IMMUNODETECTION OF MITOCHONDRIAL GLYCEROPHOSPHATE DEHYDROGENASE (MGDH) BY A POLYCLONAL ANTIBODY RAISED AGAINST A RECOMBINANT MGDH FRAGMENT PRODUCT

The mitochondrial enzyme glycerophosphate dehydrogenase (mGDH) plays a n essential role in the B-cell glucose-sensing device and its activity in islet homogenates is impaired in several animal models of type 2 d iabetes, We have now developed a polyclonal antibody, raised against a recombinant mGDH fragment product, that could be used for the immunod etection of mGDH. Total RNA was isolated from rat pancreatic islets an d used in the synthesis of cDNA, Specific primers were designed that c orresponded to the FAD binding domain of mGDH, The PCR product was pur ified and cloned into an appropriate expression vector used for transf ormation of Escherichia coli cells. The fusion protein was extracted f rom the transformed cells, further purified, and used for immunization of rabbits, The antibody recognized a single band of 72 kDa in rat is lets and testis. The recombinant mGDH product was also recognized as a single band with the expected 65-kDa reference, An ELISA procedure wa s designed for detection of antibodies against the recombinant mGDH fr agment product, The availability of the mGDH antibody opens the way to a number of further applications such as immunocytochemistry and mGDH quantification in biological material. (C) 1996 Academic Press, Inc.