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. 2021 Jul 15;13(7):1371.
doi: 10.3390/v13071371.

Dynamic Assay for Profiling Anti-SARS-CoV-2 Antibodies and Their ACE2/Spike RBD Neutralization Capacity

Thomas Phelan  1 , Jean Dunne  2 , Niall Conlon  2   3 , Clíona Ní Cheallaigh  1   4 , W Mark Abbott  5 , Raquel Faba-Rodriguez  5 , Fatima Amanat  6 , Florian Krammer  6 , Mark A Little  7 , Gerry Hughes  1   8 , Colm Bergin  1   4 , Colm Kerr  1   4 , Sudharshana Sundaresan  1 , Aideen Long  1 , William McCormack  1 , Gareth Brady  1   7
Affiliations

Dynamic Assay for Profiling Anti-SARS-CoV-2 Antibodies and Their ACE2/Spike RBD Neutralization Capacity

Thomas Phelan et al. Viruses. .

Abstract

Serological assays have been widely employed during the coronavirus disease 2019 (COVID-19) pandemic to measure antibody responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and to track seroconversion in populations. However, currently available assays do not allow determination of neutralization capacity within the assay protocol. Furthermore, commercial serology assays have a high buy-in cost that is inaccessible for many research groups. We have replicated the serological enzyme-linked immunosorbent assay for the detection of SARS-CoV-2 antibody isotypes, developed at the Icahn School of Medicine at Mount Sinai, New York. Additionally, we have modified the protocol to include a neutralization assay with only a minor modification to this protocol. We used this assay to screen local COVID-19 patient sera (n = 91) and pre-COVID-19 control sera (n = 103), and obtained approximate parity with approved commercial anti-nucleoprotein-based assays with these sera. Furthermore, data from our neutralization assay closely aligns with that generated using a spike-based pseudovirus infection model when a subset of patient sera was analyzed.

Keywords: SARS-CoV-2; antibody; enzyme-linked immunosorbent assay; neutralization; pseudovirus infection model; serology.

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Conflict of interest statement

The Icahn School of Medicine at Mount Sinai has filed patent applications relating to SARS-CoV-2 serological assays and NDV-based SARS-CoV-2 vaccines which list Florian Krammer as co-inventor. Fatima Amanat is also listed on the serological assay patent application as co-inventors. Mount Sinai has spun out a company, Kantaro, to market serological tests for SARS-CoV-2. Florian Krammer has consulted for Merck and Pfizer (before 2020), and is currently consulting for Pfizer, Seqirus and Avimex. The Krammer laboratory is also collaborating with Pfizer on animal models of SARS-CoV-2. William McCormack is founder and director of Parvalis Tx, a TCD spin-out biotechnology company and is in receipt of consultancy fees for services provided. All other authors declare no competing interests. The funders had no role in the design, execution, interpretation, or writing of the study.

Figures

Figure 1
Figure 1
SARS-CoV-2 spike and RBD expression and purification by Peak Proteins Ltd. (A) Size exclusion chromatography UV absorbance trace of purified recombinant SARS-CoV-2 spike trimer (top, Superose 6 16/60 column, fractions 19–26 pooled) and SARS-CoV-2 spike RBD (bottom, Superdex 75 16/60 column, fractions 18–25 pooled). Retention volume of molecular weight standard thyroglobulin (top) and ovalbumin (bottom) marked as dashed green lines. (B) Reducing SDS-PAGE analysis of purified recombinant spike proteins.
Figure 2
Figure 2
OD values representative of antibody reactivity in sera from pre-COVID-19 and confirmed SARS-CoV-2 patients. (A–C) Results of samples tested against RBD. (D–F) Results of samples tested against full-length spike protein. Statistical analysis was performed using Mann–Whitney U tests in GraphPad Prism. The samples represented here were taken >7 days post symptom onset and include 103 pre-COVID-19 and 91 SARS-CoV-2 positive samples. Horizontal lines represent mean values and samples were performed in triplicate. (G–J) Examples of results from longitudinal samples.
Figure 3
Figure 3
Comparison between the SARS-CoV-2 anti-spike serology assay and commercially available assays. Samples tested with the RBD and spike proteins are shown as independent assays and are comprised of IgG1 and IgM patient data. These were compared with the same samples tested by the Roche and/or the Abbott assays where n = 69 and n = 84, respectively. Samples indicative of seropositivity are shown as “+”and negative as “−“. The tiles containing “NA” indicate samples in which no data is available for that assay.
Figure 4
Figure 4
Antibody spike RBD neutralization capacity in patient sera with different immunoglobulin profiles. (A–E) Demonstrates the spike RBD neutralization capacity of patients with various immunoglobulin profiles using serially diluted serum. The blue points represent the data generated using the in vitro ACE2 binding assay and the red points represent data generated from the same sample using the pseudovirus assay. Data are the mean ± the standard deviation of triplicate samples from a representative experiment (n = 3).
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