Development and validation of IMMUNO-COV™: a high-throughput clinical assay for detecting antibodies that neutralize SARS-CoV-2

Vandergaast, Rianna; Carey, Timothy S.; Reiter, Samantha; Lech, Patrycja; Gnanadurai, Clement W.; Tesfay, Mulu Z.; Buehler, Jason; Suksanpaisan, Lukkana; Naik, Shruthi; Brunton, Bethany; Recker, Jordan; Haselton, Michelle; Ziegler, Christopher M.; Roesler, Anne; Mills, John R.; Theel, Elitza S.; Weaver, Scott C.; Rafael, Grace; Roforth, Matthew M.; Jerde, Clavin; Tran, Sheryl; Díaz, Rosa María; Bexon, Alice; Baum, Alina; Kyratsous, Christos A.; Peng, Kah-Whye; Russell, Stephen J.

doi:10.1101/2020.05.26.117549

Cited by 16 publications

(25 citation statements)

References 29 publications

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“…Tang 4 Despite widespread interest in neutralizing antibodies, methods for their detection and quantification are relatively low-throughput and limited to Biosafety Level 3-equipped research laboratories. While high-throughput methods have emerged, most rely on recombinant Vesicular Stomatitis Viruses (VSV) engineered to express a portion of the SARS-CoV-2 viral spike protein, and their subsequent entry into cell lines (12)(13)(14).…”

Section: Introductionmentioning

confidence: 99%

Association between SARS-CoV-2 neutralizing antibodies and commercial serological assays

Tang

Case

Franks

et al. 2020

Preprint

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AbstractIntroductionCommercially available SARS-CoV-2 serological assays based on different viral antigens have been approved for the qualitative determination of anti-SARS-CoV-2 antibodies. However, there is limited published data associating the results from commercial assays with neutralizing antibodies.Methods67 specimens from 48 patients with PCR-confirmed COVID-19 and a positive result by the Roche Elecsys SARS-CoV-2, Abbott SARS-CoV-2 IgG, or EUROIMMUN SARS-CoV-2 IgG assays and 5 control specimens were analyzed for the presence of neutralizing antibodies to SARS-CoV-2. Correlation, concordance, positive percent agreement (PPA), and negative percent agreement (NPA) were calculated at several cutoffs. Results were compared in patients categorized by clinical outcomes.ResultsThe correlation between SARS-CoV-2 neutralizing titer (EC50) and the Roche, Abbott, and EUROIMMUN assays was 0.29, 0.47, and 0.46 respectively. At an EC50 of 1:32, the concordance kappa with Roche was 0.49 (95% CI; 0.23-0.75), with Abbott was 0.52 (0.28-0.77), and with EUROIMMUN was 0.61 (0.4-0.82). At the same neutralizing titer, the PPA and NPA for the Roche was 100% (94-100) & 56% (30-80); Abbott was 96% (88-99) & 69% (44-86); and EUROIMMUN was 91% (80-96) & 81% (57-93) for distinguishing neutralizing antibodies. Patients who died, were intubated, or had a cardiac injury from COVID-19 infection had significantly higher neutralizing titers relative to those with mild symptoms.ConclusionCOVID-19 patients generate an antibody response to multiple viral proteins such that the calibrator ratios on the Roche, Abbott, and EUROIMMUN assays are all associated with SARS-CoV-2 neutralization. Nevertheless, commercial serological assays have poor NPA for SARS-CoV-2 neutralization, making them imperfect proxies for neutralization.

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Section: Introductionmentioning

confidence: 99%

Association between SARS-CoV-2 neutralizing antibodies and commercial serological assays

Tang

Case

Franks

et al. 2020

Preprint

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“…Our previously published SARS-CoV-2 neutralization assay relied upon virus-induced fusion of two dual split protein (DSP) reporter cell lines to generate a luciferase signal (21). To further improve assay throughput and eliminate the need for two cell lines we generated a recombinant VSV (VSV-SARS2-Fluc) encoding SARS-CoV-2 spike-Δ19CT (S-Δ19CT) in place of VSV-G, and firefly luciferase (Fluc) as an additional transcriptional unit located between the S-Δ19CT and VSV-L genes (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…VSV-SARS2-Fluc infects Vero cells via endogenously expressed ACE2 receptors (21). We hypothesized that ACE2 overexpression could enhance Vero cell susceptibility to VSV-SARS2-Fluc and thereby improve assay sensitivity.…”

Section: Resultsmentioning

confidence: 99%

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

Vandergaast¹,

Carey²,

Reiter³

et al. 2021

Preprint

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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 (VSV-SARS2-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 PRNT50% assay 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 seven 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 six-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.

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“…Donor blood samples were tested using the Ortho VITROS™ SARS-CoV-2 Total Ig assay, Abbott SARS-CoV-2 IgG assay, in-house ELISAs, and the Vyriad IMMUNO-COV™ neutralization assay as described with some modifications. (23) The IMMUNO-COV assay performed here differed from that which was described in the referenced publication in that: 1) plasma samples were heat-inactivated instead of serum samples, which is necessary due to thermal coagulation and 2) neutralization activity was quantified using neutralization end point titer (NT 100 ) method and not a standard curve.…”

Section: Methodsmentioning

confidence: 99%

Seroprevalence of Anti-SARS-CoV-2 Antibodies in a Cohort of New York City Metro Blood Donors using Multiple SARS-CoV-2 Serological Assays: Implications for Controlling the Epidemic and “Reopening”

Jin

Nesbitt

Yang

et al. 2020

Preprint

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Projections of the stage of the Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) pandemic and local, regional and national public health policies designed to limit the spread of the epidemic as well as reopen cities and states, are best informed by reproducible, high throughput, and statically credible antibody (Ab) assays. To date, a myriad of Ab tests, both available and authorized for emergency use by the FDA, has led to confusion rather than insight per se. The present study reports the results of a rapid, point-in-time 1,000-person cohort study using serial blood donors in the New York City metropolitan area (NYC) using multiple serological tests, including enzyme-linked immunosorbent assays (ELISAs) and high throughput serological assays (HTSAs). These were then tested and associated with assays for neutralizing Ab (NAb). Of the 1,000 NYC blood donor samples in late June and early July 2020, 12.1% and 10.9% were seropositive using the Ortho Total Ig and the Abbott IgG HTSA assays, respectively. These serological assays correlated with neutralization activity specific to SARS-CoV-2. The data reported herein suggest that seroconversion in this population occurred in approximately 1 in 8 blood donors from the beginning of the pandemic in NYC (considered March 1, 2020). These findings deviate with an earlier seroprevalence study in NYC showing 13.7% positivity. Collectively however, these data demonstrate that a low number of individuals have serologic evidence of infection during this first wave and suggest that the notion of herd immunity at rates of ~60% or higher are not near. Furthermore, the data presented herein show that the nature of the Ab-based immunity is not invariably associated with the development of NAb. While the blood donor population may not mimic precisely the NYC population as a whole, rapid assessment of seroprevalence in this cohort and serial reassessment could aid public health decision making.

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Development and validation of IMMUNO-COV™: a high-throughput clinical assay for detecting antibodies that neutralize SARS-CoV-2

Cited by 16 publications

References 29 publications

Association between SARS-CoV-2 neutralizing antibodies and commercial serological assays

Association between SARS-CoV-2 neutralizing antibodies and commercial serological assays

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

Seroprevalence of Anti-SARS-CoV-2 Antibodies in a Cohort of New York City Metro Blood Donors using Multiple SARS-CoV-2 Serological Assays: Implications for Controlling the Epidemic and “Reopening”

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