Screening human antibody libraries against carcinoma cells by affinity purification and polymerase chain reaction.
Bacterial scFv clones from a naïve antibody library have been isolated against cancer cell antigens with AffiSelect, a novel screening method that indirectly identifies candidate library members via an antigen reporter gene. The first step is the coating of carcinoma cell surface epitopes (antigen) with either mAbs, scFvs or phages (library members). Upon binding to a cell surface ligand, the library member generates a linking moiety. This facilitates magnetic affinity purification of the antibody-cancer cell complexes, detected by the polymerase chain reaction (PCR) using the beta-actin gene of the cancer cell as the target. Combining these well-known methods resulted in a higher resolution than a comparable cell-based ELISA method of detection. We have isolated human scFv antibodies against surface antigens of a lung carcinoma cell line. These were identified from a polyclonal mixture of phage display-enriched library clones comparing PCR patterns of the carcinoma cell line with the two negative cell types, HUVEC and peripheral blood cells (PBLs). The positive clones were sequenced and verified by FACS.
Reiersen
Herald
H
Affitech AS, Oslo Research Park, Gaustadalleen 21, Oslo, Norway. herald@affitech.com
Berntsen
Grete
G
Stassar
Marike
M
Cochlovius
Björn
B
D000199:Actins / Q000235:genetics; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D000912:Antibodies, Neoplasm / Q000235:genetics / Q000302:isolation & purification*; D000918:Antibody Specificity; D000951:Antigens, Neoplasm / Q000235:genetics / Q000276:immunology*; D001666:Binding Sites, Antibody; D002277:Carcinoma / Q000235:genetics / Q000276:immunology* / Q000473:pathology; D045744:Cell Line, Tumor; D002469:Cell Separation; D002846:Chromatography, Affinity / Q000379:methods; D042783:Endothelial Cells / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D005434:Flow Cytometry; D017930:Genes, Reporter; D006801:Humans; D007135:Immunoglobulin Variable Region; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008175:Lung Neoplasms / Q000235:genetics / Q000276:immunology*; D008280:Magnetics; D019151:Peptide Library; D016133:Polymerase Chain Reaction
D000199:Actins; D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; D000939:Epitopes; D007135:Immunoglobulin Variable Region; D019151:Peptide Library