[20] Monoclonal antibodies to vitamin B6

This chapter describes the preparation of monoclonal antibodies to vitamin B6. The advantage of monoclonal antibodies stems from the fact that they are essentially reagents, which are not subject to biological variability associated with antibodies prepared by methods involving conventional immunization of animals. As their introduction, monoclonal antibodies have rapidly become commonly used research tools. The chapter explores the applications of monoclonal antibodies to study pyridoxal 5′-phosphate (PLP), the coenzymatically active form of the six B6 vitamer forms. Identification of wells which contain hybridomas secreting antibodies to the haptenic PPxy group is made by an enzyme-linked immunosorbent assay (ELISA) procedure. For this purpose, PLP is reductively and covalently linked to a carrier protein which had not been used previously in the immunization of mice. Thus, PPxy-bovine serum albumin (PPxy-BSA)’ contains the haptenic PPxy group but no other antigenic determinants. The preparation of large amounts of monoclonal antibodies via ascites tumors production is a commonly used procedure which most often involves priming recipient mice by intraperitoneal injection of the immunosuppressant pristane (tetramethylpentadecane).