Hybridoma Cultures for Production of Antibodies: Purification and Specificity of Antibodies

During the process of characterizing the structure and function of novel antigens, it is usually necessary to create new monoclonal or polyclonal antibody reagents. However, once validated, these antibodies can be put to a multitude of experimental uses, such as detecting and quantitating antigens in cell and tissue extracts or biological fluids, purifying proteins for structural analyses, studying protein-protein interactions, and monitoring cellular differentiation. Although many well-characterized monoclonal antibodies (mAbs) to antigens of general interest are commercially available, mAbs to specialty antigens may need to be individually purified from hybridomas obtained from academic sources, or from not-for-profit cell repositories, such as the American Type Culture Collection (http://www.atcc.org ). This chapter describes protocols for producing, purifying, and verifying murine mAbs from pre-existing hybridomas.