Preparation and testing of monoclonal antibodies to recombinant proteins

Monoclonal antibodies (MAbs) are useful, often essential, reagents for the isolation, identification, and cellular localization of specific gene products and for the determination of macromolecular structure. The ability to clone and sequence specific genes has revolutionized our understanding of cellular structure and function, and the ability to prepare recombinant proteins or to synthesize peptides based on protein sequences derived from cDNA clones provides sufficient material for generating specific antibodies. The recombinant proteins may be derived from prokaryotic systems, such as E. coli, or from eukaryotic expression systems, such as Chinese hamster ovary cells (CHO) or insect cells expressing constructs in baculovirus. The eucary-otic systems are being used increasingly for expression of glycoproteins because the recombinant material is glycosylated. A number of protocols will be described here that we have used successfully with both rat (Y3 and IR983F) and mouse (SP2/0) myelomas to generate MAbs to recombinant material or peptides based on cDNA sequences.