A SMART method for efficiently isolating monoclonal antibodies from individual rhesus macaque memory B cells

Area of Science:

• Immunology
• Molecular Biology
• Primate Research

Background:

• Characterizing antigen-specific B cells in rhesus macaques (RMs) is vital for vaccine and infectious disease studies.
• Existing methods using 5' multiplex (MTPX) primers face challenges in capturing diverse RM IgV genes efficiently due to leader sequence variability.

Approach:

• Developed a novel switching mechanism at the 5' ends of the RNA transcript (SMART)-based method for amplifying IgV genes from single RM B cells.
• Optimized PCR conditions and utilized SMART 5' and 3' rapid amplification of cDNA ends (RACE) for full-length cDNA generation.
• Incorporated synthetic primer binding sites and universal 5' primers to enhance amplification of low-abundance antibody templates and simplify nested PCR.

Key Points:

• The SMART-based method enables unbiased capture of Ig heavy and light chain pairs from individual RM B cells.
• Successfully demonstrated the technique by isolating simian immunodeficiency virus (SIV) envelope-specific antibodies.
• The approach simplifies primer mixes and improves the recovery of matched antibody gene pairs.

Conclusions:

• This method enhances the isolation of antibodies from single RM B cells, facilitating genetic and functional characterization.
• It offers significant advantages over existing PCR cloning techniques for RM antibodies.
• The technique is expected to advance research in vaccine development and infectious diseases using RM models.