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A bioluminescent and homogeneous SARS-CoV-2 spike RBD and hACE2 interaction assay for antiviral screening and monitoring patient neutralizing antibody levels.
Alves, Juliano; Engel, Laurie; de Vasconcelos Cabral, Renata; Rodrigues, Eduardo L; de Jesus Ribeiro, Liane; Higa, Luiza M; da Costa Ferreira Júnior, Orlando; Castiñeiras, Terezinha Marta P P; de Carvalho Leitão, Isabela; Tanuri, Amilcar; Goueli, Said A; Zegzouti, Hicham.
  • Alves J; R&D Department, Promega Corporation, Madison, WI, USA. juliano.alves@promega.com.
  • Engel L; R&D Department, Promega Corporation, Madison, WI, USA.
  • de Vasconcelos Cabral R; Laboratório de Virologia Molecular, Departamento de Genética, Instituto de Biologia, Universidade Federal Do Rio de Janeiro, Rio de Janeiro, Brazil.
  • Rodrigues EL; Promega Biotecnologia Do Brasil, São Paulo, Brazil.
  • de Jesus Ribeiro L; Laboratório de Virologia Molecular, Departamento de Genética, Instituto de Biologia, Universidade Federal Do Rio de Janeiro, Rio de Janeiro, Brazil.
  • Higa LM; Laboratório de Virologia Molecular, Departamento de Genética, Instituto de Biologia, Universidade Federal Do Rio de Janeiro, Rio de Janeiro, Brazil.
  • da Costa Ferreira Júnior O; Laboratório de Virologia Molecular, Departamento de Genética, Instituto de Biologia, Universidade Federal Do Rio de Janeiro, Rio de Janeiro, Brazil.
  • Castiñeiras TMPP; Departamento de Doenças Infecciosas E Parasitárias, Faculdade de Medicina, Universidade Federal Do Rio de Janeiro, Rio de Janeiro, Brazil.
  • de Carvalho Leitão I; Instituto de Biofísica Carlos Chagas Filho, Universidade Federal Do Rio de Janeiro, Rio de Janeiro, Brazil.
  • Tanuri A; Laboratório de Virologia Molecular, Departamento de Genética, Instituto de Biologia, Universidade Federal Do Rio de Janeiro, Rio de Janeiro, Brazil.
  • Goueli SA; R&D Department, Promega Corporation, Madison, WI, USA.
  • Zegzouti H; Department of Pathology and Laboratory Medicine, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA.
Sci Rep ; 11(1): 18428, 2021 09 16.
Article in English | MEDLINE | ID: covidwho-1415954
ABSTRACT
Here we describe a homogeneous bioluminescent immunoassay based on the interaction between Fc-tagged SARS-CoV-2 Spike RBD and human ACE2, and its detection by secondary antibodies labeled with NanoLuc luciferase fragments LgBit and SmBit. The assay utility for the discovery of novel inhibitors was demonstrated with a panel of anti-RBD antibodies, ACE2-derived miniproteins and soluble ACE2. Studying the effect of RBD mutations on ACE2 binding showed that the N501Y mutation increased RBD apparent affinity toward ACE2 tenfold that resulted in escaping inhibition by some anti-RBD antibodies. In contrast, while E484K mutation did not highly change the binding affinity, it still escaped antibody inhibition likely due to changes in the epitope recognized by the antibody. Also, neutralizing antibodies (NAbs) from COVID-19 positive samples from two distinct regions (USA and Brazil) were successfully detected and the results further suggest the persistence of NAbs for at least 6 months post symptom onset. Finally, sera from vaccinated individuals were tested for NAbs and showed varying neutralizing activity after first and second doses, suggesting the assay can be used to assess immunity of vaccinated populations. Our results demonstrate the broad utility and ease of use of this methodology both for drug discovery and clinical research applications.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: Antibodies, Neutralizing / Spike Glycoprotein, Coronavirus / Angiotensin-Converting Enzyme 2 / SARS-CoV-2 / COVID-19 Type of study: Diagnostic study / Prognostic study Topics: Long Covid / Vaccines Limits: Humans Country/Region as subject: North America / South America / Brazil Language: English Journal: Sci Rep Year: 2021 Document Type: Article Affiliation country: S41598-021-97330-3

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Full text: Available Collection: International databases Database: MEDLINE Main subject: Antibodies, Neutralizing / Spike Glycoprotein, Coronavirus / Angiotensin-Converting Enzyme 2 / SARS-CoV-2 / COVID-19 Type of study: Diagnostic study / Prognostic study Topics: Long Covid / Vaccines Limits: Humans Country/Region as subject: North America / South America / Brazil Language: English Journal: Sci Rep Year: 2021 Document Type: Article Affiliation country: S41598-021-97330-3