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

Characterizing antigen-specific B cells is a critical component of vaccine and infectious disease studies in rhesus macaques (RMs). However, it is challenging to capture immunoglobulin variable (IgV) genes from individual RM B cells using 5 ' multiplex (MTPX) primers in nested PCR reactions. In particular, the diversity within RM IgV gene leader sequences necessitates large 5 ' MTPX primer sets to amplify IgV genes, decreasing PCR efficiency. To address this problem, we developed a switching mechanism at the 5 ' ends of the RNA transcript (SMART)-based method for amplifying IgV genes from single RM B cells to capture Ig heavy and light chain pairs. We demonstrate this technique by isolating simian immunodeficiency virus (SIV) envelope-specific antibodies from single sorted RM memory B cells. This approach has several advantages over existing methods for cloning antibodies from RMs. First, optimized PCR conditions and SMART 5 ' and 3 ' rapid amplification of cDNA ends (RACE) reactions generate full-length cDNAs from individual B cells. Second, it appends synthetic primer binding sites to the 5 ' and 3 ' ends of cDNA during synthesis, allowing for PCR amplification of low-abundance antibody templates. Third, the nested PCR primer mixes are simplified by employing universal 5 ' primers, eliminating the need for complex 5 ' MTPX primer sets. We anticipate this method will enhance the isolation of antibodies from individual RM B cells, supporting the genetic and functional characterization of antigen-specific B cells.