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

Introduction: SARS-Cov-2 (severe acute respiratory disease coronavirus 2), which causes Coronavirus Disease 2019 (COVID19) was first detected in China in late 2019 and has since then caused a global pandemic. While molecular assays to directly detect the viral genetic material are available for the diagnosis of acute infection, we currently lack serological assays suitable to specifically detect SARS-CoV-2 antibodies. Methods: Here we describe serological enzyme-linked immunosorbent assays (ELISA) that we developed using recombinant antigens derived from the spike protein of SARS-CoV-2. These assays were developed with negative control samples representing pre-COVID 19 background immunity in the general population and samples from COVID19 patients. Results: The assays are sensitive and specific, allowing for screening and identification of COVID19 seroconverters using human plasma/serum as early as 3 days post symptom onset. Importantly, these assays do not require handling of infectious virus, can be adjusted to detect different antibody types and are amendable to scaling. Conclusion: Serological assays are of critical importance to determine seroprevalence in a given population, define previous exposure and identify highly reactive human donors for the generation of convalescent serum as therapeutic. Sensitive and specific identification of coronavirus SARS-Cov-2 antibody titers will also support screening of health care workers to identify those who are already immune and can be deployed to care for infected patients minimizing the risk of viral spread to colleagues and other patients.

[1]  Kaijun Jiang,et al.  SARS‐CoV‐2 Seroconversion in Humans: A Detailed Protocol for a Serological Assay, Antigen Production, and Test Setup , 2020, Current protocols in microbiology.

[2]  Wei Liu,et al.  Two-Year Prospective Study of the Humoral Immune Response of Patients with Severe Acute Respiratory Syndrome , 2006, The Journal of infectious diseases.

[3]  P. Felgner,et al.  Analysis of Serologic Cross-Reactivity Between Common Human Coronaviruses and SARS-CoV-2 Using Coronavirus Antigen Microarray , 2020, bioRxiv.

[4]  P. Palese,et al.  Expression of functional recombinant hemagglutinin and neuraminidase proteins from the novel H7N9 influenza virus using the baculovirus expression system. , 2013, Journal of visualized experiments : JoVE.

[5]  Jing Yuan,et al.  Treatment of 5 Critically Ill Patients With COVID-19 With Convalescent Plasma. , 2020, JAMA.

[6]  D. Tyrrell,et al.  The time course of the immune response to experimental coronavirus infection of man , 1990, Epidemiology and Infection.

[7]  Young-Jun Park,et al.  Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein , 2020, Cell.

[8]  Barney S. Graham,et al.  Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen , 2017, Proceedings of the National Academy of Sciences.

[9]  S. Blomqvist,et al.  Serological and molecular findings during SARS-CoV-2 infection: the first case study in Finland, January to February 2020 , 2020, Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin.

[10]  Yongyu Rui,et al.  Heat inactivation of serum interferes with the immunoanalysis of antibodies to SARS-CoV-2 , 2020, medRxiv.

[11]  Victor M Corman,et al.  Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR , 2020, Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin.

[12]  S. Subramaniam,et al.  Broadly protective murine monoclonal antibodies against influenza B virus target highly conserved neuraminidase epitopes , 2017, Nature Microbiology.

[13]  Kyoung-Ho Song,et al.  MERS-CoV Antibody Responses 1 Year after Symptom Onset, South Korea, 2015 , 2017, Emerging infectious diseases.

[14]  Jaap Goudsmit,et al.  Human Monoclonal Antibody Combination against SARS Coronavirus: Synergy and Coverage of Escape Mutants , 2006, PLoS medicine.

[15]  C. Cunningham-Rundles,et al.  A serological assay to detect SARS-CoV-2 seroconversion in humans , 2020, Nature Medicine.

[16]  Shaoqiang Li,et al.  Development and clinical application of a rapid IgM‐IgG combined antibody test for SARS‐CoV‐2 infection diagnosis , 2020, Journal of medical virology.

[17]  A. M. Leontovich,et al.  The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2 , 2020, Nature Microbiology.

[18]  Qian Wang,et al.  Heat inactivation of serum interferes with the immunoanalysis of antibodies to SARS‐CoV‐2 , 2020, Journal of clinical laboratory analysis (Print).

[19]  F. Krammer,et al.  Antibodies to the Glycoprotein GP2 Subunit Cross-React between Old and New World Arenaviruses , 2018, mSphere.

[20]  R. Couch,et al.  Immunization with SARS Coronavirus Vaccines Leads to Pulmonary Immunopathology on Challenge with the SARS Virus , 2012, PloS one.

[21]  E. Holmes,et al.  A new coronavirus associated with human respiratory disease in China , 2020, Nature.

[22]  Andrea Marzi,et al.  Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses , 2020, Nature Microbiology.

[23]  Kai Zhao,et al.  A pneumonia outbreak associated with a new coronavirus of probable bat origin , 2020, Nature.

[24]  Malik Peiris,et al.  Molecular Diagnosis of a Novel Coronavirus (2019-nCoV) Causing an Outbreak of Pneumonia , 2020, Clinical chemistry.

[25]  Jens C. Krause,et al.  A Carboxy-Terminal Trimerization Domain Stabilizes Conformational Epitopes on the Stalk Domain of Soluble Recombinant Hemagglutinin Substrates , 2012, PloS one.

[26]  S. Harrison,et al.  Structure of SARS Coronavirus Spike Receptor-Binding Domain Complexed with Receptor , 2005, Science.

[27]  Barney S. Graham,et al.  Pre-fusion structure of a human coronavirus spike protein , 2016, Nature.

[28]  B. Graham,et al.  Cryo-EM Structure of the 2019-nCoV Spike in the Prefusion Conformation , 2020, bioRxiv.

[29]  Jing Zhao,et al.  Early Transmission Dynamics in Wuhan, China, of Novel Coronavirus–Infected Pneumonia , 2020, The New England journal of medicine.

[30]  Zhènglì Shí,et al.  Potent binding of 2019 novel coronavirus spike protein by a SARS coronavirus-specific human monoclonal antibody , 2020, bioRxiv.

[31]  Fang Li,et al.  Crystal structure of NL63 respiratory coronavirus receptor-binding domain complexed with its human receptor , 2009, Proceedings of the National Academy of Sciences.

[32]  M. Eichelberger,et al.  Analysis of Anti-Influenza Virus Neuraminidase Antibodies in Children, Adults, and the Elderly by ELISA and Enzyme Inhibition: Evidence for Original Antigenic Sin , 2017, mBio.

[33]  F. Krammer,et al.  Cross-reactive antibodies binding to H4 hemagglutinin protect against a lethal H4N6 influenza virus challenge in the mouse model , 2019, Emerging microbes & infections.

[34]  F. Plummer,et al.  Neutralizing epitopes of the SARS-CoV S-protein cluster independent of repertoire, antigen structure or mAb technology , 2010, mAbs.

[35]  P. Palese,et al.  Age Dependence and Isotype Specificity of Influenza Virus Hemagglutinin Stalk-Reactive Antibodies in Humans , 2016, mBio.

[36]  Gintaras Deikus,et al.  Introductions and early spread of SARS-CoV-2 in the New York City area , 2020, Science.

[37]  Nicholas C. Wu,et al.  A highly conserved cryptic epitope in the receptor binding domains of SARS-CoV-2 and SARS-CoV , 2020, Science.