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Observational Study
. 2020 Jul;26(7):1033-1036.
doi: 10.1038/s41591-020-0913-5. Epub 2020 May 12.

A serological assay to detect SARS-CoV-2 seroconversion in humans

Fatima Amanat  1   2 , Daniel Stadlbauer  1 , Shirin Strohmeier  1   3 , Thi H O Nguyen  4 , Veronika Chromikova  1 , Meagan McMahon  1 , Kaijun Jiang  1 , Guha Asthagiri Arunkumar  1   2 , Denise Jurczyszak  1   2 , Jose Polanco  1   5 , Maria Bermudez-Gonzalez  1 , Giulio Kleiner  1 , Teresa Aydillo  1   6 , Lisa Miorin  1   6 , Daniel S Fierer  7 , Luz Amarilis Lugo  7 , Erna Milunka Kojic  7 , Jonathan Stoever  8 , Sean T H Liu  1   7   9   10 , Charlotte Cunningham-Rundles  11   12 , Philip L Felgner  13 , Thomas Moran  1 , Adolfo García-Sastre  1   6   7   14 , Daniel Caplivski  15 , Allen C Cheng  16 , Katherine Kedzierska  4 , Olli Vapalahti  17   18   19 , Jussi M Hepojoki  17   20 , Viviana Simon  1   6   7 , Florian Krammer  21
Affiliations
Observational Study

A serological assay to detect SARS-CoV-2 seroconversion in humans

Fatima Amanat et al. Nat Med. 2020 Jul.

Abstract

Here, we describe a serological enzyme-linked immunosorbent assay for the screening and identification of human SARS-CoV-2 seroconverters. This assay does not require the handling of infectious virus, can be adjusted to detect different antibody types in serum and plasma and is amenable to scaling. Serological assays are of critical importance to help define previous exposure to SARS-CoV-2 in populations, identify highly reactive human donors for convalescent plasma therapy and investigate correlates of protection.

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Conflict of interest statement

Competing interests

Mount Sinai is in the process of licensing out assays to commercial entities based on the assays described here and has filed for patent protection.

Figures

Extended Data Fig. 1 |
Extended Data Fig. 1 |. Constructs for recombinant protein expression.
a, Visualization of the trimeric spike protein of SARS-CoV-2 based on PBD # 6VXX using Pymol. One monomer is colored in dark blue while the remaining two monomers are held in light blue. The receptor binding domain (RBD) of the dark blue trimer is highlighted in red. b, Schematic of the wild type full length spike protein with signal peptide, ectodomain, receptor binding domain, furin cleavage site, S1, S2, and transmembrane and endodomain domain indicated. c, Schematic of the soluble trimeric spike. The polybasic/furin cleavage site (RRAR) was replaced by a single A. The transmembrane and endodomain were replaced by a furin cleavage site, a T4 foldon tetramerization domain and a hexahistidine tag. Introduction of K986P and V987P has been shown to stabilize the trimer in the pre-fusion conformation. d, Schematic of the soluble receptor binding domain construct. All constructs are to scale. e Reducing SDS PAGE of insect cell and mammalian cell derived soluble trimerized spike protein (iSpike and mSpike). f Reducing SDS PAGE of insect cell derived and mammalian cell derived recombinant receptor binding domain (iRBD and mRBD). Experiments were performed six times with the same result.
Extended Data Fig. 2 |
Extended Data Fig. 2 |. Human normal immunoglobulin preparations and historic sera from HIV + patients do not react with the SAR-CoV-2 spike.
a, b, Reactivity of 21 different pools of human normal immunoglobulin (HNIG) preparations (27 different vials) to mRBD and mSpike of SARS-CoV-2. MAb CR3022 was used as positive control, three different irrelevant human mAbs were used as negative control. c, d shows reactivity of historic samples from 50 HIV + individuals to mRBD and mSpike of SARS-CoV-2. Both HNIG and serum samples from HIV + donors were collected before the SARS-CoV-2 pandemic. Experiments were performed once. MAb CR3022 was used as positive control at a starting concentration of 100 ug/ml. Of note, the experiments in A and C as well as B and D were done at the same time and their positive controls are shared and displayed in both panels. Experiments were performed once.
Extended Data Fig. 3 |
Extended Data Fig. 3 |. Isotypes and subtypes of antibodies from COVID19 patients to the soluble spike protein and microneutralization titers.
a, Mammalian cell derived spike protein was used to study isotype/subclass distribution of antibodies (n = 13 positive samples). Lines represent the geometric mean. b, Microneutralization assay (n = 12) performed with authentic SARS-CoV-2. Lines represent curves fitted using an inhibitor (log) versus response variable slope with four parameters function in Graphpad Prism. Experiments were performed once.
Fig. 1 |
Fig. 1 |. Reactivity of control and SARS-CoV-2 convalescent sera to different spike antigens.
a–d, Reactivity to iRBD (a), mRBD (b), iSpike (c) and mSpike (d). Red, green and black data points/lines show the results for sera from SARS-CoV-2-infected individuals, a convalescent serum sample post-NL63 infection and other negative control samples, respectively. e–h, Data from the same experiment as in a–d, respectively, but plotted as AUCs to obtain a better quantitative impression (control samples: n = 50 for iRDB, iSpike and mSpike; n = 59 for mRBD; convalescent samples: n = 4 for iRBD and iSpike; n = 16 for mRBD and mSpike). Statistical analyses were performed using an unpaired two-tailed Student’s t-test in GraphPad Prism. Horizontal lines represent mean values. i,j, Reactivity of the 50 negative control samples from a–h against spike protein from human coronaviruses 229E (i) and NL63 (j). k, Correlation between ELISA titers and microneutralization titers (n = 12; the three samples from negative control sera overlap and are displayed as a single point). Statistical analysis was performed using Pearson’s rank test in GraphPad Prism. The experiments were performed once. IC50, half-maximum inhibitory concentration.
Fig. 2 |
Fig. 2 |. Effect of heat treatment and serum versus plasma on assay performance.
a,b, Reactivity of paired non-treated serum and heat-treated serum samples to mRBD (a) and mSpike (b) of SARS-CoV-2 (n = 5). c,d, Reactivity of paired serum and plasma samples to mRBD (c) and mSpike (d) of SARS-CoV-2 (n = 7). Statistical analyses were performed using a paired two-tailed Student’s t-test in GraphPad Prism. The experiments were performed once.
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Update of

  • A serological assay to detect SARS-CoV-2 seroconversion in humans.
    Amanat F, Stadlbauer D, Strohmeier S, Nguyen THO, Chromikova V, McMahon M, Jiang K, Asthagiri Arunkumar G, Jurczyszak D, Polanco J, Bermudez-Gonzalez M, Kleiner G, Aydillo T, Miorin L, Fierer D, Amarilis Lugo L, Milunka Kojic E, Stoever J, Liu STH, Cunningham-Rundles C, Felgner PL, Moran T, Garcia-Sastre A, Caplivski D, Cheng A, Kedzierska K, Vapalahti O, Hepojoki JM, Simon V, Krammer F. Amanat F, et al. medRxiv [Preprint]. 2020 Apr 16:2020.03.17.20037713. doi: 10.1101/2020.03.17.20037713. medRxiv. 2020. Update in: Nat Med. 2020 Jul;26(7):1033-1036. doi: 10.1038/s41591-020-0913-5. PMID: 32511441 Free PMC article. Updated. Preprint.

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