Figure 7

High-throughput production of IgH- and IgL-expression constructs by TS-jPCR. (A) Ethidium bromide-stained agarose gels of the cognate pairs of VH and VL genes amplified from single plasma cells. Each lane contains 1 μl of a 25-μl VH or VL 5'-RACE PCR product. Representative PCR products from fifty-three samples are shown. H, heavy chain V gene fragment; L, light chain V gene fragment. (B) Agarose gels stained with ethidium bromide showing the TS-jPCR results. VH and VL genes obtained in (A) were treated with TdT and joined to the corresponding Ig-cassette. Each lane contains 1 μl of a 25 μl IgH- or IgL-expression construct. The fainter high-molecular weight bands are presumed to be a multimer of the correct band. Representative PCR products from fifty-three samples are shown. H, IgH-expression construct; L, IgL-expression construct. (C) Production of recombinant mouse monoclonal antibodies. Cognate pairs of IgH- and IgL-expression constructs (1 μl each) were directly transfected into 293FT cells. The concentration of the recombinant antibodies in the cell culture media was determined by a sandwich ELISA two days after the transfection (left panel). Antigen specificity against egg albumin is expressed as luminescence activity (right panel). The specific activity of recombinant antibodies is expressed as relative light units (RLU/s/IgG (μg)). N indicates a negative control; all incubation steps were identical except that nontransfected cell culture medium was used.