNO-COV v2.0 was comprehensively validated using data acquired from 242 assay runs performed over 7 days by five analysts, utilizing two separate virus lots, and 176 blood samples. Assay performance was acceptable for clinical use in human serum and plasma based on parameters including linearity, dynamic range, limit of blank and limit of detection, dilutional linearity and parallelism, precision, clinical agreement, matrix equivalence, clinical specificity and sensitivity, and robustness. Sufficient VSV-SARS2-Fluc virus reagent has been banked to test 5 million clinical samples. Notably, a significant drop in IMMUNO-COV v2.0 neutralizing antibody titers was observed over a 6-month period in people recovered from SARS-CoV-2 infection. Together, our results demonstrate the feasibility and utility of IMMUNO-COV v2.0 for measuring SARS-CoV-2-neutralizing antibodies in vaccinated individuals and those recovering from natural infections. Such monitoring can be used to better understand what levels of neutralizing antibodies are required for protection from SARS-CoV-2 and what booster dosing schedules are needed to sustain vaccine-induced immunity. IMPORTANCE Since its emergence at the end of 2019, SARS-CoV-2, the causative agent of COVID-19, has caused over 100 million infections and 2.4 million deaths worldwide. Recently, countries have begun administering approved COVID-19 vaccines, which elicit strong immune responses and prevent disease in most vaccinated individuals. A key component of the protective immune response is the production of neutralizing antibodies capable of preventing future SARS-CoV-2 infection. Yet, fundamental questions remain regarding the longevity of neutralizing antibody responses following infection or vaccination and the level of neutralizing antibodies required to confer protection. Our work is significant as it describes the development and validation of a scalable clinical assay that measures SARS-CoV-2-neutraling antibody titers.

Original languageEnglish (US)
Article numbere00170-21
Issue number3
StatePublished - May 2021


  • Antibody titer
  • COVID-19
  • Clinical validation
  • High-throughput assay
  • Neutralizing antibodies
  • SARS-CoV-2
  • Surrogate virus

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology


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