Journal
JOURNAL OF IMMUNOLOGICAL METHODS
Volume 478, Issue -, Pages -Publisher
ELSEVIER
DOI: 10.1016/j.jim.2019.112734
Keywords
Single HIV-1 Envelope-specific B cell sorting; Rhesus macaque antibody cloning; Monoclonal antibody production and screening
Categories
Funding
- National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (NIH) [HIVRAD P01 AI100148]
- NIH Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery (CHAVI-ID) [1UM1 AI100663-01]
- Robertson Fund of the Rockefeller University
- NIH [K99/R00 9694871]
- China Scholarship Council [201806370101]
- EMBO fellowship
- NIAID of the NIH [F31AI147458]
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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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