Cloning VH and VL genes from individual antigen-specific B cells is an attractive approach for producing monoclonal antibodies of the desired specificity. Current RT-PCR protocols, however, result in the successful identification of VH and VL gene pairs in about half of the sorted cells. Here, we demonstrate that single-cell RT-PCR is likely affected by stochastic factors, and that running PCRs in triplicate results in successful amplification of the expressed VH and VL genes in 90-100% of single sorted human B cells.
Keywords: B lymphocytes; heavy chain; immunoglobulin; light chain; single-cell PCR.