Ja. Near et al., MONOCLONAL-ANTIBODIES REACTIVE WITH A MONOAMINE TRANSPORTER PREPARATION PURIFIED FROM BOVINE ADRENAL CHROMAFFIN GRANULE MEMBRANES, Biochemical pharmacology, 48(1), 1994, pp. 87-91
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.