A modified screening method for pcDNA-1 expression libraries which is applicable to both surface and intracellular antigens. Cloning of a colon carcinoma antigen.
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.
Grimm
T
T
Institute of Immunology, University of Munich, Germany.
Johnson
J P
JP
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000912:Antibodies, Neoplasm / Q000276:immunology; D000954:Antigens, Surface / Q000235:genetics* / Q000276:immunology; D002272:Carcinoembryonic Antigen / Q000235:genetics* / Q000276:immunology; D002277:Carcinoma / Q000235:genetics* / Q000276:immunology; D002461:Cell Line, Transformed; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular / Q000379:methods*; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000276:immunology; D018076:DNA, Complementary / Q000235:genetics*; D005347:Fibroblasts; D015723:Gene Library; D007124:Immunoenzyme Techniques; D010957:Plasmids / Q000235:genetics; D014162:Transfection
D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D000954:Antigens, Surface; D002272:Carcinoembryonic Antigen; D018076:DNA, Complementary