Figure 1

A flow chart summarizing the high-throughput production of recombinant antibodies from single plasma cells. Single cell-based cDNA synthesis was performed by MAGrahd. V genes were amplified from the cDNA by 5'-RACE PCR (Day 1). The PCR products were treated with TdT for 3'-end random nucleotide tailing. The reaction products were then mixed with Ig-cassettes to generate linear Ig-expression constructs by TS-jPCR. Cognate pairs of IgH- and IgL-expression constructs were then directly transfected into 293FT cells (Day 2). The V-(D)-J repertoire and IgG subclass were determined by direct sequencing (Days 3-4). The concentration and activity of the recombinant antibodies were determined by ELISA (Day 4). Pro, promoter; pA, poly(A) site; const, immunoglobulin constant region; NNN, 3'-end random-nucleotide tail.