A rapid and efficient method for the generation and screening of monoclonal antibodies specific for cell surface...

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.