Fig. 5

Construction and screening of intact IgG molecules. a The pQMCF IgG vector was constructed using single-step CPEC joining of 4 fragments: VH, VL, promoters/leaders and vector. The antibody heavy and light chains are expressed from the resulting vector as separate proteins that assemble naturally into IgG molecules secreted from mammalian cells. b. Western blot analysis of rabbit IgG secretion from CHOEBNALT85 cells transfected with pQMCF IgG library pool DNA constructed from VH and VL regions from a rabbit immunized with mouse CD48 protein. Goat polyclonal antibody against rabbit IgG heavy chain was used for the detection of free heavy chain in reduced sample conditions (DTT+) and of the assembled IgG molecule in non-reduced (DTT-) sample. c Mouse CD48 ELISA results obtained using serial dilutions of the same sample of library pool transfection media as primary antibody. d Distribution of positive and negative clones obtained from the screening of the library pool showed in the panel c. Three positive and two negative clones selected for sequencing are indicated. e Alignment of VL domain amino acid sequences of the positive and negative clones. CDR regions are underlined