title: A rapid and efficient method for the generation and screening of monoclonal antibodies specific for cell surface antigens.
abstract: The generation of monoclonal antibodies (mAb) of desired specificity to cell surface antigens can serve as a valuable tool to study protein expression and function. However, traditional approaches to mAb generation usually involve large-scale protein purification and intensive screening, and may not result in mAb specificities to the native protein of interest. We describe a simple, inexpensive, high-throughput method for the generation and screening of hybridomas secreting mAb specific for cell surface receptors. Intact reporter cells expressing a CD3zeta-fusion receptor of the protein of interest are plated in 96-well arrays of captured, plate-bound hybridoma supernatants. A mAb to the protein of interest generates a signal leading to reporter-cell expression of beta-galactosidase, and enzyme activity can be screened in a single day using a non-radioactive substrate. Importantly, a single cell line can be used for immunization, screening, semi-quantitative affinity comparisons, and subsequent screening for physiological ligand expression, if the protein of interest is a receptor. We describe an application of this approach to generate mAb specific for a protein of previously unknown expression and undocumented function.
authors: Department of Immunology, University of Toronto, Sunnybrook Research Institute, 2075 Bayview Ave, Toronto, Ontario, Canada M4N 3M5.
authors: Aruz
authors: A
authors: Mesci
authors: James R
authors: JR
authors: Carlyle
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology*; D000918:Antibody Specificity; D000954:Antigens, Surface / Q000032:analysis / Q000276:immunology*; D017252:CD3 Complex / Q000276:immunology; D006801:Humans; D006825:Hybridomas; D007118:Immunoassay / Q000379:methods*; D047468:Immunoprecipitation; D037181:Lectins, C-Type / Q000235:genetics / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014764:Viral Proteins / Q000235:genetics / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D017252:CD3 Complex; C121704:CD3 antigen, zeta chain; D037181:Lectins, C-Type; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins