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Bio Protoc ; 11(10): e4026, 2021 May 20.
Article in English | MEDLINE | ID: covidwho-2326148


The recombinant receptor-binding domain (RBD) of the viral spike protein from SARS-CoV-1 and 2 are reliable antigens for detecting viral-specific antibodies in humans. We and others have shown that the levels of RBD-binding antibodies and SARS-CoV-2 neutralizing antibodies in patients are correlated. Here, we report the expression and purification of properly folded RBD proteins from SARS and common-cold HCoVs in mammalian cells. RBD proteins were produced with cleavable tags for affinity purification from the cell culture medium and to support multiple immunoassay platforms and drug discovery efforts. Graphic abstract: High-Yield Production of Viral Spike RBDs for Diagnostics and Drug Discovery.

Sci Immunol ; 5(48)2020 06 11.
Article in English | MEDLINE | ID: covidwho-595199


The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that first emerged in late 2019 is responsible for a pandemic of severe respiratory illness. People infected with this highly contagious virus can present with clinically inapparent, mild, or severe disease. Currently, the virus infection in individuals and at the population level is being monitored by PCR testing of symptomatic patients for the presence of viral RNA. There is an urgent need for SARS-CoV-2 serologic tests to identify all infected individuals, irrespective of clinical symptoms, to conduct surveillance and implement strategies to contain spread. As the receptor binding domain (RBD) of the spike protein is poorly conserved between SARS-CoVs and other pathogenic human coronaviruses, the RBD represents a promising antigen for detecting CoV-specific antibodies in people. Here we use a large panel of human sera (63 SARS-CoV-2 patients and 71 control subjects) and hyperimmune sera from animals exposed to zoonotic CoVs to evaluate RBD's performance as an antigen for reliable detection of SARS-CoV-2-specific antibodies. By day 9 after the onset of symptoms, the recombinant SARS-CoV-2 RBD antigen was highly sensitive (98%) and specific (100%) for antibodies induced by SARS-CoVs. We observed a strong correlation between levels of RBD binding antibodies and SARS-CoV-2 neutralizing antibodies in patients. Our results, which reveal the early kinetics of SARS-CoV-2 antibody responses, support using the RBD antigen in serological diagnostic assays and RBD-specific antibody levels as a correlate of SARS-CoV-2 neutralizing antibodies in people.

Antibodies, Viral/immunology , Betacoronavirus/immunology , Coronavirus Infections/diagnosis , Immunodominant Epitopes/immunology , Pneumonia, Viral/diagnosis , Protein Domains/immunology , Spike Glycoprotein, Coronavirus/chemistry , Zoonoses/blood , Animals , Antibodies, Monoclonal , Antibodies, Neutralizing , COVID-19 , Coronavirus Infections/blood , Coronavirus Infections/virology , Humans , Kinetics , Mice , Mice, Inbred BALB C , Pandemics , Pneumonia, Viral/blood , Pneumonia, Viral/virology , Protein Binding , Rabbits , Severe acute respiratory syndrome-related coronavirus/chemistry , Severe acute respiratory syndrome-related coronavirus/immunology , SARS-CoV-2 , Serologic Tests , Zoonoses/virology