Fig. 2.

Cloning recombinant antibodies from single B cells. Peripheral blood mononuclear cells are stained with the appropriate markers to allow B cells of the desired phenotype to be individually sorted into each well of a collection plate containing lysis buffer. The variable region of both the heavy (VH) and light (VL) chains of immunoglobulin are RT-PCR amplified from each cell, sequenced and assessed for productive rearrangements. If both the VH and VL chains were successfully rearranged, they are further amplified by nested PCR such that terminal restriction enzyme sites are attached to prepare the sequences for cloning. The amplified sequences are digested and ligated into their respective expression vectors containing the human IgG1 constant region. Natively paired VH and VL vectors are then cotransfected into mammalian cells for recombinant antibody production and purification