Overview of the technique. (A) Soluble antigen or antigen domain is expressed with a BirA tag for site-specific biotinylatation and tetramerization with fluorescently labeled streptavidin. (B) B cells are stained with tetramer and a panel of monoclonal antibodies. Tetramer-positive class-switched memory B cells (CD19⁺ CD27⁺ IgM⁻) are single-cell sorted into PCR strips. (C) mRNA preamplification is performed with T7 RNA polymerase. Single-stranded cDNA is synthesized by the use of a primer with a single-stranded T7 RNA polymerase site. Conversion to double-stranded cDNA enables an in vitro transcription reaction with T7 RNA polymerase, which provides sufficient amounts of RNA for RT-PCR from resting, recirculating memory B cells. (D) Sequencing of PCR products is carried out directly from 300- to 400-bp PCR products by the use of second-round forward and reverse primers. (E) Overlap PCR is used for construction of full-length IgG1 heavy chain and κ light sequences, which are cloned into separate vectors. These vectors are transiently transfected into CHO-S cells for expression of fully human recombinant antibodies.