Preparing Live Cells for Immunization.

Abstract

Generating monoclonal antibodies against cell-surface (i.e., membrane) proteins can be challenging because membrane and membrane-associated proteins often lose their native conformation during the purification process. This also makes fusion screening very difficult. One widely used technique to overcome this issue is to overexpress the target protein in HEK 293T cells and then immunize the host with these cells. The advantage of immunizing with native cells is that the target protein is expressed and presented to the immune system in a correctly folded form with all of its secondary posttranslational structure in place. This is essential for conformational or discontinuous epitopes, and for transmembrane proteins that weave in and out of the cell membrane multiple times. Transient or stable transfectants can be used for immunization and for screening using fluorescence-activated cell sorting, western blot, or immunoprecipitation. Although transfectants often have higher expression levels than do native cells, care should be taken to ensure that the transfectant expresses a functionally active version of the target protein, as otherwise minor folding issues or modifications in structure can result in antibodies that recognize the transfected, but not the native, protein. Care must also be taken when using cells as immunogens because numerous antigenic proteins coimmunize with the target protein. Screening hybridomas using the same cells and counterscreening them on untransfected cells will enable the selection of specific hybridomas.