A modified screening method for pcDNA-1 expression libraries which is applicable to both surface and intracellular...

A modification of the COS cell transient expression system for isolating cDNAs encoding antibody defined molecules was used to clone the carcinoma antigen defined by monoclonal antibody CORC-6. Bacteria were transformed with cDNA prepared from colon carcinoma cells and immediately divided into aliquots containing approximately 500 independent clones. The aliquots were grown for 8 h before frozen. Screening was performed by isolating plasmid from each aliquot, transfecting COS-7 cells, and staining the fixed cells with monoclonal antibodies. The aliquot giving rise to antigen expressing cells was then subdivided until a single antigen encoding colony was obtained. This approach allows a library to be rescreened and is equally applicable to cytoplasmic and surface localized antigens. Using this approach, the antigen defined by CORC-6 was identified as carcinoembryonic antigen.