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

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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Fig. 1: Reactivity of control and SARS-CoV-2 convalescent sera to different spike antigens.
Fig. 2: Effect of heat treatment and serum versus plasma on assay performance.

Data availability

The data shown in the manuscript are available upon request from the corresponding author. Nucleotide sequences of both constructs have been submitted to NCBI (GenBank IDs MT380724.1 and MT380725.1). Expression plasmids have been deposited to BEI Resources (https://www.beiresources.org/), and plasmids and sequences are also available from the corresponding author. A detailed protocol for expression and ELISA setup has been published (https://currentprotocols.onlinelibrary.wiley.com/doi/full/10.1002/cpmc.100).

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Acknowledgements

We thank Y.-Z. Zhang (Fudan University) and E. Holmes (University of Sydney) for sharing the sequence of the first SARS-CoV-2 isolate in a very timely manner. We thank J. Garlick and J. Roney (Alfred Hospital, Melbourne) for data and specimen collection, N. Aboelregal for making many different NHIG products (Mount Sinai) available and L. Martinez-Sobrido (Texas Biomedical Research Institute) for initially characterizing mAb 1C7. We are also thankful to Genewiz for speeding up gene synthesis for this project, and for being very accommodating to our needs. Furthermore, we thank D. Tidmore for help with ordering primers quickly, and finally S. Dong for commuting to New Jersey on several occasions to pick up reagents from Genewiz. We also thank the Mount Sinai Health System Translational Science Hub ‘ConduITS’ (NIH grant U54TR001433) for supporting sample collection. The work of the Personalized Virology Initiative is supported by institutional funds and philanthropic donations. This work was partially supported by the NIAID Centers of Excellence for Influenza Research and Surveillance (CEIRS; contract HHSN272201400008C to F.K. and A.G.-S.), the Collaborative Influenza Vaccine Innovation Centers (CIVIC; contract 75N93019C00051 to F.K. and A.G.-S.), Open Philanthropy, the Australian National Health and Medical Research Council (NHMRC Program Grant 1071916 and NHMRC Research Fellowship Level B (1102792) to K.K.), the Academy of Finland to O.V. and J.M.H., as well as Jane and Aatos Erkko Foundation and Helsinki University Hospital Funds to O.V. Furthermore, we thank our generous community for providing essential funds and support for our SARS-CoV-2 and COVID-19 research efforts. The following reagent was deposited by the Centers for Disease Control and Prevention and obtained through BEI Resources, NIAID, NIH: SARS-Related Coronavirus 2, Isolate USA-WA1/2020, NR-52281. Finally, we thank all of the study participants for their contribution to the research. We wish the patients with COVID-19 a speedy recovery.

Author information

Affiliations

Authors

Contributions

F.A., V.S. and F.K. conceived of and designed the study. F.A., D.S., S.S., T.H.O.N., V.C., M.M., K.J., G.A.A., D.J., J.P., M.B.-G., G.K., T.A., L.M., D.S.F., L.A.L., E.M.K., J.S., S.T.H.L., C.C.-R., P.L.F., A.G.-S., D.C., A.C.C., K.K., O.V. and J.M.H. collected data and contributed samples. F.A., V.S. and F.K. analyzed the data and wrote the manuscript.

Corresponding author

Correspondence to Florian Krammer.

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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.

Additional information

Peer review information Alison Farrell is the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

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 Pymol3. 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 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 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.

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Amanat, F., Stadlbauer, D., Strohmeier, S. et al. A serological assay to detect SARS-CoV-2 seroconversion in humans. Nat Med (2020). https://doi.org/10.1038/s41591-020-0913-5

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