In response to the SARS-CoV-2 pandemic many vaccines have been developed and evaluated in human clinical trials. The humoral immune response magnitude, composition and efficacy of neutralizing SARS-CoV-2 are essential endpoints for these trials. Robust assays that are reproducibly precise, linear, and specific for SARS-CoV-2 antigens would be beneficial for the vaccine pipeline. In this work we describe the methodologies and clinical qualification of three SARS-CoV-2 endpoint assays. We developed and qualified Endpoint titer ELISAs for total IgG, IgG1, IgG3, IgG4, IgM and IgA to evaluate the magnitude of specific responses to the trimeric spike (S) antigen and total IgG specific to the spike receptor binding domain (RBD) of SARS-CoV-2. We also qualified a pseudovirus neutralization assay which evaluates functional antibody titers capable of inhibiting the entry and replication of a lentivirus containing the Spike antigen of SARS-CoV-2. To complete the suite of assays we qualified a plaque reduction neutralization test (PRNT) methodology using the 2019-nCoV/USA-WA1/2020 isolate of SARS-CoV-2 to assess neutralizing titers of antibodies in plasma from normal healthy donors and convalescent COVID-19 individuals. Graphical abstract Image 1 Highlights • Precision, Linearity, and Specificity are essential for Clinical Assay Qualification. • Vaccine or Infection-induced humoral response magnitude can be evaluated by high-throughput ELISAs. • Neutralization of SARS-CoV-2 is the gold-standard for in vitro vaccine efficacy evaluations. • ELISA, pseudovirus neutralization and PRNT assays are Clinically Qualified for SARS-CoV-2 vaccine trials. • Positive WHO control sample of 250 ABU equals 4.7 EPT for total IgG against SARS-CoV-2 trimeric spike antigen in ELISAs.
【저자키워드】 SARS-CoV-2, neutralization, ELISA, COVID, pseudovirus, Qualification, 【초록키워드】 COVID-19, IgG, IgM, Efficacy, Vaccine, pandemic, antibody, in vitro, SARS-CoV-2 vaccine, Receptor binding domain, Antigen, Replication, RBD, IgA, lentivirus, clinical, response, Antibody titer, plasma, WHO, Neutralizing, IgG3, humoral immune response, methodology, SARS-CoV-2 antigen, convalescent, trials, ELISAs, IgG1, Pseudovirus neutralization assay, PRNT, Neutralizing titer, Image, humoral, linearity, IgG4, Abstract, Endpoint, human clinical trials, trimeric spike, plaque reduction neutralization, Complete, healthy donor, control sample, evaluate, assays, evaluated, linear, functional, magnitude, individuals, the vaccine, inhibiting, the Spike, 2019-nCoV/USA-WA1/2020, the SARS-CoV-2, 【제목키워드】 Humoral immunity, methodology, clinical sample,