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Isolation of single HIV-1 Envelope specific B cells and antibody cloning from immunized rhesus macaques.
Wang, Zijun; Merkenschlager, Julia; Chen, Spencer T; Oliveira, Thiago Y; Ramos, Victor; Gordon, Kristie M; Yao, Kai-Hui; Jankovic, Mila; Nussenzweig, Michel; Escolano, Amelia.
Afiliação
  • Wang Z; Laboratory of Molecular Immunology, The Rockefeller University, New York, NY 10065, USA.
  • Merkenschlager J; Laboratory of Molecular Immunology, The Rockefeller University, New York, NY 10065, USA.
  • Chen ST; Laboratory of Molecular Immunology, The Rockefeller University, New York, NY 10065, USA.
  • Oliveira TY; Laboratory of Molecular Immunology, The Rockefeller University, New York, NY 10065, USA.
  • Ramos V; Laboratory of Molecular Immunology, The Rockefeller University, New York, NY 10065, USA.
  • Gordon KM; Laboratory of Molecular Immunology, The Rockefeller University, New York, NY 10065, USA.
  • Yao KH; Laboratory of Molecular Immunology, The Rockefeller University, New York, NY 10065, USA.
  • Jankovic M; Laboratory of Molecular Immunology, The Rockefeller University, New York, NY 10065, USA.
  • Nussenzweig M; Laboratory of Molecular Immunology, The Rockefeller University, New York, NY 10065, USA; Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10065, USA.
  • Escolano A; Laboratory of Molecular Immunology, The Rockefeller University, New York, NY 10065, USA. Electronic address: aescolano@rockefeller.edu.
J Immunol Methods ; 478: 112734, 2020 03.
Article em En | MEDLINE | ID: mdl-31866284
ABSTRACT
Antibody cloning from single B cells is an essential tool for characterizing humoral immune responses and obtaining valuable therapeutic and analytical reagents. Antibody cloning from individuals with high serologic titers to HIV-1, Influenza, Malaria and ZIKV has led to new insights that inform vaccine design efforts. In contrast to humans and mice, less is known about antibody cloning from single B cells in macaques. Here, we describe a protocol to identify and purify single antigen-specific macaque B cells, and subsequently clone and produce macaque monoclonal antibodies. The sorting strategy requires the use of a combination of fluorochrome labeled antigens and omission of anti-IgG antibodies that can interfere with antigen binding and vice versa. Optimized methods for macaque antibody gene amplification, DNA preparation for antibody production and antibody screening by ELISA are also presented.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Linfócitos B / Anticorpos Anti-HIV / Separação Celular / Clonagem Molecular / Anticorpos Monoclonais Tipo de estudo: Guideline Limite: Animals / Humans Idioma: En Ano de publicação: 2020 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Linfócitos B / Anticorpos Anti-HIV / Separação Celular / Clonagem Molecular / Anticorpos Monoclonais Tipo de estudo: Guideline Limite: Animals / Humans Idioma: En Ano de publicação: 2020 Tipo de documento: Article