MONOCLONAL-ANTIBODIES REACTIVE WITH A MONOAMINE TRANSPORTER PREPARATION PURIFIED FROM BOVINE ADRENAL CHROMAFFIN GRANULE MEMBRANES

There have been no reports of monoclonal antibodies reactive with vesi cular monoamine transporters from any source. Western blotting and ELI SA data obtained using polyclonal serum from a mouse immunized with a highly purified bovine chromaffin granule monoamine transporter prepar ation yielded data consistent with the presence of antibodies to the t ransporter. Hybridomas produced by polyethylene glycol fusion of splee n cells from the mouse with X63/Ag8.653 myeloma cells were screened in an ELISA using partially purified transporter as coating agent. Of th e 1142 wells containing colonies, 14 were positive in the initial scre en. Hybridomas from wells testing positive were transferred to 24-well plates, grown up, and rescreened. Those still testing positive were s ubcloned, and the resulting positive wells containing single colonies were grown up and stocked. Of the 6 positive clones that tolerated fre eze/thaw (0.5% of the wells tested), 1 was IgG1 kappa, 2 were IgG2a ka ppa, and 3 were IgG2b kappa isotypes. Ascites fluid was generated in p ristane-primed BALB/c mice using hybridomas that had been cloned 2-3 t imes, and antibodies purified on immobilized Protein A. Immunoreactivi ty with a mixture of these antibodies, or with only one of them, coinc ided with dihydrotetrabenazine (TBZOH) binding activity in fractions e luted from all columns employed in the transporter purification. Antib ody from at least one of the clones was capable of removing [H-3]TBZOH binding activity from a partially purified preparation of transporter . Monoclonal antibodies exhibiting these properties have not been repo rted previously.