Identification of target membrane proteins as detected by phage antibodies.
The discovery of novel target antigens for antibody-based immunotherapy is still a major challenge. Antibody phage display is one of the technologies that is widely applied for the identification of novel cell surface molecules on intact eukaryotic cells and many reports describe the isolation of phage-antibodies binding to restricted cell populations such as cells in a certain pathological condition. However, the transition from cell-specific phage antibodies to the identification of the target antigens is still a major hurdle. Herein a method is described for the identification of these cell surface molecules using two complementary technologies. A genomic approach based on expression cloning can be used when cDNA libraries and antigen-negative cells are available. Otherwise, a proteomic approach based on small scale immunoprecipitation followed by large scale purification and mass-spectrometry-based identification can be applied. Correct identification of the antigens is confirmed using technologies such as recombinant expression of the target antigen followed by immunoprecipitation or cDNA transfection and FACS analysis.
Geuijen
Cecile A W
CA
Genmab BV, Utrecht, Netherlands.
Bakker
Arjen Q
AQ
de Kruif
John
J
D000914:Antibodies, Viral / Q000276:immunology*; D019921:Biotinylation; D002460:Cell Line; D003001:Cloning, Molecular; D005434:Flow Cytometry; D015723:Gene Library; D023281:Genomics / Q000379:methods*; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000032:analysis* / Q000276:immunology; D019151:Peptide Library; D010957:Plasmids; D040901:Proteomics / Q000379:methods*; D014162:Transfection
D000914:Antibodies, Viral; D008565:Membrane Proteins; D019151:Peptide Library